PART I

PART I I

PART II

CONTENTS

OF F ICE OF THE DIRECTOR

NAT IONAL CARIES PROGRAM

I EXTRAMURAL PROGRAMS

PART IV INTRAMURAL PROGRAMS

Dir

PART V

ec to r o f Intramural Res earch

Clinic al Den tal S ervices S ec tion

Sys tema tics Uni t

L abora tory o f Developmen tal Biology and AnomaliesConnec tive Tiss ues Se c tion

Craniofacial Anomalies Sec tion

Lab ora to ry o f B iochemis try

Pro te in Chemis try Sec tion

Enzyme Chemis try Se c tion

Lab o rato ry o f Biolo gical S truc ture

Experimen tal Morpho lo gy Sec t ion

Mole cular S truc ture Sec tion

Lab ora tory o f Microb iolo gy and ImmunologyCellular Immunology Sec tion

Humoral Immuni ty S ec tion

Microb iology Sec tion

Cl inical Immunology Sec tion

Labo ratory o f Oral Medic ine

Neurob iology and Anes thes iology B ranch

Anes thes iology Sec t ion

Neural Me chanisms Sec tion

Neuro cy tology and Experime n tal Anatomy Sect ion

Cl inical Inves tigations B ranch

Diagnos tic Me thodology Branch

O ral and Pharyngeal Development Sect ion

COLLABORATIVE RESEARCH PROJECTS

iii

REPORT OF THE D IREC TOR OF INTRAMURAL RE S EARCH

THE NAT IONAL INST ITUTE OF DENTAL RE SEARCH

Oc t ob er 1 , 19 78 S ep t emb er 30 , 1 9 7 9

The s c op e o f res ear ch in the In tramural Program encompas s e s many d if f erent

f iel d s o f b iomed ical s c ienc e a s can b e p er c e ived f rom r ead ing t h e summary

s ta t ement s o f t h e Lab o ra to ry and B ranc h Ch ief s a s well as the ind iv idual

p roj ec t repo r t s . Thes e summary s ta tement s s h ow tha t much o f the e f fo r t

i s conc erned wi th s eeking answer s to fundamental que s t ions in order to

de s ign me thod s and pro c edur e s fo r prevent ion and trea tmen t o f d i s eas es

and d i s o rd er s . T h es e s t ud ie s contr ibu t e knowledge t h a t is o f general

impo r tance t o s c ient i s t s s tudy ing d i s ea s e s no t only o f t h e mouth bu t o f

o ther par t s o f the b ody . An imal mod el s ar e us ed wid ely wh il e o t h er experi

ment s are p er f o rmed in vi t ro u s ing animal organs ,t i s s ue c el l s and c el l

component s o r c l inic al s amp l es . Exp eriment s are al s o carr ied ou t wi t h

synthe tic compound s . Inve s t igat ive d i s c ip line s pr e s ent ly repre s en t ed are

t h o s e o f b io c h emis t ry,b io l ogy ,

biop hy s ic s ,chemis t ry

,immuno logy ,

micro

b io logy , neuro p hys io logy ,and viro logy .

Las t year t h e Ins t i tu te ini t ia ted a s earc h f o r a new C l inica l D irec to r .

T h e s ear c h , cont inued t h i s y ear , was ex tend ed to countr ie s ou t s id e t h e

Unit ed S ta t e s . In Decemb er, 19 78 ,

Dr . Kar l—Ake Omne ll , Pro f es s o r , S cho o l

o f Dent is t ry,Lund s Universite t ,

Malmo,Swed en

,was o f f ered and ac c ep t ed

the po s it ion . He i s exp ec t ed to c ome on du ty la te in t h e Fal l o f 19 79 .

The Ins t i tu t e i s indeed f o r tuna te to have a p er s on o f his cal ib er and

s t and ing j o in the s taf f . As C l inica l D ire c t o r , Dr . Omne ll wil l b r ingl eader shi p and d ir ec t ion to our c lin ical d ental r es ear ch programs and

help narrow t h e gap t h a t h as ex is ted b e tween c l inical and bas ic s c ienc e

d is c ip l ines .

One o f t h e p r inc ipal f ac to r s wh ich made i t very d i f f icul t to a t t rac t qual i fied

cand ida t e s f o r t h e po s i t ion o f C linical D irec t or was a l imita t ion on t h e

numb er o f p o s i t ions tha t the Ins t i tu t e c ould make ava ilab l e to d evelop new

and expand ed p rogram e f f o r t s in the cl inic al ar ea . T h e r educ t ion in our

fu ll- t ime c eil ing o f 1 2 which was impo s ed on t h e Ins t i tu t e thi s y ear h a s

fur t h er hand icapp ed our e f f o r t s and exp ec ta t ions in th is d ire c t ion . Mor eover

and j us t a s cr i t ical the Ins titu te 's exi s t ing b as ic s c i enc e s lab ora tor ies have

had t o ab s o rb no t only the c o s t o f the 12 po s i t ions bu t al s o h ave had to come

up with t h e f our po s i tions c ommi t t ed as a minimum to the new C linica l Dire c tor .

This t o t al lo s s o f 16 p o s i tions ou t o f a re s earch s ta f f which to tal ed 145

r epr e s ent s a cu t o f over 112 in our bas ic re s ear ch e f f o r t s wh i ch have b een

and cont inue t o b e ou t s tanding ,high ly produc t ive , and in s evera l ins t anc e s

unique to N IH .

The po s it ion reduc t ion coup led with lack o f incr eas es in fund s , in s p i t e o f

rampant inf lat ion,mean that the Ins t i tu te 's Intramural P rogram is rap idly

lo s ing ground . I t i s no longer a ques t ion o f no— growth . I t is a ques t ion

o f s evere cu t—backs,even o f surviva l . Unle s s s ome thing can b e done to s top

this t rend,t h e Ins t i tu te and NIH may s h or tly lo s e s everal o f i t s highly

trained and mo s t talented emp loyees . I t s h ould no t b e nec e s sary here to

emp h as i z e the uniquenes s and advantage s o f the Intramural Programs a t an

ins t itu t ion l ike NIH . We need to do every thing po s s ib le to insure tha t they

cont inue to b e viab le and produc t ive .

The c eil ing reduc t ion has al s o a f f ec t ed ano ther o f t h e Ins t i tu te 's new

init ia tives adver s ely . Thus ,las t year the Ins t i tu te o f f ered , f o r the f ir s t

t ime , two-year appo intment f o r Cl ini cal Dental As s o cia tes under the NIH As s o c i

a tes P rogram . Ini t ial p lans were f o r the s ele c t ion o f two cand ida tes each year

f o r a to tal o f four . However,only one cand ida t e was appo inted las t year

p r imar ily b ecaus e o f lack o f qual i f ied app l i cant s . This year wi th ear lyd i s t r ibut ion o f the NIH As s o c ia tes P ro gram b ookl e t the Ins t i tu te exp ec ts

many more app l i cant s bu t may no t b e ab le to s elec t any o f them b ecaus e o f

the ce il ing cu t s .

T h e Ins t i tu te continues to exp er ienc e d i f ficul tie s in at trac t ing qual i f ied

mino r ity do c t oral cand ida te s to it s Intramural P rograms ,dif f i cul t ie s tha t

are fur t h er comp l ica t ed by t h e lo s s o f po s i t ions . A numb er o f mino r i ty

cand ida t es were given per s onal int erviews and were urged to ap p ly to the

C l inical Dental As s o c ia t e Program . None did . One den ti s t with ext ens ive

cl inical exper ience was appro ached regard ing a po s i t ion in the D ental S ervic e s

S e c t ion . He dec l ined . The po s i t ion was f illed,however , by a woman Com

mi s s ion O f f i cer , a f ir s t f o r the Ins t i tu te . Pro gr es s with res pec t to t h e

s c h edul ing o f vi s i t s by s taf f memb er s to mino r i ty ins t i tu t ions i s repor t ed

by the Ins t i tu te 's EEO C oo rd inat or .

A new S e c t ion o f Pro t eoglycan B io chemi s t ry was crea ted by the D irec tor o f

the Ins t i tu te in the Lab o rat ory o f B io c h emi s try and Dr . Vincent Has cal l o f

the Labora tory was appo int ed Chief o f t h e new s ec t ion . T h es e ac t ions which

were or ig inal ly reques t ed by the Chie f o f t h e Lab o rato ry wer e endo r s ed by

t h e Bo ard o f S c ien t i f i c C ouns elo r s fo l lowing a r eview o f the Labora t orylas t year .

In S ep t emb er , 19 7 9 , the Intramural Res earch Program s ponsored a highlysuc ces s ful f our and a hal f day Conf erenc e on B as ic Mechanisms o f Cel lular

S e cre t ion . T h e Conf erence o rganiz ed by Dr s . A . Hand and C . O l iver , Lab oratory o f B io logical S truc ture

,was at tended by over 200 p eop l e , many o f whom

were f rom fore ign countr ie s . In add it ion,rep res enta t ives o f mo s t o f the

o t h er Ins t i tu tes a t NIH and many o t h er Government Agenc ies par t i c ipa ted .

T h e mee t ing summar i z ed current knowl edge o f p ro t e in s ecre t ory mec h anisms

and their regu la t ion , addre s s ed new prob l ems,and de f ined new approaches

The e igh t— p er son Board o f S cient i f i c C ouns elo r s par t ic ipa ted in t h re e

s epara te p rogram r eviews .

_

Ad h95 _

memb e r s were added to each review group

to prov id e grea ter s c i en t i f i c exper t i s e . In S ep t emb er,the comp le t e Board

me t f or a day and a hal f to d i s cus s t h e ind ividual reviews in de ta il s and

make r ecommendat ions t o th e D irec t o r o f t h e Ins t i tu te and the D irec t o r o f

Intramural Re s ear ch .

In f i s c al 1 9 7 9 the fo l lowing NIDR S ta f f memb er s rec e ived award s and / o r

r eco gni t ion f or t h e ir contribu t ions to the Ins t itu t e 's p rogram

DHEW Di s t ingui s h ed S ervic e Award Dr . Mar ie U . Nylen ; N IH Dir ec to r 's

Award Dr . James B o sma ; NIH Mer i t Award Dr . Vinc en t C . Has c al l , Mr .

Guy R . Hawkins,Ms . Le ta S t evens on ; N IH EEO S p ec ial Ach ievement Award s

Ms . B arb ara Co r co ran , Ms . Mary Ann Wil l iams on ; Award s f rom O rgani z a tions

Ou t s ide U S PHS—NIH : Amer ican As s o c ia t ion o f Oral and Maxil lo fa cial S urgeons

Re s ear c h Re cogni t ion Award Dr . E dward Dr i s c o l l ; C ommand ing O f f ic er 's

Annual Award fo r -C ivism,Nat ional Naval Dental Center Dr . P aul H . Keyes ;

Alumni Mer i t Award,Cre igh ton Univer s i ty

,Omaha

,Neb ra ska Dr . C h arl es L .

Wit t enb erger ;

Ins titu t e s ta f f made f r equen t ap p earance s as invit ed s p eaker s,s e s s ion

chai rmen , and memb er s o f P rogram C ommi t t ee s a t b o th Na t ional and Inter

na t ional Mee t ing s ,Works h o p s and S ympo s ia . T h e fo l lowing mer i t s p ec ial

ment ion : Honorary Lec turer : Four th Annual Andr ew Mark Leppard Memo r ial

Lec ture,C o l lege o f P hy s icians and S urg eons ,

C o lumb ia Univer s i ty Dr .

Abner L . Notkins ; Invi t ed S peaker : Univer s i ty o f C op enh agen 500 Y ear sJub il e e S ympo s ium Dr . Abner L . Notkins ; Memb er

,S c ient i f i c Program

C ommi t t ee , Third Wor ld Congr e s s on Pa in , to b e h e ld 1981 Dr . Ronald Dubne r ;

Memb er , S c ien t i fic P ro gram Commit t e e ,Int erna t ional and Amer ican As s o c ia t ion

f o r Dental Res earch Dr . Mar i e U . Ny len .

Repo r t o f the C l ini cal Dental S e rvi ce s S e c t ion

O f f i c e o f th e Dire c t o r o f Int ramural Re s earch

Na t ional Ins t i tut e o f Denta l Re s earch

19 7 9

The C l ini cal Dental S e rvic e s S e c t ion furni s h e s d en tal c onsul ta t ive

service s and dental t h erap ie s fo r C l ini cal C ent er p at ien t s o f all In s t i

tuta s , o rigina t e s and carrie s out i t s own re s earch pro j e c t s and p rovide s

suppo r t and fac i l i t ie s fo r c l inica l p ro grams s pons ored b y the Denta l

Ins t i tut e 's labo ra to r ie s and b ranche s .

T h e C linical Denta l S e rvice s S ec t i on had an ac c red i t a t ion review

on Ap r il 4 , 19 79 by a rep re s en tat ive o f the C ounc il on Ho sp i tal and

Ins t i tu t ional Dent al S e rvi c e s , Amer ican Dental As s o c iat ion . C o l lab o ra

t ive re s ea rch p ro j e c t s we re reviewed by S c ient i f i c C ouns el o r s ,NIDR on

Apr il 5 , 19 79 .

During the pas t year a co o pe ra t ive e f fo rt b e twe en thi s s e c t ion and

the Depar tment o f Dent al Hygiene ,Bal t imo re Co l l ege o f Dental S urge ry ,

Unive rs i ty o f Maryland has le d t o s enio r s tud ent p ar t i c ipat ion in Dental

Cl inic pa t ient educa t ion and o rienta t ion programs . S tudent s may s e le c t

t h i s a s s i gnment a s addi t ional t ra ining in the ir Communi ty S e rv ice

P rac t i cum—Dental Hygiene Externsh ip s .

Ac t ive re s ear ch p roj e c t s ini t ia t ed by S e c t ion per s onnel inc lude

af t e r o ral surge ry ( 78—D-89 ) 2 0 1 DE 002 4 1-0 2 IR . Thi s s tudy evalua te s

t h e e f f e c t ivene s s o f f our t op i cal s o lut ions to reduce lo cal i z ed

o s t e i t i s and in fe c t ion - two adve rse s equelae as so c ia t ed wi t h to o th

ext rac t ion . The s olu t ions ( 1 ) chlo ramine— T , lx; (2 ) so d ium b i carbonat e ,

sa tura t ed so lu t ion ; ( 3 ) no rma l s al ine,

and (4 ) povidone~iodine ,

( 1% availab le io dine ) are us ed twic e da ily by the pa t ient a s t o p i ca lant i s ep t i c mouthrinse s .

evaluat ion o f the p re surg i cal and po s t surgi cal p sy cho lo gic al and

func t ional aspe c t s o f ind ividual s wi th d eve lopment al o s s eou s de fe c t s

o f the f ac e and j aws . The p sycho lo gi ca l impac t o f the surg i cal

co rre c t i on and po s t o pera t ive al t e ra t ion s in bone and t o o t h po s i t ion

are b e ing s tudied .

tha t a so dium f luoride mou thrinse t rans ien tly in crea se d t h e

vo lume o f who le sal iva in no rma l sub j e c t s . Re cent s tud ie s demons t ra t ed

that the f luo r ide mouthrins e produc ed a s ta t i s t ical ly s i gni f i cant in

crea s e in sal iva vo lume ove r b as e line when admini s t ered t o subj e c t s

xe ro s tomic due to radia t ion therapy,chemo therapy and S j o gren

'ssyndrome .

dontit is ( 78— D— 78 ) 2 01 DE 00 2 39— 0 2 IR . This s tudy compares the

e ffe ctiveness o f two home applied oral hygiene regimens in removing

subgingival too th accumulate d bac teria . Either conventional mechan

ical techniques alone or mechanical techniques in conj unct ion with

topically applie d sodium bicarbonate — sodium chloride paste" are

prescribed to pe riodonti tis patients who have had pocke t reduction

surgery performe d in one —half of their mouths .

The Clinical Dental Services Sect ion 's collaborat ion with NIDR

laboratorie s and branches involve the fo llowing proj ects :

Laboratory of Microbiolo gy and Immunology

Inve stigat ion o f cellular immunological mechanisms in

periodontal disease and the correla tion b e twe en pe riodontal

disease and histo compatibility antigens ( 76— D— 3 30 ) 2 01 DE

00205— 03 LMI .

Inve s tigat ion o f the et iology and the extra-salivary

complications o f Sj ogren 's syndrome ( 7 5—D— 54 ) 2 0 1 DE 00085— 06 LMI .

Obse rvations on the pe riodontal microbio ta o f patient swith ac tive pe riodontal lesions and with le sions contro lled

by personal oral hygiene and te tracycline the rapy ( 78—D— 7 7 )2 01 DE 00096 — 06 LMI .

Laboratory of Neurobiology and Ane sthe sio logy

A psychophysiological evaluation o f the e f fe ctivene s s o f

(and recove ry from) intravenous s e da tion for oral surge ry pro

c edure s in ambulatory patient s ( 76 —D— 148 ) 2 0 1 DE 00132 — 05 NA .

As se ssment and me asurement o f expe rimental and clinical

acute pain in a dental situation including an evaluation o f

known pha rmaco lo gical and non— pharmaco lo gical agents us ed in

pain contro l ( 7 5—D- 59 ) 2 01 DE 00 1 33— 05 NA .

Ef fe cts o f chronic ele ctric s timulation o f the brain and

narco t ic analge s ic s on human re sponses to noxious and non—noxious

stimuli : Clinica l and expe rimental evaluat ions ( 7 5—D— 4 )2 01 DE 00 784— 0 2 NA .

Tas te and its disorders 2 01 DE 00 2 12 — 03 C I .

Oral pharyngea l l ipas e ( 74— D— 1 3 ) 2 0 1 DE 00 2 18 — 0 3 C I .

The de ve lo pment and eva luat ion o f imp roved de nta l rad io

g raph sys tems , wi th emphas i s on fac t o rs in f luenc ing d iagno s t i c

pe rfo rmanc e 2 0 1 DE 0 00 6 5— 08 C I .

Lab o r at o ry o f O ra l Medic ing

The re la t ionship o f mechanic al t rauma t o o ra l aphthous

u lcer s ( 7 7-D— 9 7 ) 2 0 1 DE 00094—06 LOM .

Two pub l i ca t ions,no t inc lud ed in ind ividual prog re s s re po rt s , have

re sul t ed f rom co llabo ra t ive c l ini cal re s earch proj e c t s by thi s s ec t ion

(1 ) J . J . Baker,W . E . Wri ght

, S . P . Chan and J . J . Op penhe im : Longi tud inal

E f f e c t s o f C l inical Therapy and the Ed entulous S tat e on the Trans fo r

ma t ion o f Lympho cyt e s f rom Pat ien t s Wi th S eve re Pe rio don t i t i s . C l in .

Exp . Immuno l . (Nov . 19 78 ) 34 , 19 9— 2 05 . ( Co l labo ra t o rs : Li t tonBione tic s , Kensing t on , Md . and LMI , NIDR , N IH ( 2 ) B . J . Baum and

W . E . Wrigh t : Demons tra t ion o f Fib ronec t in a s a Maj o r Ext racel lular

P ro t ein o f Human G ingival Fib rob la s t s . J . Dent . Re s ., in p re s s .

( Co l lab orato r s : Pulmona ry B ranch , Na t ional Heart , Lung and Blo od

Ins t i tut e, NIH ) .

The C l inical Dental S e rvi ce s S ec t i on cont inue s to o f f e r a unique

and e s s en t i al oppo r tuni ty t o p rovid e a l ink b e tween ba s i c re s earch

lab o ra to ry ac t ivi t ie s , c l ini c al re s ea rch labo ra to ry ac t ivi t ie s and

the c l ini cal human di s ea s e p rob lem .

i0 c tober 19 18 to Septembe r 30 , 19 79

IT'

CF PROJECT (80 c harac te rsho logic al and Radio g

{ LAt ,LABORA TORY AND I N ST I T UT E AF F I L I AT I ON S , AND T I T L ES OF PR I NC I PAL I NV E ST I G ATORS AND ALL OTH ER

ations o f the Orthognathic Surgery Pat ient

hc su ECEAL PERS ONN EL ENGAG EO on THE PRO J ECTlP I Jame s B . SweetIOTHER : John F olio

Richard H . GracelyHarold A . Greenberg Chief Clinical Care

Jame s MaderoAlice A . Mac ynski

Donald P . Butler

None

Office of the Direc tor of Intramural Research

LClinic al Dental Services Sec tion

f lu if z iul; Ann LOCAT I O‘ NI DR ,

NIH,Be the sda , Maryland 2 02 05

The

l ob tained by tes ts,interviews ,

radio graphs , photo graphs and orthodontic study

CT 060 12 3

Dental Director IR NIDR

Chief Clin Dental Ser Sec t IR NIDR

Research Psychologis t NA NIDR

DCBR NIMH

Clin Care Cons Psychologist NIMHC linical Nurse (General ) IR NIDR

Oral Surg-Anes thesiologist NA NIDR

HUM AN T I SSU ES [3 (c ) NEIThER

o d '

gesb

ghat take place in individuals with

middle and lower facial bony defe c ts and malocclusions of the teeth af te r

cas ts taken at spe cific time inte rvals for a pe riod o f two years po stoperatively'

are compare d .

2 0 1 DE 002 13-0 3 IR

Pro j ec t Desc ription

I . Obj ec tives

Many individual s with middle and lower facial bony de formit ie s o r

severe malo cclus ion require surgical co rrec tion in addi tion to routine

orthodont ic treatment . This includes patient s with Clas s I I o r Clas s

I II maloc clusions , patient s with apertognathia , and patient s with

severe facial asymmetry .

The preoperative psycho logical s tate o f the se pa tient s i s influ

enc ed by their fac ial appearance , func tional disabili ty , and by

expec ted po st surgical changes .

Regres s ion and re laps e are common po s tope rative complications

as so ciated with orthognathic surgery .

The speci fic aims o f this s tudy are to es tab li sh the psychologicalimpac t o f the deformi t ies prior to surgery , to determine the expe c

tations f rom surgery and to correlate the findings after a 2 year

fo llow— up period . Pos tope ration change s in fac ial bone and too th

pos i tions which may occur are al so being examined .

II . Methods Employed

1 . Patient s be twe en 15 and 45 years o f age who have malo cclus ionsthat can be correc ted by surgery , or a combina tion o f surgery and

orthodont ics are examined and selected fo r s tudy .

2 . Preoperative pho tographs , s tudy mo del s and panoramic and

cepha lometric radio graphs are taken on each patient .

3 . P reope rat ively , each patient and a family member i s interviewed

by a p sychiat ri s t . Personality variable s are as se ssed by s tandard

psychological tes t s , and preopera tive expec ta tions are as ses sed by

a que s t ionnaire .

4 . Surgical pro cedures are performed in the operat ing room under

general anes thes ia us ing commonly accep ted techniques .

5 . At 3 months , 12 months , and 24 months fo llowing surgery

the bat tery o f p sycho logi cal te s t s are readminis te red and the pat ient

is reinterviewed by the psychiatris t . At this t ime , reso lution o f

pre surgical expec ta t ions and overa ll satis fac t ion o f the procedure

will be de termined .

Z01 DE 002 1 3—0 3 IR

6 . Pano ramic and cephal ome t ric rad iograp h s , s tudy model s , and

fac ia l p h o tographs wi l l b e t aken at the 3 , 6 , 12 and 24 month

int erval s to as s e s s po s t o perat ive s t ab i l i ty .

7 . At comp le t ion o f t h e s tudy a f inal eva luat ion and compar i s on

o f the resul t s wi l l b e done us ing c onvent ional bio s tat i s t i cal me t h od s .

I I I . Maj or Find ings

To da t e 1 5 sub j e c t s are in various s tage s o f t h e rapy . No

f ind ings are availab l e f o r repo r t ing s inc e th i s proj ec t en tail s

long t e rm po s t— s urgi cal evaluat ions .

IV . S igni f icance to B iomedic al Re se arch and t he P rogram o f t h eIns t i tut e

The inves t igat ion may inc reas e our unders tand ing o f pe rsonal i ty

and p syc h o log i cal pro f ile s in or t h ognat h i c surgery pat ient s . T h i swill al low a mo re tho ro ugh pre t rea tment evaluat ion and a mo re rea lis tic de f ini t ion o f the pa t ient s expe c ta t ions , which wil l re sul t in

our p rovid ing a b e t t er s e rvi ce t o the pa t ient .

V . P ropo s ed Cour s e

Con t inue unt il approximat ely f i f ty pa t ien t s have b een comp le t ed .

Pub l ica t ions : None

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Evaluation o f Surge ry and Antibac terial Agents in Contro l o f Periodont it is

N AM ES,L A BOR A TORY ANO I N STI T UT E AFF ILIATTIONS,ANO T ITLES

“

OF PR I NC I PAL INvEsTIGATORS AND ALL OT H E RPR OF E S S I ON AL P ER S ON N EL EtIG AeEO ON THE PR O JECT

PI Wi lliam E . Wright S r . S taf f Dentis t

O ther : Paul H . Keyes Dental Direc tor

Surya A . Howard Heal th Technician

(Dental )Marj orie L . Meehan Heal th Technician IR NIDR

(Dental )

[3 (b ) H UM AN T I S S U E S (c ) N E I T H ER

SUMM AR Y OF WOR K (200 wo rd s or le s s und e r lin e ke ywo rd s )

with top ical app licat ions o f sodium bicarbonate— sod ium chloride pas te to

contro l accumulations o f oral bact eria will be compared in subj ec t s wi th

thi s commonly used mode o f periodontal disease the rapy . Evaluat ion crit eriawill be ( 1 ) mo tile micros copic fo rms and white blood ce ll s ob tained from perio

dontal po cket s will be viewed and numerically es timated by phase micro scopy ,

( 2 ) gingival sulci b leeding index and ( 3 ) measurement s o f gingival at tachment s

relative to the cemento- enamel j unc tion and g ingival margin .

2 01 DE 00 21 3-0 3 IR

6 . Pano ramic and c ephal ome tr i c radiograp h s , s tudy mo del s , and

f ac ial p h o tographs wi l l b e taken a t the 3 , 6 , 12 and 24 month

int erval s to as s e s s po s t opera t ive s t ab i l i ty .

7 . At comp le t ion o f th e s tudy a f inal evaluat ion and compar i s on

o f the resul t s wi l l b e done us ing convent ional b io s tat i s t i cal me t h od s .

I I I . Maj or Find ings

To da t e 15 sub j e c t s are in various s t age s o f therapy . No

f ind ing s are avai lab l e fo r repo r t ing s ince thi s proj e c t entail s

long t e rm po s t-surgi cal evaluat ions .

IV . S igni f icance to B iomed ic al Re sear ch and the P rogram o f the

Ins t i tut e

Th e inves t igat ion may increas e our unders tand ing o f per sonal i ty

and p sycho log i cal pro f ile s in or thognathic surgery pa t ient s . Thi s

will al low a mo re tho rough pre t rea tment evaluat ion and a mo re rea lis tic de f ini t ion o f t h e pa t ient s expec ta t ions , which wil l re sul t in

our provid ing a b e t t e r s ervi ce to t h e pa t ient .

V . P ropo s ed Cour s e

Con t inue unt il approximat ely f i f ty pa t ient s have b een comp le ted .

Pub l icat ions : None

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I Oc tober 1 , 1 9 7 8 to S ept ember 30 , 19 7 9 CT 06 012 2

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Evaluat ion o f Surgery and Antibac terial Agents in Contro l o f Periodont it i s

N AM E S,L A BOR A TO RY ANO I N ST I TUTE AF F I L I A TION S , AND T I TLES OF PR I NC I PAL I NV EST I G AT OR S ANO ALL OT H E R

PROF E S S I ON AL PER S ON N EL ENCACEO ON THE PR O JECT

PI William E . Wright S r . S ta f f Dentis t

O ther : Paul H . Keyes Dental Direc tor

Surya A . Howard Heal th Technician

(Dental )Marj o rie L Meehan Health Technic ian IR NIDR

(Dental )

[3 (b ) H UM AN T I S S U E S I] (c ) N E I T H E R

S UMM A R Y OF WOR K (200 wo rd s or le s s und e r lin e k e yword s )

with top ical applicat ions o f sodium bicarbonate— sodium chloride pas te to

control accumulat ions o f oral bacteria will be compared in subj ect s wi th

this commonly used mode o f periodontal di sease the rapy . Evaluat ion criteria

will be ( 1 ) mo tile micros copic forms and white blood cell s ob tained from perio

dontal pocke t s will b e viewed and numerically es timated by phase micro scopy ,

( 2 ) gingival sulc i bleeding index and ( 3 ) measurement s o f gingival at tachment s

relative to the cemento- enamel j unc tion and gingival margin .

Z01 DE 002 39—02 IR

Proj e c t De s cript ion

I . Obj ec t ive s

The purpo s e o f thi s cl inical re s earc h p roj e c t i s to compare t h ee f f e c t ivenes s o f two home app l ied o ral hygiene regimens in r emovingsubg ingival to o th ac cumulat ed convent ional mec h ani calt ec hnique s-b rushing , flo s s ing and wat er irrigat ion ver sus (2 ) t h e

s ame convent ional me chani cal t e chnique s in conj unc t ion wi t h t op i calapp l icat ions o f

,a s odium b i carb onat e— so dium chlo ride "pa s t e P erio

dontal po cke t reduc t ion surgery wil l b e per fo rme d in two o f four

po s t erior quadrant s o f al l sub j e c t s s elec t ed fo r t h i s s tudy wi th the

d iagno s i s o f mo dera te general i z ed p er io dont i t i s .

Comb inat ions o f surgical area s ver su s nonsurg i cal area s in

conj unc t ion wi th one o f the two h ome therapy regimens wil l b e evaluated as to t h ei r ro le in e f fe c t ively as s i s t ing t h e p at ient to p re

vent or cont ro l p rogre s s ive de s t ruc t ion o f t h e per io dontal at t ac hmentappara tu s .

Me thod s Emp loyed

1 . Male o r f emale pa t ient s ranging in age f rom 20—49 year s

and demonstrat ing mode rat e general iz ed p erio dont i t i s will

b e s elec t ed fo r s tudy .

2 . P re— s tudy ba s el ine p eriodon tal parame ter s will b e re co rded ;

Evaluat ion cr i t er ia will inc lude ( 1 ) mo t ile micro s cop ic f o rms

and whit e b loo d cel l s ob ta ined fr om sub gingiva l p laque s crap

ings wil l b e numer ically e s t imat ed by p has e mi cro s c opy .

( 2 ) gingival sul c i b leed ing index and ( 3 ) measurement s o f

gingival at t achmen t s relat ive t o the cemento-enamel j unc t ionand g ingiva l margin .

3 . Sub j ec t s wil l b e randomly as s igned to per f o rm one o f two

o ral b ac t er ial cont ro l regimens ( convent ional mec h anicalonly o r convent ional mechani cal p lus daily t op ical appli

cat ions of an "ant imi crob ial s al t Random s el ec t iono f two po s t erio r quadrant s to re ceive periodontal p o cke t

reduc t ion surgery wil l b e us ed fo r al l subj ec t s .

4 . Cr i t eria evaluat ions wil l b e made b e fo re t rea tment , a f t er

surgery one month and at three month int erval s throughout

the t h ree year s tudy .

5 . Pat ient achievement goal s will b e an app rec iab l e reduc t ion

in the numb er s o f mo t ile bac ter ial f o rms and whi t e b lo o d

cel l s in their subgingival p laque s c rap ing s as de t e rmined

by phase micro s co py . Al s o a reduc t ion in gingival b leeding

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O ctober 1 , 19 7 8 to Septembe r 30 , 19 79 CT 06012 4

Effect o f V arious Preoperative and Po stoperative Rinses on Healing After

Oral Surgery

LABCRATORY Ana IN ST I T UT E AFF I L I AT I ON S , AND T I T L ES OF PR I NC I PAL IN V EST I G AT OR S AND ALL OT H E RPRuFaSS aoSAL PER SONN EL ENG AG ED ON THE PR O JECT

PI James B . Sweet Dental Director IR NIDR

OTHER : Alice A . Mac ynski Clinical Nurse IR NIDR

Donald P . Butler Oral Surg-Anesthesiologis t IR NIDR

LRATINC uulrs (if any )

i Of fip e _

o f the Director of Intramural Research

Clinical Dental Services Section

Ana LOCAT I ONIH

,Bethesda , Maryland 2 0 205

El ; MAHY L ARS :

uumrn SU B JECT S HUM AN T I S S U E S Lj (c ) NElTt

I h” H Y Ur “S KK \

200 w o rd s or le s s und e rl in e ke yword s )This clinical s tudy is designed to allow the inves tigators to evaluate the

effectiveness o f four different so lutions used topically before and afte r

oral surgery procedure s . Included will be normal saline solution , the con

ventional rinse ,which will serve as a contro l . Pos toperative localized

os te itis and infections will be re corded if they occur , as data , to be

conside re d in the overall evaluation o f the use o f topical solution for

possible prevention o f the se postoperative prob lems .

2 0 1 DE 00 24 1— 02 IR

I .O bj e c ti ve s :

The ove ra l l inc id e nce o f l o cal iz e d o s t e i t is througho ut the mouth has

been re po rt e d f rom to The inc id enc e o f lo caliz ed o s t e i t i s in

thi rd mo la r s i t e s ha s re ce nt ly be en de c re as ed with the us e o f lavage

te chnique f rom ZO~3OK t o unde r 6 2 . and the inre c t ion ra t e to unde r

Thi s sma l l pe rc en t age o f p a t ient s , h oweve r,s t i l l suf f e r wi th ext reme

po s t ope ra t ive pain . Convent ional l y , pa t ient s r ins e wi th a weak s al ine

s o lution the day fo l l owing surge ry and cont inu e f o r a few days po s t

ope rat ivel y . Th e sal ine is u se d p rimarily t o me chani c al ly c lean the

ex trac t ion wound o f any deb ri s p re s ent ; i t wi ll ad di ti ona lly re duce the

p re s ence o f bac te ria . Various o ther p re operat ive and po s t o pe ra t i ve

top i ca l s o lu t ions have be en t ried wi th variab le s uc c es s . L e ga rth and

Muns ter— Swends en rec ent ly used a chl o rhexid ine s olut ion fo r po s t o pera t ive

rins ing o f thi rd mo lar ext rac t ion s i te s and ach ie ved a 4 5% reduc t ion in

the inc idenc e o f lo c al i z ed o s t e i t i s in a s tudy invo lving 4 15 c as e s o f

mand ibula r t h i rd mo la rs . Ano t h er re cent s tudy wi th a p reo pe ra t ive rins e

o f chlo ramine-T reduc ed the incid ence o f lo ca l i z ed o s t e i t i s in man

dibular t h ird mo l ar s i t e s f rom AZ t o i . 6% . I t i s though t tha t the us e

o f c er t ain an tis e p t i c mouthrinse s c o uld achieve s imi lar o r po s s ibl ybe t t e r re sul t s , e s pe c ial ly i f us ed b o th pre and po s t o pe rat ively .

The purpo s e o f thi s p ro j ec t i s to c l inica lly te s t t h ree d i f fe ren t

s olut i ons along wi th the convent iona l sal ine us edpre and po s t o pe ra t ive ly

with o ral surge ry p ro c edure s to c omp a re the inc id ence s o f loc al i z ed

o s t e i t i s and infe c t ion ra t e to de t e rm ine if the re are advan ta ge s with

the us e o f o the r s o lut ion s a s a rins e .

I I . Me tho d s Emp loyed

An inf o rmed cons ent wil l b e s i gned on G SA S t andard Fo rm 52 2 , and

a s pe c i f i c inf o rmed c onsen t f o r thi s s tudy wil l al s o b e s i gned .

Immed i atel y b e fo re the surgi cal p ro c edu re , the pa t ient wil l rinse

wi t h 10 m1 o f a s o lut ion o f e i th er ( 1 ) so d ium chlo r ide (no rmals aline ) , ( 2 ) 12 ch l o ramine— T , ( 3 ) po vidone-iod ine ( 12 avai lab le iod ine ) ,

o r ( 4 ) s a tura t ed s od ium b i ca rb ona t e for a per io d o f one minute . The

surgery wi l l th en b e p er fo rmed in a no rmal manne r . The p a t ient wil l

rec e ive bo th o ral and wri t t en po s tope ra t ive in s t ruc t ion s fo r us e o f the

mouthr ins e fo r four t o s ix day s a f t er s urge ry . Ten mi l l ili t e rs o f the

s ame s o lu t ion us ed preope ra t ive ly wi l l b e us ed f o r one minut e bo th

mo rning and evening ; 1 20 ml wi l l be d is pe ns ed fo r t he pa t i ent 's u se

du ring th i s pe r io d o f t ime . The pa t ien t wi l l b e gin the rins e s the day

fo ll owing s urge ry and con tinue the i r us e un t il his s chedul ed po s t

o pe ra tive ob se rvat ion ap po intmen t when su t ure s will al s o b e removed .

The pa t ien t wil l b e examined f o r s igns o f l o cal i z ed o s t e i t i s o r ih

fe c tion f ive day s p o s to p e ra t ivel y ,excep t when the f i f th day is on a

2 01 DE 002 41-02 IR

Saturday or Sunday . In these cases the patient will be examined either

on the fourth o r sixth day pos toperatively .

Speci fic criteria for evaluation of a patient with localized os teiti s

will be : (1 ) a severe pain in the alveolus of a surgical site two to four

days postoperatively, ( 2 ) the presence o f a foul , grayish exudate from

the surgical site , (3 ) the presence o f a necrotic odor from the surgical

site,or (4 ) observation o f a denuded , bony alveo lus . At least two o f

these criteria will be pre sent for a diagnosis of localized os teitis

to be ma de .

Specific cri teria for evaluation o f a patient with an infection

will b e : (1 ) a swelling which persist s , increases , or appears af ter

four to f ive days pos toperatively , ( 2 ) a purulent drainage from the

surgical site, (3 ) a pulse rate significantly increased above the normal

for the patient and persis tently so elevated , or (4 ) a fever'

usually

above 38 . 30 C ,which remains e levate d even when the patient remaihs well

hydrated . At leas t one o f the above criteria will be pre sent for a

diagnosis o f pos toperative infection to be ma de .

I f an infe ction or localized o s teit is occurs it will be treated

and recorded as being present . Systemic antibiot ics will not routinely

be used postoperatively , excep t where infection occurs , and in tho se

case s ,the y will be employed immediately when the diagnosis is made .

After five days (or if the fif th day is on Saturday or Sunday ,

then either four or six days ) the surgical sites will be ob served for

he aling progres s . This data will be recorded with the patient 's po st

operative data sheets . The patient will then be discharged with

follow—up appointment s if needed . The pat ient will be ins tructed to

return if he develops any further po stoperative prob lems .

I I I . Maj or F indings

The proj ect was initiated in June 19 78 , thus insufficient data

is available fo r re porting at this time .

IV . Significance to Biomedical Research and the Program of the Institute

The results o f this s tudy can provide important information

re lat ive to a good topical rinse for use after oral surgery procedures .

Positive results will allow the re commendation o f a topical rinse to

oral surgeons and dentis ts for use in prevention of certain po s toperat iveoral surgery problems .

V . Propo sed Course

Continue proj ect until sufficient data is gene rate d to compareresults with previous s tudies .

4 - 1 6

D e p o r t o f t h e N i c r o b ia l S y s t em a t i c s S e c t i o n

N a t i o n a l I n s t i t u t e of D e n t a l R e s e a r c h

T h e M i c r o b i a l S y s t e m a t i c s S e c t i o n i s c h a r g e d w i t h

e s t a b l i s h i n g a d a t a b a nk f o r i nf o r m a t i o n d e s c r i b i n g d i v e r s e

s t r a i n s o f m i c r o o r g a n i s m s . S p e c i a l e m p h a s i s i s p l a c e d o n t h e

h um a n o r a l m i c r o b i o t a . F o r t h i s p u r p o s e , c o l l a b o r a t i v e p r o j e c t s

a r e on - g o i n g w i t h m i c r o b i o l o g i s t s d i s t r i b u t e d t h r o u g h o u t t h e

w o r l d .

A t p r e s e n t t h e r e a r e t e n s o f t h o u s a n d s o f s c i en t i s t s,

p h y s i c i a n s,

p u b l i c h e a l t h p e r s o nn e l , a n d o t h e r s i nv o lv e d i n s o m e

a s p e c t o f m i c r o b i o l o g y .T h e n um b e r o f m i c r o b i a l s t r a i n s

i s o l a t e d,

c h a r a c t e r i z e d , a n d ( i n m a ny c a s e s ) p r e s e r v e d,

b y

i n d i v i d u a l s r u n s i n t o t h e m i l l i o n s . H u n d r e d s o f m i l l i o n s o f b i t so f i n f o r m a t i o n h av e b e e n d e v e l o p e d o n t h e s e s t r a i n s . H o w e v e r

,

t h e s e d a t a a r e n o t r e s i d e n t i n a s i n g l e , c e n t r a l l y l o c a t e d

s y s t e m , p e rm i t t i n g r a p i d a n d e f f i c i e n t u t i l i z a t i o n . B e c a u s e o f

t h e l a r g e v o l um e o f i n f o rm a t i o n i nv o lv e d a n d b e c a u s e ,i n s ev e r a l

a p p l i c a t i o n s s u c h a s c l a s s i f i c a t i o n a n d i d e n t i f i c a t i o n ,

ma th e m a t i c a l m a n i p u l a t i o n s o f t h e d a t a a r e r e q u i r e d , e l e c t r o n i cp r o c e s s i n g o f t h e s e d a t a i s n e c e s s a r y .

I n c o l l a b o r a t i o n w i t h p e r s o n n e l o f t h e Ame r i c a n T y p e C u l t u r eC o l l e c t i o n ; t h e F o o d a n d D r u g A d m i n i s t r a t i o n ; t h e C e n t e r f o r

D i s e a s e C o n t r o l ; t h e V e t e r a n'

s A d m i n i s t r a t i o n a n d n u m e r o u s

a c a d e m i c m i c r o b i o l o g i s t s , s t r a i n d a t a a r e b e i n g e n t e r e d i n t o t h e

d a t a b a nk wh i c h p r o v i d e s s u c h s e r v i c e s a s : . d a t a o n s p e c i f i c

o r g a n i s m s a n d / o r g r o u p s o f o r g a n i s m s,

l o c a t i o n o f s t r a i n s w i t h

S p e c i a l c h a r a c t e r i s t i c s , i d e n t i f i c a t i o n o f u n k n o wn i s o l a t e s ,

c l u s t e r a n a l y s i s d e f i n i t i o n o f p a r a m e t e r s o f t a x a,

d a t a

m a n a g e m e n t a n d r e p o r t w r i t i n g a i d s f o r r e s e a r c h p u r p o s e s,

a i d s i n

q u a l i t y c o n t r o l o f t e s t s,

m e th o d s, a nd

'

l ab o r a t o r i e s ,a n d

c o mmu n i c a t i o n o f d a t a v i a c o mm o n f o rm a t .

D a t a f i l e s o f p r i ma r y d a t a o n a l a r g e n um b e r o f

m i c r o o r g a n i s m s f o u n d i n t h e o r a l c a v i t y a n d r e l a t e d t y p e s a r e

e s t a b l i s h e d . T h e s e f i l e s p r o v i d e a r e s o u r c e f o r a s k i n g b o t h

e c o l o g i c a l a n d e p i d e m i o l o g i c a l q u e s t i o n s o f i n t e r e s t i n d e n t a l

r e s e a r c h .

P r o g r am s h av e b e e n d e v e l o p e d a n d t e s t e d t o e n t e r , r e t r i e v e,

an d a n a l y z e th e d a t a in a v a r i e t y o f w ay s f o r e p i d e m i o l o g i c a l,

d i ag n o s t i c , t a x o n o m i c,

e c o l o g i c a l,

e t c u s e s . T h e l o n g t e rm

g o a l i s t o e s t a b l i sh a w o r l d-w i d e d a t a b a n k a t a s e r i e s o f

c o o p e r a t i n g c e n t e r s . As e x p e r i e n c e g r o w s ,b e t t e r p r o g r a m s a r e

b e i n g d e s i g n e d a n d i m p l em e n t e d .

i h e s y s t e m o r i g i n a l ly d ev e l o p e d f o r b a c t e r i a i s n ow b e ing

e x p an d e d t o in c l u d e th e y e a s t s , mo l d s , a l g a e an d p r o t o z o a . As e r i e s o f m o n o g r a p h s d e s c r i b i n g t h e e x p and e d s y s t e m i s i n v a ry in g

s t a g e s o f p r e p a r a t i o n .

Ex t e n s iv e f i l e s o f d e s c r i p t i o n s o f f i l ame n t o u s an d

p l e om o rp h i c o r g an i s m s a r e b e in g a s s emb l e d . T h e f i l e s c ov e r a l l

t h e d e s c r ib e d t y p e s o f My c o b a c t e r i a , b l e n d i n t o t h e N o c a r d i a ,

t h en t h r o u g h t h e A c t in omy c e t e s ( e s p e c i a l ly a un i q u e s e t o n o r a l

i s o l a t e s ) ,an d f i n a l ly , B a c t e r i o n ema . An ex t e n s i v e c o o p e r a t iv e

s t u d y h a s b e en in i t i a t e d t o s t u d y t h e o r a l p l e o mo r p h i c b a c t e r i a

( ma ny o f wh i ch a r e a s s o c i a t e d w i t h d i s e a s e ) . T h e s t u d y wi l lp r ov i d e a s t an d a r d s e t o f we l l c h a r a c t e r i z e d b a c t e r i a f o r t h e

D e n t a l R e s e a r c h c ommun i ty . T h e d a t a f r om t h i s s t u d y w i l l b e

i n c o r p o r a t e d i n t o t h e f i l e s o n p l e omo r p h i c o r g an i s m s . T h e s e

f i l e s a r e b e i n g a c t iv e l y U s e d i n c o l l ab o r a t i o n wi th t h e

s ub m i t t e r s o f t h e d a t a a s we l l a s nume r i c a l t ax o no m i s t s t o r e v i s e

t h e b a d ly c o n f u s e d t ax o n o m i c r e l a t i o n s h i p s o f t h e s e b a c t e r i a .

S u c h r ev i s i o n i s n e c e s s a r y t o av o i d t h e m i s i d e n t i f i c a t i o n

( l e a d i n g t o e r r o n e o u s e p i d em i o l o g i c a l c o n c l u s i o n s ) wh i ch ar e

f o un d i n s om e r e c e n t d e n t a l r e s e a r c h l i t e r a t u r e .

O t h e r f i l e s on n on- f i l ame n t o u s o r a l o r g a n i s m s ( s t r e p t o c o c c i ,

l a c t o b a c i l l i ,v e il l ion e l l a , e t c . ) a r e b e i n g c on s t r u c t e d t o s t u dy

c o r r e l a t i o n s amo ng c a r i e s a c t iv i t y , p h e n e t i c s p a n o f c h a r a c t e r s,

s e r o l o g y , s o u r c e o f i s o l a t i o n , an d h o s t d e s c r i p t i o n s .

On e o f t h e l o n g t e rm g o a l s in e s t a b l i s h in g a l l t h e s e f i l e s

a r e t h e e s t ab l i shme n t o f p r o b ab i l i ty t ab l e s t o a l l o w

c omp u t e r-a id e d p r o b a b i l i s t i c i d e n t i f i c a t i o n o f o r a l i s o l a t e s .

P r o b a b i l i ty ma t r i c e s,

f o r o n- l in e id e n t i f i c a t i o n o f b a c t e r i a

( in c l ud i ng G r am n e g a t iv e r o d s,

l a c t o b a c i l l i,

s t r e p t o c o c c i,

b a c i l l i , e t c . ) h av e b e en c o n s t r u c t e d . T h ey a r e av a i l ab l e t o

r e s e a r c h wo rk e r s f o r f i e l d u s e .

PUB L I C AT I O N S

K an e k o , T ., H auxh ur s t , J . , K r ic h e v s ky ,

M .,

a nd A t l a s ,R . M

N um e r i c a l t ax o n om i c s t u d i e s o n m i c r o o r g a n i s m s i s o l a t e d f r o m A r t i can d s ub -Ar c t i c ma r in e env i r o nm e n t s . P r o c e e d i n g s o f I n t e r na t i o n a l

S ymp o s i um o n M i c r o b i a l E c o l o g y . S p r i ng e r— V e r l a g . H e i d e l b e r g,

1 9 7 8 .

Ka n ek o , T ., K r ic h e v s k y , M . I a n d A t l a s

, R . Num e r i c a lt ax o n omy o f b a c t e r i a f r om t h e B e au f o r t S e a . J . G e n . Mi c r o b i o l .

— 1 2 5 , 1 9 7 9 .

V r i c h e v s k v ,

V

.I T h e mi c r o b i a l i n f o rma t i o n s y s t e m

A s y s t em o v e rv i ew .

( M I C RO- I S )F D A ”

v- L i n e s .— 2 2 2 , 1 9 7 0 .

W a l c z a k , C . A T h e M i c r o b i a l I n f o rm a t i o n S y s t e m C o mp l e x D a t a

An a l y s i s . F D A By— L i n e s .

- 9 5 3 , 1 9 7 9 .

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N AM ES,LA BOR AT ORY ANO I N ST I TUT E AFF I L I AT I ON S , AN D T I TL E S OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PROF ES S I ON AL P ER S ON N EL ENG AG ED ON THE PR O JECT

W i c a h T . K r i c h e v s ky C h i e f , V i c r o b ia l S y s t ema t i c s S e c . C D I R N I DR

L e s l i e L . L ov e,

B i o l o g i c a l L a b o r a t o r y T e chn i c i an O D I R K I D R

COO PERAT I NG UN I T S (if any ) 17 0

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L o s An g e l e s C A ' U n

LAB/BRANCH

D (b ) H UM AN T I S S U E S (c ) N E I T HE R

S UMMAR Y OF WOR K (200 wo rd s o r les s und e rl in e keyword s )V i c r ob i a l s t r a in d a t a a r e b e in g e n t e r e d i n t o a d a t a b a nk

wh ic h p r ov i d e s s u ch s e rv i c e s a s da t a o n S p e c i f i c o r g a n i s ms

a nd / o r g r ou p s o f o r g a n i s m s , a l o c a t o r s e rv i c e f o r s t r a in s w i th

S p e c i a l c h a r a c t e r i s t i c s,

i de n t if i c a t i o n o f unkn own i s o l a t e s ,

c l u s t e r a n a l y s i s d e f i n i t io n o f p a r a m e t e r s o f t a x a,

d a t a

m a n a g e m e nt a n d r e p o r t w r i t i n g a i ds, a id s in q u a l i t y c on t r o l ,

a nd

c o mmun i c a t io n o f da t a v ia c ommo n f o rm a t . F i l e s o f p r im a ry d a t aon a l a r g e n umb e r o f m i c r o o r g a n i s m s p r ov i d e a r e s o u r c e f o r

a s k i ng b o t h e c o l o g i c a l a n d e p ide m i o l o g i c a l q u e s t i o n s o f in t e r e s tin de n t a l r e s e a r c h .

D r o g r am s a r e b e in g d e v e l o p e d a nd t e s t e d t oe n t e r , r e t r i e v e , a n d a na l y z e t h e da t a in a v a r i e t y o f w a y s f o re p id e m i o l o g i c a l ,

di a g n o s t i c , t ax o n o m i c ,e c o l o g i c a l , e t c . u s e s .

T h e l o n g t e r m g o a l i s t o e s t ab l i s h a wo r l d-w i d e d a t a b a nk a t a

s e r i e s o f c o o p e ra t in g c e n t e r s . T h e s y s t e m o r i g i na l l y d e v e l o p e df o r b a c t e r i a is n o w b e in g e x p a nde d t o i n c l ud e t h e a l g a e , y e a s t s ,

m o l d s a nd p r o t o z o a .

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Annual Re por t of the Laborato ry of Developmental Bio logy Anomalie sNat ional Ins titute o f D e ntal Res ear ch

The maj or e f fo rts in the l ab orat ory of D eve lopmental B iology and

Anomalies are on the p revention and treatment of inheri te d and acquired

def ect s in oral-fa cial development . These proj e c ts inc lude s tudies on

the development o f facial tis sues , the p os s ib le roles of environmental

agents and o f gene t ic fact ors in oral— fac ial malformat ion , connec tive

t is sue format ion and the int e ract ion o f normal and tumor ce lls with

matrix pro teins .

Several changes have occurred in pers onnel . Dr . Hynda Kleinman was

promo ted to a permanent pos ition . Dr . J . Pennypacker , 2 01 DE 00 2 53—0 2

has lef t to as sume a pos i t ion in the Depar tment o f Zoology , Univers ityof V ermont . Dr . S . Rennard has trans f erred to the NHLBI and Dr . D .

Salomon to NCI . Ac tivit ies wil l be cont inued in their res earch areas

by o ther inves tiga tors . A contract to s tudy the dis tr ibut ion of type

IV co llagen in ora l and other tis sues with the Depar tment of Patho logy ,

Univers ity of Innsbruck has been sat is fac torily comp leted . In May ,

19 79 the ac tivi ties in the Craniofacial D evelopment S ec tion were

reviewed by outs ide s c ientis ts including rep res entat ives of the

Sc ient if ic Couns elors , NIDR . This comp letes the review of LDBA

research act ivities which b egan with the review of the Conne ctive

Tis sue S ect ion in June , 1 9 78 .

Connec t ive T is sue S ec tion

Chemotaxis Maj or advances have been made in de fining the events

invo lved in C hemotaxis . The N—fo rmylmethionine pep tides dis covered to

be chemo tact ic in this Laboratory were f ound to bind to s peci fic

recep tors on the sur face of macrophages and po lymorphonuc lear leucocytes . The interac t ion o f the chemoat tractants with the recep tor has

b een found to ac tivate enzyme sys tems which trans fer methyl group s from

S—adenosyl methionine to proteins and phospholip ids . Demethylation

react ions follow very rap idly allowing the membranes to re turn to a

quies cent s tate . Rep eated exposure to the at trac tant or to a gradi en t

o f at tractant leads to a cyclic reac t ion and a continual turnover o f

membrane me thyl groups . These methylation react ions al ter the

permeabili ty and the rigidi ty o f the cell membrane , thus p re sume ably

caus ing the orientat ion o f cytoskeletal e lements and the dire cted

mo tion of the cel l .

Bas ed on the enhanced sus cep tibility o f pers ons bearing tumors t o

inf ec tions , we have sought a tumor fac tor that impairs C hemotaxis .

The chemotac tic respons e o f cells ob tained from mice b earing a

fibrosarcoma was found to be reduced . A small pep tide with a

molecular weigh t les s than 1000 was purif ied from the tumor and found

to depres s phagocyte Chemotaxis by inhibi ting the me thyl trans fer

react ions ref erred to above . The s truc ture of the tumor pep t ide is

under s tudy . S ince reduced res is tance to inf ec tion is a maj or fac tor

in the morbidity o f cancer , it is poss ib le that an unders tanding of

this tumor fac tor will O pen new approaches to cancer chemotherapy .

F urther,such pep t ides may allow modulation of tiss ue respons e in

inflammatory disorders .

Ma lformations Mycotoxins such as the cytochalas ins are widely

dis tributed fungal products known to be ab le to induce malfo rmations

of the neural tube in embryos . We have made a sys tematic survey among

various s trains of mice and found that the sus cep tibility of these

s trains to the teratogenic eff ects of cytochalas in D varies widely .

Two strains (OEL—N and aeae ) were found to be s everal times more

sens itive to cytochalas in D than C5 7Bl or Swis s—Webs ter mice . The

susceptib ility to cytochalas in D was found to be inherited in a

recess ive pattern . While the molecular bas is for this difference is

not known and may be diffe rent for dif ferent s trains a s imilarsens itivity could occur in some humans and relate to the putative

role of fungal toxins in human malformations .

Increas ed suscept ibility to the teratogenic ef fec ts of vi tamin A has

been related to a genetic defect . In thes e s tudies , we have inves ti

gated mutant mice bearing the OEL gene . Mice homozygous for the OELgene have open eyelids and cleft palate at birth . Mice heterozygous

for the OEL gene show low levels of spontaneous malformations but a

greater suscep tib ili ty to malformations from retinyl palmitate (aform of vitamin A) than their normal li ttermates . The OEL heterozygo te

and other normal mice show equal but very high sens itivity to the

teratogenic actions of retinoic acid . S tudies with L . DeLuc a , NCI ,

indicate that mice with the OEL gene convert ret inol to retinoic acid

at a very rap id rate when compared to other mice . Thes e s tudiesSugges t that a metabolic abnormality in the OEL mice leads to the con

version of an es sential dietary factor to a highly teratogenic me tabo

lite .

Ce ll Attachment Fibronectin , a large glycoprotein , binds fibrob las ts

and other cells to collagen . Our previous s tudies localized the

binding s ite for fibronectin on collagen to a s ingle region of the

molecule coincident with the co llagenas e c leaveage s ite . Now we have

carried out s tudies of the receptors for f ibronectin on the cellsurface . Thes e s tudies show that the oligosaccharides occurring in

certain higher gangliosides such as CD and GT are able to bind tofibronectin and block the b inding of fibronectin to the recep tors on

the cell surface . This activity is dependent on at leas t two intactsialic acid res idues in the he t erO poly sac charide . Thes e s tudies

indicate that the higher gangliosides or proteins bearing s imilaroliogsac charides can serve as the cell surface recep tors for f ibronect in . The reduced binding of fibronectin by trans forme d cells couldbe due to the known defe ct in ganglios ide synthes is that accompanies

.

trans formation .

No t all connec tive tis sue cells attach to co llagen through fibronec t in .

We have f ound that chondrocy tes attach to col lagen by a dif f erent

at tachment prot ein , which we have named Chondronec t in . Chondronectin

is pres ent in serum and in cart ilage and is produced by chondrocytes

but not by other cel ls . Other cel ls may als o p roduce unique a t tachment

pro teins tha t have a speci fic af f ini ty for their cell o f origin . Such

fac tors may explain the t is s ue locali zation of cer tain cell types .

The at tachment proper t ies o f me tas tatic tumor cells from mice have b een

found to dif fer from thos e o f normal and nonme tas tasiz ing trans formed

ce lls . The metas tatic cel ls p roduce lit tle or no co llagen and f ibro

nec t in and b ind preferentially to type IV (bas ement membrane ) co llagen .

Normal cells bind equally well to types IV and I co llagen through

f ibronec t in . S ince metas tat i c cells in a populat ion of tumor cells can

b e s elec t ed for by their ab ili ty to b ind to type IV collagen , a

quanti tative in vitro ass ay may be developed to predic t metas tat ic

potential .

Bas ement Membrane Cons ti tuents Three cons tituents o f bas ement

,membranes have b een is olated from a tumor produc ing bas ement membrane .

These inc lude type IV collagen , a large glycop ro tein (laminin ) and a

heparan sul fate proteoglycan . Immuno logical s tudies sugges t tha t the

bas ement membranes in normal tis sues contain s imilar macromo lecules .

The three cons ti tuents locali ze to di f ferent regions o f the bas ement

membrane and probably have di f ferent func t ions . The type IV co llagenshave a s truc tural ro le while the proteoglycan probably controls the

permeab ility o f the bas ement membrane . Laminin may regulate cel lularinteractions .

Antib odies prepared agains t thes e bas ement membrane pro teins , as wellas o ther connective tis sue proteins , are being us ed as the bas is o f a

new analytical pro cedure which requires an enzyme linked to antibody .

The me thod is simp le , sens itive and eas ily adap ted to o ther macro

mo lecules . U s ing this method , it has been pos s ib le to measure mous e and

human f ibronec tin product ion by hybrid cells and to locali ze the gene

for human f ibronec tin to chromosome 8 .

C ranio facial Development S ec tion

Teratology An in vitro screening sys tem to detect compounds with

teratogenic act ivi ty has b een develop ed us ing di f f erentiating neuralcres t and limb mes enchyme cells . The addit ion of known animal and

human terat ogenic agents such as dipheny lhydantoin and exces s vitamin

A caus es al terat ions in the growth or di f f erentiat ion of thes e cel ls .

The s creening sys tem is als o capab le o f detect ing teratogenic c om

pounds which require metab olic modi fica tions for ac tivi ty

IT- Q R

The di fferent iation of limb mes enchyme cells into chondrocytes involvesthe loss of cell surface f ibronec tin when the cells be come committ ed

to this phenotype . Exces s amounts o f the teratogen , vitamin A , blockthis proces s by enhancing the accumulation of f ibronec tin at the

mesenchymal cell surface , thereby preventing the loss o f f ibrone c tin

neces sary for normal chondrogenes is .

Glucocor ticoids are known to caus e facial cleft ing and growth retarda

tion in exper imental animals . Previous ly , we have b een able to

demons trate,us ing various mous e s trains , a correlation b etween the

s ens itivity to glucocor ticoid-induced clef t palate and the levels of

glucocor ticoid recep tors pres ent in the develop ing cranio facial

region . In the human f etus , glucocor ticoids are known to caus e growth

retardation and are suspect ed of caus ing fac ial c lef t ing . We have b een

ab le to demons trate alterations in the glucocorti coid recep tors in

cultured dermal fibrob las ts f rom persons with a s trong familial his tory

of facial c lef ting . Thes e s tudies sugges t that alt erations in

glucocor ticoid recep tors may underli e cer tain oral facial abnormalit iesin humans . The information from thes e s tudi es may b e o f p otential

us e in genet ic couns eling for facial cle fting

C ell D if ferentiation Pep tide growth factors , such as ep idermal

growth factor (EG F ) , are know to influence the growth and di f feren

tiation o f a numb er o f cell types in culture . We have found a fetalgrowth factor in the mid and late ges tation mous e emb ryo which re

s emb les but is no t identical to ECF . This fetal growth fac tor appears

to b e involved in the growth and dif ferentia tion of ep ithelial c ellsin the developing palatal shelves . Our s tudies have als o shown that

the newborn mous e is d ependent upon EG F in the f irs t few weeks of

li fe . During this time EG F s t imulates the proli f eration and keratin

iz at ion o f the oral mucosa and epidermis which enhances too th erup tion

and eyelid opening . This ECF is apparently ob tained through the

maternal milk before synthes is b egins in the salivary gland at 3 to4 weeks of age .

The growth and dif ferentiation of mos t cells and tis sues in cultureare usually dependent upon the pres ence of serum whos e hormone

compos ition is largely unknown . O th ers have des crib ed media lacking

serum in which cells will grow . We have developed a de fined culture

medium , containing hormones and growth fac tors such as insulin and EG F

which supports the growth and dif ferent iation of mous e teratocarcinomacells , rat mammary epi thelial ce lls , fetal limb mes enchyme cells and

secondary palates . The us e o f this medium will facilitate defining

the hormonal requirements fo r a numb er of cell types in culture .

Cranial neural cres t cells are to tipot ent , in that they willdif ferentiate into a numb er o f cell types in vivo . Our s tudi es have

shown that cer tain macromolecular components o f the serum are

respons ible for influencing cres t cell dif f erentiation . In addi tion,

U - Q E

dimethylsulfoxide (DMSO ) has been found to b e a revers ib le inhib it or

o f c res t ce ll di f feren tia t ion into p igmented cells , pos s ibly by

altering the act ivity of a s erum fac tor .

The dif ferentiation of the dental papilla mes enchyme ce lls into

dentin s ec reting o dontoblas ts is dependent upon an int erac t ion o f

mes enchyme cells with the adj acent basal lamina , which is o f epithe

l ial cell origin . Tunicamy cin , a spe cif ic inhib itor of pro tein

glycosylation , r ev ers ib ly inhibits this dif ferentiat ion and prevent s

the incorporat ion of f ibronec tin into the basal lamina .

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F igueroa,A . A . and Prat t , R . M . : Autoradiographic study of macro

molecular synthesis in the develop ing ra t primary palate in vitro .

J . Embry . Exp . Morph . 50 : 145-154 , 1 97 9

F oidart ,J . M . ,

Berman,J . J . , Paglia , L Rennard , S . I . , Abe , S

Pe rantoni , A .,and Mart in , G . R . : Synthes is of fibronec tin , laminin

and s everal collagens by a liver— derived ep i thelial line .

Lab . Inves t .

F oidart ,J . M . ,

Hall , R . , Martin , G . R . and Katz , S . I . : Immuni ty to

type I I co llagen in relaps ing polychondri tis .

Symp os ium on Arthritis , Immunity and Aging , Los Angeles , CA , January10-12 , 19 79 ( to b e pub lished in a book)

F oidar t , J . M . and Kat z , S . R . : Relaps ing polychondri tis .

Archives of D e rmatol . In Pres s .

F oidart , J . M . , Reddi , A . M . : Immunof luores cent locali zation of type

IV collagen and laminin during endochondral bone formation and ef f ect

o f growth hormone upon this phenomenon .

Develop . Biol . In Pres s .

F reer , R . F . , Day , A . R . , S chif fmann , E . , Aswanikumar , S . , Showell ,

H . J . and Becker , E . L . : Further s tudies on s truc ture ac tivity

relations among chemo tactic pep t ides .

Biochemis try

Furcht , L . T . , Mo sher , D . F . , Wendels cha f er-Crabb , G . and F oidart , J .

M . : Fibronect in and typ e I procollagen f ibrillogenes is in cul tures o f

human skin fibroblas ts detec ted by ul tras truc tural immunocytochemis try .

Proc . Natl . Acad . Sci . In Press .

Furcht , L . T . , Mosher , D . F . , Wende ls chaf er-Crabb , G . and F oidart , J .

M . : Glucocor ticoids partially revers e the los s of the fib ronect in and

procollagen matrix around trans formed human cells .

Cancer Res . 39 : 2 07 7— 2083 , 19 79 .

Furcht , L . T Mosher,D . F . , Wende ls chaf er—Crabb , G . , Woodb ridge , P . A

and F oidart , J . M . : Dexamethas one— induced accumulation of a fib ronec t in

and collagen extracellular ma trix in trans formed human cells .

Nature 2 7 7 : 39 3-39 5 , 19 7 9 .

Greenb erg , Judi th H . and Oliver , C . : Dimethylsulfoxide revers ibly

inhib its the pigmentation of cul tured neural cres t cells .

Develop . Biol .

Greenb erg,J . H . and Bader , J . P . : Neural cres t cells : Temperature

dependent transformation by Rous sarcoma virus .

Cell D if ferentiat ion 8 : 1 9— 2 7 , 19 79

Greenb erg , J . H . , F oidart , J . M . and Greene , R . M . : Collagen synthe s is

in cul tures of dif f erentia ting neural cres t cells .

Dif f erent ia tion

Greene , R . M . and Prat t , R . M . : Correlation between cyclic AMP levels

and cy tochemical locali za t ion of adenyla te cyc las e during development of

the s econdary palate .

J . His tochem . Cy to chem . 2 7 : 9 24— 9 31 , 19 79

Greene , R . M . and Pra t t , R . M . : Inh ib it ion of ep ithelia l cel l death

in the s econdary palate in vitro by al terat ion of lys os ome func tion .

J . His tochem . Cy tochem . 2 6 : 1109-1114 , 19 79

Hackman , Rob er t M . and Brown , S . K . : Teratogenic s ignif icance of

shif ts in diurnal cor t icos terone pat tern of A / J mice during pregnancy .

S teroids

Harne , L . C . and Brown , K . S . : Dua l purpos e mous e cage rack for ho ldingand washing plas t ic cages and lids .

Lab . Anima l Sci .

Has s ell , J . R . , Pennypacker , J . P . , Kleinman , H . K . , Pra tt , R . M . and

Yamada , K . M . : Enhanced cellular f ib ronec tin accumulation in chondro

cy tes treated with vitamin A .

Cell 1 7 : 8 21-8 2 6 , 19 79

Hewit t , A . T . , Kleinman,H . K . , Pennypacker , J . P . and Martin G . R

Identi f ication o f an adhes ion fac tor for chondro cy tes .

Proc . Natl . Acad . S ci . USA . In Pres s .

Hirata , F . , Corcoran , B . A . , V enkatasub ramanian , K . , Schi f fmann , E . ,

and Axelrod , J . : Chemoa ttrac tants induce phospho lipas e act ivity in

leukocytes .

Proc . Natl . Acad . S ci USA . 7 6 : 2640-2 643 , 19 79 .

Huo t , R . I . , F oidart , J . M Donner,L . and S tromb erg , K . : Eff ec ts of

cul ture condi tions on the synthes is of human chorionic gonado trop in by

p lacental organ cultures .

In Vitro . In Pres s .

Kl einman , H . K ., Hewit t , A . T . , Murray

,J . C Lio t ta , L . A . , Rennard ,

S . I ., Pennypacker, J . P . , Mc G oodwin ,

E . B . , Mar tin , G . R . and F ishman ,

P . H . : C ellular and metabolic speci fic i ty in the interac tion o f adhes ion

pro t eins with collagen and with cells .

J . Supramolecular S truc ture . In Pres s .

Kleinman,H . K . , Martin , G . R . and Fishman , P . H . : Ganglios ide

inhib ition o f f ibronectin-mediated cell adhes ion to collagen .

Proc . Natl . Acad . Sci . USA . 76 : 3367-3371 , 19 79 .

Kleinman,H . K Mc G oodwin , E . B . , Kennard , S . I . and Mar tin , G . R

Preparat ion o f collagen subs tra tes fo r cell attachment . Eff ec t o f

collagen concentration and phosphate buf fer .

Anal . Biochem . 9 4 : 308-31 2 , 19 79

Lio tta , L . A Abe , S . , Rob ey , P . G . , and Martin , G . R . : Preferentialdiges tion o f bas ement membrane collagen by an enzyme derived from a

metas tatic murine tumor

Proc . Natl . Acad . S ci . USA 2 268— 2 2 72 , 19 79

Liotta , L .A . , F oidart , J .M . , Rob ey , P . G . , Martin . G .R . , and Gullino ,

P . : Identification o f micrometas tas is of breas t carc inomas by pres ence

o f bas ement membrane collagen .

Lancet 146-14 7 , 19 7 9 .

Lio tta , L . A . , G arbisa , S . , Tryggvason , K . , Murray , J . C . and Rob ey , P . G

Interaction o f metas ta tic tumor cells with bas ement memb rane collagen .

German Sympos ium on Metas tatic Tumor Growth , Grundmann , E .

Gus tau Fis cher Verlag , N . Y . , 19 79 . In Press .

Lio t ta , L .A . , G arbisa , S . , Tryggvas on , K . , and Wicha , M . : Correlat ionof metas tatic b ehavior with tumor cell degrada tion o f bas ement memb rane

collagen .

Chicago Sympos ium on Metas tas is . C risp en , R . G . 19 79 .

Lio tta , L .A . , Wicha , M . , F oidart , J .M . , Rennard , S . I . , G arbisa , S

and Kidwell , W . : Hormonal requirement for bas ement membrane collagendepos ition by cul tured rat mammary ep ithelium .

Lab . Inves t .

Mcclure , H . M . , Wilk , A . L . , Horigan , E . A . and Pra tt , R . M . : Induc

tion o f cranio fac ial ma lformation in Rhesus monkeys (Macaca mulat ta )with cyclophosphamide .

Clef t Palate J . 248— 256 , 19 79

Martin , G . R . , Brown , K . S . , Mathes on , D . , Lebowit z , H . , S inger,L .

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Introduc tion A maj or asp ec t o f inflammation is the inf lux of lenko

cytes,mainly in respons e to leukoa ttrac tants genera ted at the af fec ted

area . We have b een s tudy ing molecular events o f this chemo tact ic

respons e in vitro us ing as chemoattrac tants synthet ic fo rmylmethionylpep tides whic h may b e related to bac ter ially-produced at trac tants . We

have previous ly found that thes e p ept ides ac t at a sp ecif ic recep tor

on the leukocy te and that bo th N— formylation of the methionine and the

pres ence o f an aromatic amino acid res idue in the terminal pos ition of

a trip ep tide , are required for maximal ac tivity in a pep tide . Nuclearmagnetic res onance s tudies (J . Lois elle , Univ . Conn . Heal th C enter ,

Farmington , Conn . ) have indicated that formy lme thiony lleucy lphenyla

lanine , a mos t po tent at trac tant , has a unique conforma tion in so lution

which depends upon the formyl group . This may part ly exp lain the high

af finity o f this p ep tide for the leukocy te b inding s ite . At pres ent

we are examining the initial interaction b etween attrac tant and

cellular b inding s ite , the nature of the chemical s ignal transmit ted

to the motility appara tus to produce direc ted mo tion , and the manner

in which the chemo tact ic resp ons e is regula ted . We have als o

ini tiated s tudies on fib roblas t chemo taxis .

Methods The sources o f neutrophils were from rabb it peritoneal

exudates and human volunteers . Both as says for chemo taxis in vitro

us ing modi f ied Boyden chambers and for p ep tide—b inding to recep tors

have b een des cribed (S chif fmann , E . , Showell , H . J . , Corcoran , B .A . ,

ward , P .A . , Smith , E . , and Becker , E . L . (19 75 ) J . Immunol . 114 : 1831

Determination o f enzymatic act ivit ies such as phospho lipas eand protein methyles teras e was performed according to s tandard

procedures (Hirata e t al . , b ib lio graphy for this report ; Gagnon , C .

(1 9 79 ) Biochem . BiO phys . Res . Commun . , in pres s ) .

Resul ts Interac tion b etween at trac tant and b inding s ite With the

aid o f a contrac tor who synthes izes compounds for us we have continued

s tudies on s truc ture-ac tivity relationships o f chemotac tic p ep tides .

F ormylmethionyl- leucyl— phenylalanyl-O—Ben z y le s te r (FMe t-Leu— Phe—OB z ) ,

a les s polar derivat ive o f EMe t—Leu— Phe , has b een found to b e lo— foldmore ac tive an agonis t upon neutrophils and -fo ld more ac tive

upon macrophages than the parent tripep tide . Rep lacement o f the

formyl group with the t-butoxycarbonyl n a s eries o f b enzy l es ters

resulted in the produc tion o f weak (10 M) inh ib itors o f chemo taxis

which were , however , spec if ic in that chemo taxis to complement-derivedattrac tant was les s af fec ted than chemo taxis to synthetic pep tides or

the bacterial factor . Thes e s tudies could lead to the development o f

potent antagonis ts which may b e useful in puri fy ing the pep tide

recep tor through af f ini ty lab elling techniques . Such compounds might

als o b e effective in regulating in vivo chemo taxis .

The receptor app ears to contain an ess ential tyros ine , s ince trea tment

with tetrani tromethane , a sp eci fic reagent for tyros ine,inact ivates

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the binding S i te . This finding may explain the requirement for a

terminal aromatic amino acid re s idue fo r maximal ac tivity o f N

formylmethionine trip ep tide s .

wi th Drs . Hira ta and Axe lrod , NIMH ,we have found that chemo tac t ic

p ep tides sp e ci fica lly s timulate the turnover of N-methylate d phos

pholipids in the neutrophil . Attrac tant s enhanc e phospho lipas e A2

ac tivity , resul ting in the releas e o f bo th arachidonic ac id and

lys oleci thin f rom the ce ll membrane . Thes e findings are further

linked to chemotaxis by the obs erva tion that quina crine , an inhibi tor

o f phospholipas e , inhib i ts bo th ch emo taxis and the rel eas e o f

arachidonic ac id from the ce ll . Thes e events may partly account for

the changes in membrane charge and fluidity which accompany chemo taxis .

We have als o found tha t pep tide chemoa ttrac tants speci f ica lly s t imulate

pro tein methyles ter turnover , a resul t s imi lar to that found for

bac terial chemo taxis . The revers ib le e s terif ication o f pro tein

carboxyl groups could contribute to confo rma tional changes in pro te ins

required for the direc tionally motile b ehavior of the ce lls .

Pr eliminary experiments indica te tha t increas ed levels o f cyc lic AMPin the cells increas e the ac t ivity o f pho spholipas e A Addi tion o f

ei ther dibutyryl cyc lic AMP or pe p tide at trac tant p lus theophylline

rais ed phospho lipas e ac t ivity by 50 percent in 10 min . Chemotaxis is

part ly inhib ited (40 to 50 per cent ) when cyclic AMP accumulates . Ourresul ts rais e the poss ib ili ty tha t cycl ic nucleotide — ca taly zed

phosphorylation o f p ro tein may be invo lved in the phospho lipas e

ac tiva tion induced by chemoat trac tants . Such a mechanism may contri

bute to the regula tion o f chemo taxis .

The regulation o f chemo taxis We have b een s tudy ing the inh ib i tory

ac tion o f material derived from tumors s inc e it has b een found by

o thers that cells from tumor-b earing animals show depres s ed chemo taxis .

Ethanol extracts from a rapidly prolif era ting mous e f ib rosarcoma were

found to contain a material which inhib its chemotaxis in bo th

neutrophils and macrophages . The ma terial was no t t oxic to the cells .

Peri toneal macrophages from mice b earing the f ibrosarcoma show ed

depres s ed chemo taxis compared to cells from normal mice . We have

determined s ome properties of the tumor fac tor . G e l f il tra tion

s tudies indicated that the molecular weight of the mate rial was les s

than The anti- chemotac tic ac t ivity was des t royed by p ro teas es,

sugges t ing it was a p ep tide . Par tial puri fication o f the material by

liquid and paper chromatography resul ted in two compounds which containe d relatively high amounts o f iso leucine , leucine and glutamicacid . This ma terial does no t compete with a ttrac tant for the

ge c ep tor ,

but reduces the incorporation o f lab elle d methy l groups from Hme thyl ) me thionine int o phospholip ids o f the cell . It , therefore ,

appears to interfere with transmis s ion of the chemo tac tic s ignal

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generated by the activated re cep tor . Complete identificat ion o f this

tumor—produced inhib itor and elucida tion o f its mode o f ac tion

obvious ly would b e of great signif icance in exp laining how tumors

subvert hos t su rveillance sys tems .

Ano ther mechanism for regulating chemo taxis may b e through metabo lismo f the chemoattractants . We have previous ly showed tha t formylatedpep tides were hydrolyzed by the leuko cy tes . An al terna t ive metabo l icpathway appears to b e an oxidative one . We have now found , in

collaboration with Dr . R .A . C lark , Bos ton Univers ity Medical C enter ,

that a puri fied myeloperoxidas e sys tem derived from neutrophils ,

inac t ivated methionine—containing at trac tants such as G 5a and

formylated methionyl p ep tides . The inact ivation res ul ted from

oxidat ion o f the thioether o f methionine to sul foxide / sulfone . Pep tide

at trac tants no t containing methionine were no t a ffec ted . S imilarresults were ob tained with intac t neutrophils in the p res ence o f

attrac tants and may s ignify an important regulato ry mechanism for the

cell in controlling its own chemo tac tic respons e .

Glucocor tico ids are anti-inflammatory agents whos e mechanism o f ac tion

has b een o f broad interes t . Others have shown that thes e compounds

inhib it lysosomal enzyme releas e from leukocy tes and reduc e the

chemotac tic respons e . We have now found that a l6-hour preincuba t ion

o f neutrophils with pharma cological levels (1 pm) o f a glucocort icoid ,

fluocinolone , pro tec t s the cells agains t the degranulating ef fec ts o f

a sub sequent treatment with phorbol es ters . The lat ter compound at10 nM causes vigorous exocytos is and markedly dep res s es bo th chemo taxis

and pep tide binding in cell s no t treated with s teroid . S ince phorbol

es ters are inflammatory stimuli,a s tudy o f the counterac ting ef fect s

o f s teroids ob served here may yield some leads on their mechanism o f

ac tion and on pos t-recep tor events in chemo taxis . For example, a

phorbol es t ers are known to enhance phospho lipase ac tivity . We

plan to determine i f the s teroids promo te the synthes is o f a lipaseinhibitor .

Applications o f formylated peptides to clinical problems It is

conceivable that sys temic or lo cal adminis tration o f thes e pep tides

could be used to control appearance o f leukocy tes a t certain s i tes in

the ho s t . Such manipulation mi ght be o f bene fi t in combat tinginfec tions or ameliorating allergic inflammations . In collaborationwith L . Al tman , Univers ity o f Washington Medical Center

,we have

carried out s tudies on the survival o f thermally inj ured mi ce . The

purpos e is to det ermine whether pep tide at trac tants could s igni fi cantlyenhance the survival rate o f such anima ls which were sub sequentlyexposed to lethal dos es o f Pseudomonas . U tili zing a conj ugate o f anEMe t pep tide and sul fadiazine and mixtures o f this antimi crobial agent

and pep tide at tractant , we treated mice as des crib ed above,dres s ed

their wounds with thes e compounds,and observed their survival ra tes .

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Preliminary indications are tha t mixtures o f antimicrob ial agent and

at trac tant s igni ficantly pro tec t the mice . This mi ght b e app lied in

treatment o f burn V ic tims , in whom infec tion is a maj o r risk .

Fibroblas t chemo taxis

Others have shown that fibrob las ts res pond chemo tac t ically to lympho

cyte-derived factors tha t might be expec ted to b e formed as a

cons equence o f inflammation . We have ini tia ted a s tudy on fibroblas t

chemo taxis s ince su ch behavior may be impor tant in wound repair and

s ince our findings on leukocyte mo tili ty might be applied here . Dr .

V . G . Muller has developed an as say for fibroblas t chemo taxis uti li zingtechniques originated by A . Pos tlethwai te . The requirement for

co llagen as an a t tachment subs trate has been confirmed , but i ts

claimed role as a true at trac tant remains unclear . Fibronectin (F N) ,

pro tein involved in the at tachment o f fibrob las t s,has been shown to

b e a true a t trac tant . A mixture o f ganglios ides inhibi ted chemotaxi s

to both F N and a lymphokine speci fic for fibrob las ts . This indi cates

that reversible adherence o f the cell i s es s ential to chemotaxis and

does no t depend upon the nature o f the at trac tant . Inhib i tion o f

methylation and phospho lipase ac tivi ties depres s ed chemo taxis . The

at trac tants were found to change the turnover o f bo th pro tein methyl

es ters and methylated pho spho lip ids,events which are also

prominent in neutrophil chemo taxis .

Propos ed cours e o f pro j ec t

We plan to continue s truc ture— activi ty s tudies on pep tides in

co llaborat ion wi th contrac tors,Drs . R . Freer and E . L . Becker (N0 1-DE

In s tudies wi th the neutrophil various b lo cking group s on the

amino group o f methionine in tripep tide-O—benzyl es ters will be pre

pared and tes ted as inhib i tor s o f chemotaxis . Us ing ano ther approach,

trifluorome thy l methionine will b e incorpora ted into a tripep tide and

the lat ter tes ted for b iological ac tivi ty . We al so plan to s tudy the

macrophage pep tide recep tor . For this purpo s e a new high af fini ty

ligand , PMe t-Leu—Phe-NEZ , i s being prepared as a radiolab elled com

pound .

We intend to pursue inves tigations on the mo lecular events during

transmis sion o f the chemical s ignal triggered by the at trac tant . The

resul ts from gluco cor ticoid treatment o f cell s sugges t that a

pho spho lipase inhib itor i s increased,leading to diminished membrane

turnover . We plan to s tudy the nature o f (pro tein) and the generation

o f such an inhib itor (whether dependent upon de novo synthes is,or i ts

degradation inhib i ted by s teroid ) . We also will determine whe ther

there is an inhibi tor for the methyles teras e enzyme in neutrophil s .

The cyclic nucleo tide s tudies bear on these points , and we shall

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attempt to f ind out whether cyclic AMP enhances phospholipase via

protein phosphory lation .

We plan to identify the tumor—derived inhibitor and inves tigate more

fully its m echanism of action . Oxidative mechanisms will also receive

our attention . We plan to determine whether myeloperoxidase treatment

o f recep tor—bearing cellular fragments will inactivate the b inding

s ite . This may well o ccur s ince intact sulfhydryl groups were

previously shown to be required for maximal b inding of agonist to

recep tor .

Studies with pos sible clinical applications o f the pep tides will be

continued . I t is planned to sy nthes ize other conj ugates o f chemo

at tractant pep tides and antimicrob ial agents and then to tes t them for

ef ficacy in promoting res is tance to infection in burned animal models .

We shall also s tudy the metabo lism o f these conj ugates ( e . g .

hydrolysis ) in bo th leukocytes and bacteria .

We intend to continue s tudies on motili ty in non—phagocytic cells such

as fibroblas ts . The role of phospho lip id me tabolism. wil l be more

extens ively s tudied (exis tence of inhib itors o f phospholipase , ef fect

by levels o f cyclic AMP upon this enzymatic activity in the cell ) . We

also intend to examine fibroblas t chemotaxis in cells from patients

with abnormalities in wound healing such as diabetes .

PUBLICATIONS

Chiang , P .K . , V enka tasubramanian , K . , Richards,H . M . ,

Cantoni, C . L .

and Schif fmann , E . : Adenosyl—homocys teine hydrolase and chemo taxis .

In Creveling , R . e t al . , Transmethylation . Nor th S trong

Elsevier , 11 : 1 65-1 72 , 197 9 .

Hirata , F . , Corcoran , B .A ., V enka tasubramanian , K . ,

S chi ffmann, E

Chemoat tractants induce phospholipase activity in leukocytes . Proc .

Natl . Acad . S ci . 76 : 2640-2 643 , 19 79 .

Schi ffmann , E . , O'Dea , R . F .

, Chiang, P . K . , V enkatasubramanian , K . ,

Corcoran , B .A . , Hirata , F . , and Axelrod, J . : Role for methylation in

leukocyte chemo taxis . IGN—UCLA Sympos ium on Covalent and Non-covalentModulation o f Protein F unctions . Atkins

,D . (ed . ) Academic Press

,

N . Y . In press, 19 79 .

Schi ffmann , E . and Gallin , J . I . z Biochemis try o f phagocy te chemotaxis .

In Topics ip _

Biologic al Regulation . Horecker,B . and Stadtman , E . R .

Ac admic Press , in press, 19 79 .

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V enka tasubramanian , K ., Hirata

,F .

, Cor coran , B . A ., O

'Dea ,

R . F . ,

Gagnon, C . , Axelrod , J . and Schi f fmann

, E . : Methyles terase in

leukocyte chemotaxis . Mole c . Immunol . In pres s,19 79 .

Schi f fmann , E . ,V enka tasubramanian , K .

,Cor coran , B .

,Aswanikumar , S . ,

Day,A . R . , Freer , R . F . , Gallin , J . I . , Clark , R . A .

,Hirata

,F . , Brown

,

J .H . and Gro ss , E . : Chemo tac tic pep tides as probes o f mole cularevents in leuko taxis . 7 th International Peptide S Geo rgetownUniversi ty , Washing ton , D . C . In pres s

, 19 79

NOl DE 8 2410

issue

N AM ES,LABOR ATORY ANO I N ST I T UT E AFF I L I AT I ON S , AND T I T LES OF PR I NC I PAL I NV EST I G ATORS AND ALL OT H ER

PROF ES S I ON AL P ER SONN EL ENG AG ED ON THE PRO J ECT

George R . Mart in

OTHER : Hans-Jurgen Barrach

Jean-Michel F oidart

J . Clif ford Murray

Stephen 1 . Rennard

Pamela G . RobeyTryggvason

Mina H . Yaar

Johns Hopkins Hosp ital , Bal timore , MD ; NEI , NC I ;x—Planck-Ins ti tute fur Bio chemie , Germany

fifiqb) HUM AN T I S SU ES N E I TH E R

SUMMAR Y OF WOR K (200 wo rd s or le s s und e rlin e keyword s )

The purpos e o f this proj ect is to s tudy the formation , function and des truc tion

o f connective tissue components in normal and dis eas ed s tates . Par ticular

attention is directed toward collagen and pro teoglycan . Current aspects of

this proj ect include ( 1 ) characterization o f the ma trix components in a tumor

which produc es bas ement membrane , ( 2 ) the role .of co llagen in dis eases

and (3 ) interaction of tumor ce lls with co llagens .

Chief , LDBA

Gues t Worker

Vis iting AssociateVis iting Fellow

Research AssociateBiologis t

Visiting AssociateVis iting F ellow

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Tryggvason,Rob ey and Martin

Laminin,A Basement Membrane Glycopro tein

—‘

We have is o lated a large

glycoprotein,laminin , f rom th e tumo r wh ich is als o a maj or cons ti tuent

o f normal bas ement membranes . Laminin has a molecular weight o f about

a million daltons and contains chains of and daltons .

Laminin is not c odis tributed in the bas ement membrane with Typ e IV

co llag en s ince laminin i s found in the lamina lucida and typ e IV

collagens in the lamina densa .

Mar tin,Rob ey , F oidart , Rennard with Timpl , Max Planck Ins t itute

Bas ement Membrane Pro teogly cans Recently , with Dr . John Has s ell ,

NEI , we have isolated a proteoglycan , BMI , from the tumor that app ears

to b e localiz ed only in bas ement membranes . BMI contains about 30%

pro tein and 7OZ heparan sulfate . Antibody prepared agains t BMI reac ts

with bo th the tumor matrix and tis sue bas ement membranes . It is

likely that BMI becaus e of i ts negatively charged groups has a func

tional role in the bas ement membrane in res tric t ing the pas sage o f

acidic macromolecules .

Martin , Rob ey , Barrach , Wil z c ek , Rennard with Has s ell , NEI

Func tion o f Bas ement Membrane Components The ro le o f the bas ement

membrane components will b e tes ted by adminis tering sp eci f ic anti

bodies to fetal animals . So far we have found tha t antibo dy to typ e

IV c ollagen caus es kidney and lung damage while antibody to laminin has

no ef fec t . The ro le o f the pro teoglycan will b e inves tigated in a

s imilar fashion .

F oidart , Yaar , Brown

Tumor C ells C ertain metas tatic tumor cells produce a collagenas e that

degrades basement membrane but no t o ther co llagens . Thes e metas tatic

cells , in contras t to normal cells and nonme tas tatic tumor cel ls,at tach

pref erentially to typ e IV over types I , II , II I , and V co llagens . The

numb er o f metas tas es ob tained in anima ls is increas ed by inj ec ting the

cells that b ind to type IV collagen . These obs ervat ions sugges t tha t

the metas tatic cells rep res ent only a port ion o f the cells in the

tumor . We are develop ing a metas tatic index for tumor cells bas ed on

their ab ility to b ind to typ es IV and I co llagens and produce the type

IV collagenase .

Murray and Martin with L io tta NC I

The Ehlers—Danlos Type V I Syndrome Collagen from a patient with the

Ehlers-Danlos type VI syndrome has b een examined . This pati ent has

been found to accumula te a collagen containing a pNaZ chain . This

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indicates that the c l e aveage o f the amino terminal pep tides from the

pro al chains occurs but no t from the pro O Z . The pa tient 's cel ls

contain normal amounts o f proco llagen N— pro tease . Purified chick pro

co llagen N—pro teas e cleaved the amino terminal pep tides f rom the

pr o al but no t from the pro a 2 chains from the pati ent 's collagen .

Thes e data indicate an abnormal res is tance o f the pro a 2 chain to

procollagen N-pro tease p re sume ably due to an amino acid sub s ti tution

in the pro a2 chain .

Mar tin , S teinman with McKusick,Johns Hopkins

Fibronectin New immunoas says have been es tablished for measuring

connect ive tis sue pro teins Thes e have b een us ed to measure fibro

nec t in produc tion by hybrid cells and locali ze the human fibronec tin

gene to chromosome 8 . Further,i t has been found that the reduced

b inding o f f ibronec tin by trans formed cells i s grea tly increas ed by

dexamethasone . Pre sumeably ,dexamethasone increas es the numb er o f cel l

surface recep tors for f ibronec tin .

Rennard , F oidart wi th Furcht,U . o f Minneso ta , with Church , U . o f

Pi t t sburgh

ment as well as func tion . In our s tudies,the function o f various types

o f collagen is being inves tigated . These s tudies show that these

co llagens can be us ed as molecular markers to identi fy speci fi c celltypes . Speci fic antibodies have been prepared to these collagens and

us ed in his to logical and patho logical s tudies . De fec ts in one or

ano ther collagen are as sociated wi th cer tain dis eas es and developmentaldi s orders .

PUBLICATIONS

Ab e , S . , S teinmann , E .U ., Wahl , L .M . and Mar t in

,O . R . : High cell

densi ty al ters the ratio o f type III to I collagen synthes is by fibro

blas ts in cul ture . Nature 2 79 : 442—444 ,19 7 9 .

Cournil , I . ,Leblond

, C . P . ,Pomponio

, J . ,Hand , A . R . , S ederlo f ,

L .,

and Mar t in , G . R . : Immunohis to chemical locali zation o f proc ollagens .

I . Light microscop ic dis tribution o f pro collagen I , III and IV

antigenici ty in the rat incis or too th by an indirec t method (PAP ) .

J . His to c hem . Cyto chem . 2 7 : 1059—1069 ,19 7 9 .

F oidart , J .M .,Hall

,R . Martin

, G . R . ,and Kat z

,S . I . z Immunity to

type II co llagen in relaps ing polychondri tis . In Sympos ium on

Ar thri ti s,Immunity and Aging

,Los Angeles , CA

,January lo—lZ , 19 7 9

In Press .

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F oidar t , J .M . and Ka tz , S . I . : Relap sing polychondri tis . Archives o f

Dermatol . In pres s ,19 79 .

F oidar t , J .M . , Reddi ,A .M . : Immuno fluor escent lo cali za tion o f type IV

co llagen and laminin during endochondral bone formation and ef fec t o f

growth hormone upon this phenomenon .

“Develop . Biol . In pres s , 1979 .

Furcht, L . T . ,

Mosher,D . E . ,

wende ls chafer—Crabb ,G .

, and F oidart , J .M

Fibronect in and type I pro collagen fibrillogene sis in cul tures o f

human skin fibrob las t s de tected by ul tras truc tural immunocy tochemi s try .

Proc . Natl . Acad . S ciJ‘

USA . In pres s , 1 979 .

Furcht, L . T .

,Mosher

,D . E . , Wende ls chafer-Crabb , G . , and F oidart , J .M

Gluco cor ticoids partially reverse the los s o f the fibronectin and pro

collagen matrix around trans formed human cell s .

‘

cancer Res . 39 : 207 7

2083 , 19 79 .

F urcht , L . T . , Mo sher ,D . F . , Wende ls chafer-Crabb , G . , Woodbridg e ,

P .A

and F oidart , J .M . : Dexamethasone— induced ac cumulation o f a fibro

nectin and collagen extracellular matrix in trans formed human cell s .

Nature 2 7 7 : 39 3— 39 5 ,19 79 .

Hewit t,A . T . , Kleinman , H .K . , Pennypacker

, J . P .,and Mar tin

,G . R

Ident if icat ion o f an adhesion factor for chondro cytes . Pro c . Natl .

Acad . S ci . USA . In pres s, 19 79 .

Kleinman,H . K . , Hewi tt , A . T . , Murray , J . C . , Lio tta , L . A . ,

Rennard , S I . ,

Pennypacker, J . P .

, Mc Goodwin , E . B . ,Mar tin

,G . R .

,and Fishman ,

P . H

Cellular and metabolic speci f ici ty in the interac tion o f adhesion

pro teins with collagen and with cells . J . Supramole c . S truct . In

pres s , 1 9 79 .

Kl einman , H .K . , Mar tin , G . R . and Fishman , P . H . : Ganglio s ide inhib ition

o f fibronec tin—mediated cell adhes ion to collagen . Pro c . Natl . Acad .

Sci USA 7 6 : 336 7-3371 , 19 79 .

Kl einman , H . K . , Mc G oodwin , E .B . ,Rennard , S . I . , and Mar tin

, G . R

Preparation o f collagen sub s trates for cell at tachment . Effec t o f

co llagen concentration and phosphate buf fer . Anal . Bio chem . 94

308— 312 , 19 7 9 .

Lio t ta , L . A ., Abe , S . , Robey

, P . G . ,and Mar tin

, G . R . : Preferentialdiges tion o f basement membrane co llagen by an enzyme derived from a

metastatic murine tumor . Pro c . Natl . Acad . S ci . USA 7 6 : 2 2 68-2 2 7 2 ,

19 7 9 .

11 — 4 8

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Martin,G . R .

,Brown

, K . S .,Matheson ,

D . , Le bowit z,H .

,Singer

,L . , and

Ophaug,R . : Lack o f cytogenet ic ef fec t s in mice or muta tions in

salmonella receiving sodium fluoride . Mutation Res . 6 6 : 15 9-1 6 7 ,

Murray, J . C . , S tingl ,

G . , Kle inman , H K . , Mar tin,G . R . , and Kat z ,

Epider mal cells adhere pre fe rentially to type IV (bas ement membrane )collagen . J . Cell Biol . 80 : 19 7-20 2 , 19 79 .

Pennypacker,J . P . ,

Wilk A . L . and Mar tin , G . R . : Differ e nt ia t ion o f

me s enchyme cells into chondrocy t e s in culture : Effe c t o f tera to gens .

In Neuber t , D . , Merker , H . J . , Nau , H . , and Langman , J . Ro le

S tuttgart , 19 78 , pp . 4 11l 41 9 .

Robey , P . G . : Ef fec t o f de xame thasone on collagen me tabolism in two

strains o f mice . Biochemi cal Pharmacol . In press 1 9 7 9 .

tanley ,J . R . , F oidar t , Murray , J . C . , Mar tin , G . R .

, and Kat z , S .

I . : The epidermal cel l vhich selec tively adheres to a collagen sub~

s trate is the basal ce ll . J . Inves t . De rma tol . In pr e ss , 19 79 .

S teinmann , E . U . , Martin,G . R .

,Baum

,B . I . , and Crys tal , R . G

Synthe s is and degradation o f collagen by skin fibroblas ts from pa tients

wi th o s teogenes is imperfecta . FEBS Let ters 101 : 269- 2 7 2 ,19 79 .

S teinmann , B . and Reddi,A . H . : Changes in types I and III collagen

synthesis during matrix-induced endochondral bone di fferentiation .

Biochemical Journal . In pres s , 19 79

Stoner , G . , Toichi , K . , F oidart , J . M .,Cur tis

,H . : Identif ication and

cul ture o f human bronchial epithelial cells . In Harris,C . C . , Trump ,

B . F . and S toner , C . D . Methods in Cell Biology . New York ,

Academic Press , 1980 . In press

Timpl , R . , Martin , G .R . and Bruckner, P . : S tructure o f basement

membrane collagen obtained from a mous e tumor . In Garrone , (Ed . )F rontiers in Matrix B iology . Basel

,Swit zerland , S . Karger Verlag

,

1979 , Vo l . 7 , 130-141 .

Timpl , R . , Wick , G ., Glanville

, R .W . , and Mar tin,G . R . : Immunochemical

s tudy on basement membrane (TY Pe IV) co llagens . J . ImmunologyIn pre s s

, 1 9 79 .

Timpl , R . , Rohde , H ., Robey

, P . G .,Rennard ,

S . I . ,F oidart , J .M . , and

Mar tin , G . R . : Laminin A glycopro tein from basement membranes . J .

Biol . Chem . In pres s,1 9 79 .

LI. £1 0

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Wick,G . ,

Muller, P . U . , Timpl ,

R ., and Martin . S tudies on the

immunology o f bas ement membrane co llagen using antibody to a tumor

basement membrane . In Garron , Front iers in Matrix Biology .

Basel,Switzerland , S . Karger Verlag

,19 79 ,

Vol 7 ,pp . 120-1 29 .

Wilk,A . L . , Greenberg , J .H . , Horigan , B .A . , Pratt , R .M . , and Mart in ,

G . R . : Detec t ion o f teratogenic compounds using differentiating

embryonic cells in cul ture . In Vitro . In pres s,19 79 .

n u e lI \ \ U L U I IU IIIO L H\

'J “U u i | l g , Q “: [1 t n L J U U H I I V " H l'

tu nI

II PUBL I C HEAL TH LERVHOT ICE OF

I I N TRAM URA L RE S EARCH PRCJECT

L. ‘ M il l’

.

2

7 01 DE 0002 4— 1 3 DB

L_ _

i LR ICO b V C REU

I temberfi 9

FOJECT (so c he rfit t e r; E r

Pro ce s s es In Gene t ically Contro lled Malforma t i ons

N AM ES,LAB ORA TO RY ANO I N ST I T UT E AFF I L I AT I ON : , Au; T z iLL. OF PRILS IPAL ANO AIL OTHLR

PROF E SS I ON AL PER SON N EL EHGAG LD ON T+E q uz cr

PI Kenne th S . Brown Medical Dire c tor

OTHER : Les lie C . Harne Bio Lab Tech (Animal )

David M . Strong Bio Lab Tec h (Animal )

Marilyn Wind Pos tdoc toral F e llow

I an } Dr . W . Lena Alistihf'

DepT'

7 5F Micrbb'

ibT ogyT'

H b‘

war—d—Universi ty

‘

i

Dr .Robert B . Cranley ,

D ep t . o f P a tholo gy , St . Agnes

Baltimore ,Mary land ; Dr

.Luigi De Luca , NCI ; Dr . David Newsome , NEI ;

ly _ White , NIAMDD .

“Laboratory o f Deve lopmental Bio logy Anomali e s

Conne ctive Tissue S ec t ion

Maryland 20 20 5

PROF E S S I ON AL

6 .

CH¢ 5K A P PRO PR I ATE 80 (ES )

\0 U

1

3 (c ) H UMAN T I SS U ES Kym NEIT Hz a

I] (E I ) MINOP S IN T E RV I E N SSUMMAR . OF WOR K (200 w

ord s or le ss un d e r lin e ke yword s )

The obj ec tiv e s or this p roj e c t are to d es crib e th e ene t i c mechanisms and

and limbs,

and to ut iliz e the s e anima ls as expe rimental sys t ems for the

s tudy o f the proce s s e s o f congenital malformat ion . Mous e s trains with

he reditarv malformations as Mendelian or non—Mendelian traits have b e en

dis cove re d in this labora to ry or are ob taine d from o th e rs . Comparis on of

normal and abnorma l deve lopmen t are carrie d ou t in emb ryos_ 9 j _

time d

preparations and his tologica l s ections as we ll as b ioch emica l analys es o f

ti ssue s and o f cul tured organs and ce lls ._G ene t ic analys is involve s

se gre ga t ion analys is s e le c t io n of sub line s c ros s b re e ding o f abnormal

and norm al line s and g ene tic linkage s tudie s . Agents such as drugs ,

v itamins and hormone s are us e d as pro be s bo th for th e s tudy o f gene ac tion

in he reditary malformations and for the s tudy o f pos sib le human te ratog ens .

l')

“I

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Ob j ect ives

The obj ec tive is to determine the b iochemical ba s is o f genet ic di f fer

enc e s in malformations . Inherited di f f erences in mic e of di f f erent

geno types are b eing us ed to detec t the b iochemical mechanisms o f b irth

defects . Mutants wi th generalized def ects o f growth resul t ing in

facial clef ts and dwarf ism are b eing exp lored in detail a t the bio

chemical level . Mutants with di ff ering sensi t ivity to terato genic

agents are b eing us ed as prob es for the cellular and b iochemical bas is

o f abnormal development .

Methods

Selec ted s tocks of mice carry ing mutations producing malformations and

highly uniform inbr ed laboratory mice dif f ering in metabo lic responses

are our tes t sys tems . Timed pregnancy and genetic uni formity p ermit

sp eci fic s tudies of critical p eriods o f development and the sp ecif ic

ef fec t o f gene act ion on morphogenes is . Timed treatments with

appropriate drugs , hormones or growth fac tors act as detailed probes

of the b iochemis try o f the developmental proces s .

F indings

The findings will b e dis cus s ed f or s everal o f the genetic typ es o f mice

being inves tigated .

Op en eyelid with clef t palate oel

The mous e s train OELS has the reces s ive gene oel which p roduces the

lethal malformation o f open eye lid with clef t palate . It di f f ers

from OELN , a clos ely related normal s train , in its dif f erential

s ens itivity to teratogenes is induced by retinol . Bo th s trains are

highly and equally sens itive to retinoic acid tera to genes is . We have

b een working to delinea te the mechanism o f the di ff erential s ens i tivity .

Transplacental pas sage s tudies utili zing tritic ate d re tinoic acid and

retino l indicate tha t the drugs cros s the placenta in bo th OELS andOELN mice . Cytosol b inding recep tors for retino l in the maxillaryproces s es o f newborn mice were the same for OELS and OELN . However , we

have found in co llaboration with Dr . L . De Luca , NC I,that the s ens itive

s train metabo lizes retinol much more rap idly than the res is tant s train .

Further these s tudies indi cate that the s ens i tive animals conver t the

retinol to retinoic acid to which bo th s trains are more sens itive .

Thes e s tudies sugges t that the mal formations are due to an enhanced

convers ion o f dietary retino l to the more teratogenic retinoic acid .

The ef fect o f lowering dietary retino l levels on the incidence o f

malformations is under s tudy .

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Recurrent epilation Er

The homozygous gene Er results in fus ion of epithelial surfaces . The

oral s tructures are fus ed to each other and the limbs and tail are

fused to each other and to the trunk resulting in a pupoid fetus which

canno t breathe at birth . This defect s tarts after day 1 2 and is

as sociated with intrauterine b leeding . S teroid treatments have failed

to modify the express ion of the trait . Attemp ts to cul ture the skin

cell s have not yet succeeded . No abnormali ties o f pemphigus and

pemphigoid antigens have been detected in collabora tive s tudies with

Dr . John S tanley o f NCI . No abnormali ty o f surfactant precursor

up take or keratinz ation has been found as yet .

F orelimb defect fld

This reces sive mutant has a generalized defect of bone that resembles

some o f the types o f os teogenesis imper fecta with fractures and

c ampome lia observed in the newborn . The affected animals are born

al ive but rarely survive to weaning . His tological s tudies have no t

been diagnos tic and preliminary s tudies of bone collagen are in

progres s . Alizarin preparations show fractures o f bones in the limbs

and ribs .

Teratogenic responses o f inbred s trains

Genetic di fferences in respons es to teratogenic alkaloids which areknown to occur in foods and els ewhere in the environment might provi de

a proces s resulting in the observed irregular dis tribution o f human

birth defects . To tes t the teratogenici ty o f s ome myco toxins and

po s sible genetic di f ferences we have been collaborating with Dr . LenaAus tin o f Howard Univers ity in a s tudy of the cytochalas ins . These

sub stances produce a charac teris tic neural tube defect wheniadminis te red at the end o f the firs t week o f ges tation . Signi ficant

dif ferences among s trains and a reces s ive gene tic pat tern for

suscep tib ility to teratogenic response to cytochalas in D has beenfound . Cytochalasin E is more embryo lethal but les s teratogeni c than

D while B is an order o f magnitude les s toxic than ei ther .

F uture plans

The metabolic defect in the OELS mouse that results in sens itivi ty to

vitamin A will be characterized speci fically . We will firs t tes t the

hypo thes is that retinoic acid is formed as a resul t o f a block in

normal retinol metaboli sm . The effec t o f vi tamin A free diet on theexpres sion o f oel will al so be s tudied . S imilar extentions o f bio

chemical s tudies ou the defect in the o ther mutants are planned . In

addition the animals with defects will be us ed to tes t interactions

4 - 5 1!

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with teratogens,and among di f ferent types o f genes producing de fec ts .

New mutant s will be examined for the pres ence o f bio chemi cal defects

producing mal formation or dwar fi sm .

PUBLI CATIONS

Brown,K . S . : Genet ics o f C lef ting in the Mouse : Dis cus sion . In

Bixler,D . and Melnick

,M . Academic Pres s

,New Y ork ,

In

pres s , 19 7 9 .

Brown , K . S . : V es t ibular Anomalies as a Marker in Cle f ting : Dis cus s ion .

In Bixler , D . and Melnick,M . Academic Pres s

,New York

, In

pres s , 19 79 .

Brown , K . S . : Impli cations o f Recep tor Phys io logy for Teratogenes is .

In Bixler , D . and Melnick , M . Academic Pres s,New York

,In

pres s , 1979 .

and Diseased Tissues

N AM ES,LABORATO RY AND I N ST I T UT E AFF I L I AT I ON S, AND T I TL ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OTH ER

PROF ESS I ON AL PERS ON N EL ENG AG ED ON THE PRO JECT

PI Larry M . Paglia

OTHER : George R . MartinLucille A . OuelletteJoseph Wilczek

None

SUMMARY OF WOR K (200 word s or less und e rlin e ke yword s )

The purpo se of this proj ect is to inves tigate the regulat ion o f co llagen

biosynthes is in normal and diseased cells . Here we are concerned with

translat ional control of b iosynthesis and po s ttranslat ional s teps in helix

formation .

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The purpos e o f thi s proj e c t is to inves tigate the regulation o f

collagen biosynthes is in normal and di sease s tates . We are c oncerned

bo th with the regulation o f collagen type and the amount o f collagen

produced . Our approach involves use o f a cel l-free pro tein synthe

s izing sys tem to examine the s teps in collagen mRNA translation and

pos t trans lational modi fication .

The type and amount o f collagen pres ent in a ti ssue i s clos ely

regulated,with changes in ei ther parameter as sociated with emb ryonic

development and with dis ease s tates . The type of collagen is probably

determined by selective gene trans crip tion , however , the cell s

synthesizing collagen mus t have some mechanism for determining the

amount o f collagen present in the ti s sue and adj us ting i ts synthes is .

Recent evidence sugges ts that the amino terminal extens ion pep tide o f

procollagen,normally cleaved during proces s ing o f the molecule

,may

ac t as a feedback inhib itor o f i ts synthesis . We have inves tigated the

pos sibili ty that thi s pep tide acts at the trans lational level in a

reticulocyte lysate cell— free sys tem . The lysate ef ficiently trans

lates mRNA from collagen—producing t i ssues into procollagen . When Type

I NH — terminal pep tide was added to the trans lation reaction , pro al ( I )and pro a2 synthes is was inhib ited . This decreas e was determined by

speci fic immunoprecipitation and by gel electrophores is o f the cellfree products . The synthes is of bo th chains was equally reduced .

Translation o f the mRNA for other pro teins was unaf fected . The

analogous pep tide from Type III procollagen was also inhib itory while

pep tides derived from o ther regions o f the collagen molecule-were no t .

RNA from chick s ternum and rat chondrosarcoma has also been success fullytrans lated into Type II procollagen . Addition o f Type I prop ep tide to

these reactions inhibits Type II procollagen synthes is , while , Type II I

prO p ep tide does no t affec t trans lation o f Type II mRNA . We are

currently inves tigating the mechanism and speci fici ty o f this

propeptide inhibi tion .

Procollagen synthesi zed in the cell-free sys tem is also being analyzed

with regard to it s ab ili ty to form a native triple helix . RNA coding

for Type I and Type II pro collagen was trans lated and the resul ting

protein tes ted for he lici ty by res istance to pep s in diges tion . Ourdata indicate at leas t par tial helix formation even without

hydroxylation or glycosylation of the molecule . Cell— free synthesized

procollagen could also be precip itated with ammonium sulfate at 20%

saturation,a property o f native but no t denatured collagen . Thus ,

it appear s tha t the as sembly o f the triple helical procollagen molecule

is a pro ces s governed by its polypep tide chains . That is , the amino

acid s equence o f the chain is sufficient to es tabli sh the tertiary

s truc ture o f the prot e in .

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Signi f icance : Feedback inhibi tion of mRNA trans lation may be a means

by whi ch cells regulate collagen synthesis . Changes in disease states

may represent defec ts at thi s level as well as at the level o f trans

c rip tion . Inves tigations of procollagen helix formation provide

valuable information on the abili ty o f this pro tein to self-ass emblein vivo and provide new ins ights into the ro le o f po s t trans lationalmodi f ications to the pro tein .

PUBLICATIONS

Paglia , L . , Wilczek , J . , Diaz de Leon ,L . , Martin , G .R . , Horlein , D .

and Muller , P . : Inhib i tion o f proco llagen cell-free synthes is by amino

terminal extension pep tides . Biochemis try . In pres s , 19 79 .

Salomon , D . , Paglia , L . and V erbruggen , L . : Hormone dependent growth

o f a rat chondrosar coma in Vivo .

‘

Cancer Research . In pres s,19 79 .

Brei tkreutz ,D . , Diaz de Leon , L . , Paglia

, L., Gay , S . , Swarm , R . and

S tern , R . : His to logical and b io chemical s tudies o f a transplantablerat chondrosarcoma . Cancer Res earch . In pres s , 197 9 .

Diaz de Leon , L . , Brei tkreutz , D . , Paglia, L . ,

Zeichner,M . and S tern

,

R . : Par tial characteri zat ion of pro collagen mes senger ribonucleicacid in a murine chondro sarcoma . Connec tive T is sue Res earch . In

pres s , 1979 .

7~ In ~n v a

r a o c c .

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L h t n LTg

N OI ! CE OEp“,

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T , ; O , .vErc :

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r

Oc tober l , 1 9 7 8 to S e p tember 30 , 1 9 7 9

T IT L E OF FRU JECT ( 3 0 c hara c te rs t r

IAttachment of C e lls to Co llagen

N AM ES, LASORATORY ANO I N ST I T UT E AF F I L I ATI ON S , AND T ITLES OF PR I NCI PAL INVESTISATORS ANO ALL OTH ER

PROF E SS I ONAL P ERSON N EL ENG AG EE ON THE PRO JECT

PI Hynda K . Kle inman Re s earch Chemis t

OTHER : A . Tyl Hewit t Pos tdoctoral F e llow

George R . Mar tin Chie f ,LDBA

J . Clif ford Murray V is iting F ellow

Charlot te M . Wilkes Biologis t

4' 6 5 Y ) D e p t . of Human Biological Chemis try Genetics , The

Texas Medical Branch,Galve s ton , TX ; NCI ; NINCDS ; D ep t . o f

of Wis cons in ,

Connec tive Tis sue S ection

2§X(b ) HUMAN T I SSU E S L] (c ) N E I TH E R

UMMARY OF WOR K (200 word s or le s s un d e rlin e ke ywo rd s )

co llagenous ma tric eg .P articular attention is dir e cted towards ( l ) the

cells sy nthe s iz e or ut iliz e a t tachment prote ins dif f e rent than f ibronec tin ,

collagens and re cons t ituted collagen and ( 3) the int e rac tion of adhes ion

proteins with o the r mole cule s and with ce lls .

95 3-6040

(Re v. l0-76 ) a c; Q

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Introduct ions and obj ectives; Many cells exis t surrounded by a

collagenous matrix . The role of collagen in the tissue is probab lymore than s tructural s ince collagen matrices promo te the growth and

dif ferentiation of cells . Fibroblast i c cells do no t bind directly to

co llagen but are linked to the collagen by a large glycopro tein ,

f ibronectin . We are inves tigating the role o f co llagen and fibronectin

in cell at tachment . Our s tudies indicate that a varie ty o f co llagenattaching prot eins exis t including di f ferent ones for fibroblast s ,

chondrocytes and epidermal cells .

Methods The interac tion '

of cells wi th collagen is s tudied in vi tro .

Cells are ob tained ei ther as cell lines or from a variety o f tis sues

and separated by s tandard methods . HUman skin fibroblas ts and CHOcells were obtained from American Type Cul ture Collection , chondro cytes

from chick embryonic s ternum and a rat chondro sarcoma , hepatocy tes from

adult rat liver and epidermal cells from guinea pig epidermis .

At tachment properties o f cultured'

c ells The interac tion o f cells wi th

the collagen—attachment pro tein complex is likely to p lay a maj or rolein determining the dis tribution o f ce ll types in ti s sues . A variety

of cells are being tes ted for their at tachment propert ies , i . e . rate ,

serum dependence and collagen specif i ci ty . Cul tured fibroblas ti c cellssuch as human skin fibroblas ts , CHO

, 3T3 ,show enhanced at tachment to

co llagen and require serum fibronectin for at tachment . Cell s such as

hepatocytes derived directly from tis sue also show s erum dependence

while epidermal cells do no t . Epidermal cells probably synthes i ze

their own at tachment pro tein becaus e (1 ) they attach very s lowly , (2 )their at tachment i s no t enhanced by fibronec tin

, (3 ) they do no t make

fibronectin , (4 ) they adhere specifically to type IV co llag en , and (5 )they di f ferentiate into multilayered squamous epi thelial typ e cells on

type IV co llagen .

Murray , Wilkes and Kleinman

Chondrocytes The involvement o f f ibronec tin in the interaction

between chondrocytes and collagen in c artilagenous matrix is unlikelysince immunof luores cence s tudies indicate that fibronectin is absent

from car ti lage . For thi s reason , the at tachment o f chondro cytes to

collagen is b eing inves tigated . These cells appear to attach to

co llagen by means o f a factor o ther than fibronectin . We have named

this factor chondronectin . Ini tial s tudies on the specifici ty of

embryonic chick chondrocyte s ternal at tachment to collagen showed that,

in the prese nce o f s erum , chondrocytes attached best to type II

co llagen sub strates (60Z attachment ) , al though they also attached to

type I III and IV (3 02 ) collagens . This is in contras t

to CHO cells which bound e quallt e ll to all four types -in the

presence o f serum (Kl einman'

e t al . , 19 78,- J . Invest . Dermatol . 71

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The abili ty o f fibronectin to cross link to collagen -via blood factor

X III ( transglutaminase ) is also being explored with regard toS pecificity of collagen sub s trates . Ascaris collagen canno t partici

pate in this reaction while native collagen , denatured collagen a

chains and certain cyanogen bromide fragments of the al (I ) collagen

can . The interaction is specific in that al (I ) — CB7 and al (I ) —CB8 will

cros slink while al (I ) -CB6 will not .

Wilkes and Kleinman

Ganglioside inhibi tion of fibronectin-mediated adhesion Gangliosidesare cell surface glycolipids which are present in the lipid layer of

the cell membrane via their ceramide portion . We found that ganglioSide S

'

inhibit fibronectin-mediated cell adhesion . Only the complex

gangliosides GD1

and CT were active while GM GM and GM were

inactive . The sugar porfion o f the molecule contains the ac tivity

since ceramides ( the lipid ) alone were inactive . The sugar por tion

alone was active and oxidation o f the sialic acid res idues resulted in

loss o f activity . The gangliosides appear to be acting by bindingto the portion o f the fibronectin molecule which binds to the cellsur face . Cell adhes ion is b locked i f the fibronectin is pretreated

with the ganglioside but no t blo cked if the collagen is pretreated .

The inhibi tion will also occur after fibronectin has bound to

collagen . In addition , immunologic s tudies indicate that the

ganglios ide can direct ly bind to fibronectin . Preincubation o f the

cell s with gangliosides has no effect . Thus , gangliosides spe cifi

cally inhibit fibronectin—mediated cell—adhesi on by binding to

fibronectin through their sugar res idues . These results sugges t that

certain gangliosides may act as the cell sur face recep tor for fibro

nectin .

Wilkes and Kleinman

S ignificance Various genetically dis tinct collagens exis t in

as sociation wi th a S peci fic type of cell including chondrocytes with

type II collagen and epidermal cells wi th type IV collagen . Such

cells produce proteins that mediate their attachment . Our s tudies

explain the molecular basis o f this sorting out and the manner in

which tissues are formed from the interaction of cells with the

extracellular macromolecules they produce .

PUBLICATIONS

Kleinman , H . K ., Murray , J . C . , Mc G oodwin ,

E . B . and Mar tin, G . R

Connective tissue s tructure : The at tachment of cells to collagen .

J . Inves t . Dermato l . 71 : 9— 11 , 197 8 .

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S ilbert,C .K and Kleinman , H . K . : S tudies of cul tured human fibro

blas ts in diab etes me llitusa Changes in heparan sulfate . Diab etes

28 : 60— 64 , 19 79 .

Kleinman , H .K . ,Mc G oodwin , E .B .

, Rennard , and Mar tin , G . R

Preparation o f co llagen sub s trates for cell at tachment : Eff ec t of

co llagen concentration and phosphate buf fer . Anal . Bio chem . 94

308—31 2 , 19 79 .

Murray, J .D . , Stingl , G . , Kleinman

, H .K . ,Mar tin

,G .R . and Kat z , S . I

Ep idermal cell s adhere preferentially to type IV (basement membrane )collagen . J . Cell Biol . 80 : 19 7— 202 ,

19 7 9 .

Kleinman ,H .K . , Hewi t t ,

A .T. , Murray, J . C . , Lio tta , L . A . , Rennard , S . I . ,

Pennypacker , J . P . , Mc G oodwin , E .B . , Mar tin , G . R . and Fishman ,P .H

Cellular and metaboli c speci f ici ty in the interac tion o f adhesion

pro teins with col lagen and with cells . J . Supramol . S truct . In

pres s , 1 97 9 .

Kleinman , H . K ., Martin , G . R . and F ishman

,P .H . : Gangl ios ide inhibi

t ion o f fibronectin—mediated cell adhes ion to co llagen . Pro c . Nat l .

Acad . S ci . USA . In pres s , 19 79 .

Mosher , D . E . , S chad , P . E . and Kleinman , H .K . : Cros s—linking o f

f ibronectin to collagen by b lood coagulation factor X II Ia

J, C lin .

Inves t . In pres s,19 7 9 .

Mosher , D . F . , S chad , P . E . and Kleinman,H .K . : Inhibi tion o f blood

c ongulation factor X IIIa

—mediated cros s-linking be tween fibronectin

and collagen by polyamines . J . Supramol . S truct . In press , 19 79 .

Has s ell , J . R . , Pennypacker , J . P ., Kleinman ,

H .K . , Prat t , R .M . and

Yamada , K .M . : Enhanced cellular fibronectin accumulation in

chondrocytes treated with vi tamin A . Cell 17 : 821-826 , 1979 .

Hewitt , A . T . , Kl einman , H .K . , Pennypacker , J .P . and Mar tin , G . R

Identif ication o f an adhes ion fac tor for chondro cytes . Pro c . Nat l .

Acad . S ci . U SA . In press , 19 79 .

14 - 5 3

N AM ES,LABOR ATORY AND I N ST I T UT E AFF I L I AT I ON S , ANO T I TLES OF PR I NC I PAL I NV EST I G ATOR S AN D ALL OT H ER

PROF ES S I ON AL P ER SONN EL ENG AG ED ON THE PRO JECTPI John P . Pennypacker

OTHER : A . Tyl Hewit tKoj i Kimata

Willard A . LeeHugh V arner

None

S enior S taff Fellow

Pos tdoctoral FellowVis iting AssociateChemis tPos tdoctoral Fellow

Laboratory of Developmental B iology Anomalies

Connect ive Tis sue Section

SUMM ARY OF WOR K (200 word s or less und e rlin e keyword s )

The factors influencing chondrogenes is are under s tudy . Particularattention is being directed towards the extracellular matrix of developing

cart ilage and its relationship to normal cellular function .

In these inves tigat ions,use is b eing made of limb mes enchyme cell cultures ,

mature chondrocytes in culture,cartilage producing tumors and cartilage from

various animal mutants which may se rve as models for human chondrodys trt ie s .

2 01 DE 0025 3—02 DB

Studies On Chondrogenic Express ion in vi tro

I . Embryonic limb mese nchyme cells plated at high density~differe tiate

into cartilage within four days . However , at low de ns i ty chondro

genesis does not occur . The respons iveness of limb mese nchyme to

various growth factors was test ed in an attempt to enhance proliferation and ob tain chondrogenesis from low dens ity cultures . Orni thine

biological response to the factors . The developing limb bud in vivo

has a rise in ODC ac tivity prior to chondrogenes is ,and this is al so

true when mes enchymal cel ls are p lated at high dens i ty and allowed to

undergo chondrogenesis in culture . However,at low dens i ty the CDC

activity remained low and the mesenchyme did no t di f ferentiate . The

mesenchymal cells were found to be unresponsive to fibroblas t growth

factor , F G F at both high and low densi ty . However , they responded to

epidermal growth factor , EG F , but at low densi ty only . The EG F

ef fect did no t enhance chondrogenes is . Work is continuing to determine

if the matrix helps dic tate ECF respons ivenes s by the mes enchymal cells .

Pennypacker

II . Previous s tudies have shown that a high molecular weight fraction

of embryo extract inhib i ts chondrogenic expres s ion . The purpos e of

this s tudy is to develop an assay pro cedure for embryo extract ac tivi ty

and to utili ze i t to follow the puri fication o f the active component

o f embryo extract . A MW protein is now being characteri zed

as a marker for chondrogenic expres s ion . Preliminary evidence

sugges ts this protein is Type II procollagen and its appearance is

rapidly 24 hr ) af fected by embryo extract . S tudies are now in

progress to determine the nature o f the effect on the putative pro

co llagen .

Pennypacker and Varner

Chondrocyte Interaction wi th Collagen

Recent s tudies indicate that chondrocy te interaction with collagen is

mediated by a serum protein , designated chondronectin , whereas

fibroblas ts bind via fibronectin . The purpose o f this s tudy was to

determine whether alterations in chondrogenic expres sion are

correlated wi th changes in the requirements for at tachment to co llagen .

Vitamin A was us ed to induce a fibrobl as t— like pheno typ e in cul turedchondrocytes . Preliminary resu lts su gges t that changes in pheno type

are correlated with al tered at tachment requirements .

Hewitt and Pennypacker

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The cartilage of a new mouse mutant (car tilage matrix deficiency } was

analyzed by biochemic al and immunofluorescent techniques . This mutant

is characterized by di sproportionate dwarfism and clef t palate . The

studies indicate that the car tilage—sp eci fic pro teoglycan is absent in

the extracellular m atrix and that the primary defect involves synthes is

o f the pro teoglycan core protein .

Pennypacker and Kimata

PUBLICATIONS

Binderman , I . , Greene,R .M . and Pennypacke r , J . P . : Control of

calcification of dif ferentiating skeletal mesenchyme in vitro . S cience

In press , 1979 .

Has sell , J . R . , Pennypacker , J . P . , Kleinman , H . K Pratt , R .M . and

Yamada ,R .M . : Enhanced cellular f ibronec tin accumulation in chondro

cytes treated with vitamin A . Cell 17 : 8 21-82 6 , 19 79 .

Hewitt , A . T . , Kleinman , H .K . , Pennypacker, J . P . and Mar tin , G .R

Identification o f an adhesion factor for chondrocytes . Proc . Natl .

Acad . Sci . USA . In pres s, 1 9 79 .

Kl einman , H .K . , Hewi tt , A .T . , Murray , J . C . , Lio tta,L . A . , Rennard ,

S . I . ,

Pennypacker , J . P .,Mc G oodwin , E . B . , Martin , G .R . and F ishman

, P . H

Cellular and metabolic speci f ici ty in the interaction of adhesion

proteins with collagen and with cells . J . Supramol . S truct . In pres s,

19 79 .

Pennypacker , J . P . and G oe tinck , P . F . : Revers ible inhibition o fchondrogenic expression by cer tain hyaluronidase preparations . J .

Embryol . Exp . Morphol . In press,19 7 9 .

Pennypacker , J . P . , Hass ell , J . R . , Yamada,K .M . and Pratt

, R .M . : The

influence of an adhes ive cell surface protein on the chondrogenic

expression in vitro . Exp . Cell Res . 121 : 411—415 ,19 73 .

Pennypacker , J .P . , Wilk ,A . L . and Martin , G . R . : Differentiation o f

mesenchyme cells into chondrocytes in culture : Effect of teratogens .

In Neubert , D Merke r ,H . J .

, Nau , H . , and Langman, J . Role

Georg Thieme,pp . 411— 419 , 1378 .

4 - 6 6

c iTHSONlAN SC I E N CE I NF ORMA T I ONIRCJECT N UMB ER Do N OT us e this

tember 30 , 1 9 79

N AM ES , LABOR ATORY ANO I N ST I T UT E AFF I L I AT I ON S,AN D T I T L ES OF PR I NC I PAL I N V ES T I G AT OR S AND ALL OTH ER

PR OF ES S I ON AL PER S ONN EL ENG AG ED ON THE PR O J ECT

PI George R . Martin Chief , LDBA

OTHER : Kenneth S . Brown Medical Dire ctor

Univers ity o f Minneso ta ; Lit ton Bione tic s ; NC I ; LB

[ 1 (b ) HU M AN T I S SU E S X95] (c ) N E I T H E R

INTERV lEWS

SUMM AR Y OF WOR K (200 word s or less und e rl in e keywo rd s )

The purpos e of this proj e ct is to s tudy the act ion of f luoride on various

sys tems used to detect c las togenic or mutagenic subs tances . To date f luoride

has been examined in microbio logical assays us ed to detect mutagens and

found to be non. mntagenic , No effe cts on chromosome s tructure were notedin animals given widely different leve ls of f luoride . DNA repair af ter

X — ray was unchange by f luoride . No gene t ic ef fects of f luoride were noted

in a recess ive lethal tes t of fluoride on drosophila . The data indicate

that fluoride has no mutagenic activity

4 - 5 7

201 DE 002 75-01 DB

Fluoride has been Widely use d to suppress caries . At the leve ls re

commended CO . S-1 ppm) , no detrimental ef fects of fluoride on the

health of individuals has been no ted . At very high fluoride intakes ,

alterations in bone metabolism can occur and are associated with

degenerative changes in bone .

’

It is the purpos e of this proj ect to examine fluoride in some of the

more recently developed tes ts which detec t mutagenic or clas togenicactivity . Cons iderable epidemiological data indicate that fluoride

consumption does no t increase the incidence of cancer or b irth defects .

The tes ts described below provide c orraborative experim ental information .

In our initial s tudies , we measured the numbers of abnormal chromosomes

in mice from colonies raised on very dif ferent levels of fluoride .

Bone levels of f luoride dif fered some 450 fold . The level of

chromosomal abnormalities in bone marrow and tes tes cells were low in

bo th groups and not significantly dif ferent . The rate of s is ter

chromatid exchange was similar in animals from the two groups . Mice

give low (1 ppm) to high levels (100 ppm) of fluoride had similarlevels of chromosomal abnormali ties which did no t dif fer from the levelof abnormali ties present in animals receiving no fluoride suppliment .

Slight and no t s tatis tically signi ficant increases in KY chromosomal

dissociation were no ted in the range 2-4% in animals supplimented with

fluoride . Great variation in this parameter (up to 402 ) have been noted

in previously published s tudies and the XY chromo some dis so ciation is

believed to arise during preparation of the chromosomes for examination .

No ef fect was noted o f fluoride levels in a reces sive lethal tes t whichdetects genetic ef fects o f mutagenic subs tances .

F luoride was no t found to be mutagenic when tes ted in microbial assays

( the Ames Tes t ) .

Taken together the data show no genetic or mutagenic actions o ffluoride .

PUBLICATIONS

Martin , G . R . , Brown , K . S . , Mathison ,D .

, Lebowitz, H .

,S inger

,L . and

Ophaug , R . : Chromosome aberrations in mice and mutations in micro

organisms are unaffected by fluoride exposure . Mutation Research,

66 : -159—167 , 19 78 .

Kram ,D . , S chneider , E . L . , S inger , L . , and Martin

, G .R . : The effec ts

of high and low fluoride diets on the frequencies of sis ter chromatid

exchanges . Mutation Research , 5 7 : 51-55 , 1978 .

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2 01 DE 00149—05 DB

I . Teratogen Screening

Animal tes ts used for determining the poss ible teratogenic activity

of compounds is both time consuming and expens ive . We have developed

a rapid in vitro screening assay for teratogens us ing dif ferent iating

neural cres t cells (also s ee 2 01 DE 0025 7—01 DB Greenberg ) and limb

mesenchyme cells . Teratogenic compounds inhibit the growth and/ ordif ferentiation of one or bo th cell types in contras t to the non

teratogens tes ted so far . The teratogenic ef fect is do se dependent

and can be quantitated by measuring the amount of a dif ferentiated

cell product (i . e . melanin or pro teoglycan ) . The convers ion o f non

teratogenic compounds , such as cyclophosphamide , to teratogenic

metabolites is achieved with liver microsomes incorporated into the

cultures . F uture s tudies (previous ly 2 01 DE 00138-04 DB ) will be

d irected toward develop ing o ther cell types which can complement the

present sys tem and to develop serum-free culture media in order to

facilitate teratogen tes ting in vitro .

Hor igan (also see Greenberg 2 01 DE 00257—01 DB )

II . Palatal Development

Clef ts o f the primary and secondary palate are common and serious

b irth defect o f interes t to the NIDR . Normal palatal deve lopment

involves growth , elevat ion and fus ion of the palatal proce s ses . Anal teration in any one of these processes could lead to clef t lip and

palate .

The dis tribution of the adhesive glycopro tein fibronec tin is re

s tric ted to the mesenchymal cell surface and extracellular matrix of

the primary and secondary palate . I t appears to be involved in cellto cell and cell to matrix interactions . F ibronectin is not found on

opposing palatal ep ithelia and therefore is no t involved in epithelial

cell adhes ion . Future s tudies are aime d at identifying the gly c o

proteins which are involved in this cell to cell interaction inpalatal development .

Silver

Previous s tudies have demons trated a correlation , using various mouse

s trains , between the sens itivity to glucocort icoid-induced clef tpalate and the levels of glucocorticoid receptors present in the

developing facial region . In the human fetus , glucocor ticoids are

known to cause growth retardation and is suspected o f caus ing facial

clef ting . In preliminary experiments , we have found alteredglucocortico id recep tors in dermal f ibroblas ts from persons with a

s trong family his tory of facial clef ting . These resul ts sugges t that

4 — 7 0

2 01 DE 00149— 05 DB

an hormonal dys func tion may be associated with facial clef ting .

Sa lomon ,L ey shon

II I . Terminal D if ferentiat ion and Growth F actors Terminal

dif ferentiation of the palatal medial-edge epithelial cells is

determined ear ly in palatal development . This pro cess can be pre

c oc iously s t imulated in vitro by cyclic AMP analogs or prevented by

ep idermal growth factor (ECF ) . Our s tudies on iso lated or recomb ined

palatal ep ithelium have shown that EG F prevents cell death only when

the epithelium is in comb ination with its mesenchyme . These resul ts

sugges t that the mesenchyme may provide a neces sary macromolecular

subs trate ( collagen , f ibronectin ) which enables the epithelial cells

to respond to the mitogenic ef fects of EG F . EG F along with gluco

cortico ids may play a role in normal palatal epithelial cell growth

and different iat ion . This is supported by our recent s tudies demon

s trating the presence of an EG F — like growth factor and its recep tor

in the mid and late gestation mouse fetus .

S im , Salomon,L ey shon

The growth o f mos t cells and t is sues , such as the palate , in culture

is dependent upon the presence o f serum , al though the components in

serum are largely unknown . We have been able to develop a completelydef ined medium containing growth pept ides such as insulin and EG F

which will support the growth and differentiation of mouse terato

carcinoma cells , mammary epithelial cells , embryonic limb mesenchyme

cells and secondary palates in organ culture . Future s tudies will be

d irected toward unders tanding the hormonal requirements for various

cells and tissues in culture .

Salomon , Hor igan

IV . Too th and Limb Development

The differentiation of the dental papilla mesenchyme cells into

odontoblas ts is thought to be dependent upon an interac tion with the

adj acent basal lamina which is of ep ithelial origin . Tunicamycin , a

specif ic inhibitor of protein glyco sylation , revers ibly blo cks this

dif ferentiat ion o f mesenchymal cells into odontoblas ts . Tunicamycin

exerts its ef fect by alter ing the biochemical components esp . fibro

nectin and the ultras tructure of the basal lamina , thereby disrup t

ing epi thelial mesenchymal inte ractions . F uture s tudies are aim

ed a t characterization of the components of the basal lamina

necessary for this interaction and to determine the b iochemical

nature o f cell surface macromolecules on the mesenchymal cells which

are important for this specific interaction .

11 _ 7 T

2 01 DE 00149-05 DB

The sle ff , Horigan

The teratogen vitamin A inhib its limb chondrogene s is both in vivo and

in vitro . Previous s tudies of ours sugges ted that vitamin A exerts

its ef fect by altering f ibronectin . Recent s tudies have shown that

vitamin A prevents the lo ss o f f ibronectin from the cell surface of

mesenchymal cells which have become commit te d to chondrogenesis by

enhancing the af finity o f the cell surface for fibronectin . Future

s tudies (previous ly 2 01 DE 00135—04 DB ) are directed toward under

s tanding the role that f ibronectin plays in limb mesenchyme cell to

cell and cell to matrix interact ion bo th in vivo and in vitro .

Silver , Ley shon

The erup tion of the incisor teeth in the newborn mouse can be pre

c oc ious ly s timulated by ECF . We have found that this ef fect is

specific for EG F in that a var iety o f o ther growth pep tides do no t

elicit this ef fect . EG F appears to exer t its ef fect by increas ing

the proliferation and keratinization o f the oral mucosa . Future

s tudies are aimed at identifying the cells in the oral mucosa which

are respons ive to EG F during this neonatal period .

S im ,L eyshon ,

The sle f f

PUBLICATIONS

Diewert , V . M . and Pratt , R . M . : Selective inhibition o f mandibular

growth and induc tion of clef t palate by diazo-oxo-norleucine (DON )in the rat . Teratology . In pres s , 1979 .

F igueroa , A . A . and Pratt , R . M . : Autoradiograph ic s tudy o f

macromolecular synthes is in the developing rat primary palate in

vitro . J . Embry . Exp . Morph . 50 : 145-154 , 19 79 .

Greene , R . M . and Prat t , R . M . : Correlation between cyclic AMPlevels and cyto chemical localization of adenylate cyclase during

development of the secondary palate . J . His tochem . Cytochem . 2 7

9 24—931 , 1 9 7 9 .

Greene , R . M . and Prat t , R . M . : Inhib ition of epithelial cell death

in the secondary palate in vitro by alteration o f lysosome func tion .

J . His to chem . Cyto chem . 2 6 : 1109-1114 , 19 7 9 .

Hassell , J . R ., Pennypacker , J . P . , Kleinman , H . K . , Pratt

,R . M . and

Yamada , K . M . : Enhanced cellular f ibronectin accumulation in

chondrocytes treated with vitamin A . C ell 17 : 821—8 2 6 , 1 979

a _ 7 oL .

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Mc Clure ,H . M Wilk , A . L . , Horigan , E . A . and Pratt , R . M

Induc tion o f cranio facial malformation in Rhesus monkeys (Macaca

mulatta) with c y c lophO S phamide . Clef t Palate J . 248—2 56 ,

197 9 .

Olden, L . , Pratt , R . M . , Jawoski , C . and Yamada , K . M . : Evidence for

role o f glycopro tein carbohydrates in membrane transpor t ; Specif ic

inhibit ion by tunicamycin . Proc . Natl . Acad . S c i . 7 6 : 791—795 ,

19 79 .

Olden , K . , Prat t , R . M . and Yamada , K . M . : Select ive cyto toxicity

of tunicamycin for transformed cells . Int . J . of Cancer In pres s .

Olden , K . , Prat t , R . M ., and Yamada , K . M . : The role of carbo

hydrate in b iological func t ion o f the adhes ive glycoprotein fibro

nectin . Pro c . Natl . Acad . Sci . USA In pres s .

Pennypacker , J . P .,Hassell

,J . R . , Yamada , K . M . and Prat t , R . M

The influence o f an adhes ive cell surface pro tein on the chondro

genic express ion in vitro . Exp . Cell Res . 1 2 1 : 411-415 , 19 7 9 .

Pratt , R . M . ,F igueroa

,A . A . ,

Greene , R . M . , Wilk , A . L . and

Salomon , D . S . : Al terations in macromolecular synthe s is and funct ion

during abnormal palatal development . In Persaud T . V . N . NewPerspect ives in Teratology . MTP Pres s Ltd . , 197 9 . In pres s .

Pratt , R . M ., Yamada , K . M O lden , K . , Ohanian , S . H . and Hascall ,

V . C . : Tunicamycin-induced al terat ions in the synthes is o f sul fated

proteoglycans and cell surface morphology in the chick embryo f ibro

blas t . Exp . Cell Res . 118 : 245—25 2 , 197 9 .

Prat t , R . M . and Salomon , D . S . : Glucocor tico id recep tors and clef tpalate in mice and man . In Bixler , D . , Melnick , M . AcademicPres s . New York , In press 1979 .

Prat t , R . M ., Wilk

,A . L . , Horigan

,E . A . , Greenberg , J . H . and

Mar tin , G . R . : Screening for teratogens in vitro . In Bixler ,D .

,

Melnick , M . Academic Press . New York , In pres s 19 7 9 .

Salomon , D . S ., Gif t

,V . M . and Pratt

,R . M . : Cort icos terone levels

during midge s tation in the maternal p lasma and fetus o f clef t palatesensi tive and res is tant mice . Endocr inology 104 : 154-156 , 19 7 9 .

Salomon,D . S .

,Paglia

,L . M . and V erbruggen ,

L . : Hormone dependent

growth o f a rat chondrosarcoma in vivo . Cancer Res . In press .

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Salomon,D . S . and Pratt , R . M . : Inhibition of growth in vitro by

glucocorticoids in mouse embryonic facial mesenchyme cells . J .

Cellular Phys iology . 9 7 : 315— 328 , 197 8 .

Salomon,D . S . and Pratt , R . M . : Involvement of gluco cor ticoids in

the development of the secondary palate : A review . Differentiation

13 : 141-154 , 19 79 .

Salomon,D . S . and V is tica , D . T . : S tero id recep tors and s teroid

response in cultured L 12 10 murine leukemia cells . Mol . Cel l

Endoc rinol . l3 (1 ) : 55-71 , 1979 .

Wilk,A . L . , Greenberg

,J . H . , Horigan , E . A . , Prat t , R . M . and

Mar tin , G . R . : Detection of teratogenic compounds us ing dif feren

tiating embryonic cells in cul ture . In Vitro . In press .

O. S . D EPAR TM EN T OF

EALTH ED UCAT I ON ANO WEL

FOEL IC H EAL TH S ERVN OT I CE OF

I N T RAM URAL R ES EARCH PROJE

3 0 , 1 9 7 9

al Neural Crest Cell s

N AM ES,LA B OR ATORY ANO I N ST I T UT E AF F I L I AT I ON S

, ANO T I TL ES OF PR INCIPAL I N V E ST I G AT OR S AND ALL OT H E RPROF ES S I ON AL P ER SON N EL ENG AG ED ON THE PRO J ECT

PI Judith H . Greenberg S enior S taf f F e llow

OTHER : El izabeth A . Horigan Research Biolog ist

Robert M . Pratt , Jr . Re search Chemist

Laboratory of Developmental Biology Anomal ies

Craniofacial Development Sect ion

NIOR ,NIH , Bethesda , Maryland 2 0 2 05

HUM AN T I SS U E S I] (c ) N E I T H E R

SUMMARY OF WOR K (200 wo rd s or le ss un d e r l in e ke ywo rd s )

Cranial neural crest cell s,which contribute to the format ion of many

cranio facial s tructures,diff erent iate in culture into two o f their normal

derivat ives,neurons and pigment cell s . As an approach toward def ining

the factor s which af fect normal gpg_

abnormal neural cre st development , we

are examining various aspect s of their diff erent iat ion in culture . The

well—def ined and reproduc ible d iff erent iat ion of neural crest cultures

has enabled us to develop a system to screen for teratogens in vitro .

ZO1 DE 00 2 57—01 DB

I . F actors Af fect ing Differentiation o f Neural Cres t Cells in

Culture

Chick cranial neural cres t cells cultured in medium containing horse

serum (HS ) differentiate into cells resemb ling neurons and contain

choline acetyl trans ferase (CAT ) ac tivity , whereas those cul tured in

f etal calf serum (FCS ) or calf serum differentiate into melanocytes .

Up until the third day of culture , the pathway o f dif ferentiation of

the neural cres t cells can be changed by shift ing the type of serum

in the medium .

Dimethyl sulfoxide (DMSO ) prevents the pigmentat ion of neural cres t

cells without affect ing their growth rate or their dif ferentiation

into neuron-like cells . The inhib it ion of p igmentat ion is re

versible and involves a late s tep in melanogenes is , s ince cellsmaintained in the presence of DMSO contain preme lanosome s . Increased

concentrat ions o f FCS or o f a high molecular weight fract ion of FCS

enhances p igmentation in control cultures and part ially overcomes

inhibition of pigmentation in DMSO— treated cul tures . These resul tssugges t that DMSO may al ter the activity o f a factor in FCS thereby

preventing it from interacting with neural cres t cells to promo te

p igmentation . Experiments are in progre s s to def ine the me chanism

o f action o f DMSO .

Greenberg

II . Collagen Synthes is by Cultured Neural Cres t Ce lls

Several dis t inct tissue specific collagens exis t in the embryo .

Their synthes is changes during development and may play a role in

cell dif ferentiation and tis sue morphogenesis . Undif ferentiatedneural cres t cells , as well as p igmented and neuronal cultures ,

synthes ize types I and III collagen . When cells dif ferentiate into

melanocytes , the ratio o f collagen to non-collagenous protein does

not change , but wh en they different iate into neuronal cul tures the

ratio o f collagen to non-collagenous pro tein is markedly reduced .

These results suggest that the relative amounts o f co llagen reflec tthe pathway of dif ferentiation o f the cells and may provide a usefulcr iterion by which to as sess normal and abnormal differentiation of

these cells .

Greenberg

II I . Neural Crest Cultures as a S creening Sys tem for Teratogeni c

Compounds

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Wilk , A . L . , Greenberg , J . H . , Horigan , E . A . , Prat t , R . M . and

Martin , G . R . : Detection o f teratogenic compounds us ing dif feren

tiating embryonic cells in cul ture . In Vitro . In press .

ANNUAL REPORT OF THE LABORATORY OF BIOCHEMISTRYNATIONAL INSTITUTE OF DENTAL RESEARCH

Laboratory programs have remained nearly cons tant in both size and

content f or the pas t s everal years . The creation of the Prot eoglycan

Chemis try Sec tion this pas t year recognized the already present independence

and impor tance of tha t prog ram . The Protein Chemis try Sect ion and the

Enzyme Chemis try Section are the other two subdivisions of the Laboratory .

The number of p ersons in the Laboratory is ab out 2 5 , the exact number

varying as visitors and p ersons in pos tdo c toral~type posit ions arrive and

leave . The rat io o f research to research— support s taff is about

This rat io has increased in recent years as the numb e r of j ob pos itions

has decreased but has been made poss ible by a greater reliance on

ins truments and d isposab le supp lies . The ratio of persons in rotating

pos tdoc to ral-type p os itions and more senior vis itors to t enure research

s taff is also about This rat io ref lects a maj or element of training

in Laboratory programs .

The maj or interes t in the Enzyme Chemis try Sect ion is the family of

enzymes called transg lu tamina se s . All catalyze the same reac tion , the

f ormation o f an amide bond be tween a pept ide—bound glutamine residue and

an amine donor . The amine can be any of a variety of low molecular weight

compounds or the e— amino group of a pep tide—bound lys ine . In the latt er

case a cros s link,an E (Y -glutamy1 ) ly sine bond

,is formed between proteins

or po lypept ides . This method of stabilizat ion is well es tab lished for

f ibrin and there is evidence for the cross link in cell membranes ,

myof ib rils , s eminal p lasma prot eins and hair proteins . Recent s tudies

in the Section have shown that ano ther type of cross link can be formed

by the enzyme u tili zing a diamine or polyamine to bridge two peptide—bound

g lu tamine res idues . S ince po lyamines such as spermidine and spermine

and the diamine putres cine are important biolog ical cons tituents , this

means o f making cross links or of modifying proteins may b e important in

cer tain phys iolog ical pro cess .

In related s tudies in the Enzyme Chemis try Section , transglu taminas es

have been f ound in megakaryocytes and endometrium of uterus and in the

membranes of several cell types . This membrane—bound enzyme may be a

new and spec if ic transglutaminase .

Transg lutaminase can b e used as a means of artific a lly introducing

cros s links into pro teins . This procedure is us eful in determining the

relat ive pos ition of two pro teins in larg er b io logical s tructures . To aid

in this approach,b ifunc tional amines containing a chemically lab ile

bond have b een synthesi zed . Af ter forming a cros s link and iso lating the

c ross linked c omp lex ,.the bond can be cleaved to release the constituents

and allow their character ization .

Pro teog lycans have a protein core and a variety o f dif f er ent kinds

of po lysaccharide s ide chains . In cart ilage and certain o ther t is sues ,

proteog lycan exis ts as_

a very s tab le comp lex with hyaluronic acid , a very

long s ing le-chain polysaccharide , and link protein , a small g lycoprotein .

[I l y n

The eff orts o f the Proteoglycan Chemis try S ection are devoted to s tudying

the s tructure , biosynthesis and biological ro le of these impor tant connective

tis sue components . The sys tems currently in use are car tilage cells from

a rat chondrosarcoma , chick limb bud cells and corneal s troma . Structural

s tudies on car tilage pro teoglycan have progressed to the point where

specific fragments can be obtained that are suitable for detailed s tudies

including amino acid sequencing . I t has been found that in addition to

the well— charac teri zed glycosaminoglycan side chains,pro teoglycan from

chick l imb bud cells in culture and from the rat chondrosarcoma contains

mannose-rich oligosaccharides s imilar to those present in glycoproteins .

Biosynthe tic s tudies have . e lu c ida ted the rela tionship between the synthes is

of proteoglycan , hyaluronic acid and link protein . I t was f ound that

synthes is in serum—free cul ture is s timulated by somatomedin and to an

even greater extent by insulin . These results are impor tant bo th f or the

purpo se of providing be t ter def ined conditions for growing cells in

cul ture and also for providing a biological sys tem for s tudying the ro le

of these and other impor tant factor s that can be easily manipulated .

Pro teoglycan from car tilage can be cons idered a pro to type , bu t it is

by no means the only kind of proteoglycan . Typ ical of the po tential

varie ty of types is the f inding o f two quite diff erent proteog lycans in

corneal s troma explants . One has a s ingle chondroitin sulf ate chain and

a f ew o ligo saccharides per molecule and the o ther has one or two keratan

sulfate chains and a few oligosaccharides,while cartilage proteog lycan

may have more than 100 c ondroitin sulfate and keratan sulf ate chains per

molecule .

Emphas is in the Protein Chemistry Sec tion is on collagen s tructure .

Molecular s tructure,chemis try and biosynthes is are quite well unders tood .

Questions now being asked per tain to high level s truc ture and interac tions .

The tools being used include elec tron microscopy, quasie las tic light

s cattering,nuclear magnetic resonance and computer modelling . The

pic ture of the native collagen f ibril that is emerging is that there is a

high degree of axial order bu t that there is rapid lateral movement throughseveral or perhap s many conformational s tates . There is

,however , a

s tatis tically prefered relationship , and three dimensional order is present .

What that order is,is s till a matter of controversy . The widely—held

5—s tranded microf ibril model has been ques tioned and will a t leas t needsome revis ion .

The assembly of collagen molecules to native f ibrils in a well

def ined in vitro sys tem is a means of obtaining evidence about s tructure

that may be relevant to in vivo assembly . S tudies done in the Pro tein

Chemis try Sec tion have shown that assembly is a mul tis tep process . Longthin f ilaments that could be 5-s tranded microf ibrils are intermediates .

Their s tructure and that of even earlier intermedia tes is being studied by

light s cattering and by electron microscopy . Procedures for low beam

damage elec tron microscopy and for computer analysi s of elec tron micrographs ,

which are now nearly in p lace,are expected to aid these s tudies . In

addition , preliminary s tudies using the scanning transmis sion electron

microscopes at Johns Hopkins and at Brookhaven are promising .

III — S R

An important element in the Laboratory programs describ ed above is the

interaction b etween group s in the dif f erent Sect ions . This occurs bo th

a t the discus s ion level and in j o int proj ec ts . For examp le , collag enpro teog lycan interactions during co llagen as sembly are being studied ;nuclear magnetic resonance studies in pro teoglycan s tructure in vitro have

b een done ; and the role o f transg lu taminase in wound healing and it s

po ssible interac t ion with collagen is of interes t . All of the Laboratoryprograms a lso have active col laborat ive aspec ts with programs in o ther

parts of NIDR and NIH and with various laboratories around the world .

October 1,1 97 8 Sep t ember 3 0 , 1 9 7 9

Structural Studies on Co llag en

N AM ES , LABOR ATORY AND I N ST I T UT E AFF I L I AT I ON S , AND T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ERPROF E SS I ON AL P ER S ON N EL ENG AG ED ON THE PRO J ECT

PI Trus S enior S taf f F el low NIDR

OTHER Piez Chief NIDR

[3 (b ) HUM AN T I SSU ES l! (c ) N E I T H ER

SUMMAR Y OF WOR K 00 word s or les s und e rlin e keyword s )The primary goal of this proj ect is an under stand ing of co llagen s tructure from

the molecular to the f ibril level . Emphasis is presently on two main aspects

of the gene ral problem . 1 ) Computer enhancement and recons truc tion of electron

compute r models to s tudy and illus trate s truc tural features o f co llagen and

molecular aggregates . Each of the above two approaches has led to the develop

ment of computer sof tware that is being app lied to o ther problems in protein

this year .

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1 . Proj ect Descrip tion

The primary obj ec tive of this proj ec t is to dete rmine the s tructure of

the collagen mo lecule and o f aggregates of co llagen inc luding the na tive

collagen f ibril . Several approaches are being taken . 1 ) Compu ter

analys is (enhancement and recons truction ) o f elec tron micrographs of

collagen aggregates to reveal de tails no t obvious to the eye and to

provide quanti tative data . 2 ) Compu ter mode ling of collagen molecules

and aggregates to illus trate and tes t proposed s truc tures . 3 ) Analysis

o f co llagen amino acid sequences to search for features re lated to

higher level s truc ture . A s e condary obj ective developed during the

current year is to extend the compu ter mode ling programs developed f or

collagen to myos in,a collabora tive proj ec t with Dr . M . Elz inga

,Brook

haven National Laboratory . This is po ssible because bo th myosin and

co llagen are coiled coi ls,although the helical parameters are very

d if ferent .

Analys is of electron micrographs uses s tandard enhancement techniques ,

as deve loped a t the Univers ity of Basel and adap ted to the computer

hardware at NIH,and procedures develope d in this laboratory S pec ia lly

suited to our problem . Specifically,e lec tron micrographs are

digitized on a Perkin Elmer loloG microdensitometer . Re levant infor

mation is extracted using a F rame Buff e r disp lay , and analysis is done

using a sys tem of computer programs cal led P IC . PIC , which is an

extension of the Basel sys tem,can call over 100 operations inc luding

fas t Fourier transforms, F ourier f iltering by high and / or low pas s

f il tering,F ourier f il tering by masking

,calculation and disp lay of the

ef fec tive optical d if frac tion,inverse F ourier , analys is of periodic

s truc ture,and generation of hypo thetical or mode l elec tron micrographs .

The results of the enhancement and recons truc tion are vis ible immedia tely

on the cathode ray tube o f the F rame Buf f er and can be recorded on f ilm

by a Camera S tation . Mo lecular modeling programs f or co llagen and myosin

were develope d using an Evans and Souther land P ic ture Sys tem II inter

faced with 3 DEC compute r,F rame Buff er

,and Camera S tation . Knobs are

used to change variab les such as p itch,supe rco il , and ang les . Swi tches

allow the sele c tion of a number of viewing options . A keyboard is used

to input the one letter amino acid code for display of specif ic types of

amino acids . S ince the mod eling is es sentially a real time opera tion on

the Pic ture Sys tem,i t is extreme ly easy to s tudy dif ferent reg ions of

co llagen wi th a variety o f s tar ting paramete rs .

Development of the P IC sys tem sof tware for elec tron micrograph

enhancement and re cons truc tion has been a maj or effor t this year . The

ZO1 DE 00002— 2 9 LB

sys tem is now being used as more and be tter electron micrographs of

collagen are becoming availab le . The sys tem is also being used byo ther inves tigators at NIH wi th similar research problems .

The molecular modeling programs have be en used to produce a thirteen

minute color movie of collagen s tructure . The movie s tar ts by

illus trating molecular s tructure,shows the relationship be tween two

molecules , and concludes by depic ting the 5—s tranded microf ibrilwhich is be lieved to be a subs tructure of the native f ibril .

The myosin program has been used to s tudy the pitch of myos in along the

148 res idues in the available sequence . The pitch can be changed

dynamically along the partial sequence to accomodate sys temat ic localvariations .

To re so lve a conf lict between a recently proposed model of the co llagen

f iber based on x-ray diff raction evidence and the microf ibril model

supporte d by o ther data , we have reinterpre ted the x— ray diffraction

da ta . We have propos ed a revised model which contains compressed"

microf ibrils p lacing mo lecules on a pseudohexagonal lat tice . The unit

cell provides an accurate f it of the observed ref lections on the x—ray

diffrac tion pat tern and is cons is tent with other data .

Collagen is the maj or pro tein of connec tive tissue and is found in

various forms throughou t the body . Through interac tions with other

macromolecules such as proteoglycans , mineral , and cells,it p lays an

impor tant role in many biological processes during development and in

pathological s tates . The se s tudies on co llagen s truc ture are impor tant

to unders tanding . the s e interactions .

Approximately 90 % of the PIC sys tem has been implemented and tes ted .

While some continuing d eve lopment of new so f tware can be expected,

subs tantially more emphas is will be on use rather than development .

Initial enhanc ement . inve s tigations will be concerned wi th the ear lys tages of f ibril formation

,and attempts to identify the s tructures

pre sent . The se e arly aggrega tes wi ll be prepared as part of ano ther

proj e ct . Addi tional app lications wil l be sought for the PIC sys tem,

as data aquisition is eas ier wi th ins tallation and successfulO peration of a high resolution microdensitometer . Continuing us e ofF IG will invo lve cooperation with A . S teven on virus s tructure

,

M . Navia on immunoglobulin s truc ture,and V . Nikodem on two—dimensional

ge l quantitative analys is.

The re al time collagen disp lay sys tem will be kep t accurate to lates t

propo se d s tructure , and can be used to help evaluate more de tailed

des crip tions of co llagen . While initial work was limited to backbone

atoms , more accurate models are being buil t to include all the atomsin a particular se ction .

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October 1 , 1 9 7 8 to Sep tember 3 0 , 197 9

Phys iological Role and Metabo lism of Transglutaminase s

N AM ES,LA BOR A TORY AND I N ST I TUT E AFF I L I AT I ON S, AN D T I TL ES OF PR I NC I PAL I NV EST I G AT ORS AND ALL OTH ER

PROF E SS I ON AL PERS ON N EL ENG AG ED ON THE PRO J ECT

Chung Research Chemis t NIDR ,

[j (b ) HUMAN T I S SU E S g] (c ) N E I TH E R

SUMM AR Y OF WOR K (200 word s or le ss und e rlin e keywo rd s )

The physio log ical func tion and the mode of regulation of the transglu taminase s

are be ing s tudied . The roles o f individual transglu taminase s in the formation

of cellular membranes,in membrane-mediated s timulation of sp ecif ic cellular

funct ion , and in the regulation of tissue matrix s tabilization following

inj ury are unde r inves tigat ion .

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Ob j ectives

The -f inal phases of pro tein—tissue matrix ( s tructural pro teins ,

pho spho lipids,etc . ) inte rac tion in the "Cascade Scheme" of hemos tasis

lead to f ibrin formation induced by multi— s teps of limited pro teo lys is .

F ibrin monomer S pontaneous ly int e rac ts in a hydrophobic environment to

form polymer through an ordered process . F ibril s are formed which S how

unique 2 60 A S pac ing as charac terized by elec tron microscopy . The

s tabi lization of the se f ibrils involves enzyme catalyzed E - (Y-g lu tamyl )lysine cro s s—linking at des ignated si tes between y

—Y chain and a—a

chains . A varie ty o f primary amines may interf ere with cro ss — link

f ormation by compe ting with amino group s of pep tide—bound lys ine residues .

The trans glutaminas e s catalyze the incorporation of primary amines at

the carboxamide group o f protein and pe p tide—bound glu tamine res idues

and promo te f ormation of E— (Y r

g lutamy l ) ly sine bonds within and be tween

pro tein mo lecules . These act ive enzymes are formed : 1 ) by a limited

pro teolys is in p lasma during hemo s tas is and by an ass ocia tion wi th Ca++

ions , 2 ) by inf lux of Ca++ ions into cel lular si tes from various s timu

lations that oc cur in vivo ( tis sue transglutaminase ) , 3 ) by a ligand or

mitogen—induced activat ion of cells (membrane— bound transg lutaminase ) .

These E- (X—glu tamy l ) ly sine cross links have been found in numerous

s tabilized proteins of animal tis sue .

Als o the se cross— links may be involved in the s tabili za tion of tis sue

matrices which provide sub s traum for s elec tive adhes ion of cells and

subsequent ce ll proliferat ion (i . e wound healing ) .

The mode of transglutaminase ac tivation in the ligand- induced interac tion

of recep tor and cytoskeletal component at the cortex of ce ll and subs e~

quent endocytosis of ligand— rec ep tor complexes are currently unde r

inves tigation recep tor—me dia te d endocyto si s ) .

Extended s tud ies of the various transglu taminase s are underway with

special emphasis toward phys iological func tion and its mode of

regulation .

Ma j or F indings

In continuation of s tudie s carried out in the previous ye ars ,

inve s tigation of the biosynthe sis of protransg lutamina s e in o the r

types o f cell sys tems is underway .

Us ing f luorescent antibody technique s,me gakary oc yte s and endometrium

of u te rus were found to contain pro transg lutaminase but no t pro trans

g lu taminase—binding globulin . In this sense , they are unique and

A R 7

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College,Seoul

,Korea . Two separate methods were adop ted f or this s tudy .

1 ) F actor XIII def iciency was induced in rabbits by intravenous infusion

of IgG isolated f rom goat anti-rabbit p latelet factor XIII , and the de

f ic ient s tate was maintained by daily infus ion . Contro l rabbits were

g iven normal goat IgG . Af ter production of s tandard skin wounds , the

progre ss of epi theliali zation , f ibroblas t proliferation , and co llagenous

matrix f ormation was monitored by his to logical and immunohis tochemicalmethods . In normal rabbi ts

,epithelialization was comp lete by day 6

f ibroblas t pro lif eration by day 10 ; maximal collagenous matrix ac cmu

lation was observed on day 14 . In factor XIII— def icient animals ,

epithelialization was no t COmp le ted until day 14 ; f ibroblas t prolif e r

ation and collagenous matrix accumula tion were retarded beyond day 20 .

These observation s sugges t that factor XIII is involved in early phases

o f wound healing . 2 ) In an initial at tempt to s tudy the mode of factor

XIII in tis sue repair,the ef fec t of factor XIII on the growth of

f ibroblas t was examined in vitro . However,the addition of fac tor XI II

to the cul ture media showed no ef fect on the growth of cells .

Sensit ive and specif ic methods have been developed to detect and

quantitate trace amounts of transg lu taminas e s present in the isolated

cells . U sing peroxidase-conjugated antibody and monospecif ic anti

transglu taminase,the elec tron micros copy of hepatocytes and

lymphocytes showed c lus tering of transg lu taminase in Go lgi regions and

endop lasmic reticulum and very light dis tribu tion in the inner cellmembrane area .

Evidence has been accumulated f or the exis tence of a comp letelyd iffere nt type of transglutaminase as sociated with the membrane—rich

par ticulate fraction of cells . Iso lated membrane—rich par ticulate

frac tion from rat chondro sarcoma cells showed high S pecif ic enzymic

activity . This membrane bound transglutaminase activity was to tally

extractable with detergents . Act ivity was increased several fo ld by

a limited p ro teolys is with concomitant change of mo lecular weight from

to Several fold increase of transg lu taminase ac tivity

in the rat chondrosarcoma cells and mouse embryo cells is observed upon

treatment of cel ls with nmo le quantities of pro teases . These increases

in enzyme act ivity are closely as sociated with ligand—recep tor induced

c lus tering and subsequent endocytos is . A survey of p lasma cells,as

well as S truc tural cells,indicated that almos t all of the cell s posses s

two type s o f transglu taminase,one Which is identical wi th the tissue

enzyme and ano ther that is membrane—bound .

A comprehensive s tudy o f various transg lu taminase s has shown that thee nzymes are wide ly dis tribu ted in various cells

,in bo th the cytoso l

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dis tinc t from previous ly s tudied hepatocyte sys tems where bo th pro

enzyme and i ts binding globulin were synthe si ze d and re leased .

Endometrium of uterus p lays an important func tion in furnishing

nutrient medium and serves as a matrix for fertilized ovum implantation .

Al though the biochemical processes invo lved in ovum implanta tion are

not c learly unders tood,the pos sible involvement of covalent cros s

links has been pos tulated . This is based on clinical observation where

a high incidence of abort ion is observed in congenital factor XIII

de fic iency .

This imp lantation sys tem in vitro , may se rve to be an ef f ec tive model

sys tem to examine the early phases of cell adhes ion and adap tation of

matrix condit ions f or c e ll prolif era tion .

A method was devised for obtaining intact uterine epi thelial ce lls .

This was accomp lished by digestion of the s ter ile ever ted uteri horns

with a mixture of crude tryp s in and collagenase . Modif ied Ham 's F —12

culture med ia was employed containing calf serum and six growth

fac tors : insu lin ,hydrocortisone

,transferrin

, G HL ,somatos tat in and

thyro trop in . Ef fec t s of o ther hormones,cyclic AMP and die thyl

st ilb e st ro l on p ropaga tion were ne ga tive . F ibronect in was extremely

e ff e c tive in promot ing at tachment of the cells . Cloni z ation of

epi the lial ce lls and U WO blaS t S c fi'

ute rus has been achieved and e aCh

c ell has been maintained under the des cribed culture condi tion for up

to 6 months .

The s truc ture of lamprey fibrinogen has been the obj ec t of prolonged

inve s tigat ion because of the unusually large mo lecular we ight of its a

chain,and i ts unique subunit composi tion . Limited cro ss—linking

(only y-Y cro ss-linking ) o ccurs during the s tabili zation of lamprey

f ibrin . Extensive s tudie s on the iso lated chains o f lamprey f ibrinogen

by se dimentation e qui librium and gel pe rmeation in bo th chao tropic

s o lvent and phys iological buf fer show mole cular weigh ts o f

and f or (A) a . (B ) 8 ,and y chains , respec tively . If the

conventiona l formula t ion B (B)2

, Yz) is used to calculate the

mo lecular weight,the resul ts g ive a mass of far in exces s of

obtaine d by us . However, the data are in close agreement to

a B (B)2

, 72) f ormu lation based upon the es timated value of

mo le cular we ight . Pre liminary s tudies on CNBr— fragmented a-chain

show 1 1 p ep t ides out of a poss ible 14 per molecular weight in

f avor of the propos ed f ormulation .

The Role of F ac tor XII I in Wound Healing

Inves tiga tion of the role of fac tor XIII in wound he aling has been

initiate d in col laboration wi th Dr . 80 0 Young Lee of Catholic Medical

15 - 3 0

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and the membrane s ,and sugges ts that all may be involved in the formation

of E- (y— glutamy l ) ly sine cros slinks . These crosslinks are ess ential for

maintaining the permanent rigid s tructure of many pro tein molecules . The

f indings of transglutaminas e act ivity as sociated with cell membranes and

its enhancement by membrane s t imulators sugges t an addit ional role in

cell membrane prot ein interact ion ; i . e . , in receptor—mediated endocytos is .

The delayed epithel ializat ion and collagen format ion in experimentally

induced factor X II Ide fic ient animals , the rap id healing of wounds in

factor XIII— treated animals , and enhanced growth of f ibroblast on stab il

iz ed f ibrin plates support previous suggest ions of a vital role of

factor X II I in wound healing and t is sue repair .

Proposed Course

1 . Attent ion will be focus ed on the pos s ible in vivo interact ion

between f ibrinogen and co llagen as catalyzed by trans glutaminas es and

the poss ible re lat ionships of cross— l ink format ion to cell adhesion and

proliferation in wound healing .

2 . Studies on the b iosynthes is , release , and catabolism o f platelet

f actor XIII will be carried out us ing guinea pig megakaryocytes .

Regulat ion of fac tor XIII in rabb it uterus ep ithelial cells and its role

in ovum implantation is under inves t igation .

3 . The mode o f activat ion and involvement of membrane-bound trans

glutaminase in receptor—med iated endocytos is will be inves t igat ed us ing

lymphocytes and f ibroblas t cell lines .

TESUN IAN SC I EN CE .“F 8RMATIOH L AULAAUE. vo s. D E PART M EN T I PR OJECT N UM B E RRP L

‘ E CT‘

a-UIYIatR { to NOT us e in e sac e ) HEAU H

OEDUCATION

F‘

,

AND WELF AR E l

BL IC H EALT . SERV I CE DE— 0013 4 -0 5 LBN OT I CE OF

2 01

NTRAMURAL R ES EARCH PR O JECT

FFEHIOD COV EREO

Oct ob e r 1,19 7 8 S ep tember 3 0 ,

1 9 7 9 NO l—DE— 7 2 403

S t ructure and Biosynthes is of Pro teoglycans

N AM ES , LA B OR ATORY ANO I N ST I T UT E AFF I L I AT I ON S, AND T I TL E S OF PRI NC I PAL IN VEST IGAT OR S ANO ALL OT H E R

PROF E SS I ON AL P ER S ONN EL ENCAG EO ON THE PRO J ECTV . G . Has call Research Chemis t NIDR LB

O ther s S enior S taff F ellow NIDR LBS taff Fel low NIDR LBNIH Postdoc toral Fellow to May NIDR LBAr thr it is F ound . Fellow f rom June

L . S . Lohmander V isit ing As soc .

R .M . Mason Visit ing S c ient is t

R . L . S t evens Arthr it is F ound . F ellow

[ 3 (C ,~JmAN T I S J ES X] (c ) N E I T H ER

SUMM ARY OF (200 word s or le s s un d e rl in e ke ywo rd s )

chemistry o f proteoglycans isolated from the Swarm rat chondrosarcoma ,2 )

structure and extracellular matrix organization using chick limb bud c hondroc y

cultures, 5 ) charact erist ics and bio synthes is of proteog lycans in corneal

s troma .

(Rev. 10—76 )

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1 . Proj ect Descript ion

Background

Cartilage proteoglycans are large macromolecules (MW 1— 4 million ) in

which large,bu t variable , numbers of sulfated polysaccharide chains ,

chondroitin sulfate (CS ) and kera tan sulfate (K8 ) , are covalently

at tached to a core prot ein . Such a mo lecular architecture yie lds macro

molecules which occupy large hydrodynamic volumes in solution and which

exhibit reversible compressibili ty,charac ter is t ics that help provide

car tilages wi th resiliency and resi s tance to compress ive forces . The

core pro tein of car tilage pro teoglycans consis ts of three dis tinc t

regions . One end,referred to as the HA—binding re gion , has a port ion

of pro tein (MW about 70— 90 thousand ) which is devoid of g ly c osaminogly

cans and which interacts in a highly specif ic way with bo th hyaluronic

acid (HA ) and a pro tein (MW o f referred to as the link pro tein .

These interact ions are cr itical f or the organization of pro teog lycans

into aggregate complexes , the predominant form of the pro teog lycans in

the t is sue matrix . Adj acent to the HA-binding region is a por tion o f

the core protein,referred to as the KS —enriched region (MW abou t 25—40

thousand ) , which contains an average of about 652 of the KS chains bu t

less than 102 of the C S chains present in the intac t molecules . Dis tal

to the HA—binding region is a por tion of the core pro tein , ref erred to

as the C S— enriche d region . This latter region has a variable mo lecular

weight (f rom a few thousand to and contains more than 902 of the

CS chains but less than 3 52 of the KS chains present in the intac t mole

cules . The variable length of this region appear s to be propor tional to

the number of C S chains present on any individual pro teoglycan molecule .

The average prote og lycan mole cule contains about 100 C S chains

(average MW about per chain ) and 50 KS chains (assuming an average

of abou t 5000 per chain ) .

This program collaborates wi th : 1 ) Dr . Dennis Torchia of this labora

tory in s tudies relating to13C—NMR o f

13C— serine and

13C-g lycine

labeled pro teog lycans prepared from cul tures of chick limb bud

chondrocytes (Proj ec t 2 01 DE 0015 7 2 ) Dr . Rober t Gelman of this

laboratory in s tudies re lat ing to the interac tion of pro teog lycans wi th

collagen during f ibrillogenesis (Proj ec t 2 01 DE 002 15 3 )Dr . Gre tchen Hascal l

,Laboratory of B iological S truc ture , in s tudies on

the morphology of the Swarm rat chondrosarcoma (Proj ect 2 01 DE 00163

4 ) Dr . Arno ld Cap lan in s tudies relating to the charac teris tics of proteo

glycans isolated from the chick limb bud chondrocy te cultures ( supported

in part by a contract, NIDR NO1 DE 5 ) Dr . John Hassell , Eye

Ins titu te , in s tudie s on corneal pro teoglycans . 6 ) Dr . Bo Ni lsson ,

V isit ing F ellow,NCI

,in s tructural s tudies o f oligosaccharides on

cartilage pro teoglycans . 7 ) Dr . Robin Poole in s tudies on the immunology

o f proteoglycans . 8 ) Dr . Peter F ie t z ek in s tudies on the protein chemis try

o f link protein and proteog lycan core pro tein . 9 ) Dr . Peter Niss ley ,

LI - O’

)

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c hondroitinas e ABC diges tion and separation of disaccharides on thin

layer cellulose p lates,to determine the kinetics o f synthesis of

hyaluronic acid relative to chondroitin sulfate in the cultures us ing

3H-glucosamine as a precursor . The ratio of hyaluronic acid to

chondro itin sulfate synthes ized during a 24 hour period i s about

The d is tribution o f newly synthesized HA between cells , matrix and

medium has been s tudied . Propor tionally , in these chondrocyte cultures ,

more labeled HA than proteoglycan remains cell—asso ciated suggesting the

pos sibility that hyaluronic acid may serve addi tional functions near the

cell in addition to forming the central f ilament for proteoglycan

aggregates . Experiments are in progres s to de termine if hyaluronic acid

is synthesi zed bound to a core protein using labeled amino acids as

precursors and purifying the HA f rac tion .

c . Ef fect of cycloheximide on pro teoglycan synthes is . Cycloheximide

was used to block pro tein synthes is in chondrocytes . At times af terA

the b lock ,

J SS-sulf ate was added to de termine the kine tics of pro teo

glycan synthesis . Pro teog lycan synthes is continues , bu t at an exponentially

decreas ing rate with a tl / Z

of abou t 90 minutes . The mo lecular charac ter

istic s of pro teog lycans synthesized immediately af ter the cycloheximide

block and at times up to 6 hours later are very s imilar excep t that the

chondroitin sulfate chains gradually increase in si ze . The resul ts

sugges t tha t there is a large precursor pool of core protein in the

cells and that the ce llular mechanisms for glycosaminog lycan biosynthes is

and secretion o f proteoglycans continue in the absence of new pro tein

synthes is . This was further verif ied by pulsing cultures for 15 minutes

wi th ei ther3H—serine or

3 5S-methionine

,then introducing a cycloheximide

block . Pro teog lycans were then purif ied by dis so ciative gradients at

subsequent times af ter the block . The appearance o f bo th precursor amino

acids in comp leted proteoglycans increased af ter the cycloheximide treat

O 0 D 3 5 D O

ment W i th the same kineti cs as the decrease in S-sulfate incorporati on

O I D I I 3into proteoglycans observed in the f i rs t experiment . S ince

58

methionine is localized primarily in the HA—binding reg ion and3H-serine

primarily in the po lysaccharide at tachment region , these results are

cons is tent with a s ingle po lypep tide core pro tein for pro teog lycans .

At tempts are underway to so lubili ze the intracellular core pro tein pre

cursor poo l and to identify and iso la te these precursor s as well as

newly synthesi zed link pro tein us ing mono specif ic antisera direc ted

agains t e ither link pro te in or the HA—binding region po lypep tide .

insulin on proteoglycan synthes is . Chondrocytes maintained in serum

f ree Du lbe c c o's medium wi th high glucos e for 4 days incorporate

3 5S-sulfate into pro teoglycans typical f or cartilage bu t at a rate

only abou t 2 5 2 that of cells grown in the same medium supplemented

with fe tal calf serum . S erum free Dulbe c c o's medium containing

H Q N

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ug /ml'

or higher concentra tions of a p ep tide hormone with somato

med in activity increases the S-sulfa te incorporation into pro teo

g lycans about 502 of the dif ference be tween med ium alone and medium with

f etal calf serum . Insulin is much more ef fe c tive in s timulating

pro teoglycan synthes is wi th as li tt le as 5 ng /ml giving a s timula tion

up to 8 02 o f the d iff e rence . Exp e riments are underway to determine if

the s timula tory e ff ec ts o f the se po lypep tide hormone s are selec tive for

pro teog lycan synthe sis and if the s truc tures of the pro teoglycans are

alte red .

pro teoglycans ( Se ct ion 3b of la s t year's report ) . Monomer pro teog lycans

from chick limb bud chondro cytes were treated wi th alkaline—borohydride

to re lease glycosaminoglycans and o ligo sa ccharides from the core

pro tein . Combinations of molecular sieve c hromatography , c hondroitinase

diges tion and ion e xchange chroma tography were used to purify keratan

sulfa te , a mannose —rich oligo saccharide frac t ion and a group of small

o ligosaccharides . The keratan sulfate frac tion containe d proportions

o f sialic a c id z gluc osamine z ga lac to s e z ga la c to samine of 2 : 2 1 : 20 z l . The

galac tosamine was r ecove red primarily as ga lac tos aminito l indicating

tha t the chains were at tache d to the core pro tein by O— glycos ide bonds

be tween galactosamine and the hydroxyl groups of serine and / or

thre onine res idues . Less than re sidue s of mannose were pre sent

per galac to samine ind icating that keratan sulfate p robably does no t

contain mannose . The manno se re s idues in pro teoglycans , previous ly

repor ted by o the r inves tiga tors to be pre sent in kera tan sulfa te ,we re

shown to be locate d in a separa te class of oligosaccharides wi th a

composi tion of s ialic ac id z g luc osamine z ga la c to se z fu c o se zmannos e of

No reduced sugar mo ieties were present af ter the

alkaline-borohydride treatment sugges ting that these o ligosaccharides

are probably linked to the protein through N-glycos ide bonds be tween

g lucosamine and asparagine which are typical of g lycopro teins . The small

o ligosaccharides contained galac t osaminito l , and thus we re linked to the

c ore pro tein by the same type of linkage as the ke ra tan sulfa te chains .

The pro te og lycan from the ra t chondrosarcoma ,whi ch does no t contain

ke ratan sulfa te ,wa s found to contain the se small o ligosaccharides as

we ll as the mannos e — rich oligosaccharide s . The mannose — rich

o ligosaccharide s we re found to be locate d primarily , if no t so lely,

in the M W HA —b inding region po lypeptide . Molecular s ieve

chromatography on Bioge l P— lo se parated the small oligosaccharide

frac tion into three d is tinc t o ligosaccharides . The smalles t -was a

trisaccharide with the s truc ture

sia ly l a ( 2 6 ) galac tosyl ( l 3 ) ga lac tosaminito l .

The next larger was a t e trasaccharide

sialy l a ( 2 6 ) galactosyl ( l 3 ) gala c tosaminito l

4\ a ( 2 6 )

sia ly l

4 — 9 5

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2 . Publications

Has call,V . C . and He inegérd , D . K . : S tructure of Cartilage Pro teo

Sympos ium on G lyc oc onj uga te s (J . D . Gregory and R . W . Je anlo z ,editors )

Academic Pres s (1 97 9 ) 3 41—3 7 4 .

F altz,L . L . ,

Reddi,A . H . , Hascall , G . K . , Martin ,

D . , P ita , J . C . ,

and Has call, V . C . : Characteri s tics of pro teoglycans extrac ted from

the Swarm rat chondro sarcoma with associative solvents . J . Biol . Chem .

2 54 z l3 75—138 0 , 1 97 9 .

F al tz , L . L . ,Caputo , C . B . , Kimura , J . H . , Schrode

, J . , and Has callV . C . : S tructure of the complex between hyaluronic acid

,the

hyaluronic acid—binding region , and the link protein of proteoglycan

aggregates f rom the Swarm rat chondrosarcoma . J . Biol . Chem . 254

138 1— 13 8 7 , 1 9 79 .

Oe gema , T . R . , Hascall , V . C . , and Ei sens tein, R . : Character ization of

bovine aorta proteog lycan extracted with guanidine hydro chloride in the

p resence of pro tease inhibi tors . J . Biol . Chem . 254 z l31 2—13 18 , 197 9 .

Y anagishita ,M . , Rodbard , D . , and Has call , V . C . : Isolation and

characterization of pro teog lycans from porcine ovarian fo llicular

f luid . J . Biol . Chem . 254 z 9ll— 920 ,1 97 9 .

Heine gard , D . K . and Hascall,V . C . : The eff ects o f dansy lation and

acetylation on the interaction between hyaluronic acid and the

hyaluronic acid—binding region of car ti lage proteog lycans . J . Biol .

Chem . 2 54 z 9 2l—9 26 , 19 7 9 .

He ine gard , D . K . and Hascall,V . C . : Characteris tics of the non

aggregating pro teoglycans iso lated from bovine nasal cartilage .

J . Biol . Chem .-93 4 , 1 9 7 9 .

Kimura , J . H . , Hardingham , T . E . , Hascall , V . C . and Solursh , M

Biosynthesis of pro teoglycans and their ass embly into aggrega tes in

cul tures of chondrocytes from the Swarm rat chondrosarcoma . J . Biol .

Chem . 25 4 z 2 600—2 60 9 , 1 97 9 .

Caplan ,A . I . and Has call

,V . C . : S tructure and deve lopmental changes

in proteog lycans . (1 97 9 ) Dilatation of the U terine Cervix (F . Naf tolin

and P . G . Stubblef ield,edi tors ) pp . 7 9— 9 8 . Raven Press , New York .

Has call ,V . C . : Enamel protein biochemis try Avenues for future success .

J . Dent . Res . Special Issue B, V ol . 5 8 , 8 25—828 , 19 7 9 .

ll - Q F

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Lohmander ,L . S . ,

Hascall ,V . C . and Cap lan , A . I . : Ef fects of

4-me thy lumbillif ery l— B—D—xy 10 pyranoside on chondrogenes is and pro teo

g lycan synthesis in chick limb bud mesenchymal cell cultures . J . Bio l .

Chem . in pres s .

Hassell,J . R . ,

Newsome,D . A . and Hascall , V . C . : Characterization

and biosynthesi s of proteog lycans of corne al stroma from Rhesus monkey .

J . Biol . Chem . in p ress .

Solursh ,M Hardingham , T . E . , Has call ,

V . C . and Kimura , J . HSeparate effects of exogenous hyaluronic acid on proteoglycan synthesis

and deposition in pericellular matrix by cultured chick embryo limb

chondrocytes . Developmental Biology . in press .

Y anagishita , M . and Has call,V . C . : Biosynthesis of proteoglycans by

rat granulosa cel ls cul tured in vi tro . J . Biol . Chem . in press .

Caputo,C . B . and Rais z ,

L . C . : Degradation of F etal Rat Car ti lage

Matrix in Organ Culture : Eff e ct of S treptomyces Hyaluronidase .

Connective Tissue Research . in press .

M'THSCNIAN SC I EN CE I NF ORM A T I ON L DEPARTmENT OF I PR OJECT N UM B ERJECT . UMBLR ( Do N OT us e th is ECUCAT IC J ND

'

.sELF ARCI

i

D GBL IC HEALTR S ERV I C EN OT I CE or 7 0 1 DE— 0015 7—05 LB

i_ INTRAMURAL R E S EARCH PR O JECT I

p temb er 3 0 ,19 7 9

S tudie s on the S truc ture of Connect ive Tissue

N AM ES , L AB OR ATORY ANO I N ST I T UT E AF F I L I AT I ON S,AN D T I TL E S OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PR OF E S S I ON AL PER SON N EL ENG AG ED ON THE PRO J ECT

D . A . Torchia Biophysicis t NIDR LB

OTHERS : W . W . F leming NIH Pos tdoc toral F ellow

L . W . Je linski Staf f Fellow

D (b ) HU M AN T I S SU E S B (c ) N E I TH ER

SUMM AR Y OF WOR K (2 00 wo rd s or le s s un d e rlin e keyword s )The purpose o f this p roj ec t is to inves tigate the molecular s tructure of fibrou

pro teins and of pro teoglycans and to s tudy phosphorus chemis try in connec tive

tis sues.

The s truc tural informat ion ob tained wi ll be correlated wi th

func tion . Areas of present interes t ar e 1 ) S truc ture o f collagen and elas tin .

and inte rac tions in col lagen and elas tin f ibers . 2 ) Pro teoglycan s truc ture .

13C magnetic resonance is also being used to s tudy the molecular mobili ty of th

po lysaccharide and protein chains in the chick limb b

resonance is being used 0 probe the s truc ture of the phosphorus moieties in

connect ive tissue . 4 ) C magnetic resonance is being us ed to s tudy the extent

iand mechanism of hemoglobin § _ gelation in cell— free preparations and in

erythrocyte s . F or the se s tudie s,magnetic resonance spec trom e ters have be en

iis emble

iwhich g ive H ,

C, and P spec tra of so lids

.High power decoupling

oss—p o ariz a tion, mag ic angle spinning and solid echo experiments are all

-6040

(Rev. 10-76 )

ZO1 DE—0015 7—05 LB

Introduc tion

The goal of this work is to determine aspects of the molecular

s truc ture and interac tions of macromo lecules in connec tive tissue

and to elucidate s tructure-function relationships .

Methods

The primary research tool emp loyed is nuclear magnetic resonance (nmr ) .

Until recently,high reso lution nmr s tudies had been limited to f lexible

macromolecules s ince linewidths of rigid s tructures having high mo lecular

weights were too broad to detect . However , s tructured molecules can now

be s tudied in the s olid s tate by decoup ling the dipolar interactions

be tweenpg

o ton and C orJP nuclei . Cros s—po lari za tion can be used to

enhance C or P signals in rigid molecular lat tices where long

spin-lattice relaxation times make normal F ourier transform tech

niques imprac tical . In addit ion , cross—polari zation can be combined

with rapid sp inning of the sample about the magic axis (an axis

making an angle of with the external magnetic f ield ) to pro

duce spectra having small linewidths,com arable to tho se obtained

for s amples in solution . In the case of H nuclei , decoupling is

no t r equired and spec tra can be measured directly using a pu lsed echo

technique .

We have built two pulsed nmr spec trometers which provide solid s tate

S pectra o f H,

C,and

3 1P . High resolution cross-po larization spectra

and normal F ourier transform spectra (of C and P ) can be routinely

obtained f or samples ranging from inorganic crys tals to whole tissues ,

and magic angle spinning spec tra can be obtained f or P . Relaxationt imes in the laboratory and rotating f rames

,cros s—po lariza tion times ,

and chemical shif t aniso tropies can all be measured and provide

information about molecular orientation and molecular mot ions cover

ing the f requency range 10 to 10 Hz . Deu terium quadrupo le coup ling

cons tants are obtained from H spec tra and are sensi tive to molecular

mo tions that occur on the 10 10 Hz timescale .

Unlike P , the natural abundances of2H and

13C are low and

respectively ) . Hence , it is advantage ous to incorporate labe ledamino acids into proteins under s tudy . The presence of the label great ly

s implif ie s interpre tation of the spec tra since the H and C

re s onance s c an readily be assigned to the labeled si tes . We have

u sed biosyn thetic technique s to incorporate H and C labe led amino

acids into collagen,elas tin

,and proteog lycans .

Progress

1 . Collagen and Elas tin S truc ture . Sp ectra of chick ca lvaria collagen

f ibrils containing a C—labeled amino acid (Gly , Ala , Lys,Glu or Met )

have provided s trong evidence that rapid,aniso trop ic molecular motion

occur s in the helix backbone and in the labeled side chains . The

u- lnfl

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ZOl DE-00 15 7—05 LB

Introduc tion

The g oal of this work is to determine aspect s of the molecular

s truc ture and interactions of macromolecules in connec tive tis sue

and to elucidate s tructure—function relationships .

Methods

The primary research tool employed is nuclear magnetic res onance (nmr ) .

Until re cently ,high resolution nmr s tudies had been limited to f lexible

macromolecules s ince linewidths of rigid s tructures having high mo lecu lar

weights were too broad to detec t . However , s truc tured molecules can now

be s tudied in the s olid s tate by decoupling the dipolar interac tions

betweenpro ton and C or

JP nuclei . Cros s—po larization can be used to

3enhance C or P S i gnals in ri gi d molecular lat ti ces where long

spin—lattice relaxation times make normal Fourier trans form tech

niques impractical . In addit ion , cross-polari zation can be combined

wi th rapid sp inning of the sample about the magic axis (an axis

making an angle of with the external magnetic f ield ) to pro

duce spectra having small l inewidths ,com arable to those obtained

for samples in solution . In the case of H nuclei , decoupling is

no t r equired and spec tra can be measured directly using a pulsed echo

technique .

We have built two pulsed nmr S pec trometers which provide solid s tate

spectra o f H ,

3C ,

and3 1P . High reso lution cros s—po larization spectra

and normal F ourier transform spectra (of C and P ) can be routinely

obtaine d f or samples ranging f rom inorganic crys tals to whole tissues ,

and magic angle spinning S pectra can be obtained for P . Relaxationtimes in the laboratory and rotating f rames , cros s —po larization times ,

and chemical shif t aniso tropies can all be measured and provide

information about molecular orientation and molecular mo tions cover

ing the f requency range 10 to 10 Hz . Deuterium quadrupole coup ling

cons tants are obtained f rom H spectra and are sensi tive to molecular

mo tions that occur on the 10 10 Hz timescale .

Unlike P , the natural abundances of2H and

13C are low and

respectively ) . Hence , it is advantageous to incorporate labeledamino acids into prote ins under s tudy . The presence of the label greatly

s implif ies interpre tation of the S pec tra since the H and C

re s onances can readily be assigned to the labeled si tes . We have

u sed biosynthetic techniques to incorpora te H and C labe led amino

acids into collagen,elas tin

,and pro teoglycans .

l . Co llagen and Elas tin S truc ture . Spectra of chick calvaria collagen

f ibrils containing a C-labeled amino ac id (Gly , Ala , Ly s,Glu or Met )

have provided s trong evidence that rapid,aniso trop ic molecular motion

occur s in the helix backbone and in the labeled side chains . The

4 — l fl fl

Zol DE— 0015 7—05 LB

line shape and quadrupole spli tting observed f or the2H— S pec tra of

collagen f ibrils labeled in the methyl group of Ala conf irm the presence

o f backbone motion . These results are strong evidence that co llagen

f ibrils are not s tabili zed by a unique se t of interactions .

13C— spec tra o f cross-linked e las tin samples containing V al ,

A la or

Lys res idues labeled at backbone carbonyl carbons have shown that the

extens ible re gions o f elas tin have the kine tic freedom required of a

viscoelas ti c rubber— like network . Elas tin chain dynamics are sens i

t ive func tions of temperature and the degree of swelling . The O ppos ing

eff ec ts o f these two variables make the chain dynamic s of elas tin

virtually independent o f temperature in the 2 0 4D°

C range . These

results corre late with the macroscopic viscoelas tic behavior of elas t in

which i s also independent of temperature in this range .

2 . Proteoglycan S tructure . We have pre pared samp les of the chick

l imb bud proteog lycan monomer containing serine1C-labeled at C

8

or g lycine labe led at Ca

. Linewidths and relaxation times measured

f or the labeled carbons indicate that the protein backbone and the

point o f a ttachment of the polysaccharide to the core pro tein

undergo i so trop ic reorienta tion (as a consequence of segmental

motions ) . This reorienta tion is 3— 4 orders of magnitude fas ter than

the motion calculated f or the pro teog lycan monomer as a whole . (N . B .

This is a collaboration with Dr . V . C . Hascal l,see proj ec t

# 2 01 DE—00134-05 LB . )

3 . Phosphorous Chemis try in Connective Tissue using3 1P Mag ic Ang le

Sp inning . We have j u st compl eted cons truc tion of a probe for S pinning

samples a t the magic angle and have obtained preliminary high reso lution

sp ec tra of hydroxyapati te and other so lid samp les c ontaining P .

13 134 . C Nmr of Hemog lobin S Ge lati on . We have shown that C nmr

provides a reliable means to quantitate the amount of p olymer in a

cell— free deoxyg enated hemoglobin S g el . Preliminary s tudies , carr ied

Ou t on intac t e ry thyoc y t e s indicate that the nmr technique can be used

to f ollow p olymerizat ion in the intact cell . (N . B . This is a

co llabora tion wi th Dr . A . N . Schechter , NIAMDD . )

Interactions involving specif ically labeled si tes have been inves ti

g ated u sing the new experimental te chnique of high reso lu tion nmr

in so lids in conj unct ion wi th C , H , and3 1P-labe led tissues . New

informa tion about the mol ecular dynamics and interactions at specif ic

s ites in intac t c onnec t ive tis sue has been obtained . This inf ormation

has provided a bas is f or unders tanding the nature of the mo lecular

interac tions that determine the s truc ture and func tion of the

macromolecules inves tigated . In addi tion, C nmr is a promising method

f or inves tigating potential inhibitors of g elation wi thin intac t red cells .

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F uture Plans

The general s trategy o f using solid s tate nmr to s tudy labe led

macromolecules will be followed . However , emphasis will shif t from

u sing C labels to H labe ls to probe prote in S tructure and inter

actions . Hemoglobin S s tudies will concentrate on inves tigating the

nature of hemog lobin gelation wi thin the red cell . Magic ang le S pinning

will be used to charac terize phosphorous chemis try in connec tive tissues .

Publications

D . A . Torchia : The measurement o f proton enhanced carbon—13 T1values

D . A . Torchia and D . L . V anderHar t : High power double resonance s tudies

of f ibrous proteins , proteoglycans and model membranes . In G . C . LevyTopics in Carbon—13 NMR Spec tros copy . Wiley , New York , 19 79 ,

V ol . 3 , Chapter 3 , pp 3 25-3 60 .

J . D . Termine , D . A . Torchia and K . M . Conn : in M . U . Nylen ,

J . D . Termine Proceedings of Third International Symposium on

Too th Enamel . J . Dent . Res . Supp l . 197 8 .

J . W . H . Su therland,W . Egan

,A . N . S chechter and D . A . Torchia

Carbon— lB—proton nuclear magnetic double-resonance s tudy of

L . W . Je linski and D . A . Torchia13C/lH High power double magne tic

resonance inves tigation of co llagen backbone mo tion in f ibrils and in

solution . J . Mol . Biol . 13 2 , 19 7 9 . in press

C . T . Noguchi , D . A . Torchia and A . N . Schechter . C Nuclear magne tic

resonance quantitation of polymer in deoxyhemog lobin S gels . Proc . Nat .

Acad . S ci, 19 79 . in press

D . A . Torchia,L . W . Jelinski , W . W . Fleming and C . E . Sullivan

Mo lecular mot ions in f ibrous collagen and e las tin de termined by C1H

magnetic double resonance of 13C labeled samp les . Polymer preprints ,

1 9 7 9 . in press

4

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Zol DE-002 15—03 LB

Proj ec t Description

Obj ec tives

1 . The mechanism of collagen f ibr il formation in vi tro . The

development of a well-characterized , reproducible sys tem f or the

s tudy of collagen f ibril formation in v itro and of models of f ibril

s truc ture has made it possible to inves tigate the mechani sm ofcollagen assembly in greater detail than previous ly pos s ible . The

dependence of ass embly on co l lagen concentration and temperature has

been es tablished allowing the manipulation of assembly and the

iso lation o f separate s tages for individual s tudy .

2 . High res olution electron microscopy of collagen f ibrils and

early aggregates in assembly .

The properties of connective tissues are determined in par t by the

packing of co llagen molecules into func tional units , the native f ibril .

The precise relat ionship between monomers in a highly ordered f ibril

f or example , a tendon -is no t known . The longitudinal relationship is

known but information regarding lateral p lacement , superco iling and

possible subfibrillar units has been limi ted by the methods available .

Electron microscopy has the po tential o f providing information down to

at leas t 2 nm if certain ins trument modif ications are utilized and

preparation of the specimen bo th conserves native s tructure and

op timizes e lec tron density diff erences . The maj or purpose of this

aspec t of the proj ect is to ob tain improved elec tron microscopic

information through improved techniques and the use of in vitro

aggregates of collagen .

3 . Quasie las tic light s cattering .

Quasie la s tic light scat tering af f ords a technique f or the s tudy ofthe S ize and shape of macromolecules in so lution . This technique should

prove to be well suited for s tudies of the ear ly s tages of co llagen

assembly where small intermediates are believed to form . Development

of ins trumentat ion has been S timulated by this prob lem . The application

of the te chnique in inve s tigations into the s tructure of o ther bio logical

macromole cules is als o of interes t .

4 . The ro le o f nonc ol lagenous molecules in collagen as sembly andf ibril s tructure .

In vivo , co llag en assembles in the presence of a variety of macro

molecule s and low molecular weight subs tances . Although none of these

(excep t p e rhaps pho spha te ) appear s to be necessary for in vitro

assembly , i t is likely that they may be involved in vivo,perhap s to

u _ 1 nu

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regulate the proces s . Of par ticular interes t are pro te og lycan ,lysyl

oxidase and f ibronec tin . We initia lly propose to s tudy the ef f ec t of

pro te og lycan on in vitro assemb ly .

Me thods Emp loye d

Type I co llagen is pr epared from rat tail tendon , and purif ied and

charac terized by s tandard biochemical and biophysical methods . F ibril

s truc ture and kinetics of formation are be ing s tudied by electron

microscopy,turbidimetry

,and quasie las tic light scat tering . Increased

resolu tion of e lectron micrographs is be ing sought by sys tematically

inves tigating various prepara tive methods f or specimens and by utili zing

techniques to minimize be am damage . In addi tion to conventional trans

mis s ion elec tron microscopy (CTEM ) , we are utiliz ing s canning transmiss ion

elec tron microscopy ( STEM ) at Johns Hopkins to visuali ze heavy atom

labeled c ollagen and at Brookhaven for mass measurements o f collagen

aggrega tes . A quas ie las tic light sca ttering ins trument has been built

and i s fu lly ope rat ional . In some studies (only whe re no ted ) Type I I

collagen f rom rat chondrosarcoma has also been used .

Progres s

1 . The mechanism of collagen f ibril formation .

A set of op timal conditions f or the s elf-as sembly process has been

selec ted and used in an inves tigation of the mechanism of collagen

a s sembly . We have found that c ollagen assembly occurs via a mechanism

of a t leas t three s teps . S tep 1 ,initiation

,invo lves a temperature

d e pendent change which lead s to an unidentified intermediate that in

the second s tep spontaneously grows . S tep 2 is linear growth of

f ilaments by a temperature— independent proces s . S tep 3 i s lateral

as soc iation of f ilaments by a temperature —dependent proces s . The

concentration de pendence of the rates o f s tep s 2 and 3 , and the lack

of a measurable cri tical concentrat ion sugges t as sembly by accretion .

We have shown tha t s tep 1 is comple te in 6 min at 2 6° and is independent

o f co llagen concentration . This result sugges ts a ra te limiting con

f ormational change but does not rule ou t some f orm of early aggregation .

Co llagen trea te d with p e psin , which removes the nonhe lic al ends,makes

d is tor ted f ibrils by a S imilar mechanism to normal collagen . However ,

assembly is very much s lower and s tep 1 is markedly altered . The non

he lical ends theref ore are critically involved in a ssembly .

2 . High reso lu t ion elec tron microscopy of col lagen aggregates f ormed

early in as sembly .

Na tive col lagen f ibrils and recons tituted co llagen f ibrils have been

examined by elec tron micros copy us ing s tandard methods . Observationof co llagen in various s tages of as sembly and under various condi tions

ll - I O R

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has shown that intermediat e aggregates are formed early in ass embly

( s tep 1 ) and that the format ion of long thin filaments ( s tep 2 )precedes banded f ibril formation ( step These intermediate

aggregates and their f ilaments are dif f icult to ob serve and mus t be

dis t inguished from art ifacts . Preliminary inves t igat ions suggest

that three kinds of high resolutions studies are potent ially us eful

in this problem . (a) CTEM of negat ive ly stained preparat ions us ing

instrumental procedures for minimal radiat ion damage . (b ) STEM of

preparat ions labeled with s ingle heavy atoms at specific sites . The

arrangement of collagen molecules in an aggregate can b e deduced from

the pat tern of heavy atoms . ( c ) STEM o f unstained preparat ions t o

determine mas s . F or example,a knowledge of mass as a funct ion o f

length o f the thin filament s formed in step 2 will be important in

determining s tructure .

3 . Quasie lastic light scatt ering .

Computer programs des igned to analyze the data obtained from the light

scattering instrument have been writ t en ; analys is involves use of a

non—linear regres sion rout ine which utiliz es an on— line computer . The

direct result obtained is the dif fus ion coeff icient of the species

(molecule or aggregat e) in the\

solution being studied . From hydrodynamic

theory it is then poss ible to calculate the s ize and shape of the

molecule or aggregate .

As a control , we init ially s tudied co llagen in acid ic solut ion and

conf irmed the published value for the dif fusion coef fic ient and its

anomalous concentrat ion dependence . We found that in neutral solut ion

at 4°

C the concentrat ion dependence was normal and that our collagenpreparat ions are monomeric under these condit ions . When f ibril

format ion was init iated by rais ing the temperature,the dif fus ion

coef fic ient dropped rapidly over 5— 10 min and then reached a constant

value . This result is cons ist ent with our conclusions regarding

ste ps 1 and 2 in as sembly , described above , and places cons traint s onthe structures involved .

In collaborat ive studies with Dr . R . Nos sal ,the light scattering

instrument was used to study the mechanical propert ies of gels .

4 . The e f fect o f proteoglycan on collagen as sembly in vitro .

We have conf irmed previous reports that the rate o f type I co llagen

assembly is retarded in the presence o f a cart ilage—type proteoglycan

from a rat chondrosarcoma . Proteoglycan also b inds t ight ly to the

collagen but doe s not appear to alter the structure of the fibril .

On the other hand , proteoglycan sl ightly accelerates the as semb ly of

type II collagen and does not bind . Since type I and II collagens are

bas ically s imilar molecules and form s imilar f ibrils,this result

lI — J O F

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N AM ES,LABOR ATORY AND I N ST I T UT E AFF I L I AT I ON S, AN D T I TL ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PROF ES S I ON AL PERS ON N EL ENG AG ED ON THE PR O JECT

Chief,Enzyme Chemistry Sect ion

S taff F ellow

S taff F ellow

Others J . J . Gorman Vis it ing F ellow

Seelig NIH Postdoctoral F ellow

SUMM AR Y OF WOR K (200 wo rd s or less und erlin e ke yword s )

and body f luids is under invest igat ion . Knowledge of the catabolism of these

conj ugates has been obtained .

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1 . Proj ec t De scription

S tudies carried out ove r the past several Y ears have be en dire cted

toward c harac terization o f enzyme s termed transglutaminas e s that are

responsible for the formation of y-glutamyl amide bonds in prote ins

and po lype p tide s . The s e enzymes catalyze transf e r reac tions at the

carboxamide group of pept ide—bound L-glu tamine wi th a high degre e of

s tereospecifici ty . In the presence o f ac cep tor amines , the transf er

reac tion result s in the formation of subs ti tute d amides — COHN2

RNH2

~COHNR NH3) .

One o f the well— characteri zed covalent cross links between and wi thin

pro tein mole cules i s the c (v—glu tamy 1 ) 1y sine bond . There has been

increas ing evidence for the presence of thi s cros slink in fibrin clots ,

cell membrane,g lycerinated myof ibril s of muscle , pro te ins of seminal

plasma,nat ive wool keratin

,and the citrulline— containing pro te in

frac tion of hair . We have recently observed that the transglu taminas e s

c an catalyze formation of a hitherto undescribed cros s link . This cros s

link be tween pept ide chains is formed by means of a transf er reaction

be twe en the carboxamide group of a glu tamine residue in ea ch chain and

bo th primary amino groups of a diamine or polyamine . The wide o ccurence

and obvious impor tance of y—glutamyl amide bonds have led us to focus

at tention on the enzymes respons ible for the ir formation , as well as on

the o ccurrence and func tions of these bonds in various pro teins .

This proj ec t is in part collaborative wi th S . I . Chung proj ec t numbe r

2 01 DE— OOO4 9-08 LB .

Maj or F indings

The specifici ty of ac tivated human blood coagula tion factor XII I is

being sys tematically inves tigated . S ince,i t was known that mixed

casein is an excellent subs trate for this enzyme , purif ied a ls

B and K — caseins isolat ed from the milk of cows homozygous f or single

variant s of these c aseins were examine d as subs tra tes . B— casein ,

A variant was f ound to be by far the bes t subs tra te .

B-casein was labeled with a f luorescent amine subs tra te by use of

f ac tor X I I Ia and the labeled casein,containing a s ingle derivati zed

g lu tamine , was subj ec ted to chemical and enzymic degradation . Pep tide

mapping and se quence s tudie s identifie d the glutamine residues as

G lu— 16 7 . Solid phase syn thesis of a 15 membe r pe p tide containing the

s equence of B— casein surrounding this g lu tamine res idue s was carried

out . Thi s peptide was f ound to be an excell ent subs t ra te f or

ll T O O

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partially purified from rabbi t kidney . The enzyme is a cyclohydrolase

catalyz ing the breakdown in the f ollowing way

C N R

H N C H

COOH

Understanding of the mole cular charac teristics of the transglu taminas e s

is vi tal to de termination of the func tion of thes e enzymes in normal

and diseased tissues,pharmacological control of ac tivity , and

hormonal regulation .

The minimal subs trate struc tural requirements f or transglu taminas e s have

been defined over the pas t several years through s tudies such as those

de scribed . We now have a better unders tanding of the mechanism of

en z yme~subs trat e interactions in transglutaminase— catalyzed reactions .

The techniques used f or these s tudies , as well as ones used in de te rmin

ing specificity toward glutamine and lysine residues in macromolecular

subs trate s,mos t certainly will show similari ties , as well as dif fer

enc e s ,in the members of this impor tant group of enzymes .

Both polyamines and transglu taminas e s are widely dis tributed and bo th

may p lay important roles in control of growth and o ther bio logical

process es . The f inding that po lyamines are natural subs trates for these

enzymes is an important f irst s tep in determining the role e ach plays

in these vital proce sses . That there may be diff erences in the manner

in which the po lyamines serve as subs trates in ce ll s and in body f luidsmay contribute to our under standing of their poss ible function in con

trol of cellular and extracellular proces ses .

Until now little has been known about the catabol ism of products of

transglutaminase action . The characterization of and determina tion

o f the spe cif icity of the enzyme that breaks down y—g lu tamyl lys ine and

polyamine d erivative s will ce r tainly be of value in unders tanding thisvital pro ce ss .

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Z01 DE-00001-28 LB

Spe cificity s tudies will be continued wi th special emphasis placed on

the transglu taminas e s involved in hemo s tasis and wound healing .

Principal obj ectives will be to determine the impor tance of amino acid

side chains in close vicinity to subs trate glutamine residues and , thus ,

to design eff ective small subs trates for the enzymes .

Now that s trong evidence has been obtained that the polyamines func tion

as transglutaminase subs trates , attempts will be made to determine how

the product s of these reac tions participate in cellular and extra

ce llular proces se s . F irs t step s will involve characterization o f the

pro tein conjugates , identif ication of their cellular or extracellularlocation , and es timation of their metabolic rates and characteris tics .

Knowledge of the specif icity and dis tribution of the enzyme that breaks

down y-glu tamyl d erivatives should reveal much information about the

bas ic catabolism of the products of transglutaminase action . F or

example , can cros slinks be hydro lyzed in intac t pro teins or mus t the

pro teins be largely degrated before the cross links can be c leaved?

The answer to this ques tion is of prime impor tance to a thorough under

s tanding of the physio logical role of these impor tant enzymes .

2 . Publications

S chrode , J . , and F o lk , J . E . : Transglutaminase— catalyzed c ros slinking

through Diamines and Polyamines . J . Biol . Chem .

Schrode , J . , and F o lk , J . E . : S tereochemical aspect s of amine subs trate

attachment to acyl intermediates of transglu taminas e s . J . Bio l . Chem .

-6 61 , 19 7 9 .

G orman , J . J . , and F olk , J . E . : S tructural f eatures of glu tamine sub

s trat e s for human p lasma factor X IIIa . J . Biol . Chem . in pres s .

Gorman , J .J . , and F olk , J . E . : Transg lu taminase amine sub strates for

photochemical labeling and cleavable c ros slinking of proteins .

J . Biol . Chem . in press .

ANNUAL REPORTLABORATORY OF BIOLOGICAL STRUCTURE

NATIONAL INSTITUTE OF DENTAL RESEARCH

The research effor ts o f the Laboratory o f B io logical S tructure during the

pas t year have continued to focus on the elucidation of the s truc tural ,

chemical and func tional charac teris tics o f the hard and so f t tissues of

the oral cavity . Al though no maj or new ini tiatives have been under taken ,

cons iderable progres s has been mad e through the logical extens ion of our

exis t ing res earch proj ects . These resul ts are summar i zed below by the

general ar ea o f inves tigation , and are presented in detail in the narrat ive

s tatements of the individual progres s report s .

Apatite S truc ture and Formation

S tudies o f the chemis try , structure and formation of hydroxyapatite (HAP )and related calcium phosphate sal ts have concentrated in two main ar eas

quantitation o f the hydroxide content o f apati te ; and the charac ter i zat ion

o f calcium phosphate prec ipitation from seeded solutions . Previous resul ts

sugges ted that the early crys tall ine phases formed during the in vitro prec ipitation o f calcium phosphate were def ic ient in hydroxide . A titration

method for direc t analys is of the hydroxide content o f the precipitates

was developed . The earlies t fo rmed crys talline phas es contained 0—2 02 of

the theoretical amount o f hydroxide for HAP , while the mos t mature apat ites

s tudied rarely contained greater than 602 of the theoretical hydroxide

content . In conj unc tion with the titration method,procedures for quanti

tation o f hydroxide content were es tablished us ing infrared and laser

Raman spectros copy . Analys is by thes e methods agreed within -112 to +1 82

and 202 , respec tively,o f the chemically determined hydroxide content .

Thes e spectros copic pro cedures are under sys tematic evaluation to es tablishcondit ions for maximum reliab il ity . The s ignif icance o f the reduced hydroxide

content is no t clear ; but it indicates that hydroxide is no t required for

s tabil ity o f HAP under s imulated biological condit ions .

S ince mos t b iological calcif ication oc curs in the presence o f preexis t ing

apati te crys tals , s tudies of calcium phosphate prec ip itation reactions

focused on precipitations induced by s eeding s tab le supersaturated so lut ions

with apat ite crys tals . The course o f the reac tion depended upon the

initial degree o f supersaturation . With calcium and phosphate concentrations

equal to or greater than and mM ,respect ively , an o c tac alc ium

pho sphate— l ike intermediate occurred,as in spontaneous precip itat ion

reac tions . In contras t, with lower initial calcium and phosphate c onc en

trations ,an o c ta c al c ium pho sphate phase did no t occur . Previous s tudies

o f spontaneous precip itat ion reactions demons trated that F“and Mg

2+

af fected the l ifet imes o f the various intermediate phases . In seeded

reac tions,F“retarded reaction development at high supersaturations , while

i t accelerated the init ial reac tion at low super saturations . The eff ec ts

o f F“on the crys tallinity o f apatites was also s tudied . Whereas in

f luoridated bone the crys tals are increas ed in wid th and thicknes s but not

lI — 1 1 3

in leng th,in in vitro sys tems f luoride favored the formation of narrow

needle-like crys tals . These results emphasize the importance o f b iological

factors,such as local calcium and pho sphate concentrat ions and extracellular

matrix macromolecules,in contro lling the growth o f apati te crys tals in

V ivo .

Too th Matrix Pro teins and Mineralization

S tudies o f the matrix pro teins of fetal bovine teeth have extended the

obs ervations repor ted las t year on the chemis try and s tructure of these

unusual molecules . The ame logenins , a s eries of 8—10 prol ine-rich proteins ,

are found only in fetal enamel matrix . With increas ing f etal age or

degree o f mineralization a shift from higher to lower molecular weight

species o ccurred . The amelog enins contained 12 s ialic ac id , 12 hexosamine

and 10-152 to tal carbohydrate . The f etal ename lins are the probable pre

cursors of the mature enamel matrix pro teins . They were extracted in higher

proportion (relative to amelogenins ) from o lder fetal teeth than from

younger teeth , and the data sugges t that they are synthes ized at all s tages

o f enamel development . The ename lins were pho sphorylated , and contained

4-62 s ialic acid,4—52 hexosamine and about 2 02 to tal carbohydrate . The

b iogenes is o f these enamel matrix pro teins is b eing s tudied in hams ter

mo lar too th germs grown in culture for 2-4 days .

The dentin specif ic pro teins have been found to consis t of a maj or phospho

pro tein of Daltons,compris ing one—half to two— thirds o f the non

co llagenous matrix pro teins , 3 phosphorylated glycopro teins in the 30

Dal ton range , and 2-3 phosphorylated glycopro teins in the lO Dalton

range . The maj or dent in phosphopro tein previously has been shown to inhib it

apatite formation from synthetic calcium pho sphate solutions . Pretreatment

o f the pho sphopro tein with ionic calcium and / or collagen f ibrils reduced the

inhib itory ef f ects on apatite formation . The pho sphopro tein also inhib ited

the crys tal growth process es in seeded precipi tation sys tems . Whil e pre

treatment with calcium diminished the inhib ition , no ef fect was seen af ter

pretreatment with collagen . The dentin pho sphopro tein also inhib ited in

vitro collagen f ibrillogenes is ; this inhib it ion was greatly enhanced by

pretreatment with calcium . These results sugges t an important regulatory

role for the dentin phosphopro tein in dentinogenes is and dentin mineral

i z ation .

In a continuation o f s tudies initiated las t year microprobe methods are

b eing used to character ize in s itu the organic matrix and initial mineral

depos its in enamel , dentin and bone . These methods allow correlation of

molecular and elemental characteris tic with the his to logical s tructure .

In the enamel matrix , the maj ority of the protein appeared to be in an

antiparallel B-pleated she t t conf iguration . Additionally , an adenos ine

and / or guanos ine nucleos ide has been ident ified . In all three tissues ,

spectra ob tained from the earlies t inorganic depo sits were sugges tive o f

the magnes ium—carbonate containing mineral, huntite The

earlies t detectable phosphate was apatitic in nature,with no evidence

obtained as yet o f precursor phases . S tudies to extend these obs ervationsare currently underway .

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in length,in in vitro sys tems fluoride favored the formation of narrow

needle—like crys tals . These results emphasize the importance o f b iological

factors,such as local calcium and pho sphate concentrations and extrac ellula

matrix macromolecules,in contro lling the growth o f apati te crys tals in

V 1 V 0 .

Too th Matrix Proteins and Mineralizat ion

S tudies o f the matrix pro teins of fetal bovine teeth have extended the

ob servations reported las t year on the chemis try and s tructure o f thes e

unusual molecules . The ame logenins ,a s eries of 8— 10 prol ine-rich proteins

,

are found only in fetal enamel matrix . With increas ing fetal age or

degree o f minerali zation a shif t from higher to lower molecular weight

species o ccurred . The ame logenins contained 12 s ialic acid , 12 hexosamine

and 10—152 to tal carbohydrate . The fetal ename lins are the probable pre

cursors of the mature enamel matrix pro teins . They were extracted in higher

proportion (relative to ame logenins ) from older fetal teeth than from

younger teeth , and the data sugges t that they are synthes ized at all s tages

o f enamel development . The ename lins were pho sphorylated , and contained

4— 62 s ialic acid , 4—52 hexosamine and about 2 02 to tal carbohydrate . The

b iogenesis o f these enamel matrix pro teins is b eing s tudied in hams ter

mo lar too th germs grown in culture for 2—4 days .

The dentin specif ic pro teins have been found to consis t o f a maj or phospho

pro tein o f Daltons , compris ing one—half to two-thirds o f the non

collagenous matrix pro teins , 3 phosphorylated glycopro teins in the 30

Dal ton range , and 2—3 phosphorylated glycopro teins in the 10 Dal tonrange . The maj or dent in phosphopro tein previously has been S hown to inhib it

apati te formation from synthetic calcium phosphate solutions . Pretreatment

o f the pho sphopro tein with ionic calcium and / or collagen f ibrils reduced the

inhib itory effec ts on apatite formation . The pho sphopro tein also inhib ited

the crys tal growth process es in seeded precipi tation sys tems . Whil e pre

treatment -with calcium diminished the inhib ition , no ef fect was seen af ter

pretreatment with collagen . The dentin pho sphopro tein also inhibited in

vitro collagen f ibrillogenes is ; this inhib it ion was greatly enhanced by

pretreatment with calc ium . Thes e results sugges t an important regulatory

role for the dentin phosphopro tein in dentinogenes is and dentin mineral

i z ation .

In a continuation o f s tudies initiated las t year microprobe methods are

b eing used to characterize in s itu the organic matrix and initial mineral

depos i ts in enamel , dentin and bone . These methods allow correlation o f

molecular and elemental characteris tic with the his tological s tructure .

In the enamel matrix , the maj ority o f the protein appeared to be in an

antiparallel B—pleated she t t conf iguration . Additionally , an adenos ine

and / or guanos ine nucleos ide has been identified . In all three tissues,

spec tra ob tained from the earlies t inorganic depo sits were sugges tive o f

the magnes ium—carbonate containing mineral, hunt it e The

earlies t detec table phosphate was apatitic in nature,with no evidence

obtained as yet o f precursor phases . S tudies to extend these obs ervationsare currently underway .

Endochondral Bone Diff erentiation

The use o f the in vivo matrix-induced bone di f ferentiation sys tem has

continued to yield s ignificant data concerning the process es of car tilage

and bone cell dif ferentiation and matrix— cell interac tions . A ser ies o f

experiments es tab lished the local mitogenic ef fect o f the implanted

collagenous matrix on the hos t mesenchymal cells . Increases in ornithine

decarboxylase , a marker enzyme for cell proliferat ion,and incorporation of

3H— thymidine were ob served on days 3 and 8—9 af ter implantat ion . Radioautography revealed that the cells labeled with 3H— thymid ine were located

clo s e to the par t icles o f implanted matrix . In a related s tudy,it was

found that when the c ros s linking of newly— synthes ized co llagen was in

hibited by B— aminO prO p ionitrile , chondrogenic dif ferentiation was inhib ited

and the mineralization o f cartilage and bone was greatly reduced . These

results at tes t to the importance o f the collagenous matrix for cell growth

and dif ferentiation , and are -a f irs t s tep in unders tanding the complex

interrelat ionships between connect ive tis sue cells and their surroundingmatrix .

A variety of nutrients , hormones , metabolites , drugs and o ther subs tances

interac t d irectly or indirectly with skeletal t is sues . F or example,peri

odon tal disease is known to be complica ted by diabetes,but the precise

role o f insulin in the differentiation and metabol ism o f skeletal tissues

is unknown . S ince the matrix— induced sys tem is ideal for s tudying the

influence o f these sub s tances on the various s tages o f car tilage and bone

formation , the ef fects of exper imental diab etes and insulin on the develop

ment o f endochondral bone were s tudied . The results indicated a marked

inhibi t ion of mesenchymal cell proliferation on day 3 after implantat ion

in d iab et ic animals . The reduced cell proliferation resulted in a decreased

and delayed formation o f car tilage . The vas cular invasion prior to chon

droly sis and o s teogenes is was also decreased and delayed . F inally , diab etes

caused a reduct ion in alkaline phosphatase activity and 4 5Ca incorporation

during car tilage calcif ication and os teogenes is , which could be prevented by

adminis trat ion o f exogenous insulin .

S truc ture and F unc tion o f S ecretory Cel ls

Progres s in the s tudies of s ecretory cells during the pas t year has o ccurred

in two main areas : Gol gi apparatus— GERL s tructure and relationships ; and

identificat ion and function o f components o f the lysos omal sys tem . Acidphosphatase and thiamine pe phospha tas e have previous ly been utilized as

enzyme markers o f GERL and the Golgi saccules , respec tively . Under cer tain

conditions , however,the speci ficity of thiamine pe pho spha tas e for the

Go lgi saccules was altered ; reac tion product was pres ent in GERL and forming

s ecretory granules as wel l as the Golg i sac cules . In o ther cells GERL is

frequently considered to be specialize d endoplasmic reticulum , but the

present resul ts,which are cons is tent with previous obs ervat ions on exocrine

cells,sugges t that GERL may b e more clos ely related to the Golgi saccules

than to the endoplasmic ret iculum . An impor tant goal o f these morphological

S tudies is the elucidation o f the mechanisms o f s ecretory pro tein transport

and packaging . The localizat ion of neurophysin , the carrier protein for

vasopres sin and oxytocin , was determined in the neurosecretory neurons of

the S upraoptic nucleus , using immunocyto chemical procedures . Neuro

phy sin was present in the organelles asso ciated with pro tein synthes is and

transport,but it was no t detectable in GERL

,from which the neurosecretory

granules form . Al though these findings parallel those previous ly reported

for lacrimal acinar cells , the inab ility to detect secretory proteins in

GERL is curious . While many factors may be involved a likely explanationis the inability of the cytochemical reactants to penetrate the membrane

o f GERL .

The lysosomal sys tem has many functions in secretory cells . In addition

to the usual participation in the cellular economy through degradation of

ef fete macromolecules and organelles , under certain conditions lysosomes

s equester and degrade secretory granules , and part icipate in the retrieval

o f exces s membrane from the cel l surface fo llowing exocytos is . An addi tionalfunction s tudied in exocrine acinar cells and the neurosecretory neurons

is the up take of intravenously adminis tered exogenous ma terials . Desp ite

the marked differences in the two cell types , some s triking S imilarities

exis ted in the up take and seques tration processes . In the neurons horse

radish peroxidase (HRP ) was taken up in endocytic organelles , and transported

retrogradely to the cell body where it accumulated in secondary lysosomes .

Ci s ternae o f agranular reticulum , in continuity with the lyso somes , appeared

to funct ion in anterograde transport o f the HBF and acid hydro lases . In

exocrine cells , in addition to endocytic organelles and secondary lys osome s ,

HRP was seques tered in elongated basally-lo cated cis ternae . These cis ternae

reacted po sit ively for a lysosoma l marker enzyme , trime taphosphatase , althoughthey were generally unreactive for the classical lyso somal marker , acid

phosphatase . Thus , in bo th neuro secretory neurons and exocrine acinar cell s ,

cis ternal structures involved in the up take , seques tration and transport of

exogenous materials have been identified c y to chemic ally as part of the

lysosoma l system .

The interes t o f the laboratory in the secretory process and the commitment

o f NIDR to bas ic b iological research led to the organization of a con

ference on Basic Mechanisms o f Cellular Secretion , which was held inAnnapolis , Maryland , Sep tember 1 7-2 1 , 19 79 . The program focused on

seven maj or topics related to protein secretion , and featured formal

presentat ions by about 40 invited speakers and three pos ter ses s ions

o f contributed papers . A total o f about 200 scientis ts participated

in the conference . The c c —organizers of the conference were Drs .

Arthur R . Hand and Cons tance Oliver , and secretarial service was pro

vided by Mrs . Patricia Youmans .

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Proj ect Descript ion

Ob j ectives

The bas ic obj ec tive o f this proj ect is to ob tain further knowledgeo f the structure and function o f secretory cel ls and their organelle

sys tems . U til izing electron microscopic , cy tochemical , radioauto

graphic and bio chemical techniques , cel l ultras tructure i s corre

lated with enzyme localization , quantif ication of cellular c ons tit

uents , and glycopro tein synthes is and transport . Our ef fo rt s have

concentrated on : ( 1 ) the role of the Golgi apparatus and GERL in the

transport and packaging of secretory material ; ( 2 ) the func tion of

lysosomes in the degradation of secretory material and cellular mem

b ranes ; and (3 ) the effec ts of sialographic procedures on the s true~ture of the rat submandibular gland .

Methods Employed

Ti ssues for morphological examination are fixed by vas cular perfus ion

and prepared by S tandard techniques . Cytochemical incubations are

carried out on 50— 75 pm slices of f ixed tissue . Radioautographs o f

t issue labeled with tritiated glycoprotein precursors are prepared

by the method o f Kopriwa The procedure o f Nadler ( 19 71 ) is

util ized for grain counting and s tatis tical analys is o f EM radioauto

graphs . Biochemical determinations of protein , DNA , amylase , per

oxidase , and o ther enzyme ac tivit ies fo llow s tandard procedures .

Cy cl ic nucleo tide level s are determined by radio— immunoas say .

Our previous studies of exocrine secretory cell s demons trated that

GERL , an acid pho sphatase containing smooth membrane sys tem located

in the inner Golgi region , is intimately involved in the formation

of s ecretory granules . The membrane of GERL is continuous with that

o f the forming granules , and acid pho sphatase reac tion product is pre

s ent in the forming granules . In contras t , secretory pro teins , as

j udged by the local ization of endogenous peroxidase , are present in

the Golgi saccules and forming granules but usually are no t present

in GERL . These cytochemical results rais e ques tions concerning the

route o f s ecre tory protein transport through the various components

o f the Golgi reg ion , the origin of GERL and i ts precise relationship

to o ther organelles , and the function of acid hydrolases in secretory

granules . The following s tudies are part of a continuing ef fort to

ob tain additional information on these and o ther ques tions relatedto the secretory proces s .

Cytochemical S tudies o f the Golgi appara tus and GERL have been pur

sued using three dif ferent sys tems . The apparent role of GERL in

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s ecretory granule formation sugges ted that changes in the s truc ture

and cytochemis try o f GERL might be expected when the rate of granule

produc tion was altered . Animals were inj ected with e ither cholinergic

(p ilocarpine ) or B— adrenergic (isopro terenol ) agonis ts to induce

granule dis charge and a subsequent resynthes is o f new granules .

Lacrimal and parot id glands from animals sacrif iced at various times

after inj ec tion are currently being examine d to determine the nature

and extent o f changes in the Golgi apparatus and GERL . A preliminary

observation made on the parotid acinar cells of young animals sugges ted

that the localization o f thiamine pyrophospha tas e ac tivity was s ig

nific antly dif ferent than that found in adul t animals . Thiamine

pyrophosphatas e and acid phosphatase localizations are being s tudied

in animals o f varying ages from 2 weeks to adul t . In the young

animals , TPPas e reac tion product is present in the trans Golgisaccules , as in the adult , but is also frequently present in s truc tures

morphologically identified as GERL and forming secretory granules .

Ac id phosphatas e is als o pres ent in GERL and forming granules . These

f indings sugges t that GERL is clos ely related to and may be derived

from the Go lgi saccules , ra ther than the endoplasmic reticulum . The

third sys tem util ized is the neurosecretory neuron of the supraop ti c

nucleus o f the hypo thalamus . These neurons produce the pep tide

hormones vas opres s in and oxc y to c in , which are synthes i zed in the cell

body , packaged into granules by the Golgi apparatus and GERL , and

transported down the axon to the neurohypophys is . The neurons can

b e s timulated experimentally by adding 22 NaCl to the animal 's drinking

water . The localization o f neurophys in , the carrier protein for the

pep tide hormones , was determined by light and elec tron micros copy

us ing immunocyto chemical procedures . Light micros cope s ec tions were

used to es tablish antibody spec if ici ty and op timal s taining condi tions .

By elec tron micros copy reac tion produc t indicating the presence o f

neurophys in was localized in the nuclear envelope , endoplasmic

reticulum , Go lg i saccules,secretory granules and lysosomes . No

label ing o f GERL was observed . Thes e results are cons is tent with our

f indings for s ecretory pro teins in o ther cells . The lack o f reac tion

produc t in GERL may in part b e due to the inab il ity o f the large

antibody molecules to penetrate the membrane of GERL .

Glycopro tein localization and transport in secretory cells is also

b eing s tudied by electron micro s cope radioautography o f cells labeled

in vivo with the glycopro tein precursor H—fucose . I t is hoped that

these s tudies will provide information on the movement o f labeled

s ecretory material through the Golgi saccules and GERL , al though the

reso lution of the radioautographic method along with o ther technical

dif ficulties may prove to be the limiting factors . Initial resul ts

on paro tid acinar cells demons trate heavy labeling o f the Golgis accules up to 40 minutes af ter inj ec tion of 3H— fucose ; GERL also

appears labeled,but to a les ser degree . Analys is o f the grain counts

,

and determination o f the relative concentration o f label in the various

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2 01 DE 000 2 8— 12 LBS

organelles have no t been completed . S imilar s tudies will be carried

out on the exorbital lacrimal acinar cells , and pos s ib ly the serous

demilune cells o f the subl ingual gland .

Prel iminary EM radioautographic results on fucose uptake by pancreat ic

is let cells conf irmed that the insulin producing B—cells are much

more heavily lab eled than o ther islet cells . The localization o f

grains af ter fine—grain development procedures sugges ts that the

fucos e is probably incorporated into a membrane glycopro tein . F urther

charact erization may prove this fucose-labeled component to be a

useful marker for B—cell plasma membranes , as well as an experimental

model to s tudy membrane b iogenes is and recycling in a secretory cell .

The morphology and function of the lysosomal sys tem of secretory

cel ls has also been s tudied . The neurosecretory neurons of the supra

op tic nucleus have been utili zed to inves t igate the relationship o f

the agranular reticulum to the lysosomal sys tem . Hors eradish per

oxidase (HRP ) , adminis tered intravenous ly , is initially seques tered

in small ves icles and cup-shaped organelles of the axon terminals ,

and then transpor ted retrogradely to the cell bo dy . In the cell body

these endocytic organelles fuse with secondary lysosomes . HRP

reaction product has never been obs erved in Golgi saccules or GERL .

The s econdary lysosomes are direc tly connected to cis terns which are

morphologically s imilar to the agranular reticulum . These cis terns

appear to provide a route whereby HRP and lysosomal cons ti tuents

may b e transported anterogradely . Furthermore , on the bas is of its

hydrolase activity , at leas t a portion o f the agranular reticulum

mus t b e cons idered as part o f the lysosomal sys tem . Ano ther animal

model used to s tudy lysosomal function is the beige mouse,a homologue

o f the human Chediae igashi syndrome . The defec t in these animals

is manifes ted by greatly enlarged lysosomes in many cel l types and

abnormal releas e o f blood cell granules . Lowered intracellular levelso f cyclic GMP has been propo sed as the underly ing cause o f this

defect . The levels o f bo th cyclic GMP and AMP were compared in 10

t issues from beige and C— 57 black mice . No cons is tent dif ferences

were no ted among the tissues . However , lowered levels of bo th c GMP

and cAMP were detected in the exorb ital lacr imal gland o f the beige

mice . Based on these resul ts , a s tudy has been initiated to correlatethe time course o f s ecretory granule dis charge from the lacrimal

acinar cells with cyclic nucleo tide levels fo llowing cholinergic

(pilo carpine ) s timulation .

In a previous s tudy we had fo llowed the dis tribution and up take of

exogenous tracers introduced via retrograde infus ion of the main

excretory duct o f the paro tid gland . This work has now been

expanded in a s tudy of the ef fects o f sialographic pro cedures on

the rat submandibular gland . The main duc t is cannulated and under

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endocytosis , (b ) change s in intercellular j unction s tructure during

s timulated secre tion , ( 9 2 dif ferences between the membranes of the

endoplasmic re ticulum ,Golgi saccules and GERL , and (d ) s ites of

c lose contact of nerves with the acinar cell p lasmalemma . Add

itional ly ,we plan to begin s tudies on the lo calization of pep tide

hormone and neurotransmitter recep tor s ites in salivary glands and

o ther tis sues , us ing EM radioautographic techniques .

Publications

Hamosh ,M . , and Hand , A . R . : Development o f secretory activity in

s erous cell s of the rat tongue . Cyto logic al dif ferentiation and

accumulat ion of lingual lipase . Dev . Biol . 65 : 100— 11 3 , 1 9 78 .

Ke lly ,R . B . , Oliver , C . , and Hand , A . R . : The ef fects o f vinblas tine

on acinar cell s of the exorbital lacrimal gland of the rat . Cell

Tiss . Re s . 1 95 : 2 2 7-2 37 , 1 9 7 8 .

Bergeron , J . J .M .

5Siks trom , R Hand , A . R . , and Posner , B . I . : Binding

and uptake o f 12 I— insulin into rat liver hepatocytes and endothelium .

An in vivo radioautographic study . J . Cell B iol . 80 : 42 7— 443 , 197 9 .

Oliver , C . , Auth , R . E and Hand ,A . R . : Format ion and fate of

ethionine-induced cytop lasmic crys talloids in rat paro tid acinar

cell s . Am . J . Anat . 155 : 185-200 , 19 79 .

Cournil , I . , Leb lond, C . P . , Pomponio , J . , Hand

,A . R .

, Sede rlof , L . ,

and Mart in , G . R . : Immunohis tochemical lo calizat ion o f proc ollagens .

I . Li ght microscop ic distribut ion of procollagen I , II I and IV anti

genic ity in the rat incisor too th by the indirect peroxidase—anti

peroxidase method . J . Histochem . Cy tochem . 2 7 : 105 9-106 9 , 1 9 7 9 .

Hand , A . R . : Synthes is o f secretory and plasma membrane glycoproteins

by s triated duct cell s o f rat salivary glands as visualized by radio

autography after 3H— fucose inj ec tion . Anat . Rec . , ( in press ) .

Hand , A . R . : Cytochemical detection of peroxisomal oxidases . J .

Histoc hem . Cy tochem . , ( in press ) .

Hand , A . R . : Salivary Glands . In Bhaskar , S . N . Orban 's OralHis tology and Embryology , 9 th Edition . S t . Louis

,C . V . Mosby Co . ,

(in press ) .

Hand , A . R . : Cytochemical dif ferentiat ion of the Golgi Apparatusfrom GERL . J . His to chem . Cy to chem . , ( in press ) .

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201 DE 0002 8-1 2 LBS

Broadwell ,R . D . , Olive r

, C . ,and Brightman

, M .W . : Neuronal transport

o f ac id hydrolases and peroxidase within the lysosomal sys tem o f

organelles : pos s ib le involvement o f a S pecialized compartment of the

agranular reticulum . J . Comp . Neurol . ( in p ress ) .

Broadwel l ,R . D .

,O liver

, C . ,and Brightman , M . W . : Local ization of

neurophysin within organelles as sociated with pro tein synthes is and

p ackaging in the hypo thalamo-neurohy pophy sic al sys tem : an immuno

cyto chemical s tudy . Pro c . Natl . Acad . S ci . U SA, (in press )

Oc tober 1 , 1 9 7 8 to Sep tember 30 , 19 79

N AM ES,LAB OR ATORY AND I N ST I T UT E AFF I L I AT I ON S , AND T I TL ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OTH ER

PROF ES S I ON AL PERSONN EL ENG AG ED ON THE PRO JECTPI Hascall , Gretchen K . Senior S taf f Fellow NIDR

OTHER : Waters , Judith F . Bio logis t

Lenk , Elaine Visit ing As sociateYoumans , Patricia A . Secre tary ( s teno )De G raff , Barbara A . Clerk— typing

Floyd , S teven W . Photographer

Laboratory o f Biolo gical S tructure

Experimental Morphology S ection

S UMM ARY OF WORK (200 word s or le s s und e rlin e ke ywo rd s )This proj ec t uses electron microscopy , his tochemis try ,

and autoradiography to

s tudy the b iosynthesis o f car tilage pro teoglycans and their relationship to

collagen in cartilage matrix . Current studies include elec tron microscopy

o f the Swarm rat chondrosarcoma and cells cultured from it,including the use

o f tracers to detect s ites of uptake in the chondro cytes , and elec tron micro

s copy o f abnormal cart ilage from mice and humans .

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Z0 1 DE 00163—03 LBS

ma litie s . My s tudies on the morpho logy of the epiphyseal growth

plate show a greatly dis torted organization o f cells in the columnar

zone,but an extracellular matrix which appears nearly normal . Bio

chemical s tudies of this cart ilage by members o f LDBA,NIDR ( Z01 DE

00 201 , 00 202 ,002 03 ) indicate de c reased

'

amounts o f pro teoglycans

and co llagen,and poss ib ly an increased water content . Further ex

p eriment s to complete this proj ect await success ful breeding to pro

duce more homozygous mutants .

S tudies o f the morphology o f abnormal human cart ilage have also

been initiated . Dr . Bronwyn Bateman of Johns Hopkins Univers ityand Childrens ' Hospital has acces s to S urgical S pecimens , and in

collaboration with Dr . George Mart in (LDBA , NIDR,ZO1 DE 00 202— 02 )

i s performing b iochemical and immunological s tudies on the samp les .

Ms . Elaine Lenk (LBS ) and I are s tudying the ultras tructure o f the

tissues , in particular searching for any changes in the appearance

and dis tribution o f the matrix pro teoglycans and collagen .

Proteoglycans probably play significant roles in controlling col

lagen fibril formation , mineral ion transport , and cartilage

mineralization . These current s tudies are cons idered significant

s tep s in a broader program aimed at elucidating mechanisms of

matrix formation , compo sition and mineralization .

Publications

Falt z ,L . L Reddi , A . H . , Has call , G . K . ,

Martin, D .M . ,

Pita,J . ,

and

Hascall , Characteris tics o f Proteoglycans Extracted from the

Swarm Rat Chondro sarcoma with As sociative Solvents . J . Bio l . Chem .

2 54 , 1 375—1380 , 1 97 9 .

o f Se cre tory Cell S tructure and F unct ion .

N AM ES , L ABOR ATORY ANO I N ST I T UT E AFF I L I AT I ON S,AND T I TL ES OF PR I NC I PAL I N V EST I G AT OR S AND ALL OTH ER

PR OF ES S I ON AL PER S ONN EL ENGAG EO ON THE PRO JECT

Ol iver , Cons tance

O THER Hand , Arthur R .

Lenk , Elaine V .

Waters , Judith F .

Worrell , Dorre t te_

F .

Weatherall , Cecil ia C .

Youmans , Patricia A .

De G raff , Barbara A .

Senior S taf f F ellow LBS NIDR

Chie f , LBSVis iting As sociateBiologis t

Biologist

Bio . Aid (Biochem . )Secretary (s teno )Clerk— typing

F loyd , S teven W .

Coriell , S tevenPho tographer

Pho tographer

J . Greenberg ,LDBA

Laboratory o f Biological S truc ture

Experimental Morphology Sect ion

NIDR ,NIH

,Bethesda , Md . 2 0014

[3 (b ) HU M AN T I S SU ES (c ) N E I TH E R

SUM MARY OF WOR K (2 00 wo rd s or le ss und e rl in e keywo rd s )

Cell d issoc iation and short term culture (up to 1

These cul tures are being used to s tudy various asp

Initial emphas is is be ing p laced on morphological ,

characterization o f the cul tured cell s . Additionally , up take of horseradish

peroxidase and internalizat ion o f I labeled cell membrane are being inve s

tigat ed ,in vivo and in v itro . The events involved in ma trix depos ition by

cul tured odontob last s,and melanos ome formation in cultured neural cres t cell

are al so b eing s tudie d .

PH5-6040

(Re v. 10-76 )

ZO1 DE 00199— 03 LB S

Proj ect Descrip tion

The primary obj ect ives o f this invest igation are to es tablish pro

c edure s for short term cul ture of isolated exocrine gland acinar

cell s and to utilize these cultures in invest igations o f events

involved in the secretory process .

Methods Employed

The isolation o f the acinar cel ls involves sequential treatment of

the glands with EDTA and co llagenase—hyaluronidase solutions . The

dis soc iated cel ls are fil tered through 500 u and 25 u Ny te x fil tersand the acinar cell s separated on a Fico ll gradient . The cell s are

cultured as suspens ion cul tures at 35°

C in a humidified atmo sphere o f

5 2 C0 2 in air in Ham's nutrient mixture F -1 2 supplemented with ant i

b io t ics and the appropriate secretagogue (exorbital lacrimal , 10‘ 6M

carbamyl cho line ; pancreas 1 0“M carbamyl choline ; paro tid 10

' 6M iso

p ro te renol ) . Pro te in synthes is was asses sed by determining the amount

o f H— leucine incorporated into TCA precipitable protein .

Membrane reut ilization was studied bo th in vivo and in vitro . Fo r

in vivo s tudies,horseradish peroxidase (HRP ) ( l mg / gm body weight )

was inj ected into the saphenous vein o f adul t , male NIH Swis s mice

or Wis tar— Furth rats . Some anima l s received s ecretagogue (i so

prote renol ,20 mg/kg body weight ; pilocarpine 4 0 mg /kg body weight )

prior to or concomitant with the HRP inj ect ion . Animals were

sacrificed by vascular perfus ion o f fixative , at varying times following HRP adminis tration . The glands were excised , and sections

incubated for peroxidase activity , o r for demonstration o f various

marker enzymes , i . e . thiamine pyropho sphatase for Golgi sac cules

and trime taphosphatase and acid phosphatase for lyso somes . F or

in vitro s tudies o f membrane reut ili zat ion isolated acini were

prepared by collagenase—hyaluronidase digest ion , filtered through

Ny tex fil ters and separated on a ficoll gradient . Suspensions o f

acini were iodinated enzymatically utiliz ing a lac toperoxidaseglucose oxidase system . Internal izat ion and fate o f 12 5 1 labeledmembrane was followed by light and elec tron microscope radioautography .

Other acini were cultured in medium containing 12 HRP as a solublephase ma rker .

Major F indings

Di ssociation o f rat exorbital lacrimal glands by alternate inc ub

ations in EDTA and collagenase-hyaluronidase solut ions yielded a

cell suspension which was approximately 902 acinar cel ls and had

a viability o f about 802 as determined by trypan blue exclusion .

Af ter isolation the s ingle acinar cell s rapidly reaggregated to

4 — 1 2 8

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201 DE 00199—03 LBS

from 1 day old Sprague—Dawley rats , are culture d for up to 7 days .

The too th buds are grown on nuc leopore . filte rs in organ culture dishes

with BG Jb medium supplemented with_ penic illin , vitamin C and horse

serum . iMatrix depo sit ion was apparent by- 2 days-in vitro . The role

o f the Golgi apparatus and GERL in the syn thesis and packaging of'

matrix pro teins is currently under investigation . The effect o f'

m icrotubular inhibitors on this sys tem will be s tudied .

fiMany o f the steps involved in melanosome formation in pigmented cells

parallel tho se o f secretory granule formation . Unders tanding events

controlling melano some produc tion may yield new ins ights into the

proces s of secretory granule production . Cultured neural cres t cell s

grown in the pre sence of fetal bovine serum (F ES ) rapidly dif f e rentiat

into melanocytes , while tho se grown wi th horse serum become neuron—lik

The presence o f DMSO in the medium wi th F ES reversibly inhibi ts pig

mentat ion in these cell s , although the cell s do contain preme lano some s

Cells grown in F BS with and without DMSO will be examined at intervals

after exposure to and removal of the drug . The effect s of the various

treatments on the formation of melanosomes will be investigated us ing

electron microscopy and enzyme cytochemical localization o f tyrosinase

(a marker enzyme for melanin synthes is ) . These s tudies are being

carried out in collaboration with Dr . Judy Greenberg,LDBA , NIDR .

Sigpific anc e to Dental Research

In vitro s tudies o f exo crine gland acinar cell s should provide a

greater unders tanding o f bo th the controlling mechanisms and

synthetic pathways involved in their secretory pro ces ses . Salivary

gland secret ions are absolutely essential for maintainance of the

health o f the oral cavity . Therefore , knowledge of the normal

s ecretory proces s is critical to our unders tanding of many patho

logical condit ions which affect dental health .

Proposed Course of Proj ec t

Cultured exocrine cell s will be further charac terized with respec t

to their cellular morphology and cytochemis try,pro tein synthet ic

capacity , and response to secretagogues . Additional s tudies will

examine the dis tribut ion o f membrane recep tors, membrane reutili

z a tion , and the ro le o f microtubules and micro filaments in thes ecretory process .

Publications

Ol iver , C . , 1 9 7 9 . Isolation and maintenance o f dif ferent iated

exocrine gland acinar ce ll s in vitro . In V itro (in press ) .

2 01 DE 00199— 0 3 LBS

Oliver , C . , 1 9 7 9 . Cytochemical lo cal ization o f ac id phosphatase

and trime t apho sphatase activit ies in exocrine acinar cell s . J .

Histoc hem . Cy to c hem . (in pres s ) .

October 1 , 19 78 to Sep tember 30 , 1 9 79

N AM ES,LA BOR ATORY ANO I N ST I T UT E AFF I L I AT I ON S , AND T I TL ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OTH E R

PROF E S S I ON AL P ERSON N EL ENG AG ED ON THE PRO J ECT

F owler ,Bruce 0 . Research Chemis t NIDR

OTHER : Youmans , Patricia A . Secre tary (s teno )De G raff

, Barb ara A . Clerk- typing

None

Molecular Structure Section

p§ (b) HUM AN T I SSU E S [j (c ) N E I TH ER

SUMM AR Y or WOR K (2 00 wo rd s or less und e rlin e keyword s )

inorganic phase in teeth and bones . Infrared and Raman spec troscopy as well as

chemical me thods are employed in these studies . Methods are devised for the

preparation o f synthetic calcium apatites having controlled phys ical properties

( crystal size and perfection ) and chemical consti tuents hydroxide , fluori e ,

chloride ,carbonate

,water and acid pho sphate ) . The vibrational spectra of thes

apatites and relate d compounds are assigned and characterized . Iso topically eu

riche d apatite analogs are prepared to facil itate S pectral assignments . The

S pe ctros cop ic assignments and supp lemental spectral data ( temperature dependency

and polarization ) are then utilized to es tablish compositional and s tructural

de tails o f the apatites in ques tion which include : the type and geometry of cons tituent ions ; the site or number of sites occupied by the ions ; orientation of

ions ; chemical bonding and interact ions of ions ; and semi—quantitative e s timatio so f the const ituents present . The results for these controlled apatite systemsare then re lated to the inorganic phase in calcified tis sues

(Re v. 10-76 )

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2 01 DE 000 1 2— 17 LB S

compos ition and s tructure . Dr . J . L . Meyer o f thi s Laboratory has

developed a method for chemically determining hydroxide ion contents

in apatites and has prepared a series o f apat ites containing vary

ing hydroxide content s . These Meyer apatites had chemically de

terminad hydroxide content s o f 0 to 6 02 o f the theoretical based

on 10H/ 3PO4 as 1002 hydroxylated ,Ca/ P mo lar rat ios o f to

HPO4contents o f 1 to 1 92 o f the total P0 4 ,

C0 3 contents o f

to wt. 2 and H20 contents o f 9 to 1 2 wt . 2 . These nine teen

Meyer apat ites o f known hydroxide content s along with five high

puri ty hydroxyapati te s tandards (Fowler ) , containing about 95 2 o f

the theoretical hydroxide by chemical analyses , were used in this

s tudy.

The bas ic method used for infrared and Raman spectros cop ic

quantitation was as follows : ( 1 ) OH to P0 4 band area rat io s were

es tablished for the 952 hydroxide containing hydroxyapat ite s tan

dards, ( 2 ) a linear decrease in OH/ P0 4 ratios to zero as a func t ion

o f decreas ing hydroxide content was as sumed , and ( 3 ) hydroxide

content s o f the Meyer apat ites were computed from their respec tive

0H / PO4 rat ios on the bas is o f the s tandards . The hydroxide

s tretching band was used for bo th infrared and Raman measurements .

The v2 P0 4 band was used for Raman measurements ; the v4 P04 band ,

correc ted for the hydroxide librational band interference , was

used for infrared measurement s .

Infrared Measurement s : Important samp le preparation facto rs and

variab les to cons ider in obtaining infrared spec tra o f powders in

pres sed alkal i halide pelle ts or mulls for quantitative measure

ments are : ( 1 ) sample part ic le si ze cons is tency , ( 2 ) samp le part icleshape

, (3 ) homogeneous dispers ion o f sample part icles in pellet ,

(4 ) Chris t iansen ef fect , (5 ) pres sure induced sample changes

and sample interac t ions with pellet or mull material , and (6 ) di f

fe renc e s between index o f re frac tion o f sample and pellet or mull

material . All samples were reduced to a cons is tent small part ic le

s ize,about 2 to 4 microns , by standardi zed gent le grinding in the

dry S tate ; adverse ef fect s caused by the Chris t iansen e ffec t , band

asymmetry , were reduced at this part ic le s ize level but no t comp le telyin the hydroxide s tretching region . Effect s o f fac tor (5 ) were found

to be minimal by comparison with spec tra ob tained from a di f ferent

sampling technique , vasel ine mull s . Adverse e f fec ts o f fac to r ( 3 )and , in part , factor ( 1 ) were reduced by the OH / P04 band area ratio

method employed ; poor sample dispers ion , resul ting in an apparent

lower samp le concentrat ion , was es sential ly compensated for by

using the ratio method . In addition,the OH/ P0 4 ratio method is

es sential ly independent o f dif ferences in water and carb onate

contents o f the apat ites compared whereas with infrared measurement s

based on sample concentration , sample weight correc tions for ex

traneous cons t ituents are neces sary . The indexes o f re fract ion o f

po tas sium bromide , vaseline and the apat ites s tudied are about

and to respec tively . S ligh t asymmetry o f the

hydroxide s tretching band , caus ing quanti tat ion uncertainty,was

2 0 1 DE 00 01 2— 1 7 LB S

ob served in vasel ine mull spec tra o f the hydroxyapat it e s tandards .

This band asymmetry was minimized by us ing mul l ing fluids with

indexes o f refrac t ion o f about to However , addit ionalrequirement s fo r an accep table mulling fluid are high visco s ity ,

lack o f interfering bands in regions o f interes t and inertnes s .

A prel iminary search did no t reveal a fluid ful fi ll ing all the se

requirements,and vaseline , overall ,

was more sui table . The

po tas s ium bromide index o f re fraction is lower than ideal , but it

was used because o f i ts sui table charac teris t ic s as a pel le t

material . Crys tal shape e ffec ts on infrared band areas and inten

s it ies were no t evaluated in thi s s tudy . The reproducib ilit ies

o f hydroxide content s determined from duplicate po tas sium bromide

pelle ts and dup lica te vasel ine mulls were 5 2 relat ive or l es s .

The hydroxide content s o f the twenty— one apat i te samp les derived

from infrared OH/ P0 4 band area measurement s ob tained from potas s ium

bromide pelle t and vasel ine mull spect ra agreed within —11 to

18 , maximum ; -7 to +9 ,average ) absolute percentage uni ts from the

chemically de termined hydroxide con tent s . The hydroxide contents

derived from OH / PO4 band intens i ty rat io s deviated more - 31 to

+6 ,maximum ; — 12 to +4 , average ) from the chemical values than

those derived from area ratio s . Band intens i ty measurement s do

no t account for broadening o f spec tral bands which can be caused

by numerous fac tors in thes e apati tes whereas band b roadening i s

accounted for in band area measurements , and c lo ser agreement with

the chemical values , as expec ted ,was observed . The OH/ PO4 band

area rat io s for the mono cl inic and hexagonal hydroxyapat ite s tan

dard s agree within about 10 2 ; hence , monoc linic hydroxyapa ti te

can be us ed as an hydroxide s tandard for the hexagonal apat i tes

us ing infrared OH /PO Q area ratio s . Final measurements for di f fer

enc e s in OH/ POA band intens ity rat ios be tween monoclinic and

hexagonal hydroxyapat ite are incomple te . These resul t s show that

the infrared me thod as des cribed herein is capable o f semi— quan

tita tive determinat ion o f hydroxide content s in apat ite o f com

po s itions indica ted . Spec t ro scop ic hydroxide quanti tation in

apatite s con ta ining higher carbonate contents , addi t ional cat ions ,

and espec ially fluoride and chlor ide anions , which can cause hydroxide

b and shi f t s , area and intens ity changes , will require addi tional

evaluation .

Raman Measuremen ts : The main experimental prob lems and fac tors to

overcome or evaluate in ob taining Raman spec tra for quantitative

measurements are : ( 1 ) s ample changes , decompo s it ion and band

broadening c auSed by laser beam heating , ( 2 ) high spec t ral back

ground aris ing from fluores cence , ( 3 ) crys tal o rientation e f fect s

on hand areas and intens it ies , and (4 ) pos s ib le crys tal s ize and

shape e f fec ts on hand areas and intensi ties . To reduce lo cal ized

laser b eam heating e f fec t s and to increase Raman s ignal,the

apat ite powders were pre s sed under high pres sure into cup s in

aluminum disks . The compac ted apat it e part ic les and their int imate

4 - 1 3 5

ZO1 DE 0001 2-1 7 LB S

contac t with the aluminum facilitated heat t rans fer and reduced

localized heating . Laser light power was adj us ted to levels (N

15 0 mw at 4880 A) that caused no visually de tec tab le s amp le changes

or discoloration,and S pectra at dif ferent expo sure t imes o f laser

irradiance were compared for thermal ly induced changes . Spectra

reco rded after 2 and 20 hours laser irradiance at 150 mw were

es sent ially the same ; however,ins tantaneous samp le changes were

no t entirely excluded . Background fluores cence was reduced to

ac cep table level s by aging the samples for 4 to 16 hours in the

laser beam . Band intens ity variations aris ing from crys tal orien

ration was minimal because o f their small crys tal si ze and their

random orientat ion in the compres sed powders . Als o , ins trumentalpolarizat ion checks on the larger s ize (N 15 microns ) hydroxyapatite

s tandards did no t show crys tal o rientat ion e f fec ts . Po s s ib lespec tral e ffec ts aris ing from crys tal s iz e and shape have no t been

fully evaluated . Hydroxide content s o f twenty— four apatites were

ob tained from OH/ PO4 band area and band intens ity rat io s . The re

producibility o f duplicate runs was ab out 5 2 relat ive . A cor

relation b etween chemically de termined and Raman spec tros copicallydeduced hydroxide content s exi s ts ; however

,deviat ions from the

chemical values were , overall , greater than those determined by

infrared analyse s . The hydroxyapat ite s tandards , containing about

9 52 o f the theoretical hydroxide , showed Raman spec tros copically

deduced hydroxide contents ranging from 80 to 1 2 02 by OH/ P04 b and

area ratio measurement s . Ef fo rts to reduce and / or unders tand this

deviation are underway . At tempt s to evaluate the ef fects o f particle

s ize from spectra o f 1 0 micron and larger s ize hydroxyapat i te

crys tal s ground to smaller S ize were no t ent irely succes s ful . The

ground smaller s ize crys tal s had a higher inter fering sp ec tralbackground ari s ing from increased Rayleigh scattering

,and o ther

probable sources including impuri ties and numerous nas cent crys talsurfaces introduced by grinding . Monocl inic hydroxyapat i te was

not found to be a sui table Raman s tandard for hydroxide measure

ments in hexagonal apat ites based on OH / PO4 band intens ity ratio s .

The Raman hydroxide s tretching band intens ity o f mono clinic hydroxy

apatite is about twice that o f the hexagonal form whereas the

hydroxide band areas for bo th forms agree within about 202 . The

markedly greater Raman hydroxide s tre t ching band intens i ty o f mono

clinic hydroxyapat ite probably arises from its ordered hydroxide

ion arrangements ; hydroxide ions are disordered in the hexagonalfo rm . Po s s ible quantitat ion o f monoc linic and hexagonal domains

in hy droxapatite crys tals may be po ss ible based on the no rmalized

intens ity , width and area o f the Raman hydroxide st retching band .

Sigpific anc e to Dental Research

Characteri zat ion and ass ignment o f the infrared and Raman bands o f

apat ite containing biologically relevant ions and those o f related

calc 1 um pho sphates are es sent i al in es tabli shing corollary s tructural

lI - l S F

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October 1 ,1 9 78 to S ep tember 30 , 1 9 79

N AM ES,LABOR ATORY AN D I N ST I T UT E AFF I L I AT I ON S , ANO T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H E R

PROF E S S I ON AL PER SONN EL ENG AG EO ON THE PRO J ECT

Termine,John D . Research Chemis t NIDR

Other : Miyamoto , Maureen S .

Conn,Kathleen M .

De j t e r ,S tephen W Jr .

Belcourt,Alain B .

Lyaruu ,Donac ian M .

Lenk,Elaine V .

Arnesen , S tacey

Y anagisawa , Takaaki

Youmans,Patricia A .

De G raff ,Barbara A .

Dr . E . D . Eanes,LBS , NIDR ; ( 2 ) Dr . R . A . Gelman ,

Laboratory o f B iolo gical S truc ture

Mo lecular S tructure Sec tion

NIDR ,NIH , Bethesda , Maryland 2 0 205

[3 (b ) H UM AN T I S S U E S g] (c ) N E I T H E R

SUMM AR Y OF WOR K (200 word s or le ss und e rlin e keyword s )

t is sue pro teins are being s tudied by several techniques . Dentin , bone and

s tructural influences on act ive mineralization in b iolo gical syst ems . Spe cial

hard tissue matrix Studies .

S taf f Fel low

Research Chemis t

B io lo gical Aid (Biochem . )Vi sit ing As so c iateVis iting F ellow

Vis i ting Ass ociateSummer AidVis i ting Fel low

Secretary ( s teno )Clerk— typingPho to her

2 01 DE 00074— 06 LBS

Proj ec t Des crip t ion

The long range go al s o f thi s re search proj ec t are two fold : ( l ) to

s tudy the s t ructural and chemical propert ies o f the inorganic and

pro te in cons t ituents o f bones and teeth ; and ( 2 ) to examine crit icallythe changes tha t o ccur in thes e components during development , growth

and dis ease s ta tes . Many o f our procedure s employ bo th model and in

vi tro sys tems as well as iso la ted b iological enti ties . I t is our

hope that this mul t iface ted approach will provide a more thorough

unders tanding o f the b iolo gical mineralizat ion pro ces s .

In the pas t two f iscal years,a sequential dis so ciative extrac t ion

s cheme was devised to di f ferential ly iso late fe tal too th matrix

p ro teins . In this scheme , great care was taken to avoid p ro teolys isat all phases o f the overall extrac t ion proce s s in order to ob tain

the fe tal dentin and enamel matrix p ro teins in as nat ive and non

degraded a form as pos s ible . Further , the extrac t ion scheme was

speci fically des igned to enrich tho se t is sue pro te ins mo s t clo sely

as so cia ted with enamel and/ or dentin apatite crys tal li tes into a

s eparate pro te in frac t ion . The detail s o f these extrac tion pro

c edure s and the elec trophore ti c pro cedures devised to fo llow them

were des cribed in prior yearly repor ts . These ef fo rts had allowedus to isolate three separate pro tein frac tions from fe tal bovine

tee th : (1 ) the ame logenins , a ser ies o f pro teins present in fe talbut no t fully formed enamel that are very rich in proline ( 2 5

glutamic acid (1 7 leucine (10- 12 2 ) and his t idine (6 ( 2 )the apat ite— as sociated fe tal ename lins , a series o f acidic enamelpro teins , rich in aspar tic and glutamic acid ( 2 7 prol ine

(14 glycine (10—12 2 ) and s e rine / thre onine ( 13 and ( 3 )the dent in phos phopro teins

,extremely rich in asparti c acid ( 30— 402 )

and se rine / phosphos e rine ( 30

The fe tal ename l ins have an overal l amino acid compo s it ion which

sugge st s that they are the mos t probable pre curs ors o f tho se pro teins

that survive amelo gene s i s to pers is t in the final , fully erup ted

too th enamel matrix . This sugges t ion is supported by two addit ional

f indings ob tained this year . Firs t , the fe tal ename lins have an

extremely high af f inity fo r apat ite crys tals and are selec tivelyadsorb ed by synthet ic apat ite from mixtures o f to tal fe tal enamelpro teins ( ename lins plus ame logenins ) even in 4 molar guanidine

,an

extremely po tent dis so ciative agent . S econd , a higher propor tion

o f fetal ename lins (relative to ame logenins ) can be extrac ted from

fe tal cal f teeth relat ively late in development ( 7—8 months in utero )

than from somewhat younger ones (5-6 months in utero ) . Furthermore,

care ful analys is o f the to tal quant i t ies o f e name lins extrac ted from

develop ing bovine tee th o f di f feren t fe tal ages sugge s ts that these

pro teins are synthes ized at al l s tages o f enamel development .

2 01 DE 000 74-07 LB S

Las t year,we reported that the fetal ename lins contained three

maj or species on SDS—urea at molecular weight s o f ca .

and and that these three spec ies showed

ideality vs . known m .w . s tandards in Ferguson plo ts from SD S—urea

gels o f dif fering polyacrylamide concentrations : We have now ex

tended these elec tropho retic analyses to two o ther SDS

sys tems no t containing urea . Ferguson ideal ity was achieved vs .

known markers for the three maj or fetal enamelins in each o f these

additional gel sys tems , thus indicating that the mo lecular weights

determined for them by the SDS-electrophoret ic method were correct .

These three pro teins,which migrate to gether on ion—exchange chroma

to graphy ,s tain po s itively on po lyacrylamide gel s bo th for organic

phosphate and/ o r s ialic acid ( S tains All ) and for carbohydrate

(PAS s tain ) . On gel fil tra t ion chroma tography in 4 molar guanidine

on either Sepharo se CL—6B or S ephacryl S— 2 00 ,the fetal ename lins

also contained three maj or components at molecular weights (vs .

known m .w . s tandards ) o f ca . and and re

lative ly small amounts o f lower m .w . components at about

and Dal tons . Thes e lower m . w . fe tal ename lin comr

ponent s had a combined amino acid compo s ition almo s t identical to

that ob tained for the higher m . w . fe tal ename lin triple t , thus

sugges ting that they may b e pro teolytic breakdown produc ts o f the

larger species . Finally , we have determined chemically that the

high molecular weight fetal enamel in trip le t p ro teins contain

approximately organic pho sphorus , 4— 6 2 s ialic acid , 4

—52

hexosamine (with a gluc osamine / galac tosamine rat io o f and

ca . 202 to tal carbohydrate (anthrone me thod ) . Thus , these high

molecular weight , acidic fetal enamel pro teins,des cribed by us

for the f irs t time , are pho sphorylated,s ialo— glycopro teins .

As mentioned above , the ame lo genins are only found in develop ingenamel , being ab sent from fully erup ted tee th . Las t year , we

repor ted that the bovine fe tal ame logenins ranged in mole cularweight from on SDS urea and gave linearbut non— ideal Ferguson plo ts vs . known m .w . s tandards . We have now

extended these elec trophore tic analyses and can conf irm that these

proline—rich fe tal enamel pro teins show non— ideal SDS el e c tropho

rac ic behavior regardles s o f the gel sys tem utilized . When urea

is omit ted from the SDS polyacrylamide gels,the fe tal bovine

amelogenins appear lower in molecular weight,ranging from ca .

Dal tons . Thus , it is impo s s ible to as certain

correc t molecular weights for this par ticular se t o f fetal enamelproteins by electrophore tic methods . This is probably related to

conformational and/ or SDS—b inding anomal ies asso cia ted with theirunusually high ( 25— 302 ) prol ine contents .

As previously found by o thers , we ob served that the ame logenins

decrease in abso lute amounts isolated from tee th o f increas ing

degree o f mineral ization and pro gres s ing enamel development.

We

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ZO1 DE 000 74— 07 LB S

gel s and gel fil tration chromato graphy under di s sociat ive conditions

is ca . I t contains 6 — 72 organic pho sphorus , 12 each o f

s ial ic acid and hexosamine (with a gluc o samine / galac tosamine rat io

o f and ca . 5— 62 to tal carbohydrate (anthrone method ) . I t s

mo st abundant amino acids were serine / phospho se rine aspartic

acid lys ine glyc ine and glutamic acid

This preparation is much higher in molecular weight and more abun

dant in non— s e rine / aspartic acid component s than any o ther hereto

fore iso lated . Preliminary work with rodent dent in pho sphopro teins

(rat incisor,courtesy o f Dr . William T . Butler , Univers ity o f

Alabama in Birmingham,and hams ter molar ) isol ated us ing our

di ssoc iative,sequential extraction s cheme indicates that the

apparently high molecular weight we ob served for the f etal cal f

dentin pho sphop ro tein resul t s,at leas t in part , from spec ies

dif ferences . Our prel iminary data on the rodent dent in pho spho

pro tein indicates a mol ecular weight o f only for

this sp ecies .

In prior years,we had repor ted that dentin pho sphopro teins inhib i t

apatite formation from an amorphous cal cium pho sphate p recursor

in synthet ic extracellular fluids . We now report that this in

hibition ac t ivity is markedly af fected by pretreatment o f the phos

phopro te in with ionic calc ium and / or collagen fibrils . S ignificantly

diminished inhib ition o f de novo apatite formation was ob served

after prior expo sure o f a given phosphopro tein prep arat ion (egg

yolk pho svitin,a model pho sphopro te in ut il ized to set boundary

conditions for these experiment s , fetal cal f dent in phosphopro tein

and rat incisor dentin pho sphopro tein ) to ( 1 ) either ionic calcium

or recons tituted rat tail tendon co llagen f ib rils alone and ( 2 )combinat ions o f these two interfering agent s . In all case s , the

combination experiment yielded a grea ter diminution o f inhib i tion

activity than whe n either inter fering agent was used individually .

Inasmuch as the bulk o f dentin mineral izat ion may wel l invo lve

apatite format ion from pre— exis ting crys tal seeds , we have extended

these observations o f pho sphopro tein ef fec ts on apat ite formation

using seeded crys tal growth sys tems in a collaboration with Dr .

E . D . Eanes o f this Laborato ry . All three o f the above phospho

pro tein preparat ions provided two s tages o f inhib it ion o f apatite

crys tal growth/ rep lic a tion in seeded , metas table synthet ic so lutions .

Pretreatment o f the crys tal seeds with pho sphopro tein induced an

init ial lag phase in the crys tal growth process,probab ly as so ciated

with seed sur face phenomena , and then prolonged the sub sequent re

action period where oc tac alc ium phosphate is the predominant new

mineral phase prior to it s eventual convers ion to the final,induced

apatite produc t . A comb ination o f seed pretreatment with pho spho

protein to gether with direc t addit ion o f solub le phosphopro tein ( 17

ug/ml ) to the metastable reac tion solution completely b lo cked the

crys tal growth pro ces s at the initial lag phase s tep . In contras t

U - l A?

20 1 DE 0o0 74 w 0 7 LBS

to the re sul t s ob tained in our de novo apatite formation sys tem ,

recons ti tuted col l agen f ibril s had no ef fect on pho sphopro tein

inhibi tion o f apat ite cry s t al growth in seeded sy s t ems . On the

o ther hand,however , pretreatment o f the pho sphop roteins wi th

calc ium ions ( f inal Ca+2 concentration , ca . 2 0 uM ) did diminish

their inhib ition ac tivity toward seeded apat it e crys tal growth .

In a companion s tudy conduc ted in col laborat ion with Dr . R . A .

Ge lman o f the Laboratory o f Bio chemis try ,NIDR

, we examined the

e f fec t o f the se three pho sphopro tein preparat ions on col lagenf ibrillo gene si s in vitro . In each case , the addi tion o f s olub lephosphop ro tein inhib ited co llagen fib ril fo rmation to a s igni f icant

degre e . Once formed,however , the collagen f ibrils were identical

t o contro l s fo rmed in the ab sence o f pho sphopro te in , when viewed

in the e lec tron micro scope . In contras t to the resul t s on apati te

f ormation,p re treatment o f the interfering pho sphopro t eins with

ionic calc ium ( f inal Ca'I'2 concentrat ion

,ca . 2 0 pH ) g re atly in

creased their inhibitory ac tivity toward s co llagen fib rillo genes is .

When all these in vitro resul t s on pho sphopro tein ef fe c ts on apati te

f ormation and col lagen fibrillogene s is are taken to ge ther,a s t rong

p ic ture develop s for a po tent ial role o f the dent in pho sphopro tein

as a princ ipal regulatory age nt in bo th dentinogenes is and dentin

mineralization . Moreover,the degree o f ac tivity fo r the dentin

pho sphopro te in in these two proces ses appears to be capab le o f

modulation by calcium ions . Thus , the impo rtance o f this unusualphosphoprote in to the format ion and s t ructural integr ity o f dent in

t issue is s t rongly sugges ted from these expe riments .

Cons iderable e f for t wil l be spent in the future to con tinue these

b io chemical and b iophysical s tudie s o f too th matrix proteins . We

have made s ignif icant progress to date in b io chemically charac ter

i z ing the fetal cal f e namel pro teins , but need to puri fy the higher

molecular we ight fetal ename lin and amelo genin components further

to be t ter unders tand their s truc ture and funct ion . Careful com

p arisons at the pep tide level be tween high and low molecular weight

c omponents within each clas s S hould yield c lue s as to the ir eventual

fate in the normal enamel matura t ion pro ces s . Once these detailed

molecular inves tigations have reached this s tage o f development ,

at tent ion can be paid to the s truc tural and biophys ical p rop er ties o f

the fe tal enamel pro t eins ,e spe c ially with re gard to how they may

a f fe c t the enamel mineraliz at ion proces s . S imilarly , once a high

enough de gree of pro te in purity has been at tained , immunolo gicalmethod s c an be employed to augment these ef for t s . We have ini tiated

p rel iminary biochemical s tudies o f the human adul t enamel p ro teins

in order to find out j us t which mol e cular spe c ies survive the po s t

e rup t ive ename l maturat ion proces s and to be t ter unders tand their

s truc ture and func tion . These e f fo r t s will be complementary to

our ongoing s tudie s o f the fe tal enamel p ro teins and should enhance

our overall p ro gram in enamel pro t ein chemis t ry . At the o ther end

2 01 DE 00 74—07 LB S

o f the developmental s cale , we have initiated prel iminary s tudies

o f neonatal hams ter molars wi th an eye toward inves tigating the

b iosynthesis o f enamel matr ix pro teins in vi tro . We have found so

far that there are many s imilarities between the d if f erent enamel

pro tein frac tions o f the 2-6 day old unerup ted hams ter and fetal

calf molar s . Fur ther,following techniques developed by Dr .

Ly aruu in Ams terdam,we have es tab l ished tha t hams ter molar too th

germs can be placed in shor t term ( 2-4 day ) organ culture bo th

prior and sub sequent to the onset of normal enamel matrix produc

tion and mineralization . Thes e s tud ies will be expanded this

coming year to inves tigate the b iogenes is and fate o f the dif f er ent

developing enamel matr ix frac tions . Finally , we will continue our

s tudies,o f the dentin phosphopro teins by fo cus ing on bo th their

intr ins ic b iochemical and b iophys ical proper ties and their precis e

mode o f ac tion in dentinogenes is and minerali za tion .

Skeletal and so f t tis sue calcif ica tion dis eas es cut across a wide

swath o f med ical and dental prob lems . Or thoped ic d is eases , ar thr i tis ,

atheros clero s is,cer tain renal and endocr ine disorders , for example ,

all involve the s truc ture and metabo lism o f the skeleton . S tillfur ther

,is the need to unders tand thoroughly bo th the development

and s tructure of minerali zed tis sues bas ic to the caries , perio

dontal dis ease and cranio fac ial anomalies res earch obj ectives o f

the NIDR . The bones and teeth ar e compo s i te tissues cons is ting of

c ellular ,collagenous

,glycopro t ein , phosphopro tein and inor ganic

components . As a consequence , their phys io lo gy and pathology are

cons iderably more complicated than those o f o ther t is sues in the

body . Th is research program is des igned to s tudy in detail the

rel ationships that exis t between the various cons ti tuents of hard

tissues bo th from s truc tural and func tional po ints o f view .

Pub lications

Edi tor : Nylen , M . U . and Termine , J . D . : Pro ceedings , Third Inter

na tional_

Symposium on Too th Enamel . Washing ton,IADR/AADR , J .

Dent . Res . 58 , Sp ec . 1 8 8 . B , 1 9 79 , 358 pp .

Termine , J . D Torchia , D . A . and Conn , K . M . : Enamel Matrix

S tructural Prot eins . In : Nylen , M . U . and Termine, J . D .

Washington ,IADR/AADR , J . Dent . Res . 58 , Spec . 1 8 3 . B

,19 79 ,

pp 7 7 3— 7 80 .

Pe ckauskas , R . A Termine , J . D . , and Pullman , I . : ESR s tudies on

too th phosphopro teins us ing manganes e ions as prob es . Arch .

Bio chem . Biophy s . 193 : 186-190 , 1 97 9 .

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2 01 DE 0074— 07 LBS

o f the developmental s cale , we have initiated prel iminary s tud ies

o f neonatal hams ter molars with an eye toward inves tigating the

b iosynthesis o f enamel matrix pro teins in vi tro . We have found so

far that there are many similari ties between the d iff er ent enamel

pro tein frac tions o f the 2-6 day old unerup ted hams ter and f etal

calf molars . Fur ther , following techniques developed by Dr .

Lyaruu in Ams terdam , we have es tabl ished tha t hams ter molar too th

germs can be placed in shor t term ( 2— 4 day ) organ cul ture bo th

prior and subsequent to the ons et of normal enamel matrix produc

tion and mineralization . Thes e s tud ies will be expanded this

coming year to inves tigate the b iogenes is and fate o f the dif f er ent

developing enamel matr ix frac tions . Finally , we will continue our

s tudies,of the dentin phosphopro teins by fo cus ing on bo th their

intr ins ic b io chemical and b iophys ical proper ties and their precis e

mode o f ac tion in dentinogenes is and minerali za tion .

Skeletal and so f t tis sue calcif ication dis eas es cut acro ss a wide

swath o f med ical and dental probl ems . Or thoped ic dis eases , ar thr i tis ,

atheros cleros is , cer tain renal and endocr ine dis orders , for example ,

all involve the s truc ture and metabol ism o f the skeleton . S tillfur ther , is the need to und ers tand thoroughly bo th the development

and s truc ture of minerali zed t issues bas ic to the caries , perio

dontal dis eas e and cranio facial anomal ies res earch obj ectives o f

the NIDR . The bones and teeth ar e compos i te tissues cons is ting of

c ellular , collagenous , glycopro tein , phosphopro tein and inorganic

components . As a consequence , their phys io logy and pathology are

cons iderab ly mor e compl icated than those o f o ther t is sues in the

body . This research program is des igned to s tudy in de tail the

rel ationships that exis t between the various cons ti tuents of hard

tis sues bo th from s truc tural and func tional po ints o f view .

Publications

Ed itor : Nylen ,M . U . and Termine , J . D . : Pro ceedings ,

Third Inter

national Sympos ium ou Too th Enamel . Washing ton,IADR/AADR ,

J .

Dent . Res . 58 , Sp ec . 1 3 3 . B, 1 9 7 9 , 358 pp .

Termine , J . D . , Torchia , D . A . and Conn,K . M . : Enamel Matrix

S tructural Proteins . In : Nylen , M . U . and Termine, J . D .

Pro ceedings , Third International Sympos ium on Too th Enamel .

Washing ton ,IADR/AADR , J . Dent . Res . 58 ,

Spec . 1 8 3 . B, 19 79 ,

pp 7 7 3— 7 80 .

Pe ckauskas , R .A. , Termine , J . D . , and Pullman,I . : ESR s tudies on

too th phosphopro teins us ing manganes e ions as prob es . Arch .

Bio chem . BiO phy s . 19 3 : 186—190 , 19 7 9 .

l} _ l ll Ll

O ctober 1 , 1 9 78 to Sep tember 30 , 19 7 9

N AM ES,L A B OR A TO RY ANO I N ST I T U T E AF F I L I AT I ON S

,A N D T I T L E S OF PR I NC I PAL I NV E ST I G AT OR S AND ALL OTH E R

PR OF E S S I ON AL P ER S ON N EL ENGAG EO ON THE PRO J ECT

Eanes,Edward D . Chief , Molecular S truc ture Sec t ion

OTHERS : Martin,Garland N . Jr . Re search Chemis t

Hailer,Ar thur Chemis t

Rat tner,S teven L . Chemis t

Youmans,Patri cia A . Secre tary ( s teno )

De G raf f ,Barbara A . Clerk-typ ing

Floyd,S teven W . Pho tographer

None

Laboratory o f Bio lo gical S truc ture

Molecular S truc ture Sec tion

(3 (b ) H UM AN T I S S U E S E! (c ) N E I T H E R

SUMM AR Y OF WOR K (200 wo rd s or le ss un d e rlin e keywo rd s )The fo rmat ion and maturation o f calc ium phosphate s prepared from phys io lo gical

l ike s olutions is being s tudied with a variety o f ul tras truc tural and phys icalchemical techniques such as elec tron micro scopy , x— ray di f frac t ion , B-E—T

surface area methods,and s tandard analyt ical chemis try p rocedures . Top ics

o f current interes t include (1 ) the e f fec t o f ionic f luoride ,ma gpe sium , and

carbonate on phys ical p ropert ies o f crys tal line apat ite such as crys tallinity ,

s peci f ic sur face area,and part icle morpho lo gy , and ( 2 ) the e f fec t o f thes e ion

on the formation,growth

,and s tab ility o f precursor s to the apat ite phase .

201 DE 00088— 06 LB S

Proj ect Descrip t ion

Ob j ect ives

The purpo se o f this proj ec t is to s tudy bo th the preparat ion from

solution and the phys ico— chemic al / ultras truc tural propert ies o f

calc ium phosphate sal ts , with par ticular emphas is on the formation

and ma turation o f apat ite from physiolo gical-like solutions . The

principal obj ect ive is to acquire a more complete unders tanding o f

biomine raliz ation pro cess es , with special emphasis on the physicalchemical aspec ts o f mineral depo s it ion in vertebrate hard tis sue .

During the year covered by this report , the principal undertakings

were : (1 ) to continue s tudies on the e ffec t o f ionic fluoride and

magnes ium on the role precursor phases play in apat ite fo rmation ,

and (2 ) to init iate s tudies on the e f fect s o f fluoride , magnes ium ,

and carbonate on the physical texture o f apat i te s prepared in the

presence o f these ions . In the fo rmer undertaking , spec ial at tent ion

was directed to the formation o f non-apatitic cal cium phosphates on

the sur faces o f apatite crys tal s introduced into metas table cal cium

phosphate s olutions and the e ffec t fluoride and ma gnes ium had in

modi fying the grow th and s tab ility o f thes e precurs or phases . In

the new s tudy , part icular emphas is was given to the alterat ionsbrought about by fluoride on s uch phys ical properties o f apati tes

as spec if ic sur face area , crys tall ini ty , and crys tal shape .

Methods Employed

Synthetic cal cium pho sphate suspens ions were prepared ei ther by ( l )spontaneous precipi tat ion init iated through rap idly rai sing with KOHthe pH o f s tab le ac idic solutions of cal cium and phosphate ions to

or by ( 2 ) inocula ting supers aturated s olut ions metas tab le at

pH with crys tal s o f apati te . The s uspens ions were then aged

at either 2 5°

or 3 7°

C , the pH being maintained at with a pH

s tat . Magnes ium and / or fluoride ions were added either b efore

init iat ing precipitat ion or at various s tages during the aging

process . Reaction kine t ics were followed by monitoring solut ioncalcium , total pho sphate , hydrogen ion release

,and fluoride

consumption . S olid reac tion products were examined chemical lyand by B-E— T surface area methods

, x—ray di ffrac tion and trans

mis sion electron micro s copy .

Two methods for preparing apati tes from aqueous calcium pho sphate

s olutions are currently in general use , namely by spontaneous prec ipitation from uns table supersaturated solutions or by seeding

metas table solut ions . Work done under this proj ec t in previous

years has shown that the maturat ion o f calcium phosphate sal tsspontaneously precip itated from uns table solutions at phys iological

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2 01 DE 00088-06 LB S

o f phys iological rather than phys ico— chemical fac tors in controll ingapat ite growth in vivo .

Sigpific anc e to Dental Research

Hard t issue format ion is a complex phenomenon invo lving many poorlyunders tood p roces ses

,no t the leas t o f which is the depo s ition o f

mineral in the extracellular matrix . Becaus e o f the intimate as soc i

ation between mineral and matrix,it has proved dif ficul t to s tudy

the purely phys ical— chemical dynamics underlying this depo s ition

s tep in vivo . Consequently , to asses s the impor tance o f these

phys ical-chemical factor s it i s neces sary to thoroughly and sys tem

atic ally inves tigate the format ion and growth o f analogous cal cium

phosphate sal ts in phys iological-like synthet ic sys tems . The resul tsobtained from such experiment s during the pas t year indicate that

the degree to which b iol ogical fluids are supersaturated with respec t

to cal cium phosphate sal ts may be a cri ti cal factor in determining

whether or not phases such as OCP and ACP p lay impo r tant intermed iary

roles in the mineral ization o f hard tis sue . The fluoride— crys tallinity experiments , on the o ther hand , serve to remind us that the

dynamic s o f mineral format ion in vivo can probably never be fullydescribed in phys icochemical terms alone , but that b iological factors

such as extracellular pro tein matrices play a maj or role in direct ing

and contro ll ing this complex proces s . Hope fully such knowledge may

enable us to cons truct more appropriate model sys tems in the future

for the s tudy o f mineral depo s ition in bones and teeth and o f how

ions such as fluoride can modify this pro ce s s . Knowledge from

s uch s tudies could allow us to more completely unders tand the origin

Of the anticarious propert ie s o f this anion .

Proposed Course o f Proj ect

The current studies on the e f fec t o f inorganic anions on apat i te

formation , maturat ion , and phys ical properties will continue .

F uture emphas is will be direc ted toward examining how fluoride,

magnes ium , and carbonate , separately as well as in various combinations

, can ef fec t crys tal morphology . Additional s tudies wil lb e initiated to inves tigate the formation and s tab ility o f inter

mediate phas es in the precipitation o f calcium pyrophosphate from

aqueous solutions .

Publications

Meyer , J . L . , Eanes ,E . D . : A thermodynamic analysis of the secondary

transition in the spontaneous precip itat ion o f calc ium pho sphate .

Cal c if . Tiss . Res .— 2 16 , 19 78 .

li— 1 1I8

2 0 1 DE 0008 8-06 LB S

Eane s ,E . D . , Meyer , J . L . : The influence o f f luo ride on apat it e

fo rmation from uns tab le supersaturated solutions at pH J . Dent .

Re s .— 6 24 , 1 9 78 .

Eane s , E . D . : Enamel apat it e : chemis try,s truc ture and propert ies .

In,Nylen ,

M . U . , Termine , J . D . ( eds ) : Third Internat ional Sympo s ium

on Too th Enamel Growth , S truc ture,and Func t ion . J . Dent . Res .

5 8B : 8 2 9— 8 36 , 19 7 9 .

Eanes , E . D . : The influence o f fluoride on the seeded growth o f

apat ite from s table supersaturated solutions at pH J . DentalRe s . pres s ) .

O c tob er 1 , 1 9 7 8 to S ep tember 30 , 1 9 7 9

N AM ES,LA BOR AT ORY AN D I N ST I T UTE AFF I L I AT I ON S , AN D T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND A OT H ER

PR OF E S S I ON AL P ER S ON N EL ENG AG ED ON THE PRO JECT

Meyer , John L . Resear ch Chemis t NIDR

OTHER Hailer , Ar thur Chemis t

Lenk , Elaine Vis i ting As so ciateS elinger

,H . Andrew Bio log ical Aid

Youmans , Patricia A . S e c r e tary ( s teno )De G ra f f , Barbara A . Clerk- typing

None

Laboratory o f B iological S truc ture

Mo lecular S tructure S ec tion

NIDR, NIH , Be thesda

,Maryland 2 0014

D (b ) HUM AN T I S S U ES 5] (c ) N E I T H E R

SUMM AR Y OF WOR K (200 word s or les s un d e rlin e keyword s )

which regulate the nucl ea tion , crys tal growth and matura tion of calcium,

phosphate crys tals . This is accomplished by es t ima ting free ionic ac tivi ties

in solution for all S pec ies involved in the crys talliza tion proces s and relating

these terms to the obs erved precip itation s teps . A fur ther correlation is thenmade b e tween the compos it ion of the solution and the proper ties o f the so lid

calcium pho sphate phas e in equilibrium wi th it . The ef fec t o f crys talliza t ion

inhib itor s on the precip ita tion o f calcium phosphates is also being s tud ied in

order to elucida te their mode o f ac tion a t crys tal surfac es.

Emphas is is placed

upon inhib i tors which occur natural ly in phys io logical sys tems or which arecommon therapeutic agents .

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Z01 DE 00 16 2-03 LBS

develope d to direct ly analyze the precip itates fo r their hydroxide

content .Once correc tions were made for the presence o f the o ther

titratable ma terial s in the sol id phases (pho sphate , acid pho sphate ,

and carbonate ) it . was found that the early crystall ine calc ium

phosphate phases generally contain from 0 to 20 percent o f the

theoret ical amount o f hydroxide for HAP , which i s consis tent with

the result s o f the thermodynamic s tudies . Surpris ingly , i t was

found that even the ma ture apatitic material s al so contained les shydroxide than expected fo r pure HAP . In fac t , few crys tall inecalcium phosphate s were isolated which contained more than 5 0 per

cent of the theore tical amount . The signi ficance o f this lat ter

observation is no t clear a t present but the sugges t ion i s ma de

that hydroxyl is no t neces sary for the integrity o f the HAPs truc ture . In fac t

,the mos t s table apa titic material fo rmed

under s imulated bio logical condi tions would appear to be one with

at leas t hal f the hydroxyl pos i tions e ither vacant or filled with

ano ther S pecies , poss ibly water .

Significance to Dental Research

A knowledge o f the fac tors that influence calcium pho sphate pre

c ip itation is required for a comple te unders tanding o f the phy siolog

ical proces ses that result in hard tis sue mineralizat ion . A thermo

dynamic approach to the s tudy of cal cium pho sphate prec ip itat ion

under s imulated in vivo condit ions yields bas ic information that

can be related ul timately to c ondit ions that may exis t in vitalfluids in contac t with the mineral phase . The combinat ion o f ther

modynamic and kinetic methods can provide a better des crip tion o f

those dynamic p roces ses resul t ing in phys iolo gical and patho lo gicalcalc ificat ions or decalc ificat ions within the body .

Propo sed Course o f Pro jec t

Experiment s des igned to charac teri ze the ent ire course o f the pre

c ipitation o f calcium pho sphate from the init ial formation o f AOP

to its eventual trans formation to crystall ine hydroxyapati te willcontinue . Solutions in contact with the sol id phase will be

analyzed by chemical thermodynamic methods to ob tain information on

the nature o f the various calcium pho sphate phases fo rmed . Al lcrys talline materials will be analyzed by conventional techniques

to as sure that the thermodynamic analys is result s are cons istent

with the propert ies o f the solid phase . Crys tal growth inhibitors

are -known to affect crys tall i zat ion o f cal cium pho sphates but a

detailed analys is o f their e f fec ts on all the various phase trans i

tions has no t ye t been made . Because o f the potential b iological

s ignificance o f mineral izat ion inhib itor s on intra and extracellularcal ci fication , future work will concentrate on these subs tances with

particular emphas is on their mechanism o f ac t ion at crys tal surfaces .

N 1 F 9

2 01 DE 001 6 2—03 LB S

The relationship between the formation o f the o rganic and mineralphases in hard tis sue s will be inves tigated with part icular emphas is

placed on determining how the pre sence o f one phase af fe ct s the

init ial separat ion (nucl eat ion ) o f the o ther phase from an unstable

supersaturated solut ion . Transm is s ion elect ron micros copy will be

used to s tudy the morpholo gical re lat ionship between the organic

and inorganic phase s .

Publ icat ions

Meyer ,J . L . and Eane s , E . D . : A thermodynamic analys is o f the se cond

ary trans i tion in the spontaneous precip itat ion o f calcium pho sphate .

Cal c if . Tis s . Res . ,25 : 20 9— 2 16

, 1 9 78 .

Eane s ,E . D . and Meyer , J . L . : The influence o f f luoride on apa ti te

format ion from uns tab le supersaturated so lutions at pH J . Dent .

Res . , 57 ; 61 7-6 2 4 , 19 7 8 .

Meyer ,J . L . : Hydroxyl content o f solution— precip i tated calc ium

pho sphate . Cal c if . Tis s . Int l . , 2 7 , 15 3-1 60 , 19 7 9 .

Meyer ,J . L . and S el inger , A . H . : The ef fec t o f c itrate on calcium

pho sphate phase trans i t ions . Mineral Elect rolyte Metab . ,in pres s .

Oc tober 1 , 1 9 78 to S ep tember 30 , 19 7 9

N AM ES,LA BOR ATORY ANO I N ST I T UT E AFF I L I AT I ON S , AN D T I T L ES OF PR I NC I PAL I NV E ST I G ATOR S AND AL L OT H E R

PR OF ES S I ON AL P ER S ON N EL ENG AG EO ON THE PR O J ECT

Reddi,Akepati H . Research Biolo gis t NIDR

OTHER : Rath , Narayan C .

Weis s,Roy E .

Sullivan , NoreenMart ire , Diane

Brown , Jo sephineSmith , Edi th

Woodson , KevinYoumans , Patric ia A .

De G raf f ,Barbara A .

F loyd , S teven W .

Maryland 2 0205

Laboratory o f Bio lo gical S tructure

Molecular S tructure Sec t ion

NIDR ,NIH

,Bethesda , Maryland 20 20 5

[ 1 (b ) H UM AN T I S S U E S E] (c ) N E I T H ER

S UMMARY OF WOR K (200 word s or le s s un d e rl in e ke ywo rd s )The obj ec tive o f this p roj ect is to invest igate extracellular matrix— cell inter

actions employing an experimental sys tem o f matrix-induced endochondral bone

different iat ion . This experimental model further a f fords a method to undertake

systematic s tudies on the b iochemis try and phys iolo gy o f endochondral b one

formation . Subj ect s current ly under inves t igation are : (l ) the local mitogenic

effects of collagenous bone matrix ; ( 2 ) changes in pro line— synthetic and de

gradat ive enzymes during matrix-induced cartilage and bone development ; ( 3 )immunofluorescent lo cal ization o f type IV collagen and laminin during endo

chondral bone different iat ion and regulat ion by pi tuitary growth hormone ; (4 )

influence o f magpe sium deplet ion on endochondral bone formation (5 ) influence

o f experimental diabetes and insul in on cartilage and bone di f ferent iat ion ;

and (6 ) role o f co llagen— c ro s slinking in mineral izat ion o f bone .

Vis iting FellowPo s t— doc toral FellowPhysical Sc ience Technic ian

Biological AidS tudent— trainee

,Bio . Aid

S tudent— trainee,Bio . Aid

S tudent— trainee , Bio . AidSecretary—s teno

Clerk— typing

Pho tographer

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201 DE 00 204-03 LB S

planted collagenous matrix i s the appearance o f connect ive t is sue

mesenchymal cell s adj acent to the implanted ma trix before their

dif ferentiation into cartilage . We found that on days 3 and 8-9

after implantation there is an increase in the activity o f o rni

thine decarboxylase (ODO ) , an enzyme involved in po lyamine b io

synthes is and cons idered a ma rker for cell prol i feration . These

findings have been corroborated by incorpo rat ion o f 3H— thymidine

into acid—precip itable fractions . Autoradio graphy at the lightmicroscope level revealed mesenchymal cell s lab ell ed wi th SR— thymidine

in the vicinity o f the implanted matrix . These results imply a rolefo r collagenous ma trix in anchorage—dependent events in cell growth

and di f ferentia tion . Addit ionally , these experiments may exp lainthe lo cal nature o f fracture and wound healing responses and al soprovide a bas is fo r further s tudies in cl inical areas such as dentalimplant s and perio dontal diseases .

and Bone F ormation

Pro line , a characteris t ic const ituent o f co llagens , can be synthes ized

from either glutamic acid or o rnithine through the forma tion o f inter

mediate pyrrol ine-S—carboxylate . Prol ine may be degraded to glutamicacid . Thus mammal ian tis sues have the po tent ial for net b iosynthesis

o f pro line and for generat ion from prol ine o f urea— cycle and tri

carboxylic acid cycle intermediates . In order to charac teri ze the

t is sue-specific patterns o f prol ine b io synthes is and degradat ion in

co llagenous tissues such as cart ilage and bone , we have examined

the prol ine b io sy nthet ic and degradat ive enzymes in matrix—induced

cart ilage and bone development sys tems . Ornithine amino trans ferase

and pyrroline-S— carboxylate reductase,the biosynthetic enzymes ,

have peaks o f act ivity that correlate with maximal synthes is o f

type I I col lagen on day 7 by chondrocytes . Bo th enzymes al so are

ac tive during bone fo rma t ion . In contras t p ro line oxidase and

pyrrol ine-S— carboxylate dehydrogenase , the degradative enzymes,are

present at very low level s that do no t change as new cell typ es

appear . These findings sub s tant ia te the importance o f prol ineb io synthesis in co llagenous t is sue development . These inves ti gat ions

were done in co llaborat ion with Drs . R . J . Smith and J . M . Phang

o f the Metabolism Branch,NC I

,NIH .

The p ituitary growth hormone is es sential for normal bone developmentand mineral ization . However , the mechanisms underlying this act ion

and its target cell s are no t well de fined . The forma tion o f endo

chondral bone requires a pro gres s ion o f cell types . Vasculari zat ion,

though lit tle unders tood , is an impo rtant prerequis ite for o s teogenes is .

[ II- 1 5 6

2 01 DE 00 204—0 3 LBS

Current evidence indicates that vas cular invas ion provides the two

maj o r bone s tem cel l populations : o s teo clas t s,derived from b lood

b orne hema togenous elements (monocytes ) , and os teob las ts,probab ly

derived from vascular endo thelial cell s o r perivascular connec t ive

t is sue cell s . We have monitored vas cularizat ion during matrix

induced o s teo genes is by the immuno fluores cent l ocal izat ion o f

type IV collagen and laminin,a nonc ollagenous glycopro tein

i solated from basement membrane p roducing tumo rs . These experiments

were per formed in co llaborat ion with Dr . J .—M . F oidart ,

LDBA ,NIDR .

Thes e two antigens were lo cal ized in the vascular basement membrane

o f prol iferat ing capil larie s invading the o s teo genic p laques . Aclos e co rrelat ion was found between capillary invasion and sub sequent

b one formation as as ses sed by alkal ine phosphatase ac t ivity and5Ca incorpo rat ion into bone mineral . The vascular invas ion was

reduced and delayed in hypophysec tomized re cip ients . However , this

de fi ciency could be amelio rated by adminis trat ion o f p itui tary

growth hormone . The se resul t s sugges t that growth hormone couldmodulate o s teo genes is by regulat ing vas cul ar invas ion .

The e f fec t o f magnes ium de f ic iency on bone cell di f ferent iat ion

and b one fo rma t ion was inves t igated us ing in vivo matrix— induced

endochondral o s s i ficat ion as a tes t sys tem . Deminerali zed bone

matrix was implanted sub cutaneous ly in young ( 35 day old ) male

Long— Evans rat s tha t had been fed a semi— synthe tic Mg def icient

die t (5 0 ppm Mg ) for 7 days . Plasma Mg level s were reduced to

2 5-30 percent o f control value s at that time . Contro l rats were

p air fed the same diet , S upplemented to contain 1000 ppm Mg . The

implant s were harve sted 7 , 9 ,11 ,

15 and 2 0 days af ter implantat ionand analyzed fo r Mg and Ca content , Ca incorpo ration and alkalinepho sphatase level s . At each s tage , plaque ( imp lant s ) removed

from Mg def icient rat s showed retardation in cart ilage and bone

di f ferent iat ion and matrix calc if icat ion . Magnes ium content was

marked ly reduced when compared to the contro l p laques . His to lo gicalappearance o f the matrix-induced plaque s con firmed the re tardation

in bone development and mineral izat ion sugges ted by the chemicalindicators . Mo s t marked was the vir tual ab sence o f bone marrow

in 2 0 day old plaques in Mg dep le ted rats . These data show that

b one cell di f ferentiat ion can o ccur in a severely Mg dep le ted

environment,al though the onse t o f mineral iza tion and bone remodel

ing was delayed and bone marrow di f ferent iat ion was impaired .

Bone Di f ferent iat ion

I t is wel l known that the diabe t ic s tate resul t s in compl icat ions

o f periodontal t is sues and in generalized o s teopo ro s is . However ,

14 — 1 5 7

20 1 DE 00204—03 LB S

the precise role o f insulin in dif ferentiat ion and metabol ism o f

cartilage and bone is no t known . In order to gain further ins ights

into this prob lem we have studied the influence o f diabetes and

insulin on endochondral bone development . The e f fect o f s trep to

z otoc in-induced diabetes on dis crete s tages o f matrix— induced endo

chondral bone forma t ion has b een inves tigated . Demineral ized bone

matrix was implanted sub cutaneous ly to induce lo cal endo chondral

b one development . Three days following matrix implantation , mesen

c hymal cell proli feration was inhib i ted in diabet ic rats as

evidenced by a 6 52 reduct ion o f ornithine decarboxylase (ODC )act ivity and a 5 62 reduct ion o f [ 3H ] — thymidine incorporat ion per

pg DNA compared to non— diabet ic contro l s ; the inhib it ion was pre

vented by insul in treatment . In diabetic animal s chondrogenes is

on day 7 was reduced by 49 2 compared to control animal s as assessed

by 804incorp orat ion into p ro teoglycans . Exogenous insulin was

s timulatory to cartilage development when present during days 0

through 4 (mesenchymal cell pro liferation ) . Cal ci fication o f car

tilage and os teo genes is were monitored by alkal ine phosphatase

act ivity and 45Ca incorporation . Alkaline pho sphatase at tained

a peak value in control rats on day 11 . In diabe t ic rat s the peak

was on day 14 . In the diabet ic group Asca incorporat ion was les sthan 502 o f control throughout the duration o f the experiment . The

reduction in alkal ine pho sphatas e ac tivity and 4SCa incorpo rat ion

were prevented by insulin . Decreased in vivo endochondral bone growth

and development during diabe tes is the resul t o f (1 ) inhibition

o f insulin-dependent mesenchymal cell prol i feration (2 ) decreased

and delayed cartilage formation due to impaired mesenchymal cellprol iferation (3 ) decreased and delayed vascular invas ion p rior to

chondrolysis and os teo genes is and (4 ) reduced insul in— dependent

calcif icat ion and o s s if icat ion .

Endo chondral Bone Dif ferent iat ion

The various types o f collagens have been impl icated in cell at tach

ment , cell di f ferentiation and morphogenesi s . Cro s s— linked col lagenous matrix appears to b e a prerequis i te fo r op t imal mineral izat ion .

The availab ility o f B-aminoprop ionitrile (BAPN ) and o ther agent s

that inhib it collagen cro s s— linking enables an experimental scrutiny

o f the influence o f lathyrism on bone di f ferentiation and minerali z ation . The influence o f BAPN on mineralization during endochondralbone development was inves tigated . Mesenchymal cell pro l i ferat ion

was not affected by the lathyrit ic agent . However,chondrogenic

dif ferent iat ion on day 7 was inhib it ed by about 45-50 2 by BAPN asindicated by 35 80 4 incorporat ion into pro teo glycans . On days 9

and 14 cart ilage and b one minerali zat ion,re spe ct ively

,as indicated

by 5Ca incorporat ion , were pro foundly inhibi ted by BAPN treatment .

II- I S S

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Oc tober 1 , 19 78 to S ep tember 30 , 19 7 9 Y Ol-DE— 8002 7

N AM ES,L AB OR ATORY ANO I N ST I T UT E AFF I L I AT I ON S, AN D T I TL ES OF PR I NC I PAL I NV E ST I G AT OR S AND A OT H E R

PROF E S S I ON AL PER S ONN EL ENG AG ED ON THE PR O JECT

P I : Ca s c iani ,Francis S . S taf f Fellow NIDR

O THER : Lenk,Elaine Vis it ing As so c iate

Youmans,Patricia A . S ecretary ( s teno )

De G raf f ,Barbara A . Clerk-typ ing

Floyd , S teven W . Pho tographer

Labora tory o f Biolo gical S truc ture

Molecular S truc ture Sect ion

NIDR, NIH,Be thesda , Maryland 2 0205

[ 1 (b ) HUM AN T I S S U E S 15] (c ) N E I T H ER

SUMM AR Y or WOR K (2 00 wo rd s or less und e rlin e ke yword s )The obj ectives o f this S tudy are the identi f ication and character izat ion of

the inorganic phosphate and carbonate phases and the organic matrix o f

rodent incisor enamel , and changes in these components which occur during

mineral ization and maturat ion . Inorganic and organic pho sphate and carbonate

phases lo calized in the t is sue during development are analysed us ing spectro

s cop ic microprobe techniques . The Raman microprobe ,capable o f ident ifying

molecular species , and elemental microprobe analyses (x—ray microanalys isand ipp _

mic roprobe analys is ) used in conj unct ion allow the correlat ion o f data

ob tained from these two types o f complementary analys es . This data then

may be interpreted to discern the distribution p at terns o f calcium,pho sphate ,

magpe sium carbonate and o ther ions at mineraliz ing s ites in develop ing enamel .

2 01 DE 00 251— 02 LB S

Proj ect Des crip t ion

Th e obj ect ives o f this s tudy include the fo llowing : ( 1 ) to dis cern

the accumulation and des crib e the dis tr ibution o f inorganic (calc ium ,

pho sphate,magnes ium and carbonate ) and organic ( espec ially organi

cally bound pho sphate ) components during the mineralization and mat

uration o f hard tis sue ; ( 2 ) to correlate the accumulation o f the in

organic phase o f hard t is sue with the changes that o ccur in the or

ganic matrix,i . e . i ts concentrat ion and secondary s tructure ; and ( 3 )

to as so c iate this mineralization pat tern as des cr ib ed by ( l ) and ( 2 )above with the varying s tages o f c ellular func t ion .

Me thods

The techniques us ed in this s tudy are microprob e methods the Ramanelec tron and ion microprob es . Microprob e methods are espec iallys uited to the analys is o f thes e s pat ially heterogeneous b io log icals amples . The advantages o f these methods are : ( 1 ) through the inter

pre tation o f Raman spec tra and elemental analys es , various mol eculars pecies may b e ident if ied and correlated with an el emental analys is ;and (2 ) the resolut ion o f the methods on fresh froz en t is sue s ec tions

is approximately 5 um and therefore allows an analys is o f approximatelygms o f mater ial . As appl ied to our sampl es

,dis crete analys es

are performed on previous ly unat tainab l e samples without the disad

vantage o f s ample isolation and poo ling , and therefore the results

o f this technique may b e correlated direc tly with the his to logicals truc ture .

Samples used in thes e s tud ies

Two aspec ts o f b io logical mineral ization which are o f interes t to

this s tudy are the charac teri zat ion o f the matrix and the init ialcrys tall it e at the s it e of mineral i zat ion and the changes which o ccur

in thes e phas es as the tis sue matures . Therefore , sampl es which

contain thes e areas o f interes t as dis crete , eas ily ident if iab le reg ions

are suitable to the s tudy . Thes e includ e the fol lowing : (1 ) the con

tinuously erupt ing rat incisor in which bo th amelogenes is and dent ino

genes is may b e s tudied ; ( 2 ) the epiphys es in embryonic tib ia as a sys t em

f or b o th chondrogenes is and os t eogenes is ; and (3 ) matrix—induced bone

plaques,which allow the s equential s tudy o f dis crete s tages of endo

chondral bone formation such as depos i tion and calc if ication o f the

car tilage matrix during chondro genes is , dis so lution o f the car t ilage

during chondro lys is,bone matrix formation and m ineral ization , and

have remo deling . F ur ther exper imental de tail s of this sys tem may b e

found in the repor t o f A . H . Redd i (2 01 DE-00204

4 - 1 5 1

2 01 DE 002 5 1-02 LBS

Sample Preparation

All sampl es cons is t o f thin s ec tions cut from fro zen unf ixed t issue

as des crib ed in las t years repor t . The pro to col , which allows

precise analys is o f mic rore gions , is the following : (1 ) 5— 30

pm fro zen s ections cut on a cryo s ta t ar e trans ferred to the appropri

ate Raman microprob e subs trate ( either l ithium fluorid e or sapphire )and air dried . Al ternate s er ial s ect ions are s tained employing con

v entional his tolog ical procedures ; ( 2 ) pho tomicrographs of the s ec

t ions are recorded ; ( 3 ) Raman microprobe data are co llec ted on spe

c ific areas (normally 5 um in diameter ) o f the s ec tion ; (4 ) the

s ame sec tion may then b e carbon coated for x e ray microanalys is and / orion prob e analys is o f the same reg ions ; and (5 ) thes e s ections may be

fur ther s tained for his to log ical s tudy .

Raman analys is o f a reg ion in thes e samples resul ts in a spec trum

charac teris tic o f the components pres ent . From a priori knowledge o f

the maj or components , the to tal spectrum mus t b e reso lved or decompos ed

into the spec tra o f the various components . This has b een accomplished

by ob serving the spec tra o f bo th pure materials and tho s e s eparated

from tissue through b io chemical techniques . Once the spectral

features of thes e components have b een as s igned or determined,changes

in concentration may b e followed by compar ison o f the results from

one reg ion to ano ther .

Major F indings

Inorganic Components

1 . In ra t incisor enamel , dentin and embryonic bone,in reg ions o f

very low mineral izat ion , the earl ies t phosphate mineral phas e detec ted

has a v1 P— O s tret ching mode at 9 60 cm“l

. Th is is in good agreement

with the v1 for an or thophosphate ion in the apatite lat tice . Com

parison of the Raman spec trum of thes e regions with the Raman spectrum

o f wel l charac teri zed phosphates such as o c ta c alc ium pho sphate or

dicalcium phosphate reveals no evidence for their pres ence as precursor phas es to b io logical apati te .

2 . In mineraliz ing enamel , within a few micrometers f rom the amelo

b las t , port ions o f the Raman spec trum o f the mineral huntite , Mg3Ca (CO3 ) 4 ,

have b een consis tently ob s erved . In thes e regions the phosphate

intens ity is ei ther very weak or nonexis tent . Mg Ca (co3 ) 4 is a

mineral with a unit cell very s imilar to that o f apatite,with carb onate

ions in nearly equivalent po s it ions o f the phosphate and hydroxyl

ions in the apatite lat tice . This s imilarity in the crys tal s truc

tures would permit , as our preliminary in vitro s tudies sugges t,the

epitaxial growth o f pho sphate m ineral on a core o f carbonate . The

as so ciation o f this carbonate phas e with magnes ium and the resulting

4 — 1 6 2

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2 01 DE 002 51-02 LB S

1 .S amples o f the mineral huntite wil l b e prepared . These will b e

us ed as s eeds upon which calc ium pho sphate wil l b e allowed to grow

from metastable so lutions . This material will b e analyz ed spec tro

s c opic ally to determine if the phosphate changes the s tructure of

the under lying huntite via a solid s tat e reac tion . I f this were to

o ccur, i . e . suff icient carbonate and phosphate were to interdif fuse

to eliminate the s tructural at tributes o f huntite whil e maintaininga high concentration o f carbonate in the orig inal s eed material , i t

would explain the lo ss o f the huntite spec tra and account for the

carbonate spectrum o f mature hard tis sue .

2 . Bas ed on the relative intens ity of the Raman b ands as soc iated

with (a) the antiparallel B-pleated sheet , (b ) the nucleos ide, ( c )

the organic phosphate , (d ) the inorganic phosphate , and (e ) the car

bonate component,their concentrations will b e mapped out his to

logically in the four mineraliz ing t is sues des crib ed above .

3 . From prel iminary experiments on mineraliz ing car t ilage , it has

b een determined that the sulfate component o f the pro teoglycans o f

car tilage has a very dis t inct ive spec trum . Thes e s tudies will be

continued , especially in the matrix-induced pl aques o f Dr . A . H . Reddiand growth plate cartilage .

4 . Ion prob e analys es are to b e continued to determine the Mg : CO3ratio s in mineralizing t issues .

Publications

Cas c iani , E . S . , Et z , E . S . , Newbury,D . E . and Do ty

, S . B . : RamanMicroprob e S tudies o f Two Mineraliz ing Tissues : Enamel o f the RatIncisor and the Embryonic Chick Tib ia . S canning-Elec tron Micros copy /1 9 79 / I I 383-391 .

Cas c iani , F . S . and Etz , E . S . : Raman Microprob e S tudy o f Bio logicalMineralization in S i tu : Enamel o f the Rat Incisor . Proc eed ings of

Microbeam Analys is S o ciety (in press ) .

14 — 1 6 14

Summary S tat ement

Laboratory of Microb iology and Immunology

Nat ional Inst itute of Dental Research

The maj or emphas is in our res earch programs has remained virtually unchanged

since FY 19 78 . Our‘

ac t ivit ie s inc lude the f ol lowing areas o f inves t igat ion

1 ) The role of solub le med iat or s d er ived f rom lymphoret icu lar c ell s in the

induct ion of immunity and inf lammat ion,2 ) the r egulat ion o f connect ive

t is sue met abo l ism by c ellular and humoral immune mechan isms,3 ) c l inical

immuno log ical s tud ies on aut o immune and per iodontal d is eas es,4) genet ic

det erminant s in the ho st respons e to bact er ial endo toxin,5 ) the mo lecu lar

b iology , phys iology and taxonomy of oral bact er ia and 6 ) mechanisms of

microb ial pathog en ic ity ult imat ely r elat ed to per iodontal d is eas es and

dental car ies . A more det ailed d escr ipt ion of the spec if ic res earch

relat ed t o thes e ar eas can b e found in the indiv idual report s . The

fo llow ing is a resume o f our progres s dur ing th e past year .

Although the microb ial c ompo s it ion of dental p laque is complex,curr ent

res earch in our Microb io logy S ect ion has est ab l ished that highly spec if ic

int eract ions occur among c erta in of the res id ent microf lora . St rept ococcu s

sangu is is one of the f ir s t spec ies of bact er ia to co loniz e the enamel

port ion of the t ooth sur face . Last year we report ed that a numb er of

at tach t o S . sangu is while s tra ins o f Act inomyces israel ii d id not . In

expand ing this study to include over 100 human iso lat es of act inomycet es,

a total of s ix b iochemically d is t ingu ishab le patt erns of coaggr egat ion

have b een ident if ied .

A . israeli i,an inhab itant of the more anaerob ic environment o f p eriodontal

plaque,do es , however

,int eract with certain gram negat iv e g l iding bact er ia

( Cyt ophaga S p . ) that are al so found in such plaqu e . The interact ions

ob served between these two group s o f organ isms are spec if ic too , and Cytophaga

sp . do not coaggr egat e with A . vis co su s or A . naeslund ii . The Cy tophaga

sp . isolat ed f rom periodontal p laque al so exhib it a uniqu e and sp ec if ic

af f inity for co loniz ing the roo t surfac es of t eeth . In v itro as says

revealed that d emineralizat ion of the t ooth sur fac e result ed from such

co loniz at ion . Recent stud ies have shown that the init ial at tachment o f

the organisms t o hydroxyl apat ite sur fac es is med iat ed by certain l ipo

prot ein component s conta ined within the out er membrane o f the bac t er ial

cel l .

Other eff ort s are b eing d irect ed t owards def in ing the enzymat ic mechan isms

respons ible for the regulat ion of sugar metabo lism and glucan b io synthes is

in oral s trept oco cc i . Concert ed s tudies have shown that sucros e is

metabo l iz ed by an induc ibl e sucro s e— d ependent pho sphoenolpyruvat e phospho

transferas e syst em which t ransport s and s imultaneou sly pho sphorylat es the

gluco s e mo iety o f sucros e . Th e resultant sucros e 6-phosphat e is then

cleaved to fructo s e and gluco s e 6-pho sphat e by a const itut ively synthes iz ed

sucros e 6— pho sphat e hydrolas e . B iochemical s tudies ind icat e tha t the sucro s e

6— pho sphat e hydro las e is probably respons ib le for the intracellu larinvertas e act iv ity report ed by other workers in Streptococcu s mut ans

and other oral s trepto cocc i .

11 - 1 51 1?

b iosynthes is and s ecret ion o f glucosyltransf era s e is be ing stud ied .

Product ion of the extracellular gluco syltransf erase complex is completely

inhib ited by cerulenin , an inh ib itor of fatty acid b iosynthes is . This

ant ib iot ic has no ef fect on DNA,RNA or prot e in synthes is

,but does inhib it

cellu lar incorporat ion o f ac etat e . The secret ion of gluco syltrans f erase

appear s t o b e clo s ely relat ed to long chain fat ty ac id synthes is .

By encod ing the informat ion neces sary for . the trans cr ipt ion of enzyme

b iosynthes is on plasmid s,bact er ia gain the advantage of b eing able to

produce s ignif icantly higher levels of thes e pro te in cataly st s than microb es

that carry such informat ion chromo somally . The genet ic d ispo s it ion of

informat ion that codes for the pro duct ion of enzymes catalyz ing the metabo lism

of two impor tant d ietary sugars ,sucro se and lact os e

,are current ly b eing

invest igat ed . In certain strep tococc i and lactobac ill i one or more of

the enzymes respons ib le f or the dis s imilat ion of lacto se appear s t o be

plasmid borne . Preliminary evidence suggest s that sucro s e metabol ism may

al so b e plasmid mediat ed in thes e same organisms . Plasmids appear to play

a maj or ro le in the metabo lism of other carbohydrat es in the Lancef ieldgroup N strept ococc i and probab ly play a minor role in the transmis s ion

of t et racycl ine res istance in oral streptococc i .

Three d ist inct approaches ar e b eing us ed by the Cellular Immuno logyS ect ion to inves t igate the bas ic ho st defens e mechanisms that are invo lved

in mount ing inf lammatory r eact ions in d iseas es such as periodontal dis ease

and rheumato id arthrit is .

Lymphocyt es,monocyt es and cell l ines s ecret e a numb er of med iators

which modulat e cellu lar par t ic ipat ion in inf lammat ion . These med iator s

are recovered from the supernatant s o f cultured ce ll s . Iso lat ion and

characteriz at ion of thes e fact ors should increas e our unders tand ing and

cons equent capac ity to manipulat e inf lammatory react ions . The fact or s

that are b eing invest igat ed inc lud e lymphokines and cytokines such as

lymphocyt e der ived mitogenic factors (LHE) ,os teoclas t act ivat ion fac tor

(OAF ) , monocyt e derived lymphocyt e act ivat ing fact or (LAF ) and tumor

necros is f act or (TNF ) , as well as f ibrob last cell l ine der ived co lonyst imulat ing factor ( s ) (CSP ) and interferon ( IF) . These factors are hormone

like glycopept ides ranging in MW from and are b io log ically

very act ive at low (ng ) concentrat ions . A maj or effort is und erway to

use cell lines to produce some of th es e factors in quant it ies which are

suff ic ient f or iso lat ion , charact er izat ion and to obtain ant ibod ies to

them . LAF obtained from a mouse cel l line has b een pur if ied by a factorof

Cons iderable progress i s be ing made in def ining the b iolog ical rol e in

ho st d efense o f some of these med iators . A numb er of these molecules

amplify the aff erent l imb of t he inf lammatory response . For example,LAF

has b e en found to st imulat e the pro lif erat ion as well a s func t ional maturat ion

of thymocytes and lymphocyt es . It promot es ant ibody product ion and enhances

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factors that presumab ly are respons ib le for the phenomenon of

endo toxic ity . Thes e factors includ e pro st agland ins , LAF , IF ,CSF and

serum amyloid as soc iat ed prot ein,an acute phas e reactant . Normal macrophages

also can b e act ivat ed by LPS to kill tumor cell s and to s ecret e tumor

necros is factor (TNF ) which is tox ic for tumor cell s . Thus LPS is an important

inducer o f inf lammatory react ions and stud ies of it s eff ects eluc idat e

bas ic mechan isms involved in host react ions to inf ect ious organisms .

The Humoral Immunity S ect ion has ext ended it s invest igat ions concerning

immunological mechanisms in the r egulat ion of connect ive t i s su e metabo l ism .

Synthes is of collagen , a maj or const itu ent of connective t is sue , may b e ,

in part,under immunolog ical cont rol . A f actor ( s ) pres ent in the super

natants of st imulated lymphocyt es and macrophag es enhances the synthes is

of collagen as well as non— co llag en prot ein by f ibroblas t s which sub sequent lyundergo cell div is ion . Del ineat ion of the ear ly cellu lar events lead ing

to the product ion o f thes e prote ins is receiving maj or emphas is . CAMPand prostag landin are impl icat ed in th is act ivat ion s equenc e s ince within

a few hours aft er exposure to ac t ive lymphocyt e or macrophag e supernat ant s,

both intracellular CAMP and media pro stagland in level s are elevat ed in

cultures of f ibroblast s . Charac terizat ion o f this "f ibrob las t act ivat ing

fact or" and it s relat ionship t o other b io log ically act ive moleculesder ived from lymphocytes and macrophag es are pres ent ly b eing invest igated .

Immunological respons es may als o contr ibute to the catabo l ism o f

connec t ive t issue . The prev ious ly d escribed dependence on pro s taglandin

synthes is for the product ion of collag enase by act ivat ed macrophages ha s

now been shown to b e relat ed to an elevat ion o f CAMP in thes e cells .

This requirement for pro staglandin synthes is can b e bypas s ed by the

exog enous addit ion o f the cycl ic nuc leot ide analog ,d ibutyryl CAMP .

In addit ion , agent s such as cholera toxin which elevat e CAMP at pharmac ologic al concentrat ions enhanc e the product ion o f col lagenas e by ac t ivat ed

macrophages .

The humoral immune syst em also contr ibut es to the des truct ion of

connect ive t issue . Evid ence has now b een ob tained which suggest s that

complement act ivat ion by immunog lobulins r eact ive with cell sur fac eant ig ens pres ent in bone has b io log ical cons equ enc es in addit ion to the

init iat ion o f pro stagland in synthes is with re sultant destruc t ion o f the

bone . This ef fector sy stem al so inhib it s th e growth o f bone as d et ermined

by de cre ased synthes is o f both co llagen and non—co ll agen prot ein . Ofint erest is the find ing that although the inh ib it ion of bone growth by

complement su ff icient s erum is accompan ied by enhanced pros tagland in

synthes is , these two event s are appar ent ly unrelat ed s ince ag ent s which

block the product ion of the prostagland ins do not res tore the growth ofbone .

S tudies in collaborat ion with the Microb io logy S ect ion have concentrated

on delineat ion o f the mechan isms by which microorganisms adher e to other

microorganisms and mammalian cells . Int erac t ions b etween st rains o foral act inomycet es and strep to cocc i occur via lect in— like s it es on one

sp ec ies wh ich react with carbohydrat e recept or s on the other spec ies .

Curr ent work in this S ec t ion is focu s ing on the immunochemical charact er

iz a tion of the lec t in involv ed in these reac t ions and has id ent if ied the

carbohydrat e spec if ic it ies to b e lac to s e and B—methyl galac to s id e .

In collaborat ion with our Cl in ical Immuno logy S ect ion monoclonal ant ibod ies

have b een produc ed .against the bac ter ial st ructures which carry the

lect in b inding s it es thu s prov id ing reagents which will d ef init ively

C harac t er iz e the c ell wall component s invo lved in adherenc e . In add it ion ,

thes e lat t er s tud ies may b e extremely helpful in def ining mor e pr ec is ely

the ro le of ant ibodies in ho st res is tanc e through their int erac t ions

w ith c oaggregate s of dental p laque bac ter ia .

The Cl inical Immunology S ect ion cont inues to invest igat e genet ic and

immuno log ical fact ors in the pathophys io logy of Sn ren's Syndrome .

Dur ing the past y ear the current knowledg e about this d iseas e was r ev iewed

at one o f the Combined C linical Conf erences and will be publ ished in the

Annals o f Int ernal Med ic ine . Clinical stud ies demonst rated that Sn ren's

Syndrome either alone or as soc iated with sy st emic lupus erythemato sus

ar e C lo s ely related , but d if f er from Sn ren's Syndrome as soc iat ed with

rheumato id arthr it is . Thes e d isea ses also have diff erences in the

d ist r ibut ion and spec if ic ity of the autoant ibodies present in the pat ient 's

s erum . Pat ient s with Sn ren's Syndrome have auto— ant ibodies d ir ec t ed

to S S— A and S S—B ant ig ens , but no t to RANA ; in contrast pat ients with

rheumato id ar thr it is as soc iat ed with Sn ren's Syndrome produc e ant ibodies

t o RANA but not S S— A or SS—B ant ig ens . These two group s of pat ient s

have common genet ic markers (B— lymphocyt e Ia—marker s Ia— l7 2 and Ia

but d iff er in some other immunog enet ic markers (HLA—B8 ,HLA—DRW3 , HLA

DRW4 and Ia The result s sugg es t that the lymphocyt ic inf il trat ion

of the sal ivary glands may dep end upon the Ia— immune response genes .

Immuno log ical stud ies are also att empt ing t o bet ter def ine the patho log icalmechanisms in Sn r en

's Syndrome . The sera of these pat ient s have

immune complex es and ther e is def ect iv e in v ivo cl earance of immune

complexes . The pat ients with the mo st s evere def ec t ive C learance have

the mo st prominent d is eas e . S tud ies with lymphocyt e subpopulat ionsdemons trate normal to tal lymphocyt es , T and B c ell number s although

ther e is a decr ea sed relat iv e proport ion of T c ell s among pat ient s with

ex traglandular d is ease . This was found to be due to a serum factor

which rever s ib ly b locked the expres s ion of the IgG F C rec ep t or of the T

c ells . However,unlike the suppr es sor cell def ec t in pat ient s with

syst emic lupus erythemato sus,ther e ar e no abnormal it ies in mitog en g enera t ed

suppres s ion . Th is d if f er enc e in immunoregulat ion between sy st emic lupus

erythemato su s and Sn ren's Syndrome may explain the d if f er ences in the

s ever ity and expr es s ion of these autoimmune react ions .

G

Studies are at t empt ing t o def ine the role of methylat ion reac t ions in

the rel ea se of histamine from human ba sophil s and mast cells . Methylat ionreac t ions mediated by S-adeno syl—L—methionine play an important role in

a number of b iolog ical r eact ions inc luding bact er ial and human monocyt e

chemotax is . Ut il iz ing a number of inhib it or s of methylat ion , st rong

evidence was f ound that methyla t ion reac t ions p lay an important ro le in

fl - J F Q

the early st eps of IgE—mediated histamine secr et ion from human basoph ils .

In stud ies with cultured mast cel l s ; the methylat ion st ep was found to

be due t o the methylat ion of phospho l ip ids in the membrane .

An act ive program has b een init iat ed in the Cl inical Immuno logy S ect ion

to develop a numb er of monoclonal hybr idoma ant ibodies . The fu s ion o f

S p leen cells from immuniz ed mice with a myeloma cel l line result s in

C lones o f cells producing antibodies to which the mic e had prev ious ly

been immuniz ed . Ut iliz ing this techno logy , monoc lonal IgE hybridomas have

b een produced . Ant ibod ies t o cell surface F c recep tors have also b een

made and will be ut iliz ed in funct ional stud ies of thes e recept ors .

Ant ibodies o f s everal C lones have also been prepar ed to bact er ial surface

components and these can be ut il iz ed in stud ies o f bact er ial coaggregat ion

as alluded to earlier . Thes e mono clonal ant ibodies ar e , therefore,very

us eful reagent s in a numb er of our immunolog ical and microb iological

stud ies .

J? J

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OBJECTIVES 2 01 DE 0045 08 LMI

The role o f mediators produced by human macrophages and lymphocytes in

the af ferent limb o f the immune response is b eing inves t igated . Thes e

endogenous nonspecific mediators appear to be es s ent ial in act ivat ingand amplifying lympho cyte mediated immunological react ions . In addit ion ,

o ther invest igators are obs erving that LAF is physico chemically inseparablefrom macrophage der ived endogenous pyrogen and from the monocyt e derived

factor that s timulates "ret icular" fibrob last s in the synovial t issues

o f inflamed j oint s to produce large amounts o f pros taglandins and

collagenase . Therefore LAF also appears to s t imulate non— lympho id cellsin the hypo thalamus and j oint s and may play a pivo tal role in acute as

well as C hronic inflammatory react ions . Invest igat ions o f the nature

and mechanism o f act ion o f these vital mediators in in vitro tissue

culture model s may provide the requis it e unders tanding to permit eventualtherapeut ic manipulat ions o f the signal s init iat ing and augment ing

inflammatory react ions .

METHODS

l . LAF act ivity is operat ionally def ined by it s mitogenic ef fect on

CBH/He J thymo cytes . The ef fect o f supernatants o f human mononuclear

cell , mouse peritoneal cell or fibroblast cul tures or p art ially purif ied

chromatography fractions of such supernatant s in st imulat ing or enhancing

thymo cyte proliferation is determined .

2 . Mediators such as LAF produced by macrophages and LMF produced by

human lymphocytes are also as sayed for their ef fect s on :

a) Thymocyte dif ferent iation e . g . , Changes in expres s ion o f Ly t—l

and Ly t-2 markers on thymocyt es us ing the fluore senc e act ivated

cell sort er (FACS ) .

b ) mitogenic eff ect s on human C irculat ing thymus derived (T) and

bone ma rrow derived (B ) lymphocyt es .

c ) Enhancement of immunoglobulin product ion by human B cells .

d ) Intracellular level s of CAMP and c GMP .

3 . In co llaborat ion with Dr . S teven Miz e l human LAF and LMF act ivit ies

are b eing physico chemically character iz ed us ing Sephadex , s ephacryl ,

phenyl sepharos e , DEAE cellulose , hydroxylapatite and po lyacrylamide gel

(PAGE ) Chromato graphy .

MAJOR FINDINGS '

Agent s that act ivate human macrophages to produce increased lymphocyte

act ivat ion factor (LAF ) act ivity were inves t igate d . Proj ect s that have

b een completed indicate that dialys able extract s o f leuco cyte s which

also contain trans fer factor,the synthe t ic adj uvant muramy l dipept ide

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(MDP ) , various extrac t s o f mycobact er ial cell wall s,st rains o f corynebacterium

parvum with adj uvant act ivity , the co carc inogen phorbo l myr is t ic acet at e

(PMA ) and the pro ces s o f in fect ing mice with malaria all act ivat e macrophage

t o produce LAF .

“S tudies of serum factor ( s ) that act ivate macrophage LAF

p roduct ion have not been succes s ful in ident i fying the respons ible serum

component and have been dis cont inued . The ob servat ion that murine

co lony s t imulat ing factor (s ) (CSF ) induce macrophage LAF product ion is

act ively being inves t igat ed in co llaborat ion with Dr . Robert Moore . CSF

i s a macrophage growth factor that is produced by either f ibroblas t cel ll ines or lect in s t imulated lympho cytes . CSF s timulates no t only macrophage

p roliferation , but al so promot es their maturat ion so that they either

p roduce LAF o r become capable o f responding to other agent s that induce

LAF p roduct ion .

A mul t ip lic ity o f ef fect s o f LAF on lympho id cell s is b eing s tudied

1 ) In co llaborat ion with Dr . Bonnie Mathie son (NIAID ) , we are finding

that LAF is directly mito genic only for the minor subpopulat ion o f

thymo cyte s that is no t agglutinated with peanut agglut inin (PNA The

PNA sub-population is presumed to compris e the medull ry cor t isone

r es is tant thymo cyte s . In contrast the maj ority o f PNA (presumablycor t ical ) thymocy tes requires two s ignals to be in induced to prol iferate .

PHA and LAF t ogether synergis t ically st imulat e PNA t hymo cytes , but have

no ef fect no t by themselves .

2 ) Studies in conj unct ion with Dr . Northo f f o f a bat tery o f sugars have

revealed that allos e compet it ively and revers ibly inhib it s LAF induced

thymo cyte pro liferat ion . This C lue as to the nature o f recep tors for

LAF i s being used to prepare af finity co lumns for further pur if icat ion

o f LAF .

3 ) The fluores cence ac t ivated cell sort er (FACS ) is be ing us ed by Dr .

Mathieson to detect Changes in the surface marker s o f thymo cytes incubated

with LAF cont aining fract ions . We are ob serving a dramat ic inc reaie in

the expres s ion of Ly t—l and decrease in Ly t

-2 on bo th PNA and PNA

thymo cytes af ter overnight incubat ion . The relat ionship o f the factor

that induces this thymocyte dif ferent iat ion to LAF is b eing ascertained .

4 ) Dr . Douglas Olson (Meloy Labs ) has been assay ing cyclic nucleo t idesl evels in lymphocytes incubated by us with a wide variety o f st imulant s .

These s tudies have revealed that : (a ) Although LAF containing supernatant s

s t imulate cons iderabl e increases in thymocyte C AMP levels , this was

a ctually due to pro st agland ins rather than the LAF present in such

supernatant s . (b ) Even though human LAF containing supernatants alsos t imulate rap id and dramat i c in crease s in the c GMP level s o f human

mononuclear C ell s ,this was actually due to a serum component (s ) rather

than due to the LAF present in the supernatant s . (C ) A study by L . Rauppwith L . Lum and M . Blaese (NCI ) has revealed that the C AMP level s o f

2 01 DE 0045 O8 LMI

human T c ell subpopulat ions with F C recep tors for IgG (TV ) are raised to

a much greater degree by a variety of agent s than the levels in the

larger subopulations that bears F C receptors for IgM (Tu) .

5 ) Part ial purification o f human LAF by sephadex Chromato graphy yieldstwo p eaks o f act ivity in the and mw range . The lower mw

LAF when mixed with human serum is part ially converted to the higher mw

f orm o f LAF . The higher mw human LAF is mito genic for human peripheralb lood T but no t B lymphocytes . However , it can not rep lace the require

ment for viable macrophages in ant igen induced T cell pro liferat ion .

LAF therefore funct ions as a secondary ampl ifying fac tor rather than as

an init iator of lymphocyte act ivation .

6 ) In collaboration with Dr . F . GMelig-Mey ling , we found that only the

low mw human LAF can subs t itute for macrophages in facilitat ing pokeweed

mitogen (PWM) induced polyclonal immunoglobulin product ion by human B

lympho cytes . S ince PWM is T cell dependent , the low mw LAF presumablyp romo tes T cell helper funct ions .

We have observed with Dr . G . S chechter (VA Hosp tial ) that the macrophages

from anergic patients with Hodgkins dis ease are abnormal in their in

vitro LAF p roduct ion . They produce inhib itors including pro s taglandins

However , the relat ionship o f these ob servat ions to the diseas e pro ces s

are as yet unreso lved .

We are al so s tudying lymphocyte derived mitogenic fac tors (LMF ) produced

by concanaval in-A (Con-A) and antigen ( S treptolys in-O , SLO ) act ivated

lymphocytes . LMF is similar to LAF in that it st imulates PNA thymocyt e

proliferat ion and is eluted in the and mw regions from

Sephadex . Bo th LMF and LAF are des troyed by chymotryps in and SDS .

However , LMF exhibit s a number o f dif ferences from LAF : (1 ) I ts product ion

by human T lymphocytes is macrophage dependent . Its product ion especiallyin response to Con A is inhib it ed by T suppress or cells . Therefore the

product ion of LMF can be enhanced by inhibi t ing suppres sor cell funct ions

with prolonged incubation or drugs cyme tidine ) . (2 ) LMF is les s

suscep tib le to the inhib itory ef fect of allos e than LAF . (3 ) Unlike LAF ,

both the higher and lower mw LMF are mito genic for human peripheral T

lymphocytes . In addit ion , the lower mw LMF (unl ike LAF ) is mitogenic

for human B lymphocytes and is al so capable o f support ing the growth o f

cytotoxic T lymphocyte lines (CTLL ) .

S IGNIFICANCE TO DENTAL RE SEARCH

The central role of cell mediated and humoral immunity in periodont al

d is ease has been do cumented . Macrophages as well as lymphocytes and

p lasma cells part icipate in C hronic gingival inf lammat ion . In vitro

t is sue culture studies can be used to dis sect the cellular interact ions

and to study the exogenous and endogenous s ignals tha t contribute to

this inflammatory state . Thus , bo th unfrac t ionat ed gingival plaque

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2 0 1 DE 0045 08 LMI

Oppenheim , J . J . , Togawa , A . , Ghedid ,L . , and Mi z el , S . B . :

Component s of mycobacteria and muramy l dipep tide with adj uvant

act ivity induce LAF . Cell . Immunol . In press . 19 7 9 .

Northo ff , H . and Oppenheim , J . J . : Regulat ion o f the product ion o f

lect in induced mitogenic fac tor (LMF ) by human T lymphocytes in

Proc . o f the 13th Leuco cyte Cul ture Conf . Ed . G . Kaplan .

Pub . Elseview No . Holland N . Y . In pres s .

Oppenheim , J . J . : Lymphocyte interact ion with macrophages : In

duct ion o f LAF pro duct ion . In pro c . o f VII I internat . Meet ingo f the RES Soc iety Press . 1 9 78 . In press .

Fox , P . G . and Oppenheim , J . J . : Cel l Mediat ed Immunity for Text

book o f Microb iolo gy with Dental Applications . J . Mc Ghe e ,

and S . Michalick . Pub . Harper and Row . In pres s .

wyler , D . J . , Oppenheim , J . J . and Koont z , L . C . : The influenceo f malaria infec tion on the elaborat ion o f soluble mediators

by adherent mononuclear cell s . Inf ect . and Immunity , 24 : 1 51

15 9 , 1 9 7 9 .

Togawa , A . , Oppenheim , J . J ., Kirkpatrick , G . H . : Ab ility o f dialy

sates containing trans fer factor to induce lymphocyte act ivating

factor by human mononuclear cell s . Cell . Immunol .-141 ,

19 7 9 .

Kabir , S . , Opp enheim, J . J . , and Ros ens treich , D . L . : Murine B

cell mitogenic propert ies o f corn pro t eins . Cell . Immunol . 42

394-404 , 19 7 9 .

Oppenheim , J . J . , Koopman , W . J . , Wahl ,L .M . and Dougherty , S . F . :

Pros taglandin E rather than LAF produced by act ivat ed human

mononuclear c ells s t imulat es increas es in murine thymo cyte CAMP .

Cell Immunol . In press .

Oppenheim , J . J Moore , R . ,GMelig

-Mey ling , To gawa , A . , Wahl ,

S . , Mathieson , B . , Dougherty , S ., and Cart er , C . : Ro le o f

cytokine and endotoxin induc ed monokines in lympho cyte pro lif era

tion , dif ferentiat ion and immunobl obulin production . In regulatoryRole o f Macrophages in Immunity . Eds . E . R . Unanue and A . S . Ro senthal .

Acad . Pres . In press .

Oppenheim , J . J . , Northof f , H . , Greenhill , A . , Mathieson , B . J

Smith , K . and Gill is , S . : Prop er ties o f human mono cyt e derived

lymphocyte act ivat ing factor (LAF ) and lymphocyt e derived mitogenic

f actor (LMF ) . In The Biochemistry o f Lymphokines ,A . De Weck ( ed . )

Pub . Acad . Press , N . Y . In pres s .

Ep stein , L . B . , Goldblat t ,D . , Y u , K . , Dean , J . H . , Herberman , R . B . ,

Oppenheim , J . J . , Rockl in , R . E . and Sugiyama , M . : Mediator product ion

in human mononuclear cell s by strains o f corynebac terium parvum

In The Bio chemis try of the Lymphokines A . De Weck (ed . ) Pub . Acad .

Pres s NY In pres s .

4 - 1 7 6

ZOl DE— OOl3l—05 LMI

OBJECTIV E S

The principal obj ec tives o f this research proj ect are : 1 ) to purify

and C haracter iz e the ant igen nonspec ific lymphokines which modulatethe act ivat ion o f the humoral and cell—mediated immune responses and

2 ) to det ermine the mechanism-o f-act ion o f these so luble factors .

METHODS

S tandard in vitro lymphocyt e culture methods for the induct ion and

as say o f antibody and cell—mediat ed immune responses were ut il iz ed

including the enumeration o f ant ibody forming cell by the Jernehemolyt ic plaque— forming cell (P F C ) assay and the Cr release assay

for quant itat ion of cytotoxic lymphocytes . S tandard cel l s eparation

pro cedures were used to prepare purif ied preparat ions o f B cells ,

T cells , and macrophages .

MAJOR FINDINGS

IL We have previously shown that Con A— s t imulated mouse sp leen cellsproduce a solub le factor (des ignated TME ( IL which exhib it s an apparent

molecular weight of approximately and enhances thymocyt e proliferat ion ,

ant ibody synthesis , and the induction o f cyto toxic lymphocytes . We have

now examined the interact ion between macrophages (MO ) and T cell s in the

product ion of IL 2 and found bo th cell types to be required . Thus , from

these types o f experiment s it has b een impo s s ible to determine the cell

of—origin o f the factor . However , a number o f exper iment s have shown

that the ab ility of purif ied T cell s to produce IL 2 fo llowing s t imulat ion

with Con A is res tored by the add it ion o f nanogram amounts o f phorbo l

myr is t ic acetate (PMA) . Al though the mechanism by which PMA replaces

the requirement for MO is unclear , the results C learly demons trate that

the cell-of—origin of IL 2 is the T cell . Recent experiments al so indicate

that the MO dep endenc ey during the production o f IL 2 can be replacedby supernatants from MO cultures . These result s indicate that act ivat ion

o f T cell s to the product ion o f IL 2 requires at leas t on "po s it ive"

s ignal from the MO and that this s ignal is provided by a soluble product

o f the MO .

F urther s tudies with PMA on unfract ionated spleen cells indicate that the

addit ion of 100 nanograms o f PMA to Con A— s t imulated spleen cell cul tures

enhances the product ion o f IL 2 by 6 to 10 fold . This ob servat ion provides

a s ignif icant advance in our ef fort s to ob tain suf f icient material to

cont inue the purificat ion and charact er izat ion studies .

On the in vivo oduc tion o f L te Ac tivatin Factor

I t has previously b een reported that BCG infected

mice that are adminis tered LP S release into their serum a P F C helper

factor and a tumor necrot iz ing factor . The two factors have been reported

to c o-purify sugges t ing molecular ident ity . We also conf irmed the presence

o f a tumoricidal act ivity in BCG /LP S serum which is presumably ident ical

to tumor necrot iz ing fact or . We therefore inves t igated the b io chemical

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Zol DE OOll3l—5 LMI

amount of IL 2 synergiz ed with IL 1 , maximal synergiz ing late-act ing

TRF act ivity was pres ent in a poo l o f fract ions which containeds ignif icantly les s IL 2 . Further resul t s indicate that IL 2 is

res is tant to pH inact ivat ion whereas at leas t a port ion o f the

lat e—acting synergiz ing TRF act ivity is s ens it ive to pH Thus ,

on the bas is of funct ional and b iochemical evidence , we have concludedthat IL 2 and late-act ing TRF are dis t inct molecular ent it ies . Thequest ion as to the relat ionship b etween TRF and other lymphokines is

under inves tigat ion . In this regard , the pH— sens itive TRF was

found to elute from phenyl sepharos e in fract ions r ich in immune

inter feron .

Finally,another factor which is neither IL 2 , TRF , or inter feron has

been separated on phenyl sepharose . This lat ter factor ( s ) exhib it s no

intrins ic PFC helper ac tivity and does no t synergiz e with IL 1 , but

does enhance the synergism that one ob serves b etween IL 1 and TRF . The

ident ity o f this factor is currently under inves t igat ion .

culture supernatant s were b iochemically analyzed for fac tor ( s ) that

restored the macrophage dependency of alloant igen specif ic cytotoxic T

lymphocyte development . Throughout a sequential C hromatographic

purif icat ion scheme DEAE cellulo se , and PAGE ) factors

that res tored the macrophage func t ion in killer cell development al soc o-eluted with the factor that s t imulated thymocyt e pro liferat ion .

Based on the cellular source (macrophages ) , molecular weight (approximatelyC harge heterogeneity exhib ited on DEAE cellulo se and po lyacrylamide

electrophoreses , and thymocyt e mitogenic act ivity , the factor sub s titut ing

for macrophages in the development o f killer T lymphocytes was ident ical

to IL 1 (LAF) .

S IGNIFICANCE TO BIOMEDICAL RE SEARCH

The inf lammatory response is regulated by a complex mult icomponent network

which includes as its act ive component s ant igen , ant ibody , complement ,

macrophages , helper and suppres sor T cells , and B cells . These regulatorycomponent s serve to control the init iat ion , express ion , maintenance ,

and suppres s ion o f the inflammatory response . S ince periodontal d is ease

is characterized by an inflammatory response within the gingiva and

periodont ium , an unders tand ing o f the regulatory mechanisms involvedshould allow us to intervene in the course o f the inflammatory pro ces s .

S ince lympho—mac rophate interact ion is mediated in part by solub le fac tors

produc ed'

by these cells and s ince the cellular interact ion is a vital part

o f the regulatory network , the elucidat ion o f the mechanism— of-act ion o f

the solub le factors is crucial to our unders tanding o f inflammat ion .

ZOl DE— OOl 3l— 05 LMI

PROPO SED COURSE

In the suc c edding year experiment s will b e conducted in an e ffor t to

1 ) determine if IL 1 is capab le o f replac ing the requirement for MOduring the product ion o f IL 2 ; 2 ) determine the target cell o f bo th

IL 1 and IL 2 in ant ibo dy synthes is and cyto toxic cell induc t ion

sys tems and 3 ) charac teri ze and ident ify the nature o f TRF and the

c o-factor which fac il itat es the synergism between TRF and IL 1 .

PUBLI CATION S

1 . S imon , P . L J . J . Farrar , and P . D . Kind . 19 7 9 . Bio chemical

relat ionship between murine immune inter feron and a killer

cel l helper fac tor . J . Immuno l . 1 2 2 : 12 7— 1 32 .

Hor ton , J . E W . J . Koopman , J . J . Farrar,J . Fuller-Bonar

,

and S . E . Me rgenhagen . 19 7 9 . Part ial purif icat ion o f

a bone— resorb ing factor elaborated from human allogeneic

cul tures . Ce llular Immunol . 43 : 1— 10 .

Mc G he e , J . R J . J . Farrar , S . M . Michalek,S . E . Mergenhagen ,

and D . L . Ro s enstre ich . 19 7 9 . Cellular requirement s for

l ipopo lysaccharide adj uvant ic ity . A ro le for bo th T lymphocytesand macrophages for in vitro respons es to part iculateant igens . J . Exp . Med . 149 : 79 3—807 .

Farrar , J . J P . L . S imon , W . L . F arrar , W . J . Koopman , and

J . Full er-Bonar . 19 79 . Role o f mitogenic factor,lymphocyte

act ivat ing factor , and immune inter feron in the induct ion

o f humoral and cell—mediated immunity . Ann . N . Y . Acad .

S ci . , in pres s .

Farrar , J . J . , M . L . Hil f iker , S . B . Miz e l , and J . Fuller—Bonar .

1 9 7 9 . Are all helper factors for ant ibody synthes is also

mitogenic for T cell s ? Pro c . Second Int l . LymphokineWorkshop . In pres s .

MHnne l , D . N . , J . J . Farrar , and S . E . Mergenhagen , 19 7 9 .

B io lo gical and b io chemical C harac ter izat ion o f cyto toxic

fac tor from mur ine macrophages . Pro c . S econd Int l . Workshop ,

in pres s .

Octob er 1 , 1 9 78 S ep temb er 30 , 1 9 79

ical Ac t ions of Bac terial Endotoxin In vivo and I

N AM ES,L ABOR A TO RY AND I N ST I T UT E AFF I L I AT I ON S , ANO T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND OT H ER

PROF E S S I ON AL PER S ON N EL ENG AG ED ON THE PRO J ECTP I : Ros ens treich , David L .

Other : Mergenhagen , S tephan E .

Weedon , LyndaVogel , S tefanie N .

MHnnel , Daniela

Moore , Rob ertWahl

,Larry

Wahl , Sharon

Oppenheim , Joo s t

COO PER AT I NG UN I T S (if any )Dr . Jean D . S ip e NIAMDD , NIHDr . Carl T . Hans en Divis ion o f Res earch , NIHDr . Zanvil Cohn Ro ckef eller Univers ity , New York , N . Y .

Laboratory of Microbiology and Immunology

[ 1 (b ) HUM AN T I S SU E S Q (c ) N E I TH ER

S UMM ARY OF WOR K (2 00 wo rd s or le s s und e rl in e ke yword s )The LP S-unres

pons ive C3H/ He J mous e strain has been ut il ized to help elucida

the mechanism o f action o f bact erial endo toxin (LP S ) . Thes e mice po s s es s a

defec t in the LP S— respons ivenes s o f their T and B lymphocyt es , macrophages , and

f ibroblas t s that is due to a mutat ion in a s ingle , auto somal c o-dominant gene .

The b io logical ef fect s o f LP S are cell mediat ed because the interac tion o f LP Swith lymphocy tes and/ or macrophages renders C3H /He J mice suscep t ibl e to LP S

by the adop tive trans fer of bone marrow cells from LP S-sens itive mice . C3H /HeJ

immunos timulant. Endotoxic ity may be med iated by a number of LP S— induced acute

phase reactants such as pro staglandins,lymphocy te act ivat ing factor ,

interferon

The LP S gene may al so p lay a

tro l the ab ility o f macrophages

ll as the ab ility o f macrophages

t e c tabl e in the s era of LPS— s ens itive animals .

vital phys iological role s ince it app ears to con

.t o manifest LPS— induced tumor c e l l killing;as we

(Re v. 10-76 )

Medical Of ficer—ResearchChief , Lab . Micro . Immunol .

Biologis t

Po st— Doc toral FellowVis it ing Fellow

S taff FellowSenior S taf f Fellow

Invest igatorMedical Of ficer-Research

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ZOl DE 001 67—03 LMI

the ability to produce serum amyloid as so ciated pro tein . This serum

f actor appears to be a macrophage produc t s ince LP S—s t imulated C3H/HeNmacrophage culture supernatants also res tored to C3H/ HeJ mice the abil ityt o produce SAA . These f ind ings strengthen the hypo thes is that many o f

the ef fect s o f endo toxin are mediated by the init ial act ion o f LP S on

some lympho id cell , presumably the macrophage .

3 ) Another approach has been taken to elucidate the cel lular interact ions

involved in med iat ing endotoxic ity . Infect ion o f C3H /HeJ mice with BCGprior to LP S adminis tration rendered them LP S sens it ive as measured in

vivo by LP S— induced lethality and hypoglycemia , as well as the product ion

o f serum interferon and SAA . In contrast , BCG-infect ion increased ant i

LPS ant ibody product ion only minimally . The ab ility o f BCG to induce

LPS s ens it ivity in C3H/HeJ mice demonstrat es that LPS respons ivenes s in

C3H/HeJ mice is not due to an ab so lute def ect in this s train,but rather ,

a partially rever s ible st ate of hypore sponsivene s s . These f indings alsosuggest that BCG infect ion acts pr imarily to enhance the LP S sens it ivity

o f T cell s and/ or macrophages rather than B lymphocytes . S ince , BCG has

been shown to be a po tent macrophage ac tivat ing agent and appears to

exert its act ion through T lympho cyt es , studies are ongo ing to determine

the role of T cell-macrophage interact ion in mediat ing endo toxic ity . We

have found that if one incubates LP S —unrespons ive C3H/HeJ macrophages in

the presence of a concanavalin A— s t imulated spleen cell supernatant (CS )they will produce lymphocyte act ivat ing factor in response to LPS ,

sugges t ing that a T C el l derived product can render an LP S-unrespons ivemacrophage , LPS—respons ive . Furthermore , pur if ied T cells from LP Srespons ive mice ( including BCG-infect ed C3H /HeJ mice ) , co cul tured with

C3H/HeJ macrophages plus LP S,will produce a cul ture supernatant capable

o f caus ing thymocyt e prol iferat ion . These data st rongly implicat e T

c e llv mac rophage interact ion in endo toxin respons ivenes s . Final ly , work

has b een completed which C learly demons trates that in vitro LPS — inducedadj uvanticity can be rest ored in LP S unrespons ive B C ell cultures

by co culture with responder T cell s and responder macrophages . Taken

collect ively , these dat a support the no t ion that T—macrophage interact ion

i s es sent ial for bo th the humoral and cellular manifes tat ions o f LP Ss ensit ivity . In collaborat ion with Dr . Carl Hansen , we have Characteriz ed

a potentially useful new mouse strain,the C57Bl/ lOSC N We

found that this athymic mouse strain was al so LP S unrespons ive and

b ehaved comparably to the C3H /HeJ mouse strain in vivo and in vitro .

This s train may provide a power ful new tool in the fur ther examinat ion

f or T cell part icipat ion in the init iation o f LP S-induced t is sue dama ge .

4 ) In addition to the LP S defect exhib it ed by C3H/HeJ macrophages,

these cells lose , upon culturing , the ab ility to bind and phago cyto s e

opsonized erythrocytes . Increased phago cyt ic ab ility has long been one

measure o f increased macrophage dif ferent iation . This defect,like the

2 0 1 DE 001 6 7— 03 LMI

LP S def ec t , can be reversed by expo s ing the macrophages to a concanaval inA- s timulated sp leen cell supernatant . Addit ionally

,we have found that

this de fec t can be reversed by dibutyryl— cyclic AMP . The relat ionshipb etween the maintenance o f F C receptor capacity and LPS sens i t ivity willb e ac t ively pursued .

5 ) S ince there app ears to be an int imate relat ionship between macrophage

LPS— sens it ivity and macrophage ac t ivat ion , s ignif icant ef for t has been

put for th to purify macrophage ac t ivat ing factor (MAF ) , a lymphokine

capable o f ac t ivat ing res ident peritoneal macrophages to become tumor

ic idal or capable o f kil ling intracellular paras i tes . The puri fication

s cheme employed result s in a 75— fold puri f icat ion and cons is t s o f an

ammonium sul fat e prec ip itat ion followed by Chromato graphy on Sephadex

G 100 and DEAE cellulo se . Pur if ied murine MAF exhib it s a molecular

weight o f approximately daltons and exhib it s C harge heterogeneity .

Purif ied MAF preparat ions will act ivate res ident peritoneal macrophages

to produce detect abl e level s of H O and to cause the intracellular

killing o f the paras i te Tgypanosoma cruzi . In ad dit ion to C haracteri z ing

the molecular nature o f an extremely important lymphokine . these s tudies

should help elucidate the relat ionship between LP S sens it ivity and

macrophage ac t ivat ion . Our studies to date s trongly sugges t a vitalro le for the LP S gene in ma intaining ho s t def ense mechanisms and the

homeos tat ic control o f the immune sys tem .

S IGNIFICANCE TO BIOMEDICAL RESEARCH

Bacterial endo toxins are ub iquitous in nature . Because o f their unique

ability to ac t ivate B lympho cyt es and macrophages , it i s po s tulated that

they may be es s ential to the no rmal maturat ion o f the immune sys t em .

Furthermore,because o f their diver se propert ies , they have b een impl icat ed

as C ausat ive factor s in certain patho logical condit ions such as gram

negat ive shock . Our find ings are therefore important to biomed ical

r es earch for two reasons . First , they help us to under s tand the pathways

by which B lymphocyte s are induc ed to grow and divide . S econdly , they

help us t o unders tand the patho genes is o f the toxic ef fect s o f LP S , and

sugges t po ss ible modes o f therapeut ic int ervent ion .

One o f the maj or goal s o f the NIDR is to understand the et iology o f

p er iodontal d is ease (PD ) and to explore methods to prevent or eliminate

this cond it ion . S ince PD i s due to chronic inflammatory re sponses in

the gingiva,and s ince endo toxins from oral flora are abundant in the

mouth,it is important to understand how these endo toxins act ivate

lympho id cells to incite and prolong the immune respons e that resul t s in

PD .

PROPOSED COURSE

l ) The role o f macrophages in mediating the ef fects o f LP S will be

2 01 DE 001 6 7-03 LMI

pursued . Our maj or thrus t will be to evaluat e the ext ent to which T

lymphocyte-macrophage interact ion underlies LP S sens it ivity and how this

respons ivenes s is related to macrophage act ivat ion . Having p er fected

our t echniques for the adopt ive trans fer of lymphoid cells, we will now

attempt to trans f er specif ic cell populat ions such as macrophages and B

and T lymphocyte populat ions to determine specif ic cellular requirements

for LP S s ens it ivity . We will measure LP S sens itivity in vivo by measuring

lethality , hypoglycemia , and the ab il ity to produce ant i-LPS ant ibodies

in response to LP S and by measuring the appearance o f int erf eron , CSE ,

or SAA in the serum following LP S administrat ion .

2 ) We will try to det ermine the extent to which macrophage act ivat ion

and LPS s ensit ivity are related , using F c — recep tor funct ion as a marker

for macrophage di f fer ent iat ion . We will pursue an analys is o f the

act ive agent ( s ) in concanaval in A s t imulated spleen cell supernatant

which correct s bo th the LP S defect and the F C receptor defect in C3H/HeJmacrophages .

3 ) The role of speci fic macrophage product s in manif es tat ions o f LPSs ens it ivity will be pursued . The po tential ro le o f LAF as the inducer

o f serum amylo id as so ciated prot ein will be ass es sed .

PUBLICATIONS

Ro senstreich ,D . L : The biological func t ion o f the LP S gene : In

Biology of the Inbred Mouse . S . Morse Academic Pres s .

N . Y . , N . Y . P . 45 7 . 1 9 78 .

Ro sens treich ,D . L . ,

Vogel, S . N . Jaques ,

A . , Wahl , L . N . , Scher , I .

and Me rgenhagen ,S . E . : Dif ferent ial endo toxin sens it ivity o f lympho

cytes and macrophages from mice with an X -linked defec t in B— cell

maturation : J . Immunol . 19 78 .

Ro senstre ich ,D . L . , Vogel , S . N . , Jaques , A . R . , Wahl , L .M . and

Oppenheim, J . J . : Macrophage sens it ivity to endotoxin : genet ic

contro l by a s ingle codominant gene . J . Immuno l . 1 9 78 .

Ruco , L . P Melt zer , M . S . , and Ros ens treic h , D . L . : Macrophage

act ivat ion for tumor cytot oxicity : control o f macrophage tumoricidalcapacity by the LP S gene . J . Immunol . 1 9 7 8 .

V ogel , S . N . , Hansen , C . T . and Ros ens tre ic h : Charact erizat ion o f

a c ogenitally LP S—res istant , athymic mouse s train . J . Immunol .

1 2 2 : 6l 9 , 19 7 9 .

V o gel , S . N Marshall, S . T . , and Ros enstreich , D . L . : Analys is o f

the ef fect s o f lipopolysaccharide on macrophages : Dif ferent ial

phago cyt ic responses o f C3H /HeN and C3H/HeJ macrophages in vitro .

Inf e c . Immun . 1 97 9 .

Mc Ghe e , J . R . ,Michalek

, S . M . , Moore , R . N Mergenhagen , S . E . and

Ro senstreich ,D . L . : Genet ic control o f in vivo sensitivity to

D T H E

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Octob er 1 ,1 97 8 S ept emb er 30 ,

19 7 9

N AM ES,L ABOR ATORY ANO I N ST I T UT E AF F I L I AT I ON S , A ND T I T L E S OF PR I NC I PAL I NV E ST I G ATOR S AN D ALL OT H ER

PROF E SS I ON AL PER SON N EL ENG AG ED ON THE PRO JECT

PI : Miz el ,S teven B . S enior S taf f F ellow

OTHER : Miz e l ,Diane Chemist

B en-Zvi,Amo s Vis it ing Assoc iat e

Sando,Julianne Pos tdoct oral Fellow

[j (b ) HUM AN T I S SU E S a (o ) N E I TH E R

e b een

X with a yield of Chemical stud ies have revealed the importance o f argenine

groups in LAF act ivity . B iolog ical studies have demonst rat ed that LAF enhances

l) thymocyt e prol if erat ion ; 2 ) ant ib ody synthe s is _ip_

v itro ; 3 ) the g enerat ion of

5 ) the ggnerat ion o f stab le E— ros et te forming human T cell s and 5 ) col lagenas e

and prostaglandin product ion by rheumat oid sypovial cells . Thus LAF may play

an important , if not es sent ial ro le in T cell act ivat ion for humoral and c ell

mediat ed immune respons es as well as in inf lammatory respons es such as in thej o ints o f pat ient s with rheumato id arthrit is .

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OBJECTIVES

The maj or obj ec t ives of this ongo ing proj ect are : l) to pur ify to

homog eneity the macrophag e—der ived Lymphocyt e Act ivat ing Fact or (LAF )which app ears to p lay an important , if not es s ent ial ro le in T lymphocyt eact ivat ion ; 2 ) to b iochemically C harac ter iz e the LAF ; 3 ) to prepare a

mono spec if ic ant is erum to the pur if ied LAF ,and 4 ) to charact er iz e the

S p ec trum of b io log ical act iv it ies of LAF and the mechanism s that r egulat eLAF product ion .

MAJOR FINDINGS

1 . B io chemical S tud ies on LAF

We have cont inued to us e the mur ine macrophag e cell line, P388D as a

c ell sourc e o f LAF and phorbo l myr is t ic ac etate (PNA ) as the s t imulant

for LAF product ion and s ecr et ion . The maj or f ind ings are as follows

1) Part ia lly pur if ied LAF was prepar ed by ammonium sulfat e frac t ionat ion ,

and C hromatography on DEAE cellulos e,S ephacryl 8 200 ,

and phenyl S epharo s e ,

and elec trophor es is in 102 Tr is— glyc inat e discont inuou s po lyacrylamid eg els . The increa s e in specif ic ac t iv ity was approximat elyfo ld with a yield of At a minimum

,the LAF was 5% pur e

,b eing one

of 13 pro t eins det ect ed by two d imens ional polyacryl amid g el electrophor es is

and staining with a method sens it ive t o ng prot e in /mm gel . Aproj ec t ed spec if ic ac t iv ity of l—3 x 10 unit s /mg prot ein was es t imat ed

for a pur if ied preparat ion of LAF . Us ing pr eparat iv e iso e le c tro f ocu s ing

and high p erformance l iquid C hromatography we should soon approach this

valu e . Of maj or importanc e is our ab ility to now as soc iat e a s ingleprot ein band detec t ed on the gels with the LAF b io log ical ac t ivity .

This is a new development in the f ield of lymphokines .

2 ) Inj ec t ion o f rabb it s w ith highly pur if ied LAF d id no t result in the

produc t ion of act ive ant i—LAF sera . We ther ef ore examined the pos s ib il ity

that avian s might be a mor e responsiv e sp ec ies . We have rec ent ly obtained

s era from chickens immuniz ed with LAF in complet e Freund 's adj uvant .

Although of relat ively low pot ency (act iv e l / lO dilut ion) thes e s era

exhibit ed some inhib itory ac t iv ity on LAF—med iat ed thymocyt e pro lif erat ion ,

wher eas the prol if erat ion in respons e t o an unrelat ed T lymphocyt emit ogenic fact or was unaf f ect ed . We are ther efore cont inuing to boo s t

the chicken with LAF in an ef f or t to increase the t it er of the s erum .

The ac t iv e mater ial in this s erum is no t ab sorb ed by the target thymocyt es

and copur if ies with IgG .

3 ) S tud ies on the chemical nature of LAF have b een cont inued . In

pr eviou s stud ies,we demonst rated that LAF was relat ively res istant to a

number of endopep t ida ses,includ ing chymotryp s in

,papain , and tryp s in ;

reduct ion and alkylat ion ; and irrevers ible denaturat ion by urea and

sod ium dod ecyl sulfat e . The s e result s were int erpret ed to indicat e that

LAF was a relat ively inf lex ible polypep t id e . In order to more pr ec is ely

def ine the st ruc tural requir ement s f or LAF act ivity , we init iat ed studies

on the pos s ible ro le o f S p ec if ic amino acid s . LAF act ivity wa s decr ea sed

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in a t ime dependent manner by the argenine-spec if ic mod ifying ag ent ,

phenyl glyoxal,ind icat ing the funct ional importanc e of arg enine in LAF

act ivity . The ess ent ial arg inine res idu es mod if ied by phenyl glyoxal

must be in the int ernal port ion of the molecule , perhaps in the active

s it e,s ince their pres ence on the external reg ions of LAF should have

made them acces s ible to tryps in—med iat ed C leavag e and LAF inact ivat ion .

Pheny lgly oxal-treated LAF was not only les s act ive as a st imulant for

thymocyt e pro l if erat ion,but was also les s act ive in the induct ion of in

vitro ant i— sheep red b lood cell p laque f orming cell responses and the

enhancement of rheumato id synovial c ell collagenase product ion ( see

proj ec t report on ACTIVATION OF RHEUMATOID SYNOV IAL CELLS BY LYMPHOCYTEACTIVATING FACTOR) .

In contrast to pheny lglyoxal t reatment of LAF ,diethy lpyroc arbonat e

modif icat ion o f hist id ine res idues result ed in only a 2 52 reduct ion in

LAF act iv ity,sugg es t ing that hist id ine res idues may p lay a les s cr it ical

rol e in the act ion of LAF . Cyanog en bromide treatment of LAF did not

g enerate any act ive f ragments,sugg es t ing that the act iv e port ion of LAF

may be dependent on the total integr ity of the mol ecule or alt ernat ively ,

methionine groups are lo cat ed in the act ive s it e of LAF .

I I . B io log ical Stud ies on LAF Act iv ity

In previous studies , we Charac teriz ed the eff ect s of P388D c ell der ivedLAF on thymocy te pro lif erat ion . In more recent s tudies

,we have f ound

that LAF may play a mor e g eneral role in T lymphocyt e ac t ivat ion and

also in non lympho id c el ls act ivat ion of the inf lammatory response . Ourmaj or f indings are as fo llows

1 ) In conj unction with Drs . J . J . Farrar and M . L . Hil f iker of our

laboratory we have f ound that LAF synergiz es with several other lymphokines

(at least two) in the induct ion of T c ell-dependent in vitro ant ibody

r esponses . The nature of these factors and their r elat ion to LAF are

curr ent ly b eing invest igat ed .

2 ) The in vitro pro lif erat ion of pr imed mur ine lymph node lymphocyt es

(LNL ) in respons e to the so luble ant ig en ovalbumin (OV A) wa s dependent

upon the presence of per itoneal adherent cells . Restorat ion of OVAinduced LNL prol if erat ion could be achieved by addit ion of highly purif iedLAF . LAF did no t st imulat e LNL prol if erat ion in the abs ence of the

priming ant ig en or T c ell s . Furthermor e,t reatment of LNL with ant i

macrophag e s erum completely blocked the ab ility of these cells to respond

t o OVA LAF , sugg est ing that the res idual macrophag es in the LNL populat ion

were nec es sary to provid e an addit ional funct ion or s ignal,pos s ibly

ant ig en presentat ion , in conj unct ion with LAF . Thes e data ther efor e

support the two s ignal hypothes is of macrophag e—med iat ed lymphocyt e

act ivat ion and demonstrate the ab ility of LAF to provide one of these

s ignals . F urthermor e , these result s indicat e that one component o f the

amplify ing eff ect of LAF on antigen— spec if ic immune react ions may invo lveT c ell pro lif erat ion ;

3 ) In conj unct ion with Dr s . W . F . Farrar and J . J . Farrar in our laboratory,

we have found that LAF init iat es the act ivat ion of alloant ig en— specif ic

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ZOl DE— 00209— 03 LMI

enhanc ed by a factor obt ained from rabb it mononuclear c ell s . In V iew of

our result s,it is qu it e pos s ible that this fact or is also related to

LAF . Thus LAF may p lay a general ro le in in vivo inf lammatory react ions .

III . S tudies on the Regulat ion of LAF Produc t ion

In prev ious stud ies,we danons trat ed that the act ivat ion of LAF product ion

and secret ion by phorbo l myr ist ic acetat e (PMA) a tumor promo t er and

inf lammatory ag ent,invo lved the intracellular conver s ion of LAF from a

high molecular weight,relat ively inact ive form to a low molecular

weight form that was subsequently secr et ed by the macrophag e . At present ,

Dr . Jul ianne Sando,a po stdoct oral f ellow who j oined our laborat ory in

Apr il,197 9 ,

is engag ed in studies on the pr ec is e meghanism (s ) by which

PMA act ivat es the macrophag e to release LAF . Us ing H— PNA , we have

establ ished the pres ence of a b inding receptors on P388D c ell line

macrophag es . The b ind ing of H—PMA to the target cells can be blocked

by PNA—analogs in direct proport ion to their tumor promo t ing and inf lamma toryac tiviti s . For example

,the inact ive tumor promot er

,phorbo l , d id not

inhibit H—PMA b inding,but phorbo l didec anoat e and phorbo l dib en z oate

which are act ive inf lammatory agent s did block H—PMA b inding t o P388D

sel ls . These result s indicat e the pharmacolog ical S pec if ic ity of the

H-PMA b inding . We ar e also exam ining the eff ect of PMA antagonist s ,

such as ret ino ic ac id , on PMA- induced increases in LAF product ion . Such

compounds should provide addit ional tools with which to probe the fundamental

mechanisms involved in macrophag e ac t ivat ion .

S IGNIF ICANCE TO BIOMEDICAL AND DENTAL RESEARCH

Although it is well accept ed that the immune sy st em plays a dominant

role in a variety of inf lammatory reac t ions,the under lying mechanisms

are not well understood . S everal proj ect s which have been undertaken in

the LMI /NIDR , are unit ed by a common ef fort to under stand the role ( s ) of

lymphokines in immunological funct ion . It is only through an und erstand ing

o f macrophag e and lymphocyt e funct ion at the cellular and subc ellu larand molecular level s that we will be able to rat ionally manipulat e the

immune syst em f or therapeut ic purpos es . In add it ion,the under stand ing

o f the mechanism ( s ) by which PMA ,a pot ent inf lammatory ag ent

,act ivat es

macrophag es would prov id e a foundat ion for a mor e general under standing

of inf lamma t ion , esp ec ially as it appl ies to per iodontal dis ea se .

Our studies on the role of LAF in lymphocyt e and synovial c ell act ivat ion

cl early rais e the po s s ib il ity that this factor may be a crit ical in v ivo

r egulatory control mechanism in inflammat ion and immune reac t ions,both

humoral and cellular .

The increa s ing at t ent ion g iven to lymphokines at int ernat ional meet ing s,

such as the Second Int ernat ional Lymphokines Conf erenc e at which IC ha ir ed a sess ion on macrophag e and lymphocyt e act ivat ion

,and in new

j ournals , such as Lymphokine Repor t s (J . J . Oppenheim and I b eing member s

of the Ed itorial B oard) , is ev id ence of the increa s ing awarenes s of the

importance of these sub stances in inf lamma t ion and human c ell funct ion .

lI 1 0’)

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PROPOSED COURSE

1 . S tud ies on the pur if icat ion and C haract er iz at ion of LAF wil l be

cont inued . We ar e current ly select ing for const itut ive mutant s of

P388Dlc ell s tha t make increased amount s of LAF .

2 . As pur if ied LAF b ecomes available we plan to sequ enc e the fac tor

and po s s ibly synthes iz e the ac t ive por t ion of the molecul e .

3 . Stud ies on the preparat ion of ant is era wil l b e cont inued u s ing

C hickens as a sour c e of serum .

4 . S tud ies on the b io log ical ac t ions wil l be cont inued and ext ended .

We int end to probe mor e deep ly into the mechanism ( s ) by which LAF act ivat es

T c ell s in conj unct ion with ant ig en . In add it ion,we p lan to examine

the synovial f lu id of pat ients with rheumato id ar thr it is for the pres ence

of LAF . The level s of LAF will then b e correlat ed with the patho log ical

s ituat ion in the j oint s .

5 . The mechanism o f ac t ion of PMA on LAF roduct ion will be stud ied in

cons id erable detail . B inding stud ies with H— PMA should lead to the

iso lat ion of the component ( s ) involv ed in the trigger ing o f macrophag e

act ivat ion and LAF r elease .

PUBLICATIONS

l . Miz al,S . B . , Ros enstreich ,

D . L . , and Opp enheim , J . J . 1978 .

Phorbo l myr ist ic ac etate st imulat es LAF product ion by the macrophag e

c ell line, P3 88D

1. Cell . Immunol . 40 : 230— 23 5 .

2 . Miz e l ,S . B . 197 9 . Phys icochemical C harac t er iz at ion of lymphocyt e

act ivat ing fact or (LAF ) . J . Immunol . 12 2 : 21 67-2 17 2 .

3 . Miz e l,S . B and D . L . Ros enst reich . 197 9 . Regu lat ion of lymphocyt e

act ivat ing fact or (LAF ) produ ct ion and s ecret ion in P 388D1c ell s

Ident if icat ion of high mo lecular weight precursor s of LAF .

J . Immuno l . 12 2 : 2 17 3— 2 1 7 9 .

4 . Togawa,A J . J . Oppenheim and S . B . Miz e l . 1 97 9 . Charact er izat ion

o f lympho cyt e act ivat ing fac tor (LAF ) produced by human mononuclearc ell s : B iochemical relat ionship of high and low mol ecular weight

forms of LAF . J . Immunol .,12 2 : 2 11 2-2 118 .

5 . Miz el,S . B . 1 97 9 . B io chemica l and b iolog ical charact er iz at ion of

lymphocyt e act ivat ing f act or (LAF ) produc ed by the mur ine macrophag e

c ell line, P388D

lAnn . N . Y . Acad . S c i in pr es s .

6 . Niz el , S . B . 197 9 . B io chemical charact er ist ic s of macrophag e der ived

lympho st imulatory f ac tors . In Mol ecular Med iators of CellularImmunity : Charact er iz at ions and Act ions . J . W . Hadden and

W . E . S t ewart II . Ed s . Humana Pr es s . Cl if ton,N . J .

,in pr es s .

11 - 1 9 3

13 .

14 .

2 0 1 DE— 002 09—O3 LMI

Oppenheim,J . J . ,

A . Togawa,L . Chedid and S . B . Miz e l . 197 9 .

Component s of mycobac t er ia and muramy l dipep t ide with adj uvant

act iv ity induc e lymphocyt e act ivat ion fac tor . Cell . Immunol .,

in press .

Miz el,S . B . ,

J . M . Dayer , S . M . Krane , and S . E . Merg enhag en .

1 97 9 . In v itro st imulat ion of rheumato id synov ial c ell co llag enas e

and pro staglandin product ion by lymphocyt e ac t ivat ing fact or

( Int erleuken Manuscr ip t in preparat ion .

Miz e l ,S . B . and A . B en-Zv i . 197 9 . S tudies on the ro le of lymphocyt e

act ivat ing fact or ( Int erl eukin 1 ) in antig en— induced lymph nod e

lymphocyt e prol if erat ion . Manu scr ipt in preparat ion .

Mi z e l,S . B . 197 9 . S tudies on the purif icat ion and structure

funct ion relat ionship s of lymphocyt e act ivat ing fact or ( Int er leukinMol . Immunol .

,in pres s .

Farrar , W . F . , S . B . Miz e l,and J . J . Farrar . 197 9 . Init iat ion

o f alloant ig en sp ec if ic cytotoxic T cell responses by lymphocyt e

act ivat ing fact or ( Interleukin Manuscr ipt in pr eparat ion .

Niz el , S . B . 197 9 . Charact er ization of lymphocyt e act ivat ing fact or

obtained from the mur ine macrophag e cell line , P388D In

Proceed ings of the S econd Int ernat ional Lymphokine Conf er ence .

A . L . deWe C k , ed . Acad . Pres s,N . Y in pr es s .

Farrar , J . J . , M . L . Hilf iker,S . B . Miz e l and J . Fuller-Bonar .

1 97 9 . Are all help er fac tors for ant ibody synthes is also mitogenic

f or T c ell s ? In Proc eed ings of the Second Int ernat ional LymphokineConf er ence . A . L . deWe C k , ed . , Acad . Pres s . , N . Y in pr ess .

Ro senstreich , D . L . and S . B . Miz e l . 197 9 . S ignal requir ement s

for T lymphocyt e act ivat ion . I . Replac ement of macrophag e funct ion

with phorbo l myr ist ic acetate . J . Immunol . in pres s .

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Zol DE7 002 38-02 LMI

OBJECTIVE S

The maj or obj ect ive o f this proj ect is to invest igate the nature o f

regulatory mechanisms funtioning in an inflammatory response to controlproduct ion o f b iologically ac t ive mediators . Part icular emphas is is

placed on understanding the interact ion b etween macrophages and bacterialendotoxin resul ting in product ion o f these mediator s .

MAJOR FINDINGS

During the pas t year several aspect s o f macrophage responses to LP S have

been invest igated in in vitro cul ture sys tems . The resul t s o f these

experiments are summarized later in this sect ion . Several in vivo

experiments , however , have also been performed which merit at tent ion

Primarily these in vivo s tudies have fo cused on the cellular source o f

both CSF and interf eron in endo toxin treated mice . S ince ma crophages

have b een shown to produce bo th mediators in vitro it has been as sumed

that these cell s are a main source o f both CSF and interf eron in vivo .

In our studies an att empt was made to val idate these conclus ions . For

this purpos e a syst em ut iliz ing responder and nonresponder C3H mice was

employed . The C3H/HeJ s train produced neither inter feron or CSF when

C hallenged with endotoxin while the C3H/HeN mice responded to endo toxin

in normal fashion producing det ectable serum level s o f bo th mediator s .

Permanent chimeras were made between these mouse s trains by a cqmbination

o f lethal irradiat ion ( 85 0R) and bone marrow recons t itut ion (10 cellsi .v ) , and after 4 weeks the chimeric mice were C hallenged with endotoxin .

Briefly , the irradiated C3H/HeJ mice recons t ituted with responder C3H/HeNbone marrow produced bo th CSF and interf eron . While these resul ts do

not conclus ively prove that macrophages are the maj or source o f interf eron

and CSE in endotoxemic mice , they C learly demonstrate that the lymphoret icularsystem contro ls the product ion of bo th factors in response to the toxin .

Def inite progress has also been made in unders tanding regulatory mechanisms

funct ioning to influence mediator product ion by endo toxin-s t imulatedmacrophages . In general PCB has been found to be suppres s ive while CSF

appears to augment macrophage funct ions . Result s ob tained with PG E are

summarized as follows

1 ) Addit ion o f indomethacin (an inhib itor o f pros taglandin synthes is ) to

res ident peritoneal leuko cytes and to their adherent subpopulat ions(approximately 852 macrophages ) at the time of endo toxin challengeresul ted in a marked enhancement of CSF product ion . S ince indomethacin

neither influenced the as say for CSF nor inhib ited product ion o f suppres s ive

factors by endotoxin-tr eated cell s,these result s were taken to indicate

that pros taglandins produced by endo toxin-s t imulated cell s suppres s CSE

product ion . This was conf irmed by adding exogenous pro s taglandins to

endotoxin-s timulated cul tures in the presence o f indomethacin . PG E , but

not PGE reversed the enhancement o f CSF product ion by indomethacin .

II— I S E

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S ince CSF funct ions to enhance clonal prol iferat ion and dif ferent iat ion

o f granulocyte—macrophage s tem cell s , these resul t s sugges t that RG B , by

suppres s ing CSE product ion , may serve to limit the myelopo iet ic response

elicited by inf ect ion and / or inflammation .

2 ) Phys io logical concentrat ions o f PGE have also b een found to inhib it

intgrf eron produc t ion by endo toxin— s t imulated macrophages . As l it tle as

1 0 M PG E or P G E inhib i ted inter feron produc t ion by approximately SOZ .

Addit ion o f indomethacin to endotoxin— st imulated macrophages tended to

increas e the interf eron yield suggest ing that endogenous P G E synthes is

may also serve to limit interferon produc t ion as wel l as CSF .

As s tat ed ear lier , CSE unl ike PGE has been found to b e s t imulatory

rather than suppres s ive in thes e studies . Summar ies o f our result s with

CSF are dis cuss ed as fo llows

1 . Macrophages treated with L9 9

cell condit ioned medium (LCM) and

supernatant from concanaval in A (Con A) s t imulated sp leen cell s produced

lymphocyte act ivating fac tor (LAF ) . Bo th o f these condit ioned media

contained C SF and macrophage growth fac tor (MCF ) ac t ivit ies . Treatment

with anti—CSF ant is erum inhib ited LAF product ion s t imulated by the LCM ,

suggest ing that CSF was the agent respons ibl e for inducing LAF . Moleculars ieve chromatography o f bo th the LCM and Con A sp leen preparat ions

resulted in elution pro files , indicat ing that CSF and MCF are ident icaland that CSF /MCF direct ly s t imulat es macrophages to produce LAF . This

ob servation is the f irs t to dat e demons trat ing that CSF serves a direc t

aff erent func tion in the immune response .

2 . CSF r ich LCM has been found not only to direct ly s t imulate macrophage

LAF product ion but al so to augment LAF product ion by endo toxin— s t imulatedmacrophages . This enhancement has been demons trated with macrophages

from normal mice , the PU— S , macrophage cell l ine and al so with

C3H/HeJ mouse macrophages which are normally unrespons ive to bact erialendo toxin . Al though prel iminary in nature

,thes e result s suggest that

CSF enhances the response o f mur ine macrophages to LP S .

3 . The ef fect o f bact er ial endo toxin on CSF (MCF ) induced macrophage

pro liferation has b een inves t igated . Macrophage co lony format ion in the

presence o f LCM was s ignif icantly inhib it ed by bac ter ial endo toxin .

This inhib it ion,however

,was b iphas ic in nature , i. e . , the inhib itory

ef fec t o f endotoxin increas ed with increases in CSF do sage while smalldos es o f LP S in the presence o f minimal CSF t ended to increase macrophage

pro liferat ion . The inhib itory ef fec t o f LP S on macrophage prol iferat ion

was not due to cyto toxicity and was no t mediated by pro s taglandins .

Although it is impo s s ib le at pres ent to relate these f indings to phys iolo gical

s ituat ions , these resul t s (as in 2 above ) suggest that CSF sens it iz es"

macrophages to the ef fect s of endot oxin .

4 . The ef fect on CSF or inter feron product ion by endo toxin s t imulated

macrophages has also been inves t igated . Unl ike LAF , s imultaneous

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addition o f LCM and endotoxin to macrophages tended to suppres s interferon

product ion . Preincubat ion o f macrophages with LCM for 7 2 hr , however ,

s ignificant ly increased inter feron production by these macrophages .

These preliminary resul t s again sugges t that CSF sens it iz es

macrophages to endo toxin .

A s imilar sys tem sugges ting enhanced respons ivenes s o f CSE treated

macrophages has b een ut il iz ed by treating LCM— C loned macrophages with

endo toxin . Macrophages propagated with LCM CSF (MCF ) were found to be

exquis itely sens it ive to bacter ial endo toxin . On a per cell bas is ,

these cells produced at least 10 t imes as much interf eron in response to

endo toxin as control exudate macrophages .

S IGNIFICANCE TO BIOMEDICAL RE SEARCH

Macrophages expos ed to inflammatory s t imul i such as bacter ial endotoxin

are known to produce sub s tances which have profound ef fect s on surrounding

t is sues and o ther cell s o f the immune sys tem . The experiments des cribed

above represent an at temp t to ascertain regulatory mechanisms funct ioning

to control macrophage responses to endotoxin . In br ief,two separate

controlling factors have been tentat ively ident if ied . E pro staglandinshave been found to limit macrophage CSF and interf eron product ion whileCSF has b een shown to directly st imulate LAF product ion . Preliminarydata sugges t ing that CSE enhances endotoxin respons ivenes s o f macrophages

have also been ob tained . These ob servat ions may prove to be o f part icular

importance since bo th PGE and CSP are product s o f inflammatory and

immunological react ions . Tentatively CSF and PCB may act to regulate

these responses by serving as either enhancing or l imit ing S ignal s .

PROPOSED COURSE

The roles o f CSF and PCB as po tent ial regulators of macrophage funct ions

will be inves t igated . With regard to PG E , s tudies will be init iated to

determine if the suppress ive ef fect on CSF and int erferon product ion is

mediated by alterat ions in intracellular cyclic nucleot ide levels .

Preliminary observat ions regarding CSF ac t ivit ies will b e invest igated

extens ively in an ef fort to det ermine if the act ive endotoxin— enhancing

principal in L cell condit ioned medium is indeed CSE .

S ince macrophages o ccupy a central role in virtually all immune responses ,

b iochemically C haract er iz ed CSF preparat ions will be utilized to determine

if CSF , through it s macrophage st imulat ing propert ies , can affect either

the init iat ion or express ion of immune responses in vitro . Experiment s

will alSo b e des igned to inves t igate a po tent ial ro le for CSF in influencing

macrophage— f ibrob last interact ions . Product ion o f fibrob last ac t ivat ing

factor and/ or collagenase by CSF st imulated macrophages would sugges t a

cooperat ive amp lication mechanism between macrophages and fibroblas t s inwound healing and repair .

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October 1 , 19 7 8 through September 30 , 1 97 9

N AM ES,LA BOR ATORY AN D I N ST I T UT E AFF I L I AT I ON S , AND T I T L ES OF PR I NC I PAL I NV EST I G ATO R S AND ALL OT H ER

PR OF E S S I ON AL P ERS ON N EL ENG AG ED ON THE PRO JECT

PI J . T . Hof feld Dental Of f icer

OTHER : Z . Metz ger Vis it ing Fellow

J . J . Oppenheim Med ical Of f icerS . M . Plunka COSTEP Dental Of f icerP . Fox Dental Of f icer

Laboratory o f Microb io lo gy and Immunolo gy

Cellular Immunology

NIDR ,NIH

,Bethesda , Maryland 20205

HUM AN T I SS U ES N E I T H E R

SUMM AR Y OF WOR K (200 wo rd s or le ss und e rl in e ke yword s )

(OBERG) (viz ,superoxide

,hydroxyl

,s inglet oxygen and hydrogen peroxide ) in

inflammat ion by using three in vitro model s o f inflammatory cellular interac ti

We have S hown that the radical s cavenger , reduced glutathione is a maj or po s it

det erminant o f the ab ility o f murine spleen cell s to produced an ant ibody

respons e,in vitro . We have found that the enhancement o f the ant ibody re spon

can b e mimicked by the addit ion o f other known scavenger s o f ODF RC . Macro

st imulated in vivo , can suppres s lect in— induced 1

synthes is and s cavenger s o f ODF RC has provided evidence that the suppress ion

o f lympho cyte pro liferat ion by macrophages is produced by a synergy o f

pro staglandins and ODFRC ; s imilar experiment s have provided evidence that the

suppres sion o f fibrob last prol iferat ion by macrophages is mediated by ODF RC alo e

Thus ODFRC appear to be important in the modulat ion o f inflammatory cellular

interactions .

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(Rev. 10—76 ) LI 2 r.) 0

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OBJECTIVES

1 . Demonstrat e the deleterious ef fect s on mammal ian cell s , in vitro o f

spontaneous ly generat ed ODF RC .

2 . Delineate the interact ions o f pro s taglandins and ODFRC in inflammat ion

model sys tems , in vitro .

3 . Def ine bo th ef fect ive st imulant s for the product ion o f and s cavenger s

against the eff ec ts o f ODFRC .

4 . Modulate the negat ive ef fect s o f ODF RC by the use o f agent s which

either neutralize the act ivity o f or st imulate the product ion o f ODF RC .

METHODS EMPLOYED

1 . In vitro model syst ems

a) Primary ant ibody response o f murine S pleen cell s,as de s crib ed

by S chre ier and Nordin .

b . Lect in— induced prol if erat ion response o f murine sp leen cell s ,

as init iated by concanavalin A .

C . S erum—induced pro li ferat ion o f mouse dermal fibrob las t s,

as des cr ibed by Marcelo e t al .

2 . As say for glutathione : the spectro fluorometr ic methods o f Hissin

and Hilf were used .

3 . S t imulat ion , in vivo , o f suppres s ive macrophages : intraperitonealinj ect ion o f heat killed bact er ial cell s (primarily Corynebacterium

parvum) or thioglycolate bro th .

4 . As say o f primary ant ibody response : Cultured cell s were collectedand washed . The numb er o f antigen— sp ecif ic ant ibody forming cel l s was

determined by the plaque— fo rming cell as say o f Jerne and Nordin .

5 . As say o f lympho c y t e / fibroblast pro l if erat ion response : Cul tured

cell s were puls ed with tr it iated thymidine s ix hours (lymphocytes ) or

twelve hour s ( f ibroblas t s ) prior to t erminat ion o f the culture period .

The cells were co ll ected on f ib erglas s fil t ers and the incorporated

radiolabel was determined by a sc int illat ion counter .

MAJOR FINDINGS

Macrophages and polymorphoneuc l ear leukocytes produce exces s ive quantitit e s

of oxygen— der ived free radical compounds (ODF RC ) as a mechanism o f

kill ing phago cyto sed cell s . Bo th in the normal resp ir ing cell and in

the s t imulated phagocyte , the probable sel f— des truc t ive ef fect o f ODF RC

is prevented by endogenous scavenging mechanisms (glutathione redoxcycle

, superoxide dismutase , catalase , a to copherol,trans f err in

c aerulop lasmin ,

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The primary ant ibody response is apparent ly the mo st sensit ive in vitro

model which we have cho sen to use , s ince an ef fect on any of the requisite

complex cellular interact ions will diminish antibody product ion . Acrit ical variable in this sys tem is the part icular commercial lo t o f

fetal cal f serum (F CS ) used in the culture medium . Us ing a quantitativelysuper ior culture syst em , we have conf irmed and extended our earl ier

f indings concerning the funct ion o f the radical scavenging glutathioneredox cycle in this model . We have shown that the reduced glutathione

(G SH ) content o f F CS is a maj or det erminant of the antibody response o f

murine spleen cells , in vitro . F urthermore , the enhancement o f this

ant ibody response by 2-mercap toethanol ( 2—ME ) is mediated by G SH in FCS .

We have propo sed that the act ion of G SR in this sys t em is the pro tect ion

o f the responding cell s from endogenously— produced ODFRC and from

autoxidat ion . In support of that propo sal , we have shown that spec if ic

scavengers of ODF RC can mimic the G SH-mediated enhancement ef fect and

can act addit ively with subop t imal dos es o f 2-ME .

The intraperitoneal inj ec tion o f heat— killed bact er ial cell s or thioglyco lates t imulates the murine macrophage populat ion to become act ivated".

These act ivated cell s produce cop ious amount s o f pro s taglandins

and ODF RC , as well as demons trating many other enhanced metabo lic funct ions .

These cells are also capable o f suppres s ing the lect in— induced prol iferat ion

o f lymp choy te s . We have conf irmed the f indings o f o thers that pro s taglandins

contr ibute to this suppres s ion , but we have also found that radical

scavenger s and inhibitors o f pro s taglandin synthes is act synergis t ically

to prot ect agains t suppres s ion of lymphocyte prol iferat ion . We are now

def ining the func tional interrelationship s o f ODFRC and pro s taglandins .

In the s implest in vitro model , serum— induced f ibroblas t pro liferat ion

act ivated macrophages can al so suppress pro liferat ion . We have found

that this suppress ion d if fers from the suppres s ion o f lymphocytes in

that f ibroblast suppres s ion canno t be prevented by inhib itors o f pros taglandinsynthes is but rather , can b e prevented by scavengers o f ODF RC .

In Hodgkin 's disease o f humans , lect in— induced lymphocyte pro l iferat ion

is suppress ed . This suppress ion is at tributable to the macrophage s in

the responding cell populat ion . In some cases , pro staglandin produc t ion

by these macrophages causes the suppres s ion o f lymphocyte prol iferat ion .

In co llaborat ion with Dr . Geraldine S checht er , we have found that in

certain cases , the suppress ion is attr ibutable to ODFRC .

In collaboration with Dr . Philip Fox , we have been inves t igat ing the

po ss ible involvement of suppres s ive macrophages in human periodontal

diseases . We have some early indicat ions that such macrophages are

present in some cases .

1! — 7 0 2

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Tumor Cell Derived Immunosuppres s ive Factor s

N AM ES,LA BORATO RY AND I N ST I T U T E AFF I L I AT I ON S , AND T I TL E S OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PROF E SS I ON AL PERS ON N EL ENG AG ED ON THE PRO JECT

PI : Miz e l ,S t even B . S enior S taff Fellow

OTHER : Miz e l , Diane

Farrar,Will iam F . Post—doctoral Fellow

Hilf iker,Mary Po st—doctoral Fel low

[j (b ) H UM AN T I S SU E S m (c ) N E I T H E R

SUMMARY OF WOR K (200 wo rd s or le s s und e rlin e keyword s )Murine sarcoma virus (Mu SV ) transf ormed mous e

immunosuppres s ive fact ors ( ISF ) . The part ially purif ied IS F inhib it ed

phytohemagglut inin (PHA) and concanavalin A ( Con A) induced thymo cyt e

ip _

v it ro splenic ant i- sheep red blood cell plaque forming cell r espons e,and the

generat ion of alloant igen spec if ic cytotoxic T cell s . The ef fect of I SF on

thymo cyt e pro l if erat ion was not read ily revers ib le and requ ired only a 4 hr

expo sure of the thymocytes t o I SF in order to inh ib it cell prol if erat ion .

Although ISF shared several b iochemical propert ies with a Mu SV -transformed c ell

der ived sarcoma growth factor ( SG F ) ,e . g . ,

ac et ic ac id solub il ity,s ens it iv ity

to d ithiothreito l , the two fact ors cou ld b e reso lved by g el f iltrat ion on Bio

Gel P— 60 . Two p eaks o f I S F act ivity were f ound with apparent molecular weight sof and

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OBJECTIV ES

The maj or obj ec t ives of th is new proj ec t are l ) to pur ify and Charact er iz e

the immunosuppres s ive fact or s ( I S F ) produced in vitro by mur ine sarcoma

v irus (Mu SV ) — transf ormed mouse f ibroblas t s, 2 ) to evaluat e their c ellular

targ et s , bo th in v itro and in vivo,and 3 ) to manipulat e in vivo immuno log ical

ac t ivity .

MAJOR FINDINGS

l ) Mur ine sarcoma v iru s (MuSV ) transf ormed mou se f ibroblas t s produc e

po t ent in v itro immunosuppres s iv e fact ors ( I SF ) . The part ially purif ied

ISP inhib it ed concanaval in A and phytohemagglut inin and lymphocyt e

ac t ivat ing factor-induced thymo cyt e prol if erat ion,lipopoly sacchar id e

induced sp leen cell pro lif erat ion,the in vitro splenic ant i— sheep blood

c ell p laque— forming c ell res pons e,and the generat ion of alloant ig en

S p ec if ic cyt otoxic T c ell s . The ef f ect of ISF on thymocyt e pro l if erat ionwas no t read ily revers ibl e and requir ed only a 4 hr exposure of the

thymocy tes to I SF in order to inhib it cell pro l if erat ion .

2 ) Preliminary s tudies on the in vivo ac t iv ity of ISF indicat e that ISP

g iven to mic e i . p . or i . v . can inhib it the mitog en respons iveness of

S p leen cells t es t ed in v itro 2-3 days later .

3 ) Al though ISF shared several b iochemical propert ies with a MuSV

transformed cell—der iv ed sarcoma growth fac tor (S G F ) ,e . g . ,

ac et ic ac id

so lub ility and s ens it iv ity t o de thiothr eit ol ,the two fac t or s cou ld b e

reso lved by g el f iltrat ion on B io—Gel P 60 . Two p eaks of IS F act iv ity

were f ound with mol ecular weight s of and

S IGNIF ICANCE TO BIOMEDICAL AND DENTAL RESEARCH

Immunosuppres s iv e fac t ors have been descr ibed in the s erum of tumor

b ear ing humans and animals . The resu lt s of our studies have lead us t o

the conc lus ion that at l ea st some of the immunosuppres s ive factors ( ISF )may be act ively produc ed by the tumor c ells themselves . The study of

thes e fac tors is ther efor e of some impor tanc e s ince such factors wou ldobv iously contribut e to the in v ivo surv ival of tumor c ell s . However ,

ano ther impor tant aspec t of such fac tors is their us e ( theor et ical at

pr es ent ) t o regulat e und es irable immune respons es as , f or exampl e , in

inf lammat ion (p er iodontal dis eas e) or auto immune d isease .

PROPOSED COURSE

1 ) S tud ies wil l b e cont inued on the pur if icat ion and charact er iz at ion

of the IS F .

2 ) The c ellular targets of I SF will be prec is ely def ined .

3 ) In vivo s tud ies with ISF in mice will be expanded to inc lude the NewZealand B lack st rain of mou se which ha s b een used as an experimental

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model for the study of the immunopatho logy of autoimmune diseas e .

At t empt s will be made us ing ISF to mod ify the dis eas e stat e .

PUBLICATIONS

l . Miz el , S . B . , J . E . LeLarc o , G . J . Todaro , W . F . Farrar and

M . L . Hil f iker . 197 9 . In vitro product ion of immunosuppres s ive

factor s ( ISF ) by murine sarcoma v irus transformed mous e f ibroblas t s .

Manuscr ipt in preparat ion .

7 0 5

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Zol DE—002 60—Ol LMI

OBJECTIV ES

To ident ify and map the genes that control r es is tance to common inf ect iou s

organisms in mic e esp ec ially tho se such as Herp es s imp lex viru s-l , that

ar e involved in oral dis eas e . To det ermine the mechanisms by which

these genes control immunologic proc es ses .

MAJOR F INDINGS

Us ing r ecomb inant inbr ed (RI ) mous e s trains and backcro s s linka g e analys es ,

we hav e mapped a number of g enes that contro l natural res is tanc e .

1 ) R . Tsu sugomu shi . A s ingle,autos omal dominant gene contro ls res is tanc e

t o this organism . We have mapped this g ene t o Chromosome 5 , extremely

C los e to the marker rd,and have des ignat ed this gene RIC with r and 8

representing the respect ive res is tant and su sc ept ibl e al leles . Mic e are

killed within 4 hour s by very high numbers of this organism ( tox icdeath) . Suscep t ibility to t oxic death is also controlled by RIC , ind icat ing

a g enet ic mechanism that funct ions very early af t er inf ect ion .

to this organism,I ty and LPS . LPS unrespons ive mic e ar e extr emely

su sc ep t ible to this organism,and backcro s s linkag e analys is ind icat ed

that res is tanc e wa s contro ll ed by the LPS g ene . S tud ies us ing adopt ive

transf er of c ells or pas s ive ant ibody , s trongly sugg es t tha t this gene

act s through a cellular ( p erhap s macrophage) mediat ed mechanism .

Us ing RI strains we have mapp ed the second g ene Ity , to chromsom e 1 .

Pr eliminary analys is sugg est s that this gene also controls res istance toanother

'

organism, the f lag ellat e paras it e,L . Donovoni .

3 )-Herpes s implex viru s 1 . Res istanc e to this organism has been

previously f ound to be polyg enic .

' RI s train analys is has conf irmed thisf inding .

Other genes . We hav e also mapp ed another po lymorphic g ene locus of

mice , SAS—l . This g ene is located in the middl e of Chromosome 1 . The

funct ion of this gene or it s s erum produc t rema ins unknown . However,it

has proven to be an extremely useful marker f or other genet ic mapp ings tudies .

S IGNIF ICANCE TO B IOMEDICAL RESEARCH

Peop le vary tremendously in their susc ep t ibility to inf ect ious dis eas es

including those caused by oral agent s such as HSV— l or the bac t er ial

ag ent s respons ible for per iodontal dis eas e . The reasons for this var iat ion

in suscep t ibil ity are no t known . Charac t eriz at ion of the genes that

contro l res istance to inf ect iou s organisms will fac ilitat e ident if icat ion

of individuals suscept ib le to any g iven dis ease and enable the developmentof appropriat e prevent ive or therapeut ic measures .

Zol DE— OOZOO— Ol LMI

PROPOS ED COURS E :

The mechanisms contro ll ed by RIC,LP S and Itv will be inves t igat ed now

that the genes hav e been mapped .

We will try to ident ify the exac t number and locat ion of the HSV — l

r es is tance genes . Mechanism s tudies will then be pursu ed .

PUBLI CATIONS

l . Ro s ens tre ic h ,D . L .

,M . G . Groves , H . A . Hof fman and B . A . Taylor .

Locat ion o f the SAS — l locus on mouse c hrom som e l . Immunogenet ic s

in pr es s .

2 . Ro sens tre ic h ,D . L .

,M . C . Groves

, A . O'Brien and B . A . Taylor .

Genet ic control o f natural res istance . Proc eedings of the Dahl em

Workshop ( in pr es s )

Zol DE-002 60— Ol LMI

OBJECTIV ES

To ident ify and map the genes that control res is tanc e to common inf ect iou s

organisms in mic e esp ec ially tho se such as Herp es simp lex viru s—l , that

ar e involved in oral dis eas e . To det ermine the mechanisms by which

these genes contro l immunologic proc es ses .

MAJOR F INDINGS

Us ing r ecomb inant inbr ed (RI ) mouse s trains and backcro s s linkag e analyses ,

we have mapped a number of g enes tha t contro l natural resis tance .

1 ) R . T su sugomu shi . A s ingle,autosomal dominant gene controls res is tance

t o this organism . We have mapp ed this gene to Chromosome 5 , extremelyC los e to the marker rd

,and have des ignat ed this gene RIC with r and 8

represent ing the respect ive res istant and su sc ep t ibl e alleles . Mice are

killed within 4 hour s by very high numbers of this organism ( tox icdeath) . Suscep t ibility to toxic death is al so control led by RIC , indicat ing

a genet ic mechanism that funct ions very early af t er inf ect ion .

to this organism,I ty and LPS . LPS unrespons ive mic e are extremely

su sc ep t ible to this organism,and backcros s linkag e analys is indicat ed

that res is tance wa s controlled by the LPS gene . Stud ies us ing adopt ive

transf er of c ells or pass ive ant ibody,strongly sugg est that this gene

ac ts through a cellular ( perhap s macrophage) mediat ed mechanism .

Us ing RI strains we hav e mapped the second g ene Ity , to chromsome 1 .

Pr elim inary analys is sugg est s that this gene also c ontro ls'

r e sistanc e toanothe r

'

organism, the f lag ellat e paras it e,L . Donovoni .

3 )'Herpes s implex virus 1 . Res istance to this organism has been

previously found to be polyg enic . RI s t rain analys is has conf irmed thisf inding .

Other genes . We hav e also mapp ed another polymorphic g ene locus of

mice , SAS— l . This g ene is located in the middl e of chromo some 1 . The

funct ion of this gene or it s serum product rema ins unknown . However,it

has proven to be an extremely useful marker f or other genet ic mapp ings tudies .

S IGNIF ICANCE TO BIOMEDICAL RESEARCH

Peop le vary tremendously in their susc ept ib ility to inf ect ious dis eas es

including thos e caused by oral agents such as HSV —l or the bact er ial

ag ent s r espons ible for per iodontal dis eas e . The reasons for this var iat ion

in suscept ib il ity are no t known . Charac t eriz at ion of the genes that

contro l res istance to inf ect ious organisms will facilitat e id ent if icat ion

of individuals suscept ib le to any g iv en dis eas e and enabl e the d evelopmentof appropriat e prevent ive or therapeut ic measur es .

41 - 2 0 8

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Octob er 1,19 7 8 S ept emb er 3 0 , 197 9

N AM ES,L A B OR ATO RY AN D I N ST I T UT E AFF I L I AT I ON S , ANO T I T L ES OF PR I NC I PAL I NV E ST I G A T OR S AND ALL OTH E R

PROF ES S I ON AL P ER SON N EL ENG AG ED ON THE PR O JECT

PI : Ros enstreich , David L S enior Surgeon

COPI : Weinb lat t , Anit a Po st—doctoral F ellow

OTHER : G ingras,S t even B io Tech

[3 (b ) H UM AN T I SS U E S pg (c ) N E I T H E R

in order t o pro lif erate in vitro in

will replac e macrophag es and enabl e T cell s to pro liferate in respons e to a

mitogenic but not an ant ig enic st imulus . T cell s pr imed with PMA will alsoprolif erat e in respons e to a solub le produc t der ived from the cu lture of ant igen

pulsed macrophages : We ar e current ly examining th e product in the cultur e

supernatant of ant ig en puls ed macrophages to s ee if it exhib it s the theore t ic allpred icted propert ies o f

"proces s ed ant igen" ant igen spec if ic ity and

g enet ic res tr ict ion in it s ef fect s or if it is a non spec if ic T-C el l tr igger ingmo lecule .

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OB JECTIV ES :

To und ers tand the mo lecu lar S igna ls requ ired t o ac t iva t e T cells .

MAJOR F INDINU S

T lymphocyt es in v it ro will no t prol if erat e unles s supp lied with macrophag e

help ; ei ther in the form of an int act cell or with a so luble produc t

d er ived f rom macro phag es ,£ ~ C e ll act ivat ing fact or (TAF ) . Macrophag e

acc es sory func t ion fo r mit og enic ac t ivat ion c an be rep laced complet ely

by PMA . PMA by it s elf is non—mitogenic f or pur if ied gu inea pig T c e ll s,

and seems to ac t as a s econd ac tivat ing signal .

RMA will no t enabl e T cells to r e s pond t o sp ec if ic ant ig ens in vitro .

Th is is presumably becau s e o f an addi tional requirement for a macrophag e

Ia mol ecu le relat ed s ignal tha t must b e asso c iat ed with the ant ig en .

This add it ional mo l e cu le wa s p roduc ed by col lect ing the cultur e supernatant

of macrophag es tha t had b een pu ls e d with sp ecif ic antig en . This ma t er ial

is undet ec tabl e in culture supernatant s or intrac ellularly until 2 hou rs

of culture . It is maximal at 4- 2 4 hour s of culture .

S IGNIF ICANCE TO BIOMEDI CAL RESEARCH

Act ivated T lymphocy tes ar e an e s s ent ial component of almo s t all immune

reac t ions . Ident if ica t ion o f the s ignal s tha t are requir ed to act ivat e

thes e cells will fur ther our under standing o f the immune pro c es s,

and

may lead to the development of pharmacolog ic ag ents spec if ical ly d es igned

t o enhance or suppress immunity .

PROPOSED COURSE

1) B iochemical ba s is of lymphocy t e act ivat ion . Us ing the two def ined

compounds that ar e requir ed t o act ivat e T lympho cytes , PMA and a plant

l ec t in , we will at t emp t to d e f ine the mechanisms underly ing lymphocyte

tr igg er ing . The e f f ect of each agent will be examined individual ly or

in comb inat ion ,for it s ef f ect s on b iochemical pathway s known to be

involved in cell div is ion — i . e . cycl ic nucleo t id es , nuc lear ornithine

d ecarboxylas e,membrane pho sphol ipid s .

2 . Macrophage proc es sed ant igen . The product in the culture supernatant

of ant ig en puls ed macrophag es wil l be examined t o s e e if it exhib it s the

theor et ica lly pred ic t ed propert i e s of ”proc es s ed ant ig en ant ig en

sp ec if ic ity and genet ic rest r ic t ion in it s eff ec t s . B iochemical

charac t er izat ion w il l b e per f ormed to C haract e riz e and pur ify this

mater ial . Finally,this mater ial will b e analyz e d in the manner propo sed

in part 1 t o ident ify it s mechanism of act ion .

PUBLICAT I ONS

l . Ro s ens t re ic h ,D . L . and S . M . Miz e l . The part ic ipat ion of macrophages

and macrophag e c el l line s in the act ivat ion of T— lympho cy te s by

m itogens . Immunolog ica l Rev iews 40 : 102 — 13 5 ,19 7 8 .

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Ros enstreic h ,D . L . and S . M . N iz el . S ignal requ ir ements for T

lymphocyt e ac t ivat ion I . Rep lac ement o f macrophag e funct ion with

phorbo l myr ist ic ac etat e . J . Immunol . in pres s .

Ro s ens treic h ,D . L . , A . Weinb lat t and L . Weedon . The us e of synthet ic

compounds to rep lace the macrophag e requirement for T— lymphocyt e

ac t ivat ion . Proceed ings of the Brook Lodg e Workshop on Macrophag e

F unct ion . ed A . Ro s enthal and E . R . Unanu e,in pr es s .

Ros enstre ic h , D . L . The macrophag e In The macrophag e in the c ell

b iology of immunity and inf lammat ion . J . J . Oppenheim,M . Pot t er

and D . L . Ro senstre ic h ed s in pres s .

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2 01 DE 00046—09 LMI

OBJECTIVE S

The ongo ing res earch in this laboratory focuses on unders tanding the

mechanisms whereby the immune sys tem may modulate connect ive t is sue

metabol ism . Connective t is sue metabo lism is markedly altered in many

inf lammatory les ions . The c o-exis t ence o f inf lammatory cells and

connect ive t issue cell s in such les ions led us to inves t igate whether

lymphocyt es could inf luence f ibrob las t funct ion .

In addit ion to lympho id cel ls , macrophages play a prominent ro le in the

immune response . We are invest igat ing how lymphokines and other agent s

including the synthet ic adj uvant , muramy l dipep t ide , act ivate these

phago cyt ic cell s to migrate , to releas e b io lo gically act ive monokines ,

and to increase their b io chemical funct ions as primary perpetrators o f

the inf lammatory response .

Another cell which becomes func t ionally modif ied in inflammatory les ions

is the epithelial cell . Us ing gingival ep ithel ial cell s we are at temp t ing

to def ine tho se agent s , bo th immuno logically— derived and nonspecific ,

which may inf luence these cel ls to regenerate the ep ithel ial surfaces

af ter an interrup t ion in their cont inuity o ccurs .

METHODS EMPLOYED

Methods ut ilized to inves t igate the aforement ioned obj ec tives includealready publ ished pro cedures for macrophage chemotaxis , lympho cyteculture , prol if erat ion , lymphokine product ion , pro s taglandin as say ,

cycl ic AMP and GMP determinat ions , f ibrob last cul ture , analysis o f

collagen format ion , and ep ithel ial cell culture .

MAJOR FINDINGS

Lymphocytes expo sed to spec if ic ant igen respond by producing numerous

effector molecules . One such recently des cribed molecule , f ibrob las t

act ivat ing factor (FAF ) , exer ts it s ef fec t on quies cent f ibroblas t s by

caus ing these cells to prol if erate and to increase their synthes is o f

pro tein . T lymphocytes from dinitropheny lated ovalbumin (DNP—OA ) immunized

guinea pigs , when challenged with the ant igen in vitro , produced s ignif icant

amount s o f this med iator within 24— 48 hr . Addit ion o f these supernatant s

to sub c onfluent cultures of guinea pig dermal f ibrob las t s in serum— free

medium result ed in f ibrob last pro liferat ion as determined by trit iated

thymidine uptake and by an increase in cell numbers . This lympho cyte

derived mediator can al so modulate other f ibrob las t act ivities . Within4 6 hr af ter exposure to FAF

,f ibroblas t s synthes ized and released greatly

enhanced levels o f pros taglandins into their culture medium . FAF also

induced elevat ions in intracellular cyclic nucleo t ide ( CAMP ) levels . As ignif icant increase in pro te in synthes is o f bo th the collagen and

nonc o llagenous types occurred prior to the maj or peak o f f ibroblas tpro liferation .

2 01 DE 0004 6- 09 LMI

In addit ion to T cell s , guine a pig p e rit fi ne al macrophages s t imulated

with lipopo lysaccharide or muramy l dipep t ide produced a so luble factor

which act ivated f ibroblas t s to prol iferate . S t imulated macrophages , bu t

not contro l cell s , releas e d this factor which was measurable ear ly aft er

act ivat ion (1— 2 hr ) and increased during the sub sequent 244 8 hrs . This

factor is nondialyz ab le , heat s table and has a molecular weight of 40

Addit ionally , a small dialyzab le molecule was found in these

supernatant s which inhib it ed thymidine uptake but no t cell divis ion .

Further C haract er izat ion of these molecules is in progres s . I t thus

app ears that bo th macrophages and lympho cyt es when appropr iately act ivated

release soluble molecules that can regulate f ibroblas t funct ion . These

f ibroblas t act ivat ing fac tors may contr ibute to the f ibro t ic reac t ions

as so c iated with C hronic inflammatory les ions .

Fur ther inves t igat ion into the mechanisms o f macrophage act ivat ion

revealed that muramy l dip ep t ide , lipopolysaccharide and lymphocyteder ived macrophage ac t ivat ing fac tor which induced f ibrob las t ac t ivat ing

factor produc t ion also ac t ivated the macrophages to produce co llagenase .

Collagenase product ion was dependent upon an increase in pro s taglandin

synthes is which in turn elevate d intracellular levels o f CAMP . That

this elevat ion in cyc l ic AMP may contro l macrophage act ivat ion will be

fur ther explored by det ermining it s ro le in modulat ing the product ion o f

b io lo gically ac t ive med iators such as the f ibrob las t act ivat ing factor .

In l ight o f our f inding s that lymphocytes and macrophages can influence

f ibroblas t func t ion , we have explored an in vivo model o f delayed hypersens it ivity

hyper sens it ivity for po ss ible interact ions between lympho id cell s and

f ibroblas ts . Inf ec t ion o f mice with Schis to soma mansoni result s in

granuloma format ion in the liver which is then fo llowed by hepat ic

f ibro s is . The caus e o f this f ibro s is , which result s in death o f the

animal s,is unclear . Our inves t igations sugges t that the l iver granulomas

which form in respons e to the s chis to somal infect ion s t imulate f ibrob las t

proliferat ion with the releas e of a soluble molecule ( s ) and thus may

play a ro le in the development o f hepat ic fib ro s is in S . mansoni infect ions .

We are currently at t empt ing t o de f ine the cel lular source o f this f ibrob las t

s t imulat ing med iator within the granuloma .

In related s tud ies,the ro le of inf lammatory cell s on ep ithelial populat ions

is b eing explored . Following inj ury ep ithelial cell s alt er their normal

b ehavioral pat t erns and begin to migrate , to phago cyt ize and to divide .

I t appear s that inf lammatory leukocyt es may be , in part , respons ib le for

influencing these cell s to divide through the transmis s ion o f a soluble

s ignal ( s ) . F urthe r analys is of such a mechanism is underway .

S IGNIF I CANCE TO BIOMEDI CAL AND DENTAL RESEARCH

Cellular immune phenomena are the basis for the development of many

C hronic inf lammatory le sions . There are certain pathologic condit ions

including s cleroderma,pulmonary f ibro s is , and schis to somias is which

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have been charact er iz ed as cell-mediated which lead to exces s ive connect ive

t is sue format ion or f ibro si s . It is po s s ible that the C hanges in the

connect ive t is sue could b e mediat ed by the local product ion and releaseo f lymphokines by ant igen activated lymphocytes . Lymphokines appear to

regulate fibrob last funct ion sugges t ing a pathway for enhanced connect ive

t is sue metabol ism includ ing co llagen format ion .

Chronic inflammatory les ions which are C haracterized by an inf il trate o f

mononuclear cells may also be des truc t ive to the surrounding connect ive

t is sue as in rheumato id arthrit is and per iodontal dis ease .

In connect ive t is sue destruct ion , immunologic event s may init iat e the

pro cess with the ul t imate degradat ion actually brought about by inflammatory

cell s such as the macrophage through the product ion and release o f

pro teolyt ic and t is sue degrad ing enzymes .

Act ivated lymphocytes may al so be largely respons ible for the product ion

o f C hemotac t ic st imul i which at trac t macrophages to s it es o f inf ect ion

or inflammation . Once lo calized at such a s it e these macrophages may b e

act ivated to produce a number o f enzymes and to release soluble factors

(pro s taglandins , f ibrob last act ivat ing factor which influence

o ther cells . I f cont inuous ly act ivated as in C hronic inflammatoryles ions l ike periodontal disease and rheumato id arthrit is , these cell smay contribute to the patho logic t is sue des truct ion by the release o f

lysosomal enzymes , co llagenase,and pro s taglandins . Thus , through an

under standing o f the way these cell s are tr iggered , it may b e po s s ibleto contro l or modulat e their func t ion .

Macrophages may also play a prominent rol e in enhancement o f immunologicalevent s as so ciated with the use o f adj uvant s . The adj uvant ef fect s o f

mycobacteria can be replaced by chemically def ined iso lates o f the cellwall s including a wat er solub le fract ion (WSA) and by the synthet ic

analog , N— acetyl-muramy l— L-alanyl—D— isoglutamine (MDP ) which is the

minimal s tructure required for adj uvant ic ity . These clearly def ined

compounds can direc tly ac tivate macrophages sugges t ing that immunologicadj uvanticity is related to enhanced macrophage funct ion . The act ivat ion

o f macrophages may then in turn prec ip itate a mul t iplic ity of o ther

act ions result ing in enhanced immune responses .

Mechanisms regulat ing re— ep ithel ial izat ion fo llowing an inflammatoryles ion have b een undef ined and l it t le explored in the pas t . I f inflammatoryC ll s can regulate epithel ial cell b ehavior , it may enable manipulat ion

o f this sys t em to encourage or to delay the re-ep ithel izat ion process to

favor the resolut ion o f certain patholo gic s ituat ions .

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ZOl DE-OOO46-O9 LMI

Mccarthy , J . B . , S . M . Wahl , J . Rees , C . E . Olsen ,

A . L . Sandberg and L . M . Wahl . 1 979 . Regulat ion o f macrophage

collagenase produc tion by 3'-5 cycl ic adeno s ine monopho sphate .

In pres s .

Wahl , S . M . , L . M . Wahl , J . B . Mccarthy , L . Chedid and

S . E . Mergenhagen . 19 79 . Macrophage act ivat ion by myco

bact er ial water soluble compounds and synthet ic muramyl

dipept ide . J . Immunol . 122 : 22 2 6 .

Oppenheim , J . J . , A . Togawa and S . M . Wahl . 1979 . Immunologicalro le o f macrophage and lymphocyte derived amplif icat ionfactor s . In The Cell Biology and Immunology of Leukocytes .

(M . Quastel , ed . ) Academic Pres s , New York . In pres s .

Wahl , S . M . 1 97 9 . Role of inf lammatory cell s , lymphokinesand monokines in wound healing . In The Cell B iology o f

Immunity and Inf lammat ion . (J . J . Oppenheim , D . Rosens treich

and M . Pot ter , Els evier North Holland Biochemedic al

Pres s , New York . In pres s .

Wahl , S . M . and L . M . Wahl . 1979 . Lymphokine modulat iono f connect ive t issue metabol ism . N . Y . Acad . S c i . In

Pres s .

Wahl , L . M . , C . E . Ols en , S . M. Wahl , J . B . Mccarthy ,

A . L . Sandberg and S . E . Mergenhagen . 19 79 . Pro s taglandinand CAMP regulat ion of macrophage involvement in connect ive

t is sue des truct ion . N . Y . Acad . S c i . In pres s .

wahl , L . M . , S . M; Wahl and J . B . Mccarthy . Adj uvant act ivat ion

o f macrophage funct ions . In Regulatory Role o f Macrophages

in Immunity . (E . R . Unanue , and A . S . Ro senthal ) . In press .

Oppenheim , J . J . , R . Moore , F . G'Melig

—Mey ling , A . Togawa ,

S . M . Wahl , B . Mathieson , S . Dougherty and C . Carter . 1979 .

Role o f cytokine and endo toxin induced monokines in lymphocyteprol iferat ion

,dif ferent iat ion and immunoglobulin product ion .

In Regulatory Role o f Macrophages in Immunity . (E . R . Unanue ,

and A . S . Ro senthal , In pres s .

wyler , D . J . , S . M . Wahl , A . W . Cheever and L . M . Wahl .

19 79 . Fibroblas t st imulat ion in vitro by soluble product s

o f i solated schistosomal egg granulomas . In pres s .

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Wahl , S . M . , L . M . Wahl and S . E . Mergenhagen . 19 7 9 .

Lymphokine and monokine regulation o f fibrob last funct ion

In B io chemical Charact erizat ion o f Lymphokines . Pro c . o f

the Second . Int l . Lymphokine Workshop (A . L . DeWe C k ,

Academic Pres s , In pres s .

Oct ob er 1,19 7 8 S ept emb er 3 0 , 19 7 9

N AM ES,L AB OR ATORY ANO I N ST I T UT E AFF I L I AT I ON S, AND T I TL ES OF PR I NC I PAL I NV E ST I G AT OR S AND ALL OTH E R

PROF ESS IONAL PERSONNEL ENG AG ED ON THE PRO JECT

PI : Sandb erg,Ann L . Res earch B iolog ist

OTHER : Wahl,Larry M . Res earch B iolog ist

Pa z ole s ,Pamela P . Chemist

ty o f Connect icut H ealth Cent er

in turnus es r esorpt ion of the b one . A cell s imilar t o a monocy t e may b e re sponsiBler the elevat ed levels of pro stagland ins found in these cultures 3

t ivat ion by immunoglobul ins st imulat es pro stag landin product ion by macrophages

culture . Immunoglobu l in f ragment s which are incapable of d irect ly act ivat ing

es e cells but which act ivat e the alt ernat ive comp lement pathway ar e ef fect ive

this r egard . Following int eract ion of the component s of the comp lement sy st emth m acrophage membranes , enhanc ed incorporat ion of pro stagland in precur sor s

to pro stagland ins occur s . These stud ies demonstrat e that both st ero id as well

inhib it s the growth of bone in organ cultur es of

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ZOl DE—OOO6l- O6 LMI

To determine if macrophag e act ivat ion requires bo th the interact ion of

complement with the macrophag e membrane and binding of ant ibody t o the

F C r ece ptors, F ( a b

') fragment s were u t iliz ed . These immunoglobul in

preparat ions which interac t only with the cellular ant ig ens and act ivat e

only the alt ernat ive complement pathway were as eff ec t ive as intact

ant ibody in init iat ing complement dependent prostaglandin synthes is by

macrophag es . Thus,the funct ion o f ant ibody in complement dep endent

pros taglandin synthes is by thes e cells appe ars to be only to init iate

complement act ivat ion at a cell surface . Complement markedly enhanc ed

the incorporat ion of the pro staglandin pr ecursor into pros taglandins bymacrophag es treated with F (ab

')2fragm ents .

S im ilar result s were obtained in cultures of f etal rat bones in that in

the pres ence of complement and a C hidonic ac id,prostagland in levels

were elevated and,in addit ion

,Ca release from the bones was s ignif i

c ant ly increas ed . These results sugg est that complement alt ers the cell

membrane suf f ic ient ly to allow acces s of the pros taglandin pr ecur sor to

the enzymes invo lved in the biosynthes is of pro staglandins . These

eff ects wer e abolished by the addition of the prostaglandin synthetaseinhibitor , indomethacin .

Another non— st ero id ag ent,f lu f en emic ac id also inhibit s the complement

dependent enhanc ement of arachidonic ac id st imulat ion of pro staglandin

product ion and bone resorp t ion . Numerou s mechanisms hav e been pos tulat ed

for the eff ects of the glucocor t ico id,hydrocort isone

,on pros tagland in

synthes is . Its inhibit ion of the complement mediated elevat ion of

prostaglandin levels as well bone resorp t ion in cultures containing

arachidonic ac id sugg es t s that one s it e of act ion of this pharmacolog icalag ent is the inhibit ion of the incorporat ion of prostagland in precur sor sinto prostagland ins .

Another ef f ect of complement on bone metabolism has also been def ined .

In addit ion to it s role in inducing bone destruct ion, omp l emen t also

inhibit s bone growth . Det ermination of the uptake of H prol ine in

organ cultur es of rat calvaria has demonstrated that ant ibody act ivat ionof complement inhibits the synthes is of both co llagen and non— collagen

prot ein . This eff ect is also dependent on the lat e complement components ,

at least through C6 . Although complement and antibody stimulate pro staglandin

synthes is in rat calvaria no correlat ion exist s between the product ionof this agent and inhibit ion of bone growth . Indomethac in and fluf enamic

acid inhibit the synthes is of prostaglandins but ar e completely inef f ect ive

in restoring bone growth in cultured rat calvaria . Thu s , C may have a

dual funct ion in the regulation of bone metabo lism : it not only induces

re sorp tion of bone but in addit ion,inhib it s the growth of bone .

S IGNI F ICANCE

In c ertain inf lammatory les ions such as per iodontal dis ease and rheumato id

arthr it is , d e struc t ion of conne ct ive t issue is a common f ind ing . Elevated

levels of pro stagland ins have been found bo th in inflamed g ing ivae and

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the synovia of pat ient s with rheumato id ar thr it is . Pro s tagland ins are

known to b e pot ent bone resorb ing ag ent s . S inc e complement ac t ivat ion

ha s now b een shown to result in enhanc ed synthes is of pro stag land ins by

b ones in organ culture as wel l as by macrophag es,the complement sy st em

is impl icat ed as a contribut ing fac tor t o the des truct ion of connect ive

t i s sue . In add it ion ant ibody init iat ion of complement act ivat ion can

also prevent the synthes is of new connect iv e t is su e element s .

PROPOSED COURSE

S tud ies will b e cont inued to inves t igat e the mechanisms by which immunolog ical

ac t ivat ion o f the comp lement syst em inf luences connect iv e t i ssu e metabo l ism .

In inv es t igat ions of bo th bone r esorpt ion and st imu lat ion of pros tag land in

synthes is in macrophag e cultures iso la t ed components of complement wil l

b e ut il iz ed to d ef ine their int erac t ion with membranes . The spec if ic ity

o f the ant ibod ies requ ired f or init iat ion o f thes e complem ent dep endent

ev ent s will al so b e examined . Complement int eract ion with macrophages

and other cell s will also b e examined to det ermine if this typ e of

c ellular s t imulat ion can result in the produc t ion o f med iat ors other

than pro s tag land ins which inf luenc e connec t ive t is su e metabo l ism . A

po s s ibl e d ef ec t in pro stagland in produc t ion by complement st imulat ed

macrophag es f rom o st eop et ro t ic mice wil l also be examined as will the

eff ec t s of compl ement and ant ibody on resorpt ion of bones from thes e

animal s and their normal l it t ermat es .

PUBLICATIONS

l . Wahl,L . M Ols en

,C . E . ,

Wahl,S . M .

,Mc Carthy , J . B . , Sandb erg ,

A . L . and M ergenhagen ,S . E . Prostaglandin and CAMP regulat ion of

macrophage invo lvement in connect ive t is su e destruc t ion . N . Y .

Acad . S c i . 19 7 9 . In pres s .

2 . Mccarthy,J . B .

,Wahl

,S . M .

,Rees , J Ols en , C . E . , Sandb erg ,

A . L . and Wahl,L . M . Regu lat ion of macrophage co llagenase

product ion by 3'— 5

' cyc lic ad enos ine monopho sphat e . J . Immunol .

1 9 7 9 . In pres s .

Larry Wahl

N AM ES,LABOR ATORY ANO I N ST I T UT E AFF I L I AT I ON S , AND T I TL ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PR OF E S S I ON AL P ER SON N EL ENGAG EO ON THE PRO JECT

PI Wahl , Larry M . Research Biologist

OTHER : Olsen,Charles E . Po st—Doctoral Fellow

Wahl,Sharon M . Research Microb iologis t

Sandberg ,Ann L . Research Bio logis t

Mccarthy , James Microb io logist

Winter,Chr ist ine Microb iolo gist

V . A . Ho sp ital

SUMMARY OF NOR K (2 00 wo rd s or les s und e rlin e ke yword s )The purpo se o f this proj ect is to s tudy the role of the immune syst em in

connect ive tissue metabol ism . We have demons trated that the prostaglandins

control the product ion of collagenase by endotoxin and lymphokine ac tivat ea

Addit ionally , dru

pho sphodie S t era s e

been shown to modulate bo th pro s taglandin and collagenase product ion of

endotoxin act ivated macrophages . High concentrat ions of these CAMP enhancing

agent s significantly inhib it the product ion o f prostaglandin and collagenase

whereas both are st imulated by lower concentrat ions of these drugs . Studies

the product ion of pro staglandins and in the production o f suppre s sons cfif lympho

cyte prol i ferat ion . The lower product ion o f prostaglandins by macrophages

from the os teopetrotic rat s may in part be one o f causes for the hype rprolife r

s in the animals s ince prostaglandins are known 0 pup ress

n _ o o 4c . A.

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2 01 DE 002 1 6— 03 LMI

SIGNIFICANCE TO BIOMEDICAL AND DENTAL RESEARCH

Chronic inf lammatory les ions such as periodontal disease and rheumato id

arthr it is are associated with the des truct ion o f connect ive t is sue . Ourpresent s tudies have fo cused on furthering the under standing o f the

cellular events leading to the degradat ion o f connect ive t is sue . These

s tudies have indicated that pro staglandins and cycl ic adenos ine mono

phosphate have es sent ial ro les in the product ion o f collagenase by

macrophages . Thus , any way in which the product ion of pro s taglandinsand/ or C hanges in cyclic nucleo t ide l evel s may be modulated would

po tentially ef f ec t the degradat ion o f connect ive t issue .

The s tudies involving the o st eopetrot ic rat model indicate that there

are defects in the immune sys tem which may be related to the pathology

o f this d isorder . Charact erizat ion o f the exact manner in which these

def ect s contr ibut e to exces s bone format ion will increase our unders tanding

o f the ro le o f lympho cytes and macrophages in bone resorp t ion .

PROPOSED COURSE OF STUDY

Inves t igations into the mechanisms o f macrophage act ivat ion leading to

the product ion o f collagenase will cont inue . S ince activat ion occurs

co inc ident with a C hange in cell morpho logy the emphas is will b e on

experiment s dealing with changes in cell membrane which will include

determinat ion o f lip id compo s it ion , changing the lip id compos it ion o f

the membrane by adding various lip ids and determining the role o f the

cyto skeleton on cel l membrane funct ion . The po ss ib il ity o f subpopulat ions

o f macrophages , based on siz e , exis t ing in guinea p ig peritoneal exudate

which have dif f erent capab il it ies in their product ion of pro s taglandins

and collagenase will al so b e examined . Bone resorp t ion s tudies ut il iz ingthe o s t eopetro t ic too thles s rat will concentrate on the apparent defect

in the macrophages o f these animal s . The product s secret ed in vitro by

macrophages from os teopetro t ic rat s and their normal littermates will be

compared for their ab il ity to s t imulate bone resorpt ion and f ibroblas tpro liferat ion .

PUBLICATION S

l . Me rgenhagen ,S . E . , Rosenstre ich , D . L Mc Ghe e , J . R . , and

Wahl,L . M . Po t ent ial role o f lymphore c ticular cell s In the

Ho s t Response to Endotoxin . Immunology , Ed . Gergely , Medgyes i

and Ho llan, 19 78 . pp 431—44 7 .

2 . Wahl , S . M Wahl,L . M . , Mc Carthy ,

J . B Ghedid , L . and

Me rgenhagen ,S . E . Macrophage act ivat ion o f mycobacterial water

so luble compounds and synthetic muramy l dipep t ide . J . Immunol .

12 2 : 2 22 6— 22 31 , 1 9 7 9 .

3 . Ab e , S . , S teinmann , B . U . ,Wahl

,L . M . and Mart in , G . R . High

cell density alt er s the rat io o f type I I I to I co llagen synthes is

by f ibroblas ts . Nature . 2 7 9 : 44 2-444 .

ll .

12 .

2 01 DE 002 1 6— 03 LMI

S chechter , G . P . , Wahl , L . M . and Oppenheim,J . J . Suppres sor

monocytes in human disease . A Review . Eighth Pro ceedings o f

the Re c t iculoendo tho liel ial So ciety . In pres s .

Wahl , L . M . , O ls en , C . E Wahl,S . M . ,

Mccar thy ,J . B . ,

Sandberg,

A . L . and Me rgenhagen , S . E . Pro s taglandin and CAMP regulat iono f macrophage involvement in connect ive t issue des truct ion .

New York Acad . S ci . 19 7 9 . In pres s .

Wahl , S . M . and Wahl , L . M . Lymphokine modulat ion o f connect ive

t i s sue metabol ism . N . Y . Acad . S c i . 19 7 9 . In pres s .

Wahl , L . M . , Wahl , S.M . and Mc carthy ,J . B . Adj uvant act ivat ion

o f macrophage func t ions . In Regulatory Rol e o f Macrophages in

Immunity . Academic Pres s (Ed . Unanue and Rosenthal ) In pres s .

Oppenheim , J . J . , Koopman , W . J . ,Wahl , L . M . and Dougherty ,

S . F . Pro s taglandin E2rather than LAF produced by mononuclear

cell s st imulat es increases in murine thymocyte CAMP . Cell .

Immuno l . 1 97 9 . In pres s .

Wahl , S . M . , Wahl , L . M . and Mergenhagen , S . E . Lymphokineand monokine regulat ion o f f ibrob las t funct ion . In B iochemical

Charac ter izat ion o f Lymphokines . Pro c . o f the Second Int l .

Lymphokine Workshop (A . L . D eWe C k , Academic Pres s . In

pres s .

Moore,R . N . , Urbaschek , R . , Wahl , L . M . , and Mergenhagen ,

S . E . Pro stagland in regulat ion of colony s t imulat ing factor

product ion by lipopo lysaccharide s t imulated murine leukocytes .

Infe c . Immun . In pres s .

Mccarthy ,J . B Wahl

,S . M . ,

Rees , J . , Olsen , C . E . , Sandberg ,

A . L . and Wahl,L . M . Regulat ion o f macrophage co llagenase

product ion by 3'—5

' cycl ic adenos ine monopho sphat e . In pres s .

Wyler,O . J . ,

Wahl,S . M . , Cheever , A . W . and Wahl , L . M .

Fibrob las t s t imulat ion in vitro by so lub le product s o f isolated

s chis to somal egg granumolas . In pres s .

11

N AM ES,LA BOR A TO RY AND I N ST I T UT E AF F I L I A T I ON S , A ND T I T L E S OF PR I NC I PAL I NV ES T I G AT OR S AND ALL OT H E R

PROF E S S I ON AL P ER S ON N EL ENGAG EO ON THE PR O J ECTPI : Cisar , John 0 . S enior S taf f Fellow

Other : Kol enbrander , Paul Senior S taff FellowBerg

,Shelley Mi crob iologis t

hmar Gabriel , Georgetown Univers ity

{ 9 (b ) HUM AN T I S S U E S (c ) N E I T H E R

S UMM AR Y OF W OR K (2 00 word s or les s und e rlin e keyword s )S tudies are cont inuing to examine mol ecular mechanisms by which S pec ies o f o ral

act inomy cet es adhere to o ther p lague bact er ia and to mammal ian cell s . Lect ins it es which are inhib it ed by lacto s e are as so ciated with the surface fimbriae

These s it es mediate coaggregation o f the ac tinomycet es with certain oral

s trep to co cci and hemagglut ination o f the act inomycetes wi th neuraminidas e

treat ed human erythro cy tes . Thus,int eract ions of a lect in—carbohydrate type

may contr ibut e si gnif icantly to the dis tr ibut ion o f cer tain bac ter ia within

the o ral cavity .

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2 01 DE 0025 4— 02 LMI

number o f galacto se-speci f ic plant lect ins have been s creened for their

ability to agglut inate strains of S . sanguis and S . mitis . Ricinuscommunis agglut inin— 1 20 (RCA-12 0 ) reac ted only with tho se s trep to co ccal

s trains which gave lacto se revers ible coaggregation react ions . This

correlat ion sugges t s that RCA-1 20 binds the strepto co ccal recep tor

involved in lacto se-rever s ibl e coaggregat ion .

Pro teinaceous f ibrils (i . e . f imbriae or pil i ) which cover the sur face o f

act inomycete cell s have been isolated from three act inomycete s trains .

These s truc tures appear to carry lect in binding s it es for adherence .

Two-dimens ional immunoelectrophores is o f each f ibril preparat ion has

revealed two antigens . I t is no t known yet whether each ant igen is

a d if ferent type of f ibril and whether each is as so ciated with lect inact ivity . Various approaches are being employed to answer these ques tions

including the use o f mono clonal ant ibodie s agains t the f ibrillar antigens .

At pres ent , monoclonal antibodies have been prepared agains t one o f the

f ibrillar ant igens o f A . viscosus TI4V , and this ant igen has been found

to po ss es s lect in ac t ivity .

S IGNIFICANCE TO BIOMEDICAL AND DENTAL RESEARCH

Sp ecif ic microb ial adherence,though widely recogniz ed as the init ial

event in many ho s t-parasite relationships , remains poorly unders tood

at the molecular level . The result s of thi s proj ec t indicate that surface

as soc iated lect ins are present on cer tain oral bact er ia and that these

molecules may funct ion in the att achment o f microorganisms to each o ther

and to muco sal sur faces . These po s s ibil ities provide support for the

concept that interact ions o f a lect in— carbohydrat e type represent a

s ignif icant and general mechanism o f microb ial adherence . A bet ter

unders tanding of microb ial adherence at the molecular level may suggest

new and useful approaches for the control o f cer tain bacterial infect ions .

PROPOSED COURSE

S tudies will cont inue towards C haracter iz ing the l ect in b inding s it es

on A . vis co sus T14V and o ther act inomycet es . These ef fort s will fo cus on

S trep to co ccal carbohydrat es from selected s trains will be pur if ied by

aff inity Chromatography on ROA—1 2 0 columns . I solated mat erial will be

s tudied to determine whether it rep resent s the st repto coc cal receptor in

lacto se—revers ible coaggregat ion .

Studies to examine certain ef fect s o f neuraminidase on immune cel ls willb e undertaken .

h 2 3 fl

PUBLI CATIONS 2 01 DE 002 5 4— 02 LMI

Ci sar , J . O . and A . E . Vatter . 1 9 7 9 . Surface f ibrils (Fimbriae )

Cisar , J . O P . E . Kolenbrander and F . C . Mc Intire . 19 7 9 . Spec if ic ity

o f coaggregat ion react ions between human oral st rep tococ ci and

Immun .— 7 5 2

Co st el lo , A . H . , J . O . Cisar , P . E . Ko lenbrander and 0 . Gabriel .

1 9 7 9 . Neuraminidase-dependent hemagglutinat ion o f human erythrocyt es

by human s trains o f Act inomyces viscosus and naeslundii .

Infec t . Immun . ( In pres s ) .

N AM ES, L A BOR ATO RY ANO I N ST I T UT E AF F I L I AT I ON S, AN D T I T L E S OF PR I NC I PAL I NV E ST I G AT OR S AND ALL OTH E RPR OF ES S I ON AL P ERS ON N EL ENGAG EO ON THE PR O J ECT

PI : Witt enb erg er , Charles L . Res earch Microb iologist

COPI : S t . Mart in , Edward S enior S taff F el low

OTHER : B eaman , Alfred J . B iolog istWo lf

,Anitra B io log ist

S UMM A RY OF W OR K (2 00 wo rd s or le s s un d e rlin e keyword s )

in oral bact er ia cont inue t o b e under invest igat ion . Current ly, S p ec ial emphas is

is placed on 1 ) C haracter iz at ion of the enzyme gluco sylt rans f eras e ( GTas e )

enzyme complex has b een reso lved into two catalyt ically act ive component s . One( G Tas e I ) , produc e s a wat er insolub le po lymer from sucro s e while the other ( G Tas e

S ) catalyz es the synthes is o f a water solub le po lymer from suc ro s e . Mutant s have

been isolat ed that are mis s ing the G Tas e I component . Product ion of the extra

cellular G Tas e compl ex by S . salivarius is compl et ely inh ib it ed by c erul enin .

This ant ib io t ic has no eff ect on DNA,RNA or prote in synthes is , but do es s evere l

inhib it cellular incorporat ion of acetat e . I t app ears that GTas e secret ion

is s omehow C los ely linked to the de novo synthes is o f long C hain fat ty ac ids .

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(Rev.-76 ) LI 2 3 2

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o f fat ty ac ids in the s ecretory proc es s .

The ant ib iot ic cerul enin is known to be a potent and sp ec if ic inhib it or

of bact er ial B—keto—acyl thioest er synthetase . This enzyme catalyz es

the condensat ion of fat ty acid—acyl carrier pro t ein with malonyl—acyl

carr ier pro t ein,which result s in fatty ac id C hain elongat ion . We have

found that cerulenin severely inhib it s acetate incorporat ion by S .

sa livar ius and complet ely blocks the ab il ity of the organism to produc e

G Tas e . In contrast,the ant ib io t ic has litt le or no ef f ect on the

synthes is of DNA,RNA or pro t ein . It appear s therefor e that c erul enin

inhib it s G Tas e produc t ion by int erf er ing with long C ha in fat ty ac id

synthes is . Thes e result s further suggest that the inhib it ory ef f ect is

at the level of enzyme secret ion rather than enzyme synthes is .

We have cons idered that cerulenin might exert it s ef f ect by cau s ing an

altera t ion in the fa tty ac id compo s it ion of cell membrane lip id s . Such

an alterat ion could af f ect membrane f lu id ity and thereby pr event s ecret ion

of the enzyme . This was t est ed by growing the organism in med ium containing

a concentrat ion of cerulenin that inhib it ed G Tas e product ion . Thes e

cell s were then harvest ed,washed free of the ant ib io t ic

,and test ed for

their ab il ity to produce GTase in fr esh medium . It was found that cellsgrown in the presenc e of cerulenin produced GTas e as well as c el ls grown

without the ant ib iot ic . Thu s , any membrane lip id alt erat ions tha t may

have occurred dur ing growth of the organism in med ium containing cerulenin

have no ef f ect on the abil ity of tho s e cells t o subsequ ent ly synthes iz e

and s ecr et e the enzyme . Mor eover,c erulenin has been found to ex ert it s

inhib it ory ef f ect more or les s immed iat ely af t er add it ion to cells

produc ing the enzyme . This too mil itat es against the ant ibio t ic ac t ing

to caus e ext ens ive alt erat ion of the fat ty ac id compo s it ion o f / membrane

l ipid s .

Taken together,the availabl e da ta are more cons ist ent with the hypo thes is

that cerul enin blo cks G Tas e produc t ion by inhib it ing some po st— translat ional

ev ent involv ed in it s s ecr et ion from the cell . This event ( or event s )appears to requir e d e novo long cha in fat ty ac id synthes is and we are

current ly explor ing the pos s ib il ity that this may b e coupled to a covalent

mod if icat ion of a secretory form of G Tas e .

S IGNIF ICANCE TO BIOMEDICAL AND DENTAL RE SEARCH

These stud ies on the mechanism of G Tas e s ecret ion and it s regulat ion in

oral microorganisms are under taken with a broad view toward advanc ing

our stat e of knowledg e concerning the means by which the cell coordinat es

and controls it s diver se b iochem ical ac t iv it ies . Such informat ion isC learly of broad b iolog ical S ign if icance . Mor e S pec if ically ,

however ,

certain of the microorganisms under invest igat ion have been st rongly

impl icat ed as et io log ical ag ent s of oral d iseas es . It is further antic ipat ed

ther efor e , tha t this work will lead to a more comprehens ive under stand ing

o f how thes e bac t er ia succ es sfu lly co loniz e and subs ist in the complex

oral eco sy st em . Result s f rom our studies on gluco sylt rans f erase s ecret ion

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by oral strep tococc i may also prov id e ins ight into pos s ibl e means of

ar t ific ally contro ll ing it s product ion .

PROPOSED COURSE

In g eneral , our stud ies on exoenzyme s ecret ion by member s of the oral

microf lora will b e cont inu ed . Sp ec ial emphas is wil l b e placed on reso lv ingthe ro le o f fat ty ac id s in the secret ion of the S . sal ivar iu s glucosyl

transf erase . Att empt s will also be made to mor e fully C haract eriz e the

iso lat ed ind iv idual components of the enzyme complex .

PUBLICATIONS

1 . Marucha,P . T . , Key e s ,

‘

P . H .,Wit tenberg er

,C . L . and London

,J .

A rap id method f or the id ent if icat ion and enumerat ion o f oral

Act inomyc es . Inf ect . Immun . 2 1 : 78 6—7 91 ,197 8 .

2 . Crow,V . L .

,and Wit t enberger , C . L . S eparat ion and propert ies

o f NAD and NADP—d ependent glyc erald ehyd e—3—pho sphat e dehydrog enas es

from St r ept ococ cu s mutans . J . B io l . Chem. 2 54 : 1134— 114 2 ,197 9 .

3 . S t . Mart in,E . J and Wit t enberg er

,C . L . Charact er izat ion of a

pho spho enolpyruvat e— d ependent sucro se pho spho transf era se sy st em in

Inf ect . Immun . 865—8 68 ,1 97 9 .

October 1 , 1 9 7 8 S ep tember 30 , 19 7 9

N AM ES,LA BOR ATORY AND I N ST I T U T E AF F I L I AT I ON S , ANO T I T L ES OF PR I NC I PAL I NV EST I G ATOR S AND ALL OTH ER

PROF E S S I ON AL PER S ON N EL ENG AG ED ON THE PRO JECTP I : London , Jack Research Microbiologis tOther : Chace , Nina M . Chemis t

Ce le sk , Roger Gues t workerMccab e , Rober t Bio Tech

[3 (b ) HUM AN T I S SU E S [5 (c ) N E I T H E R

SUMMAR Y OF WOR K (200 wo rd s or le s s und e rlin e ke ywo rd s )

bacillus casei and S trep tococcu s avium via a pathway unique to these micro

and the ensuing hetero f ermentat ion of the sugar alcohol s resul ts in the pro

duct ion o f lact at e,aceta te and ethano l . Regul at ion by gluco se is no t st ringent

and several o f the enzymes involved in pent itol d is similation cont inue to be

synthesiz ed while gluco se is metabo liz ed .

Gram negat ive iso lates from sub ging ival p laque depo s it s have been C haract eriz ed

teeth,so f t ening and demineralizing the roo t s tructure in the pro cess . The ini

t ial at tachment o f the organism to hydroxylapat ite appear to be mediated by

certain pro tein and lip id component s o f the cell's outer membrane .

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into the cells to provide inducer for the synthes iz e of pent ito l—Pdehydrogenase while the cell s metabol iz e and grow at the expense o f the

hexose .

At t empt s to s tudy and C haract er ize the syst ems regulat ing the dis s imilat iono f p entitols by trans ferr ing the genes coding for the pent itol PTS and

dehydrogenase us ing plasmid pAMB as a vector or modi fying extant trans format ion

pro cedures were unsucces s ful . Neither xylitol or rib itol ut ilizationwas expres sed in related , recip ient s train of bac teria that were init iallyunabl e to ut iliz e p ent ito ls . However , pAMB was shown to be freely

t ransmiss ible among s trep to co cci , pedio cocci and lactobacilli .

pocket and their bind ing propert ies . A group of 22 morphologicallys imilar gram negat ive bacter ia have been isolated from the ap ical area

o f the periodontal po ckets and C haract erized phys io logically and genet ically .

These thin , flexuous rods are mo t ile by gliding and exhib it limitedsac charolyt ic ab ilities ferment ing only four to s ix sugars . They grow

both aerob ically and anaerob ically and dif fer from Capnocytophaga sp . in

that they exhib it no CO requirement for growth . This group o f bact eria

c lo sely res emble facultat ively anaerobi c members o f the genus Cytophaga

and are now cons idered to be member s o f that genus .

S ix o f seven s trains tes t ed to date have exhib ited the ab il ity to adhere

to and colonize the too th roo t surfaces o f human teeth in in vitro

experiment s . The areas benea th the coloniz ed por tions o f the roo t

mutans coloniz ing teeth . The pro ces ses dif fer in that glucan— l ikepo lymer s do no t appear to be involved in the adhes ion o f the Gram negat ive

organisms .

Model sys tems employing sphero idal hydroxylapat ite beads ( SHA) and

s ized , powdered roo t fragment s have been devised to study the mechanism

o f adherence o f the Cytophaga sp . to roo t sur faces . The adsorp t ion o f

these bacteria to roo t powder or SHA appears to involve outer membrane

protein and l ip id component s by virtue o f the fac t that adsorpt ion

could b e blocked or inhib it ed by the fo llowing treatment s . (1 ) Heat ingat 70

0C f or 15 min reduced binding by SOZ . ( 2 ) Expo sure o f the cell s to

pro teinases and pho spho lipase C and D reduced binding by 802 . ( 3 ) Binding

was blo cked by pretreat ing the SHA or roo t powder with endo toxin or

l ipopolysaccharide extract ed from the Cytophaga sp . cell wall s . (4 )Binding was also blo cked by pretreat ing the sub strate with outer membrane

ves ic les is olated and pur if ied from culture supernatants .

PROPO SED COURSE OF RE SEARCH

A . The regulatory mechanisms controll ing pent itol metabol ism willb e elucidat ed by creat ing mutant s o f L . casei and S . avium blocked in

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e ither hexo se or pent itol ut ilization and determining the effec t s o f

add ing po t ent ial inducer and / or repres sor subs trates to growing cultures

o f the mutant s . The p athway o f rib ito l,D— arabito l and xylito l dis s imilat ion

in S . avium wil l be C harac ter ized and the prevalence o f pent ito l— ut il iz ingbacter ia in oral cavity will b e determined .

B . Comp le te C haract erizat ion of hydroxylapat ite b inding components

in outer membrane o f Gram negat ive bacter ia . An at t empt will be made

to iso lat e and C hemical ly def ine component s in the cell wal l respons ible

for at tachment . The phys io lo gical study of enzymat ic react ions involved

in trans it ion from anaerobic to aerob ic s tates o f growth will b e cont inued .

The source or reservo ir for Cytophaga sp . and es t ab lish relat ionship

b etween o ral Cytophaga sp . and so il forms will be det ermined .

PUBLI CATIONS

l . Marucha , P . T . , Keyes , P . H . , Witt enberger , C . L . , and London , JA rapid method for the ident if icat ion and enumerat ion o f oral

2 . Gibson,E . M . ,

Chace , N .M . , London , S . B . , and London , J . : Trans fer

o f p lasmid—mediated ant ib io t ic res istance from s trep to cocci to

lac tobac illi . J . Bac ter iol , l3 7 : 6l4— 6l9 , 1 9 79 .

3 . Johnson, J . L . ,

Phelps, C . F . , Cummens , C . S . , London , J . and Gas s er ,

Inter . J . Syst . Bact . In pres s .

4 . Ce lesk , R . A . ,Mccabe , R .M . and London , J . : Colonization o f the

cementum sur face o f t eeth by oral gram negat ive bact eria . Infect .

Immun . In pres s .

N AM ES,L A BOR ATORY ANO I N ST I T UT E AFF I L I AT I ON S, ANO T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PR OF E S S I ON AL P ER S ON N EL ENG AG EO ON THE PRO J ECT

PI : Bruce M . Chassy Research Chemist , LMI , NIDR

Other : Port er , E . V . Chemist , LMI , NIDR

Hull , E . B io logical Lab . aide , LMI , NIDR

Rokaw , E . Chemis t , LMI , NIDR

Laboratory o f Microb io logy and Immunology , NIDR

Laboratory o f Microb iology

I] (b ) HU M AN T I S SU ES (c ) N E I T H E R

SUMMARY OF WOR K (2 00 wo rd s or le ss un d e rl in e keyword s )The metabo lism of sucros e and lacto se ha s

‘

b een stud i ed b ecause o f these sugars

involvement in support ing the format ion o f dental caries in conj unct ion with the

ported into S . mutans cells by the act ion o f a PEP z PTS act ivity with the

product ion of intracellular (S6P ) . S 6P has been C hemicallysynthes iz ed

,and an enzyme catalyz ing it s oly sis to gluco se—6—pho sphate

and fructos e has been ident if ied and pur if ied . It is apparent ly ident ical with

the previously described intracellular invertase . A fructokinase that catalyz es

the ATP— dep endent pho sphorylation o f fructo s e has b een C haracter ized as well .

In L . casei it has been S hown that lacto se is transport ed into the cell by a_

specif ic lacto se PEP-PTS ; the result ing lacto se— 6—pho sphate is cleaved by

pho sp ho—B—galac to s idase t o gluco se and galacto s e— 6— pho sphat e . It has been shown

that these f irs t twO s teps o f the metabo l ism o f lacto se are plasmid-coded .

Several d is t inct lactose plasmids have been ident ified and their genet ic

po t ent ial and inter—relatedness is under invest igat ion.

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ZOl—DE—OOO42— 9 LMI B

it s C hemical synthes is has no t previously been repor ted . In this study

a chemical means for the synthesis of S 6P has been developed . Usingc yanoe thy lpho sphate and dic y c lohexy l

— carbodiimide in pyridine , sucro se

was pho sphorylated to produce a mixture o f isomers compris ed o f 62 2 S6P ,

2 42 S 6'R and small amount s o f o ther isomers . Homogeneous S6P was ob tained

by ion exchange C hromatography on Dowex— l— C hloride employing a linearly

decreas ing gradient o f so dium borate in ammonium C hloride . The result ingS6P , represent ing a 2 72 yield based on sucro se , was C hromatographicallyand analytically pure . Enzymat ic analys is es tablished a minimum content

o f 972 S 6P .

B . Characterizat ion o f S 6P hydro lase and it s ident ity with intracellularinvertase .

Extra cms o f S . mutans 6 715—10 ( cultured on sucro se) hydro lyz ed S6P to G 6P

and f mnxose . I t was found that S . mutans s trains E49 , BHT , SLl , and LM— 7

had s imilar act ivity . Cell s o f these s trains cultured on glucos e or

ruc tose contained S6P hydrolas e ac tivity , although level s were 2 to 5

fold lower than tho se ob served af ter growth on sucro se . Extract s o f S .

mutans 6715—10 cell s were subj ect ed to DEAE-cellulo se and Ultragel ACA

54 column C hromatography to pur ify the S 6P hydrolase . A 562 recovery o f

a 71-fo ld pur if ied enzyme was ob tained . Polyacrylamide gel electrophores is

revealed that the preparat ion was ess ent ially homogeneous . The enzyme

has a pH op timum of in ME S buf fer , no metal ion requirement , is

quite s table and has a molecular weight o f

It was ob served that the previous ly des cribed intracellular invertase

c o-purif ied with S6P hydrolas e . The rat io o f S 6P hydrolase to invertase

act ivity remained constant throughout the purif icat ion . In addit ion ,

bo th act ivit ies c o—migrated in a single band upon disc gel e le c trophoere sis .

The ident ity o f the two enzymes is further indicated by the ob servat ion

that sucros e is a compet it ive inhib itor o f S6P hydrolys is with a Ki o f 8

mM . S ince the Km for S6P hydro lys is is mM and the Km for sucros e

hydro lys is 90 mM , the enzyme should b e cons idered a S 6P hydrolase rather

than an invertase . As such it represents a previous ly uncharacteriz ed

enzyme . The ac tion o f S 6P hydrolase provides G 6P for entry into the

glycolyt ic pathway and liberates fructo s e .

C . Purif icat ion and propert ies o f fructokinase from S . mutans

During growth on sucro se,intracellular fructo se is produced by the

act ion o f S 6P hydro lase on S6P . Previous s tudies in this laboratory had

demonstrated the presence o f a manno-fructokinase catalyz ing the ATPdependent pho sphorylation o f fruc to se or mannose in extract s o f S .

mutans . The enzyme has b een highly purif ied,but ins tab ility has prevented

the iso lat ion o f a completely homogeneous protein . The Km for fructos e

pho sphorylation is mM and for mannose mM . The molecular

weight , determined by gel f il trat ion,is The two pho sphorylat ing

act ivit ies c o-purify . F ructo se is a compet it ive inhib itor o f mannose

u- z LI9

Zol DE— OOO4 2 — 9 LMI B

pho sphorylat ion and mannose is a compet it ive inhib itor o f fructos e

pho sphorylat ion . In addi tion , the two act ivit ies c o—migrate during dis c

gel electrophores is . These resul ts indicated that the same pro tein

catalyz es bo th ac t ivit ies . The ac t ion o f this kinas e would enable the

fructo s e mo iety o f sucro s e to be as s imilated by glycolys is . Moreover,

under growth condit ions where the pho spho-transport sys tem for fruc to s e

or manno se is inact ive ( e g : high subs trate concentrat ion or low pH as

has b een ob served for gluco se—PTS activity ) the enzyme would al low

pho sphorylat ion o f manno se or fructo s e that entered the cell via apermeas e .

D . Purif icat ion and propert ies o f glucokinase from S . mutans

Under condit ions o f high sub s trate concentrat ion or low pH the gluco se

PTS o f S . mutans can be inact ivated . A highly spec ific glucokinase has

b een iso lated f rom cell s of S . mutans during this study . The enzyme

would provide an alternat ive pathway for pho spho— ry lation o f gluco se

facil itat ing it s entry into the glyco lyt ic pathway . Glucokinase would

also serve to pho sphorylate any intracellular gluco se that accumulates,

such as would o ccur during the fermentat ion o f lacto se or mal to se . The

kinas e has been pur if ied to homogeneity by DEAE—cellulo se , Ultrage l ACA

54 and ATP-af f inity ge l column Chromatography . It has 3 Km for gluco se

pho sphorylat ion o f mM , a molecular weight o f ( SD S gel electrophores is

and gel f il trat ion ) , and requires ATP and divalent metal ion for act ivity

I t exhib its o rdered B i Bi react ion kinet ics typical o f several o ther

ATP—dependent glucokinases previously studied .

E . Plasmid— determined B—galac to s ide metabo lism in Lactobacillus casei

subsp . casei .

Previous s tudies in thi s laboratory had shown that lo s s o f the 2 3 mdal

p lasmid , pDRIlOl ,from L . casei DR1002 result ed in lo s s o f the ab il ity

to ferment lacto s e . This same lo s s o f lac to se fermentat ion has been

ob served with f ive addit ional s trains o f L . casei af ter treatment with

agent s known to cause delet ion or curing o f p lasmids . L . casei DR1004harbors a s ingle mdal plasmids which is ab sent in lacto se negat ive

mutant s,while L . casei DR1005 lo ses a 36 mdal lacto se plasmid when it

lo s es the ab ility to ferment lact ose . L . casei DR1005 lac variant s

retain the 4 and 7 mdal crypt ic plasmids found in the parent s train . I t

has b een det ermined tha t lacto s e fermenting strains o f L . casei sub sp .

casei have a lact os e— PTS for the transport and pho spho-ry lation o f

lacto se . The lac to se— 6— pho sphate thus formed is cleaved to galacto se— 6

pho sphate and gluco se by phospho-B—galac to s idase . The galacto syl moietyo f lactos e

,as we ll

'

as galacto se it sel f , has been shown to be metabol ized

by the tagato se— 6— pho sphate pathway rather than the C las s ical Lelo irpathway . Lo s s o f these lacto se plasmids result s in the lo s s o f the

ab ility t o ferment al l B- galacto s ides ( eg : lactulo se , lac tobionic acid ,

methyl—B— galacto s e ,and lacto se ) . Lac isolates are devo id o f lacto se

PTS act ivity as wel l as pho spho— B— galacto s idase .

'

I t is propos ed that

4 — 2 4 3

Zol DE-OOO42—9 LMI B

the s truc tural genes for at least part o f the lacto se-PTS and for pho spho- B

galactos idase reside on the lacto s e plasmids , while the genes o f the

tagatos e— 6— pho sphate pathway appear to be Chromasomally encoded .

Other trait s as so ciated with the lacto s e plasmids have not b een es tabl ished ,

however lac variants o f all three strains dif fer from their parent

s train in res is tance or s ensit ivity to arsenite , arsenate , C hromate and

cadmium ions . Cell wall analys is has revealed no dif ference in cellwall compos it ion . No evidence for conj ugal funct ions , pro teases , ant ib io t ic

res is tances or res is tance to UV radiat ion has been found to b e as sociated

with the lactos e plasmids . Preliminary att empt s at res trict ion enzyme

mapping o f the plasmid DNA have shown that no obvious homology exis t s

b etween the three lac to se plasmids isolated from three s trains o f L .

casei .

S IGNIFICANCE TO BIOMEDICAL RE SEARCH

The mechanism o f carbohydrate ut il iz at ion by oral microbes has doub tless

b een an important selec tive criterion in their evolut ionary development .

These s tudies underscore the diverse and complex adap tat ion o f these

organisms to ef f ic iently ut il iz e carbohydrates . Ult imat ely , such studies

S hould allow us to unders tand the economy o f thes e organisms and may

sugges t C hanges that can be made in the oral environment that wouldfavor S hift s in the ab ility o f the eco sys t em to support a b enign rather

than a pathogenic micro flora .

Cons idering the demonstrated ro le o f plasmids in coding for pathogenic

po tent ial , c onfering antibio t ic res is tance , as well as coding for ant igen

and bac terioc in product ion in o ther bacteria , it would be o f great value

to know if such relat ionship s exis t b etween plasmids and the oral micro flora .

In addit ion , a study o f the dis tr ibut ion , funct ion and relatednes s o f

these plasmids may contribute to our unders tanding o f bact er ial evolut ion,

spec ial izat ion , and adaptat ion in an emerging and C hanging eco sys tem .

PROPOSED COURSE

1 . To C haracter iz e fully S6P hydrolase in S . mutans 6 715-10 and tocompare it to S6P hydro lases iso lated from o ther S . mutans s trains .

2 . To conduct a comparison o f S 6P hydrolase and extracellular invertase

from S . mutans , as well as to ass es s their relat ive phys io lo gical ro les .

3 . To develop a genet ic trans fer syst em such as trans format ion,conj ugat ion

or transduct ion for the analys is of lacto se plasmid gene product s .

Lactobacillus phages being isolated in this laboratory may b e explo it edin developing a transduct ional syst em . AS an alternat ive , this obj ect ive

may b e met by ut iliz ing a cell— free DNA— directed pro t ein synthes is

sys tem . Cloning lactos e plasmid genes into suitable ho s t organisms is

also a po s s ib ility .

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Octob er 1,197 8 S ept emb er 30 ,

197 9

N AM ES,LA BOR ATORY ANO I N ST I T UT E AF F I L I A T I ON S , AN D T I T L E S OF PR I NC I PAL I NV EST I G AT OR S AN D ALL OT H E R

PROF E S S I ON AL PER S ON N EL ENG AG ED ON THE PR O J ECT

PI : Donkers loot , Jacob A . Res earch Microb iolog i st

OTHER : Harr , Rob ert J . B io Lab Tech (Micro . )Flatow , Ursula Microb io log ist Tech

Hull , Eunic e M B io Aid

[3 (b ) HU M AN T I S SU ES [X (c ) N E I T H E R

SUMM AR Y OF W OR K (2 00 wo rd s or les s und e rlin e keyword s )The pos s ib ility that an endog enou s endo-dextranas e contr ibut es to the st ructural

strep to cocc i is current ly under invest igat ion . F or th is purpo s e,Strept ococcus

sal ivarius mutant s that produc e either more or les s d extranas e have b een

iso lat ed . Whereas the h igh-d extranas e mutant synth es iz ed about the same amount

of inso lub le g lucan and produc ed adherent depo s it s like the parent,the low

dextranase mut ant produc ed more s olub l e glucan and d id not form adherent

depos it s . Two dextranase— def ic ient mutant s o f S . mutans 67 15— 13 have b een

id ent if ied and thes e also synthes iz ed more so lub le g lucan . In order to

determine wheth er thes e phenomena are causally related,the amount s o f the

transf erase enzymes produc ing so lub le and inso lub le g lucan will b e

s ible ro le of plasmids in the phys io logy of S . mutans is b eing

As a resul t of th es e stud ies,two new spec ies of c ircular DN

from S t rain LM— 7 . The larg er one is a 3OMdal covalent lynd the smaller one is a 2Mda1 C ircular spec ies

,which is not a

h appears t o or ig inat e from the prev ious ly descr ib ed 3 . 6Mdal

Zol DE-OOO43— O9 LMI

OBJECTIVES

The ongo ing research ha s two g eneral obj ect iv es,which will be discu s s ed

s equent ially .

1 . One f oca l p lane of our at t ent ion has been to increas e our und er standing

of the enzymo logy of insolubl e glucan synthes is by oral st rep tococc i .

The insoluble glucan synthes iz ed by S . mutans is complex becau s e it is

highly branched and contains s equ enc es of bo th d— ( l 6) and a— ( l 3 )l inked gluco s e res idues . At leas t two funct ionally d ist inct gluco syltrans f eras e

(GT) act iv it ies ar e present in many st rains, G T— I and G T— S ,

that catalyz e

the synthes is of an inso lubl e and a solubl e glucan,respec t ively . G T— I

med iat es the synthes is of a relatively linear d- ( l 3 ) g lucan,and G T—S

a pr imar ily a— ( l 6 ) — linked glucan that is probably branched . Thus , it

is evid ent that the nat ive inso luble glucan is more complex than the

product synthes iz ed by G T— I . S everal strains of S . mutans secr et e a

third enzyme,endo—d extranas e

,and it has b een sugg es t ed that the act ion

of this enzyme might account for the gr eat er complex ity of the nat ive

inso lubl e glucan . Th is dext ranase pref erent ially hydrolyz es d — ( l — 6)l inked glucans to isomalto s e sacchar ides . If this enzyme ac t s on the

po lymer synthes iz ed by G T— S ,and if the product s ar e re— incorporat ed

during glucan synthes is,the synthes is of an inso lubl e g lucan similar to

the nat ive one can be conc eived . This year we have begun to t est this

hypo thes is by study ing mutants with alt ered dextranase act iv ity .

2 . The s econd general obj ec t iv e of our stud ies has b een to C haract eriz e

the s tructure and funct ion of p lasm ids in oral st rep to cocc i . S o far ,

the maj or thrust o f our eX p e rimental ef fort ha s been to crit ically

examine the pos s ib il ity that a p lasmid contro ls glucan synthes is by S .

mutans .

METHODS EMPLOYED

For mos t of our stud ies on glucan biosynthes is , S . sal ivar ius 2 597 5 ,

rather than S . mut ans,has b een us ed because it produces much higher

l evels of GT and b ecaus e this organism has been employed in several

o ther s tud ies in our laborat ory .

Thr ee new method s were used : ( 1) becaus e the previously descr ibed in

v ivo method to det ect d extranas e act ivity was not s ens it ive enough for

S . sal ivar iu s,a new method wa s developed ( see below) . A st aining

method was develop ed which allows part ial C haract er izat ion of the glucan

produc t s mad e in gels by G T— I and GT— S af t er electrophor et ic separat ion .

( 3 ) A new method,rec ent ly developed by Don LeBlanC and Linda Lee of our

sect ion,has been used t o examine S . mutans for plasmid DNA .

Other method s have b een descr ib ed pr eviously .

MAJOR F INDINGS

B ecaus e S.sal ivar iu s 2 5 97 5 d id no t S how dextranase ac t iv ity on the

pr ev ious ly d es cr ib ed t es t agar,a more sens it ive test medium was developed

II _ Q II7

ZO1 DE-OOO4 3-O9 LMI

for this stra in . To prove that this new test agar wa s , in general , mor e

sens it ive,a survey was conduc ted . It showed that several oral st reptococc i

that had prev iously been report ed as dextranas e—negat ive , did po ss es s

ac t iv ity,b ecaus e they decoloriz ed the new , blue-dextran containing

agar . This med ium was then us ed to det ect dextranase mutant s af t er

mutagenes is of S . salivar ius . S everal mutant s producing mor e and one

mutant producing les s dextranas e than the parent wer e isolat ed . The

high dextranase mutants wer e s imilar to the parent with respec t to the

amount of insoluble glucan synthes iz ed and the format ion of adherent ,

plaque— like,depo s it s with sucro s e . In contrast

,the low dextranas e

mutant synthes iz ed a soluble glucan rather than an insolubl e glucan , and

did not form plaqu e— like depo s it s .

AS a r esult of a survey for dextrana se act iv ity among a s er ies of previouslyisolat ed mutant s with alt ered glucan synthes is

,two other dextranas e

def ic ient mutant s wer e ident if ied . These mutant s or ig inat e from S .

mutans 67 15-13 and hav e b een f ound to synthes iz e mor e so lubl e glucanthan their parent

,and to b e non-car iog enic for smoo th enamel surfac es .

To s ee whether the increas ed synthes is of solubl e glucan in the S .

mutans mutant s was d ir ect ly due to the dextranase def ic iency , GT-I and

G T— S act ivit ies wer e as s es sed aft er elec trophor et ic s eparat ion . Prel iminary

ev idence from act ivity sta ins sugg es t s that the increas ed synthes is of

soluble glucan in thes e mutant s is due to an increas ed level of GT-S

act iv ity , rather than the dextranase def ic iency as such . S imilar exper iment s

ar e planned for the S . sal ivar iu s mutant .

Our stud ies on the pos s ibl e ro le of p lasmids in the phys io logy of S .

mutans ar e cont inu ing . We have pr eviously S hown that , although the

ab ility to form inso lubl e g lucan and to form adherent,p laque— l ike

depo s it s i s lo st at relat ively high fr equ ency from S . mutans LN—7 , this

is not accompanied by the lo s s of the megadal ton plasmid pAM7 .

However , the po ss ib il ity ex is t ed tha t ano ther p lasmid that could no t be

d et ect ed by the method s then in use,was controlling this event . B ecaus e

a new technique to det ect p lasmids in st rep tococc i had j us t been worked

out , it was appl ied to S . mutans LM—7 . Agaro s e el ectr ophor es is showed

two DNA bands that had not b een s een prev iously . The lower mob il ity

sp ec ies could be pur if ied by Os Cl-ethid ium bromid e c entr ifugat ion , and

appear s to be a covalent ly C los ed C ircular molecule of about 30 megadal ton .

However , the small er S pec ies was lost upon s imilar c entr ifugat ion .

Because of this unexpect ed result,X was studied in mor e detail . Electron

microscopy showed that X had a molecular weight of 2 mill ion . However ,

desp it e s everal s earches,a covalent ly C los ed mol ecule of this S iz e was

never found , and it was f inally dec ided that the X mol ecules s een af t er

agaros e electrophor es is were in a labile , op en C ircular , conf igurat ion .

Further work S howed that if the adj ustment of the cell lysat e to pH

which is part of the p lasmid iso lat ion proc edure , was omit t ed ,X

was no t ob served . In the next experiment we obs erved that X was obtained

in good y ield by subj ect ing pAM7 to pH This shows that pAM—7

contains alkal i-s ens it ive S it es . Whether these S it es repr es ent modif ied

ba ses that are alka li- sens it ive,or shor t str et ches of RNA , and how the

conver s ion of pAM7 to X takes p lac e,is under invest igat ion .

Int — 2 4 8

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O ctober 1 , 19 7 8 through S ep temb er 3 0 ,

N AM ES,L A B OR AT ORY AN D I N ST I T UT E AFF I L I A T I ON S , ANO T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT HE R

PROF ES S I ON AL P ER S ON N EL ENG AG ED ON PRO J ECT

Keyes,Paul H . Dental Director NIDR

OTHER : Howard,Surya A . Health Technician NIDR

(Dental )Mccabe ,

Robert M . B io . Lab . Te ch . (Mic ro ) NIDR

Microb iology Sect ion

[3 (b ) HUM AN T I SS U E S (c ) N E I T H E R

difand amoebae . Populat ions o f actinomy ces also appear to dif fer . Som

mot ile rods that have b een isolated and studied appear to b e S trains

Only a few o f the highly prevalent sp irochital populat ions have been isolated .

Amoebae have been iso lated,but maintained with dif ficul ty . As determined by

phase contrast micro s copy, populat ions assoc iat ed with disease can be shifted

to S uppress the microb iot a not reliably controlled by the f irs t two measures .

2 01 DE 0009 6— 05 LMI

OBJECTIVES

This proj ec t is des igned to provide answers to the following ques tions

1 ) Do es the micro f lora as so ciated with disease dif fer from that as soc iated

with heal th ? Are the dif ferences merely quantitat ive (as mo s t dent is t s

b el ieve ) or do they also dif fer qualitat ively? Can the use o f a phase

contras t micro s cope in the dental operatory help in the diagno s is and

control o f periodontal diseases ? Can sp iro chetes,mot ile rods

,amoebae

and o ther bac teria that are prevalent in les ions be isolated and s tudied ?

Can target populat ions be ident if ied and eliminated by appropriate

therapy ? What happens to teeth af ter bact erial contro l has been attained

and maintained ?

MAJOR FINDINGS

Samples o f bact er ial plaque removed from the gingival crevices of persons

with no evidence o f per iodontal inf lammation reveal a loo s ely s tructured

network o f f ilament s co lonized by coc ci and o ther small forms . Nosp iro chetes , amoebae , or larger gliding and sp inning rods have been

det ec ted . Very f ew white blood cells have been present . Older persons

with marginal g ingivit is have harboured more dense populat ions o f colonizedthreads . Sp inning rods have been prevalent and in mo s t ins tances a few

sp iro chetes have been found . A few very long ( 50 u+) S piral rods have

occas ional ly been present . Highly turbulent surfaces o f spiro chetes and

larger gl iding rods have no t been ob served . By contras t , samples removed

from roo t s o f t eeth undergo ing autogenous excis ion reveal highly turbulent

sur faces . S tat ic mas ses o f cel ls are colonized by mill ions o f sp iro chetes

and gliding rod s that beat together , of ten synchronous ly , set t ing up

wave upon wave o f pul sat ing act ivity . Numerous amoebae usual ly glideover the rapidly moving ends of sp iro chetes and flexing rods . Whileb lood cell s

,in numbers too numerous to count , as semble in the spaces

per ipheral to the turbulen t surfaces .

A number o f s olut ions can cause almos t immediate ces sat ion o f all mot il ity

and also "coagulat ion o f the intracellular s truc tures o f larger microorganisms ,

e . g . : solut ions o f so dium b icarbonate , sodium C hloride , sodium fluoride ,

sodium periodate,aluminum Chlorate , z inc phenol sulfonate , magensium

sul fate, C hloramine—T ,

glycerol , et c . I f proper ly del ivered , any o f

thes e agent s S hould b e benef icial adj uvant s for suppres s ing turbulentpopulat ions . All have been used as aid s to oral hygiene , some for

centuries .

Mod if ied irrigator t ip s and various applicators are being tried in the

C linic and by pat ient s to improve del ivery o f medicament s to C lear ,

"det oxify and decontaminate roo t surfaces and C ircumradicular spaces .

Evaluat ions o f the C l inical s tatus o f pat ient s , by all convent ional

paramet er s o f as ses sment,indicat e that periodontal les ions abate or

arres t when bacter ial populat ions are reduced to levels the periodontalt is sues tolerate , as indicated by low counts o f white blood cell s in

C ircumradic ular spaces .

2 01 DE 0009 6-05 LMI

Teeth malpo sitioned b efore being brought under microb iological control,

by pres sures from high edematous t is sues , have tolerated repo s it ioning

without apparent lo ss o f additional t is sue .

S IGNIFICANCE TO BIOMEDICAL AND DENTAL RESEARCH

These s tudies are des igned to explore relat ionship s b etween the microb ialflora and the d is ease s tate o f the ho s t . I t is ant ic ipated that

informa t ion derived from such s tudies will ult imately be useful in

formulat ing rat ional approaches to the diagno s is and treatment o f periodontaldis ease .

PUBLICATIONS

l . Marucha , P . T . , Keyes , P . H . , Wit tenberger , C . L . and London ,

J . A rap id method for the ident if icat ion and enumerat ion o f

oral Inf ec t . Immun . 21 : 786-7 9 7 . 1 9 78 .

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Zol DE— OOl7 4—O4 LMI

OBJECTIVES

The long— range goal of this proj ect remains the eluc idat ion of the role

o f bact er ial p lasmids in the pathog enes is , ecology and taxonomic s tatus

of the oral microb ial f lora , with part icular empha s is on the st reptococc i .

This repor t summar iz es the r esu lt s of stud ies aimed at : 1 ) develop ing a

rap id procedure for iso lat ing p lasmids from st rep tococc i ; 2 ) d etermining

the ef f ect s of tet racycl ine therapy on the oral microb ial f lora ; 3 )evaluat ing the role of p lasmid s in res istance of the oral strept ococc i

to t etracycl ine ; 4 ) d et ermining the genet ic and molecular propert ies of

a delet ed res istance p lasmid from a Str ept ococcu s st rain ; and 5 ) eluc idat ing

the ro le of p lasmids in the metabo lism of carbohydrates by stra ins of S .

lact is .

METHODS EMPLOYED

A new pro cedure,d escr ib ed b elow

,for iso lat ing p lasmids from members of

the g enus Str ept ococcu s wa s developed dur ing the pas t year . Otherwis e,

all pro cedures used in these studies were standard microb ial or mo lecularb iolog ica l t echniques , with mod if icat ions apprO pr ia t e for adaptat ion to

s trep tococc i and s trep to coccal p lasmids .

MAJOR FINDINGS

in Str eptococc i . St andard p lasmid iso lat ion t echniques have either been

too expens ive and t ime—consuming,or totally inad equat e

,for p lasmid

screening of larg e number s of S tr ept ococcus iso lates . By employ ingcr it ical st ep s from published enrichment procedures

,and incorporat ing

'

t echniques which enhanc e the lys is of oral st rep t ococc i , we have d evelopeda proc edur e for the iso lat ion and det ec t ion of p lasmid DNA from v irtuallyall S pec ies of s trep toco cc i . The procedure is rapid , r eproduc ibl e and

inexpens ive , requir ing neither rad io iso t opes nor dens ity grad ient s f or

preliminary scr eening purpo ses . We have used this t echniqu e to screen

more than 150 strains of st reptococc i,includ ing 10 dif f er ent oral

spec ies iso lat ed from C linical sourc es . Plasmid s have been demonstrat ed

in member s of all sp ec ies t est ed . S ev eral oral iso lat es pr eviou sly

thought to be d evo id of p lasmid DNA,f ollowing analy s is by standard

t echniques , wer e S hown by our procedure to harbor one or more plasmidspec ies . We have also succ es sfully employed the proc edure for preparat ive

purpo ses , start ing with cell s from 4 00 t o 800 ml of C ulture .

2 and year s tudy , des igned to determine the ef f ect s of short- term

t et racycl ine therapy on the facultat iv e anaerob ic subg ing ival microbial

f lora of p er iodontal pat ient s,ha s been complet ed . The data ind icat ed

that virtually any sample of human subg ing ival p laque,whether obtained

b efore or af t er ant ibio t ic therapy,wil l contain tet racycl ine res istant

bac t er ia . In the major ity of pat ients studied the res istant populat ion

repres ented a small perc entage of the total pr ior to treatment . However ,

the propor t ion of the populat ion res is tant to t etracycl ine increased

II - Q RAI

Z0 1 DE— OOl74— O4 LMI

dramat icall y ( 10 to SOC— fold ) aft er two weeks of therapy . Withdrawal

of t he treatmen t result ed in a S ignif icant ( 3 to 100— fold) decrea se in

the res istant popu lat ion within 60 days . The maj or ity of t etracyc l ine

r es istant iso lat es ob tained at all thr ee sampl ing t imes wer e id ent if ied

1 0 sp ec ies wer e ident if ied , with S . sanguis ( 62 2 ) and S . mit is ( 192 )p redom inat ing . There were no S ignif icant diff er ences in the dist r ibut ion

of S p ec ies isolat ed befor e , af t er or fol lowing the ces sat ion of t etracyc linetherapy .

The plasm id enrichment proc edure has been us ed t o screen 124 of the

t etracycl ine r es istant iso lat es for the pr esence of plasmid DNA . P lasmid s

wer e d et ec t ed in only 2 12 of the stra ins exam ined . In two of the plasmid

containing st rains it was po ss ibl e to correla te the tet racyc line res istance

pheno typ e with the presence of a S p ec if ic p lasmid S p ec ies . The resu lt s

sugg est that tetracyc l ine res ist ance among the oral strep tococc i is

g enerally no t p lasmid-med iat ed .

’

This is part icular ly s ignif icant b ecau se

t etracycl ine res istanc e is probably the mos t common plasmid—med iat ed

drug r es istance found in nature and,hist orically

, has almo st univer sally

b een p la smid—borne among bact er ial iso lat es from C l inical sourc es .

Unusual Proper t ies of a Delet ed Res istance Plasmid . We have previously

d escr ibed the mol ecular and genet ic proper t ies of a transmiss ibl e res istance

p la smid, pAMBl ( B) , or ig inally iso lated from a

gtrain of S . f aecalis .

The B plasmid has a mo lecular weight of 17 x 10 mediat es res istance to

ery thromyc in and l incomyc in,and can be transf erred between several

S p ec ies of st rep to co cc i by conj ugat ion .

S . angino sus s train DR1501 ( 8) has b een used as a univer sal donor of the

B p lasmid in conj ugat ion exper iment s . We have r ec ent ly iso lat ed a

var iant of this st rain,DR1501 /BR1 ,

which is no long er abl e to donat e

the B p lasmid but has retained the res istance phenotypes .

Plasm id DNA from S . angino su s st ra in DR1501 /BR1 wa s f ound to pos s es s the

f ollowing prO p ert ie S : 1 ) In agaro s e gels , NA band s correspond ing tg

p lasm id s hav ing mol ecular weight s of 7 x 10 . ( sp ec ies A) and 10 x 10

( S pec ies B ) wer e obs erved,but no 17 M dal band cou ld be seen . 2 ) A

p lasmid— les s culture of S . anginosus st rain DR1501 could b e trans formed

by either spec ies A or spec ies B DNA . All trans formant s were res ist ant

to erythromyc in and l incomycin but cou ld no t transf er the res istance

trait s in conj ugat ion experiment s . All transformed C lones examined

contained bo th S pec ies A and spec ies B plasm id DNA , even though only one

o f the two spec ies had b een us ed in the transf ormat ion . 3 ) Both sp ec ies

A and B wer e covalent ly clos ed C ircular ( C C C ) plasmids , based on dye

buoyant dens ity gradient analyses . 4 ) Analy sps of rest r ict ion endonucleas e

dige s tgreveal e d a

6mol e cular weight of 8 x 10 for each S pec ies , not

7 x 10 or 1 0 x 10 The restr ict ion pat t erns of each spec ies wer e

id ent ical,each miss ing the same fragment s from the or ig inal 17 M dal

B p lasmid . 5 ) Cont our length measurement s of r elaxed C irc l es of bo th6

sp ec ies wer e id ent ical,prov id ing a mol ecular weight est imat e of 8 x 10

6 ) The nat iv e C C C molecules had very diff erent conf igurat ions , as det ermined

by el ec tron micro scopy . Sp ec ies A appeared as a t ight ly coiled ros et t e

1 ike mol ecule,while spec ies B had a relat ively normal C C C conf igurat ion .

ZOl DE—OOl7 4—04 L MI

Thes e r esult s sugg est tha t S . anginosu s st rain DR1501 / DR1 carr ies a

s ingle p lasmid from which a 9 M dal segment of the B plasmid has been

delet ed,and that at least one funct ion required f or conj ugat ion has

b een lost . The rema ining 8 M dal s egment st ill po ss es s es rep l icat ive

func t ions and ant ib iot ic res istanc e trait s , and can ex ist in two d iff er ent

and unusual , C C C conf igurat ions .

Among strains of S . lact is the metabol ism of gluco s e,mannos e

,lacto se ,

galacto s e,sucro se and fructos e is init iat ed by a pho spho enolpyruvat e

d ependent pho sphotransf eras e syst em (PTS ) . The act ivit ies of the sugar

S p ec if ic component s of the PTS f or f iv e of thes e carbohydrat es app ear to

b e p lasmidd m ediat ed . In one strain of S . lac t is , DR12 51 , the ab ility t o

metabo liz e lacto s e , galact os e and sucro s e , and the corresponding PTS

act iv it ies , wer e correlated with the presenc e of specif ic plasmids .

Lact o s e and galacto s e— PTS act ivit ies wer e bo th dependent on a 32 M dal

plasmid in this s train,while lo ss of sucro s e-PTS act ivity was accompanied

by the lo s s of a 28 M dal p lasmid . A s econd strain of S . lac t is,

DR12 52 , was found to b ecome def ect iv e in it s abil ity to grow on gluco s e

at a high spontaneous frequency . Thes e gluco s e-def ect iv e isolat es were

also manno se—def ect ive , in that growth rat es on either subst rat e wer e

cons id erably S lower than in the parent s train,and f inal growth y ields

were only half o f that obtained with the parent . This dual ef f ect was

as soc iat ed with the los s of a 23 M dal p lasmid by stra in DR12 52 . In yet

a third strain of S . lac t is,DR1253 ,

the ab il ity t o grow at the expens e

o f D— xylo se , a non— PTS sugar,has tentat ively been correlat ed with the

pres enc e of a M dal plasm id . All of thes e resu lt s sugg es t that

plasmids p lay an extremely impor tant ro le in the metabo lism o f S .

lact is , a Lanc ef ie ld group N S trepto co ccus .

S IGNIFICANCE TO BIOMEDICAL RESEARCH

The contribut ions of p lasm id s to the pathog enes is , ecology and taxonomic

status of s everal groups of bact er ia have been well— document ed . Examplesof p lasmid-med ia t ed bact er ial func t ions which have had C l inical s ignif icance

ar e as f ollows : 1 ) The widespr ead occurrenc e of p lasmid—med iat ed mult ip le

ant ib iot ic r es istanc e cont inues to frust rate at t empt s to trea t numerous

bact er ial inf ec t ions ; 2 ) the ent erotoxins respons ibl e for many cases of

human and animal d iarrhea ar e now known to b e p lasmid-cod ed ; 3 ) the

ability of pathog enic co liforms t o at tach to int est inal ep ithel ial

c ells , and thu s rema in in a proper eco log ical set t ing for expr es sion of

pathog enic t rait s , is also p lasmid— assoc iat ed ; and 4 ) certa in p lasmidmediat ed metabo l ic propert ies

,such as lacto se ut il iz at ion in Salmonella

has lead to confus ion in the diagno s is of cer tain dis ea se stat es .

Result s fr om this laborat ory,as wel l as o thers

,have shown that p lasmid s

may p lay s imilar rol es among the str ep tococc i,at least with respect to

ant ib iot ic res is tance and metabo l ic trait s .

The overall comparat ive stud ies on the molecular bio logy and g enet ic s of

s treptococcal p lasmids are expected to contribut e to a bet t er understanding

of these extrachromo somal element s in general . Furthermore,such studies

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Zol DE-OOl74— 04 LMI

LeBlanc , D . J . , Hawley , R . J Lee , L . N . and S t . Mart in,E . J .

Conj ugal" transf er o f p lasmid DNA among oral strep tococc i .

Proc . Nat l . Acad . S c i . U . S . A . 7 5 : 3483 ,197 8 .

L eBlanc , D . J Hawley , R . J ., Lee , L . N . and S t . Mart in

,E . J .

Ant ibiot ic— r es istance p lasmids among the oral st rep to cocci .

In Parker , M . T . Pathog enic Streptococc i . Re edbooks ,

Surrey , England . p . 28 6 , 197 9 .

iP ERIOD COV ER ED

Oc tober 1 , 1 9 7 8 through Se ptember 30 , 19 7 9

Various Member s o f the

N AM ES , L AB OR A TO RY ANO I N ST I T U T E AFF I L I AT I ON S , AND T I T L ES OF PR I NC I PAL I NV E ST I G AT OR S AND ALL OT H E RPR OF E S S I ON AL P ER S ON N EL EN G AG ED ON THE PR O JECT

PI Edward J . S t . Mart in Senior S taf f F ellow

OTHER : Evelyn M . Gib son Bio logist

g (b ) HU M AN T I S SU E S [g] (e ) N E I T HE R

S UMM AR Y OF WOR K (2 00 word s or les s un d e rlin e ke ywo rd s )

using mutant analys is t echniques . Two enzyme syst ems for ammonia as s imilat ion

have been ident if ied : glutamat e. dehydrog enas eand the coupled react ions o f

glutamine synthetas e and glutamat e synthase . The primary pathway for sucro se

ut ilizat ion has also b een det ermined . Catabol ism o f sucro s e is init iated

by an induc ib le phosphoenolpyruvate—dependent sucro se pho spho trans f erase sys t em

followed by an intracellular sucro se 6— pho sphat e hydro lase .

Zol-DE—002 08—3 LMI

OBJECTIVE S

The general obj ect ives o f this proj ect is to use mutant iso lat ion and

analys is techniques to examine the funct ion and regulat ion o f severalkey metabol ic react ions present in cariogenic microorganisms . More

specif ically , we have under taken the study o f ammonia as s imilat ion and

sucro se catabo l ism in S . mutans .

MAJOR F INDINGS

Ammonia ass imilat ion . We have previously reported that several s trains

o f S . mutans are capable o f growth in a C hemically def ined medium without

the addit ion o f an exogenous supp ly o f amino acids . An enzyme,glutamate

dehydrogenase , that is capable of f ixing free ammonia to form amino

acids was detected in these strains . However,examinat ion o f glutamat e

dehydrogenase—negat ive mutant s revealed that they were s t ill capab le o f

growth in the absence o f amino acids . Us ing these mutant s , we have

ident if ied a second method for ammonia as s imilat ion in S . mutans . This

pathway is composed o f two enzymes , an ATP— driven glutamine synthetase

and a NADH-C oupled glutamate synthase , that can funct ion together to f ix

free ammonia for the bio synthes is o f amino acids . The levels o f allthree enzymes are regulat ed by the availab il ity o f amino acids . This

proj ect has been completed .

Sucro se-catabol ism . We have ident ified a phosphoenolpyruvat e— dependentsucro se pho spho trans f erase ac t ivity ( suc— PTS ) in S . mutans . The product

o f this react ion is sucro se 6—pho sphate ( suc— 6— P ) . Mutant s that are

mis s ing either suc— PTS activity or the ab ility to hydro lyze suc— 6—P ,

were isolated . We have used a mutant that is cons t itut ive for the

synthes is o f suc—PTS act ivity but mis s ing suc— 6—P hydrolase act ivity to

synthes ize subs trate amount s o f suc— 6— P . Using the purif ied sub s trate ,

we have found that suc— 6— P hydro lase is a cons t itut ive enzyme with a Kmfor suc— 6-P o f mM .

SIGNIF ICANCE TO BIOMEDICAL AND DENTAL RE SEARCH

The cariogenic po tent ial o f oral microorganisms res ides in their ab ilityto ef fect ively co lonize and pro l iferate on dental surfaces where they

produce acid fermentat ion product s from available carbohydrates . The use

o f mutant analys is will permit us to det ermine which o f the metabo lic

react ions pos sessed by these microb es might b e important to their succes s ful

coloniz at ion of a compet it ive and chang ing environment , such as the oral

cavity , and p ermit expres s ion o f their pathogenic po t ent ial .

PROPO SED COURSE OF STUDIES

Work on the pathways o f sucro se ut il izat ion will now fo cus on enzymes

that are capab le of hydro lyz ing sucro se . In order to examine these

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Oc tober 1 , 19 78 Sept ember 30 , 19 7 9

N AM ES,L A BOR ATORY ANO I N ST I T U T E AF F I L I AT I ON S , AN D T I TL ES OF PR I NC I PAL I NV EST I G ATOR S AND ALL OTH E R

PR OF E S S I ON AL PER S ON N EL ENG AG ED ON THE PR O JECT

PI : Ko lenbrande r , Paul E . S enior S taff Fel lowO ther : Cisar , John 0 . S enior S taf f Fellow

S tradley , An Microbiologis t

D (b ) H UM AN T I S SU E S Q (c ) N E I T H E R

S UMMAR Y OF WOR K (200 wo rd s or le ss un d e rlin e keyword s )The propert ies o f oral ac tinomy cetes which aff ect the pot ent ial o f these bac

t eria t o partic ipate in cell-cell int eract ions with o ther oral microorganisms

are under inves tigation . Many o f the patt erns o f cell— cel l interac tions b etween

the oral ac tinomycetes and oral s treptococc i have b een elucidated . Resultsfrom a recent inves t igat ion using f reshly isolated oral s trep toco cc i and

act inomycet es not'

only confirmed earlier result s with s tock s trains from

national culture co llect ions but also revealed addit ional a tterns of coa

t ion reac t ions . Currently,an extens ion o f the type of inves tigation which

des crib ed the coaggregat ion pat terns between oral act inomycet es and s trep to c o c c

phage isolat es has b een ob served . It i s expect ed that further invest igat ion

of bact erial cell— cell interac tions will lead to bet ter unders tanding o f the

natural mechanisms required to maintain these oral microorganisms in their

ecologi cal niche .

OBJECTIVES Zol DE— 002 7 3— Ol

The overall focus o f this proj ect is to ident ify the mechanisms employed

by oral bacteria in their int erac tions with o ther bac teria and ho s t

t is sues . This repo rt present s the result s of our inves t igat ions deal ingwith

l ) cell— cell interact ions (co aggregat ion ) between oral actinomycetesand s trep toco cci .

2 ) b inding properties and spec if icity o f coaggregat ion between oralCy tophaga isolates and act inomycetes

.

3 ) requirement for neuraminidase act ivity for hemagglut inat ion o f

human erythrocytes by act inomycete iso lates.

METHODS EMP LOYED

All t echniques used are s tandard methods rout ine to the type of s tudyconducted .

MAJOR FINDINGS

group s o f oral st rep toc occ i and two group s o f act inomycet es based upon

the p at terns o f coaggregat ion between members o f each type of bacteria .

Coaggre gations o ccurred frequently when human s trains o f Act inomyces

sanguis o r S . mit is , but were infrequent or did not occur at all with

o ther oral ac t inomycete s and s treptococc i . These experiment s were

conduct ed us ing primarily laborat ory sto ck strains obtained from the

Center for Di s eas e Cont rol and the American Type Culture Co llect ion

(ATCC ) . Subsequent studies us ing more than 300 freshly iso lated oral

s trains b oth conf irm the previous findings for each o f the S ix groups

g iven above and expand the number o f groups . One new group o f st reptoco cci

and two new group s o f act inomycet es have been found . In addit ion ,

ano ther pat tern o f coaggregat ion al ready not ed in the init ial s tudies

was again observed with the new oral isolates . This lat t er pat t ern was a

Since i t was the only act inomycete which exhibited that coaggregat ion

p at tern in the initial study , it was not well do cument ed . With the

discovery o f four new iso lates with the same coa ggregat ion pat te rn , it

will be pos s ibl e to define more care fully this important group o f oral

act inomycet es . It is S igni ficant tha t wi th such a large number o f fre sh

oral i solates , so f ew new coaggregat ion patt e rns we re det e ct ed . These

resul t s sugges t a great deal of speci fic ity is operat ive in cell— cellinterac t ions between oral bacteria .

ind icat ed that none o f these coaggregat ed with Act inomyce s israelii

ZOl DE-002 7 3— Cl

s trains . However , test ing o f 18 freshly iso lat ed Cytophaga strains

revealed that these strains coaggregate only with A . israel ii and not

s ignif icant ly with A . vis co sus or A . naes lund ii . Int eract ions between

members o f the lat ter two species and oral st reptococc i form the bas is

o f the coaggregat ion patt erns des cr ibed in the above sect ion . Dis t inct

coaggregat ion pat terns between the Cytophaga isolat es and A . is raelii

s trains were ob served . S ince only four A . israel ii s trains were immediatelyavaliable for this study , addit ional strains have b een reques ted from

appropriate sources in order to complet e this study . Freshly iso lat edA . israel ii s trains will al so be tes t ed . I t is expected that non—random

coaggregat ion pat terns will be obs erved S imilar to the resul t s ob tained

b etween oral s treptococ ci and A . vis cosus and A . naes lund ii strains .

Already , the sp ecif ic ity of cell— cell interac tions between oral bact eria

i s evident .

l ) by the ab sence of coaggregat ion between oral streptoco cci and

A . israel ii2 ) by the interact ion between oral Cyt ophaga iso lates and only A .

israel ii s trains

and oral st rep tococci

4 ) by the limited number (i. e . specif icity ) of coaggregat ion pat t erns

b etween greater than 300 freshly iso lat ed act inomycetes and

s trep to co cc i

The b inding propert ies of these Cyt ophaga iso lates and A . israelii with

respect to bind ing to roo t powder and sphero idal hydroxyapat it e are

current ly under inves t igat ion in collaborat ion with Dr . Roger Cele sk and

Dr . Jack London o f this section . I t appear s that pre c oating either o f

the above sol id sub strates with Cyt ophaga cel ls decreas es the b inding

ef f ic iency o f A . is raelii as compared to binding o f A . israel ii to

uncoated subs trate. This resul t conf irms the exist ence o f cell— cellinteraction already ind icat ed by coaggregat ion experiment s between these

two group s o f bac ter ia . It also po int s toward the po tent ial for demons trat ing

a compet it ion between these two group s o f bact eria for sol id subs trat e

S it es .

erythrocyte s by act inomycete isolates . In addition to bind ing to other

bacter ia,oral ac t inomycet es are able to hemagglut inate human erythrocytes .

In collaborat ion with Dr . John Cisar o f the Humoral Immunology Sect ion ,

we have found that hemagglut ination oc cur s af ter a period o f incubat ion

at 370C unles s the erythrocytes have been pretreated with neuraminidase .

In the latt er case hemagglut ination occur s immediat ely . Nearly all o f

the act inomycet e st rains test ed ( 2 5 o f 26 tes ted ) produce neuraminidase .

Part o f the act ivity is cell—bound and the remainder is in a solubleextracellular form . Only the so luble form is ef f ect ive in pretreatment

o f erythrocytes for hemagglut inat ion with act inomycete cell s , and an

absolute requirement for neuraminidase ac tivity for hemagglut inat ion has

b een demons trat ed . The nature of the binding o f act inomycete cells to

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N AM ES,LA BOR ATO RY AND I N ST I T UT E AFF I L I AT I ON S , ANO T I TL E S OF PR I NC I PAL I NV EST I G AT O R S AND OT H ER

PR OF E S S I ON AL PER S ON N EL ENG AG ED ON THE PRO JECT

PI : Siraganian , ReubenCOPI : Hook , Will iam A .

Barsumian , Edward

Morita , YutakaOTHER : V lagopoulos , Triphon

Wahn,Hans Ulrich

Basc iano ,LuAnn

Chief , Clinical ImmunologyResearch Microb io logis t

Vis iting Fellow

Vis it ing FellowGues t Worker

Gues t Worker

Microb io logis t

q b ) HU M AN T I S S U E S D (c ) N E I T HE R

SUMM AR Y OF WOR K (2 00 wo rd s or le ss un d e rlin e keyword s )

o f the immuno log ical mechanisms involved in inf lammat ion . Among the his tamine

releas ing agents employed are IgE antibody , environmental ant igens , and

between IgE c ro s slinking ,basophil desens it iz ation and his tamine release were

analysed by kinet ic s tudies . Release o f inf lammatory mediators from culturedmas to cytoma C e lls is being compared with mechanisms of release from mas t

cells and basophils .

2 0 1 DE 00034— 11 LMI

I . Pro j ec t Descrip t ion

Basophil s from atop ic individual s can be desensi t i zed by incuba

t ion with antig en under non—releas ing cond itions . Cell s from human

subj ec t s were desens i ti zed in vi tro with two dif ferent environmental

ant igens and then chall enged with the same antigens and anti— IgE .

The ra te o f c el l des ens it ization varied with the antigen concentrat ion .

Wi th concentrat ions of ragweed antigen in the ascending port ion o f the

hi s tamine release curve , desens it ization was slower than with supra

op t imal amount s o f antigen . The reduc tion o f his tamine release af ter

desens iti zat ion was propor tional to the time o f incubat ion . The

degree o f de sens it izat ion at l to 37°

C was proport ional to the ineu

bation t emperature . The leukocytes of mo s t subj ects te s ted showed

non-specif ic de sens it iza tion to two or more antigens . Des ens it iza t ion

o f the cell s o f a f ew other subj ec ts showed only a one—way de sensi

t iz ation . Only one ind ividual was found that showed S pec if ic desen

sitiz a tion toward two antigens , i . e . cells were desens it iz ed speci

f ic ally to bo th ant igens . The result s ind icate that in vitro desen

sitiz ation o f human basophils is predominant ly non— specific and this

S pec if ic i ty i s not dependent on ant igen concentrat ion nor on incuba

t ion temperature .

B . Mathematical model s for analys is o f recep tor c ros slinking and

A ma thematical model for relat ing the number of ant igen— induced

I gE C lus ter s on the bas ophil plasma membrane to the amount o f his

tamine released was developed . The method is valid at low do ses o f

ant igen where,it is shown ,

the number of bound antigens and the

number of IgE c lus ter s are linearly proport ional to the to tal coneen

trat ion o f antigen . Cross linking of receptors on some port ion of the

sur face is cons idered to be a neces sary condit ion for transmis s ion o f

a S ignal that leads to degranulat ion . No assump tion is made about

whether the degranulat ion thus st imulated is local or global .

Analys is of the low— do s e po rt ion o f the do se-response curve , however ,

indica te s the following : 1 ) Degranulat ion is lo cal , and the maximum

numb er o f lo c al_

c ente rs per cell is small (N IO ) . 2 )The number o f IgE- C lus ters required to init iate such local release is

small bu t variabl e and depend s upon the cell source and ant igen

valence.The result s varied between three and eight for Con A , AgE ,

and antiIgE . 3 ) These number s represent thresho lds for release ; i . a . ,

to a good approximation some minimum number o f ant igens in exces s o f

one mus t b e mul tip ly bound before gpy_

his tamine can b e released .

This sugges ts a high degree o f co operativity in the b iochemical

Z01 DE 00034—11 LMI

sequelae to C lustering . 4 ) Only a small fract ion of C lus ters are

transmit t ing a release S ignal . This suggest s , and is C ons is tent with

the idea , that C lus ters are interac t ing with some other membrane mole c ule and that only tho se that are bound to the o ther molecule are

b io lo gically ac tive . 5 ) With respect to transmit t ing a s ignal for

his tamine release the number o f independent C lus ters appears to b e a

more fundamental quant ity than the to tal number o f cros s-linked recep

tors ; i . a . , there is lit tle dif f erence in ac tivity as the s iz e o f a

C luster increases due to increase in l igand valence .

Analysi s was per formed of doseresponse and desens it izat ion o f

rabb it b asophil s by a variety o f ant igens . Unlike the response o f

human basophils , the his tamine release curves rarely ri se and fall in

a symmetric fashion , but more of ten either rise to a p lateau , peak ,

and then fall to a non— z ero plateau , or exhib it b imodal ity . In tho se

cas es in whic the curves peak , preincubat ion with antigen in the

ab sence o f Ca has a smaller ef fec t at the high and low concentrat ion

por t ions o f the curve than it does toward the center , where the in

b ib itory e ff ect is maximum .

The result s can be exp lained by a S imple model in which : 1 ) The

s ignals for release and desens it izat ion bo th increase as the c onc en

tra tion o f re ce ptor cro ss-links increases and 2 ) the signals reach a

pla teau they have a maximum value ) when the number of recep tor

cro s s—links b ecomes suf f ic iently large . By changing the parameters

o f the model , the balance between desens it izat ion and release changes ,

and all the ob served pat t erns can b e reproduced . In part ic ular , if

bo th s ignal s saturate (reach their maximum value ) at a l igand c onc en

tration b elow that of maximum cros s-linking , the histamine releasecurve will plateua . I f the re lease s ignal saturates but the inhibi

tory s ignal cont inues to increase as lgand concentration increases

toward it s op timum value , the his tamine release curve wil l be b imodal .

Many of the parameters can b e ident i f ied with tho se that aff ect

recep tor clus tering . These include the aff inity of IgE for the

ant igen the number of IgE molecules per basophil ,IgE hinge flexi

bility , membrane fluidity,and ant igen concentrat ion and valence .

This ident ificat ion permit s a variety o f add it ional predic t ions . Onesuch predic t ion i s that the dif f erence between a dos e-response curve

that p lateaus and one that is biphas ic and unimodal ref lec t s eas ier

des ens itizat ion o f C ell s respons ible for the lat ter curve and / orthat have a smaller numb er o f cro s s— links . Bo th predict ions are

readily tes table experimentally .

Basophils from rabb it s sens iti z ed to penicillin release his ta

mine when S t imulated with bi— or mul t ivalent penicillin derivatives

but not by the monovalent hapten . Monomer inhibit s release by either

bi or mult ivalent antigens . Kinet ic studies revealed his tamine

release curves which were s igmoidal , having delays in the onset o f

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2 01 DE 00034—11 LMI

tomatoes . The resul t s indicate that there frequently is no correla

t ion b etween a C linical his tory o f hypersens it ivity to food and the

ant igen-induc ed in vitro hi stamine release from human leukocytes .

The intriguing observat ion was al so made that a sizeable proport ion

o f subj ec ts without symptoms of food hypersens it ivity S how IgE-mediated

his tamine r elease with the food allergens . This suggest s that humans

may po s sess a control mechanism to prevent an allergic response to

inges ted food ant igens .

F . Histamine release by cultured masto cytoma cells .

Mas tocytomas induced in mice by Abel son murine leukemia virus

were adap ted to in vitro cell culture . The mas to cytoma cell s po s ses s

granules containing his tamine and sero tonin and are useful for studies

o f the mechanisms o f his tamine secret ion . Four mas to cytomas were ob

tained from four dif ferent mous e strains : Balb / C , CX BG , CXBI and CB

ABM—4 . The histamine content , IgE and IgG-mediated his tamine re

leas ing ab ility of the various cell l ines has b een s tudied . Work is in

progres s toward def ining o ther pheno typic characteris tics o f these

mouse mas tocytomas .

The rat basophil ic leukemia (RBL ) cell l ine originally ob tained

by Eccles ton is unrespons ive to IgE-mediated histamine release . How

ever , ir has been po ss ible to ob tain variant s of the RBL lines which

behave l ike normal animal cell s in this property . The releaser and

non—releaser clones thus ob tained have a C hromo some number rangingfrom 65— 75 . Bo th po s ses s the same IgE receptor and their IgE recep

tor as soc iat ion C ons tants are s imilar . However , IgE dis so ciates

S lower from the non—releaser cell l ines . Such REL clone s ob tained

from the parental tumor ce ll cons titut e a homogeneous population of

cells that can b e utili z ed to study the complex phenomenon o f

his tamine release .

G . Prepara t ion o f hybridomas .

IgE-produc ing hybrid l ines were ob tained aft er hybr idiz ing mouse

myeloma (NS-1 ) and sp leen lymphocytes from BALD / C mice paras it ized with

were succes s fully trans ferred to mice and the as c ites fluid ob tained

from the tumor— bearing anima l s contained large quantit ies o f IgE .

This monoclonal IgE and subsequently a homologous ant i-IgE will allows tudies o f the interact ion of IgE with it s receptors on mas t cells .

Monoclonal antibodies were al so ob tained agains t rat F C recep tors ;this will allow us to s tudy the role o f such recep tors on mas t cells ,

and to compare the IgE and IgG recep tors on these cell s .

11- 2 7 0

2 0 1 DE 00034 — 11 LMI

H . S tudies o f inhib it ors and enhancers o f hist amine release

The eff ect o f various compounds on the IgE—mediated , ant igen

induced his tamine releas e from RBL-l HR+ (2H3 sub line ) ~ was inves t i

gated . The cell s were sensit iz ed with IgE , prein cubated with various

pharmacologic agent s and challenged with ant igen . The metabo l ic ih

hibitors antimycin A , dinitrophenol,sodium az ide and po tas s ium

cyanide inhib it ed his tamine releas e espec ially in the ab sence o f

gluco se while 2— deoxy gluco se had minimal or no ef fect . Agents which

reac t with micro tubules had variable ef fec t s ; cyto chalas in B and D

enhanced ant igen— induc ed release , where agcyto chalas in A had a bi

phas ic ef fect : low C oncentrat ions 10 M enc hanc ed and higher

leve ls inhib it ed releas e (10 5M) . Vinblas t ine and co lchic ine inhi

b it ed his tamine rele ase whereas D 0 showed mild enhancement . Com

pounds known to increase intrac e lIular CAMP levels inhib it ed release

only at high concentrat ions (e . g . 502 inhib itory leyzls with dibu

tyryl CAMP , aminophyl line and isopro terenol >5 x 10 M) . Cholera

toxin caused enhancement at 100 ug /ml . Other compounds which had no

ef fec t on his tamine release included dibutyryl c GMP , CAMP and car

bachol . Release was inhibi t ed by the pro teas e inhib itors TP CK or

TLCK and by hetrazan . In general , the releas e mechanism from RBL— l

HR+ c ell s appears to have dif ferences from that of normal mas t cell s .

I . His tamine rel eas e by G 5a and C3a anaphylatoxins o f human

serum .

The ac t ivat ion o f human serum complement by incubat ion with

microb ial cel l walls,including dental plaque , generates anaphyla

toxin (C5 3 ) which releas es hi s tamine from human basophils . These

cel ls al so po s s es s recep tors for C3a , ano ther anaphy latoxic pep tide

derived from human s erum . On a molar bas is , C5a is approximately

1000 t imes more ac t ive in releasing his tamine than C3a . The S pec i

fic ity o f the recep tors for pur if ied C5a and C3a were s tudied by cel l

de s ens it izat ion exp eriments . I t was found that leukocytes de sensi

tiz ed with human C5a reta ined their s ens it ivity to release by puri

f ied human C3a . Smiliarly ,cel ls desens it ized with the chemo tac tic

pep t ide fMe t— Leu—Phe reta ined full sens it ivity to his tamine rel ease

by bo th C5a and C3a . The re sul t s support the concep t that sepa

rate,sp ecif ic rece p tors for C5a and C3a are present on human

basophil s .

I I.S ignif icance to Biomedical Research and the Program o f NIDR

The purpos e o f the proj ect is to s tudy the immuno lo gic release

o f mediators such as his tamine which re present the ef fe c tor mechanisms

by which IgE ,aller gens ,

or microb ial cells may int erac t with lenko

cytes or s erum to caus e inflamma tion . Desensi t izat ion mechanisms

2 0 1 DE 00034-11 LMI

and pharmacologic agent s are s tudied in vitro for their po tent ial use

in contro l o f inflamma tory re sponse s .

Experiment s will pursue further pathways and regulatory mechanisms

of immunologically-induced his tamine release .

IV . Publicat ions .

1 . Siraganian , R .P . and Har z ard, KA . : Mechanisms o f

mouse mas t cell act ivation and inac t ivation for IgE

med iated h is tamine release . J . Immuno l . 1 2 2 : l 7l9

1 7 25 , 197 9 .

DeLisi, C . and Siraganian , R . F . : Receptor cros s—linkingand his tamine release . I . The quant itat ive dep endence

o f basophil de granulation on the number o f recep tor

double t s . J . Immunol .—22 9 2 , 19 7 9 .

De Li s i , C . and Siraganian , R . F . : Recep tor cros s

l inking and his tamine releas e . II . Interpre tat ion

and analysis of anamolous dose res ponse patt erns .

J . Immunol .-229 9 , 19 79 .

Siraganian , R . P . and Metzger , H . : Evidence that the

Mouse Mas tocytoma" cell line (MGT—l ) is o f rat origin ,

J . Immunol . 19 78 .

Siraganian , R . P . and Sandb erg , A . L . : Characterizat ion

o f mous e allergens . J . Allergy Cl in . Immuno l . 6 3

435 , 1979 .

Siraganian ,R . P . , Baer , H . , Hochs tein , H . D . and May ,

J . C . : Allergenic and b io logical ac t ivity o f commerc ialpreparations of hous e dus t extract . J . Allergy and Clin .

Immunol . ( In pres s ) .

Hernandez—Asens io , M . , Hooks , J . J . , Ida , S . , Siraganian ,

R . P . and Notkins , A . L . : Interf eron— induced enhancement

o f IgE—mediate d histamine release from human basophil s

requires RNA synthes is . J . o f Immunol . 1 2 2 : l 601-1603 ,

1 9 79 .

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2 01 DE 00085—6 LMIProj ect Des cript ion

To determine the pat hophys iology and et iolo gy of S j 6 gren's

syndrome .

II . Methods

1 ) Cl inical s tudies

a . S tudy o f the cl inical manifes tations o f d gren's

syndrome in t h e presence or absence o f rheumato id ar thrit is . This

s tudy was conducted through retrospect ive analys is of the medicalrecords o f 43 pat ients .

b . S tudy o f the relat ionship o f sys temic lupus erythema

tous ( SLE ) to Sj 6 gren's syndrome . This study was done in 24 c ons e c

utive pat ients with SLE who were evaluat ed for C l inical , laboratoryand his tologic findings o f Sj 6 gren

’s syndrome . Each pat ient answered

a ques t ionnaire and underwent a complete eye examination,paro tid

flow rate , parot id s c int igraphy and b iopsy of the lab ial salivaryglands .

2 ) Laboratory s tudies

a . HLA typ ing Lymphocytes from 2 2 pat ients with Sj 6 gren s

syndrome alone and 11 pat ients with Sn ren'S syndrome and rheumato id

arthrit is were typed for HLA—A ,-B ,

-C ,-DRW and Ia—like B— lymphocyte

ant igens . The s tudy was done in collaborat ion with Dr . Dean Mann .

b . Serologic s tudies S era from 24 pat ient s with SLE ,

who were tes ted for C l inical and his tolo gic findings o f d gren's

syndrome , 1 7 patient s with Sn ren'S syndrome alone and 12 pat ient s

with Sn ren's syndrome in the presence of rheumato id arthrit is were

tes ted for rheumato id fac tor by the bentonite flo cculat ion tes t , anti

bodies to nat ive DNA by ammonium sul fate prec ip itat ion tes t and anti

bodies to cellular antigens S S—A , SS—B and RANA by immunodif fus ion .

c . C irculat ing immune complexes and the F C recep tor function

o f RES in Sj 6 gren's syndrome .

These s tudies were done in collaborat ion with Dr . M . Frank 's

laboratory . The presence o f C irculat ing immune complexeswipg

t es t ed

in the serum o f 54 patient s with Sj bgren's syndrome by the I—Clq

b ind ing as say and the Raj i cell rad io immunoas say . Fur thermore , the

compos it ion of immune complexes was examined . The F c recep tor func

t ion o f the RES was inves tigated in 19 pat ient s with d gren's syndrome

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by measuring the rate of e l iminat ion of inj ected C hromium labeled IgG

s ens it iz ed autologous e ry throcyte s .

d . Immunoregulat ion in Sn ren's syndrome These s tudies were

done in co l laboration with Dr . Pauc i 's laboratory . Peripheral T and B

lymphocyt es were enumerated by standard techniques . The immunoregulatoryT— cell subpopulat ions were identified on the bas is o f the presence o f F c

recep tor s for IgG or IgM immunoglobul ins . The B—lympho cyt e spontaneous

ac t ivat ion was de f ined ac cording to the ab ility o f the peripheral B

lymphocytes to produce plaques to the hap ten TNP . In addition the

number o f C irculating intracytoplasmic lg containing B— C ell s were

enumerated . The B— cell func t ion was evaluated on the bas is o f their

respons e to po lyclonal ac t ivator pockweed mitogen . The suppre s sor

func t ion o f t h e T— cells was ass es sed by the their ab ility aft er con

canaval in-A s t imulat ion to S uppre s s pockweed mitogen driven cul tures .

III . Maj or Findings

as so ciated with o ther auto immune diseases .

Sn ren's syndrome occurs as an entity alone or in as so c iat ion

with o ther auto immune diseas es ,primarily rheumato id arthriti s or

sys temic lupus erythema to sus .

Our retrospect ive cl inical s tudy on the C l inical manifes tat ions

o f Sj bgren's syndrome in the absence and presence of rheumato id

ar thr it i s revealed that the two ent it ies have quite dis t inc t C l inicalfeatures . Pa t ients with S j é gren

's syndrome alone had a S ignif icantly

great er frequency o f recurrent paro t id gland enlargement , Raynaud 'sphenomenon

,purpura

,lymphadenopathy

,myo s it is and renal involvement

desp it e the fac t that the sal ivary his topathology was ident ical in both

group s o f pat ient s .

S tudy on t h e inc idence o f autoan t ibo dies to ce llular ant igens

S S-A , S S-B and RANA revealed that pat ient s with S j bgren's syndrome

alone have a high incidence o f autoantibod ies direct ed to S S—A and

S S-B ant igens and no t to RNA . In contras t partients with rheumato id

ar thrit is and Sn ren's syndrome produce antibo dies to RANA and no t

to SS-A and S S— B anti gens . Thus patients with t gren's syndrome alone

in addit ion to cl inical d if ferences with pat ient s with d gren's

syndrome and rheumato id arthrit is po ss es s a serologic dicho tomy . To

inves t igate if this dichotomy occurs on the grounds o f genet ic dif fer

enc e s we s tudied the asso ciat ion o f the se groups with cer tain HLA

ant igens.

We found tha t pa t ients with Sn re n's syndrome alone i s

highly a s so ciated with HLA—B8 ,HLA—DRW3 , Ia— l7 2 , Ia— 350 and Ia-715

while S j bgren's syndrome in the presence of rheumato id arthrit is

ZOl DE 00085-6 LMI

is as soc iated with HLA-DRW4 Ia-l7 2 and Ia-350 . Thus these two groups

of pat ients have common genet ic markers (Ia—17 2 and Ia-350 ) and

dif ferent ones HLA-88 , HLA—DRW3 , HLA—DRW4 and Ia-715 . The lympho

c y tic infil trate o f the minor salivary glands may depend on the de

gree as sociated with Ia—1 72 and Ia-350 while the gene (s ) asso ciated

with HLA-B8 , HLA—DRW3 and Ia-715 may fulf ill a ro le in pat ient s with

d gren's syndrome alone s imilar to that played by rheumatoid arthrit is

and its as soc iated ant igen HLA—DRW4 in pat ients with Sn ren's syn

drome and rheumato id ar thrit is . These observations have promp ted us to

evaluate the C haract erist ics o f Sn ren's syndrome in pat ient s with

sys temic lupus erythemato sus . We evaluat ed 24 patients with sys temic

lupus erythemato sus for C linical (kerato conj unct ivit is S icca , xero

s tomia) , his tologic , and serologic evidence o f Sn ren's syndrome .

Cl inical abnormali t ies suggest ive of Sj 6 gren's syndrome in pat ients

with syst emic lupus erythematosus were ident if ied by paro t id scan

ques t ionnaire lab ial b iopsy Schirmer 's tes t (332 ) and

paro t id flow rate The mos t s triking find ing , however , was that

pat ient with sys temic lupus ery thematosus‘

and abnormal lab ial b iopsy

had antibodies to SS—A cellular ant igen and to immuno globulinsS imilar to tho se s een in pat ient s with Sj 8 gren

's syndrome alone . Thus

in three auto immune d iseases asso ciat ed with focal lymphocyt ic infil

trate o f the salivary gland s dis tinct ant ibody respons es to dif ferent

cellular ant igens can b e demonstrated . We interpret these findings to

suppor t the hypo thes is that d gren's syndrome alone and S j 6 gren

's

syndrome in syst emic lupus erythematosus are C lo sely related and each

dif fers from Sj figren's as soc iated with rheumato id arthrit is .

We have des cribed previously that three pat ients with Sn ren's

syndrome developed immune complex glomerulonephrit is . Thi s prompted

us to s tudy if immune c e omplexe s c irculate in patient s with d gren's

syndrome and their physiochemical propert ies . The sera of 54 patients

yigh S j 6 gren's syndrome were tes ted for immune comp lexes with the

I-Clq b inding assay and the Raj i rad io immunoas say . Eighty-f ive

percent o f these s era were posit ive by ei ther as say for immune com

plexes . This was true o f all cl inical subcategories and regardles s o f

whether or no t typ ical s igns and symptoms of immune complex mediated

dis eases were pre sent . The two as says showed concordance of 902 in the

detec t ion o f C irculat ing immune complexes . The amount o f immune come

plexe s as det ect ed by the Raj ii cell as say was not correlated with

the t iter o f rheumato id factor,but a po sit ive asso ciat ion was found

be tween the Clq binding act ivity and the t iter o f rheumato id fac tor .

To eliminate the po s s ibility that rheumato id fac tor may comprise the

ma j or part of the c irculat ing immune complexes in pat ients with S j 6

gren's syndrome , s era were pretreated with 2~merCaptoe thanol to dis

soc iate the p entameric IgM rheumato id factor or with solid phase pro tein

LI _ ? 7 FZB k

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2 0 1 DE 00085— 6 LMI

Neither patients with glandular nor extraglandular diseas e mani

f e s ted increased number s of in vivo act ivated circulating lymphocytesas det ermined by spontaneous anti-TNP PF C . However

,pat ients with

glandular di s ease had reduced numb ers o f PWM— induced anti— SRBC PF C

(p as compared to normals and pat ients with glandular disease .

Of no te was the fact that de spit e the modulation o f T subpopulat ion

by the serum fact or in pat ients with extraglandular di s ease ,thes e

pat ients manifes ted normal Con A— generated suppressor cell s o f PWM

induced P F C responses in allogeneic c o-cul tures . This was unlike the

suppres sor cell def ect previously des cribed in this sys t em with SLEpat ient s . The discrepancy was at tributed to the fact that the T

defec t was revers ib le as well as the fact that Con A—generated sup

pres sor cell s are no t limit ed to the T sub set s . Thus,these s tudies

have demonst rat ed revers ible abnormali t ies in T cell s with extra

glandular S j bgren's syndrome which are no t as so c iated with suppres sor

cell defect s . The di screpancy between these f indings and the immuno

regulatory defec ts demons trate d in SLE may explain the dif ference in'

s everity o f the auto immune expres sion in these dis eases .

IV . Publicat ions

1 . Kas san S S , Thomas TL , Mout sopoulos HM , Hoover R,Kimberly RP ,

Budman DP , Co s ta J , Decker JL and Chus ed TM : Increased risk

o f lymphoma in sic ca syndrome . Ann . Inter . Med -89 2 ,

19 7 8 .

2 . G o t tdiene r JS , Moutsopoulo s HM , Decker JL : Echocardiographic

ident if icat ion o f c hardiac abnormal ity in s cleroderma and

related disorders . Amer . J . Med .-398 , 19 79 .

3 . Mout S O poulo s HM , Webber BL , V lagopoulo s TP, Chused TM

,

Decker JL : Dif ferences in the cl inical manifes tat ions o f

s icca syndrome in the presence and ab s ence of rheuma to id

arthr it is .

— 7 36 , 19 7 9 .

4 . Chused TM , Mout sopoulos HM , Sharrow S , Hansen CL : Evidence o f

a primary B-lymphocyte abnormaility in NZB mice . In genetic

control o f auto immune disease . NR Ros e , PE Biga z z i ,NL

Warner , (eds ) Els evier , Amsterdam 19 7 9 , pp 1 7 7-19 1 .

5 . Hooks JJ , Mout sopoulos HM , Geis SA , S tahl NI , Decker JL ,

Notkins’

AL : Immune inter feron in the circulat ion o f pat ients

with auto immune dis eas e . New Engl . J . Med .

'

301 : 5—8 , 1 9 7 9 .

6 . Tauro g JD , Moutsopoulo s HM,Ros enberg Y J , Chus ed TM , S t ein

berg AD : CBA/ N x-l inked B-cell defect prevents NZB-B- cellhyperac t ivity in P

1mice . J . Exp . Med . 150 : 3l—43 , 19 7 9 .

ll .

1 2 .

1 3 .

14 .

2 0 ] DE 00085— 6 LMI

S imon BF ,Mou t so pou

l os HM : Primary amylo ido s is resembl ing

s ic ca syndrome . Arthrit is and Rheum . In pres s 19 7 9 .

Mout sopoulo s HM , Mann DL , Johnson AH, Chus e d TM : Genet ic

d if ferences b etween primary and secondary s ic ca syndrome .

New Engl . J . Med . In pres s 19 7 9 .

Lawley TJ , Moutsopoulos HM , Kat z S I , The ofilopoulos AN ,

Chused TM , Frank MM : C irculat ing immune complexes in

Sj agren's syndrome . J . Immun . In pre s s 19 7 9 .

Hamburger MI, Mout so poulo s HM , Lawley TJ ,

F rank MM : Ade fec t in ret iculoendo thel ial sys tem F C rece ptor func tion

in Sj ogren's syndrome . Ann . Inter . Med . In pres s 19 7 9 .

Mou t sopoulos H M, Kl ippel JH , Pavlidis N , Wolf R0 , Sweet JB ,

S t einberg AD ,Chu F C , Tarpley TM : Correlat ive his to logic

and s ero lo gic f indings o f s icca syndrome in pat ients with

sys temic lupus erythematous . Arthr . Rheum . In pres s 1 9 7 9 .

Reiner t sen JL ,Shaefer ES , Brewer HB , Mout sopoulo s HM

S icca—l ike syndrome in type— V hyperl ipopro teinemia . Arthr .

Rheum . In pres s 197 9 .

Moutsopoulo s HM ,Fauci AS : Immunoregulat ion in S j figren

's

syndrome . Inf luence of serum fact ors on T— cell sub populat ions .

J . Cl in . Inves t . In pres s 19 7 9 .

Mout sopoulos HM (moderator ) : Sj ogren 's syndrome . Current

is sues.NIH conf erence . Ann Inter . Med . In pres s Dec . 1 9 7 9 .

N AM ES,LA BOR A TORY AND I N ST I T UT E AF F I L I AT I ON S, AN D T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PROF E S S I ON AL P ER S ONN EL EN G AG ED ON THE PRO JECT

PI Siraganian ,Reuben Chief, Clinical Immunolo gy NIDR

COP I : F ox ,Phil ip C . Clinical As soc iate NIDR

OTHER : Fischler,Cynthia Med ical Technician Micro . NIDR

[j (b ) H UM AN T I S S U E S [ 1 (c ) N E I T H E R

SUMM ARY OF WOR K (2 00 wo rd s or les s und e rl in e keyword s )

The contro l mechanisms o f IgE product ion are under s tudy ut il iz ing a mouse

model sys tem . At tempt s are being made to pur ify mur ine I gE and develop a

radio-immunoassay capab le o f quantitat ing the small amount s o f IgE present in

mouse s erum (es t imated to be les s than ug/ml ) : for this purpo se anIgE

—hydridoma has b een produced . Mice expo sed to nebulized antigens are

incapable o f an IgE response When later immunized with the same antigen . The

mechanism o f this phenomenon is under inves t igation .

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2 0 1 DE 00114—05 LMIII I . S ignificance

Mediator release from ma s t cell s and basophil s is an important

amp lif ication sys tem in immunological inj ury . IgE ant igen inter

ac t ion i s the s pec ific tr igger for the release o f histamine . The

present s tudies are an at temp t to b etter unders tand the mechanisms

which control the induct ion and syn thes is of IgE . The mouse model is

ideal f or this type o f s tudy because o f the extens ive previous s tudies

on cellular interactions in t h is animal and the availab ility of mult iple inbred s trains .

IV . Prepos ed Cour se

1 . S tudies will determine the characteris t ic o f the phenomenon

o f IgE suppres s ion induced by exposure to nebuliz ed antigen . The

cellular bas is o f this suppres s ion will b e inves tigated .

2 . Purif ied mouse IgE will b e used in s tud ies o f the immuno

b io logy o f the IgE sys t em; e . g . s tudies on the interac t ion o f IgE

with mas t cell .

30 , 1 9 79

r Immuno logical Mechanisms in Periodontal Disease

N AM ES , LA B OR A TO RY ANO I N ST I T UT E AFF I L I AT I ON S,AN D T I TL ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PR OF ES S I ON AL P ER S ON N EL EN G AG ED ON THE PR O J ECT

Fox,Phil ip C . Clinical Ass o ciate NIDR LMI

Wahl,Sharon M . Res earch Microb io logis t NIDR LMI

Oppenheim,J . Medical Of f icer NIDR LMI

Hook,Wil l iam A . Research Microb iolo gis t NIDR LMI

Siraganian ,Reuben Chief

,Clinical Immunology NIDR LMI

Met zger,Zvi Vis it ing F el low NIDR LMI

(b ) H UM AN T I SSU E S (c ) N E I T H E R

SUMM A R Y OF WOR K (200 word s or les s und e rl in e keyword s )

A group o f pat ient s with periodonto s is is b eing s tudied to ident ify cel lular

and adultperiodont it is and individuals without periodontal patho logy . A

a famil ial pat tern o f this di sease is be ing inves t igated .

ZO1 DE 002 05— 3 LMI

PROJECT DESCRIPTION

I . Ob j ect ive

The s tudy group cons i st s of individual s with periodontal di sease,

inc luding a sub—group of young peop le with periodontos is,a severe

lo calized j uvenile form of periodont it is . The aim o f this proj ect is

four-fold

1 ) To determine the immunologic s tatus o f pat ient s with

periodontal diseas e .

2 ) To identif ty sp ec if ic immune reac t ions in patient s with

dif ferent forms of periodont it is .

3 ) To attempt to dif f erentiate periodonto s is from o ther periodon

tal d is eas es (periodont it is and j uvenile periodont it i s ) in a

sys temat ic manner .

4 ) To inves t igate familial pat terns o f periodonto s is .

I I . Method and Des cr ipt ion

All pat ient s are screened for underlying medical condit ions or

vitamin def icienc ies . A full oral exam is carried out which includesdental radiographs and a complete periodontal evaluat ion . Immune

function i s s tudied ut il i z ing s everal assay syst ems including lymphocytetrans formation , lymphokine produc t ion , mono cyte and neutrophil chemo

taxis and histamine release . Gingival fluid is analysed for cellularcomponent s . Dental plaque is evaluated for it s ' antigenic po tent ialin these pat ients . Immune responses to pure cul tures o f other oralmicroorganisms are s tudied .

Groups s tudied include individuals with adul t and j uvenile perio

dont itis of varying sever ity and normal s . Chemo tact ic ac t ivity was

s tudied in response to : 1 ) part ially purif ied C5a derived from zymo

san ac t ivated s erum ; 2 ) crude bacterial factor de rivd from E . Coli ;and 3 ) fMe t-Leu—Phe . Result s of the chemo tact ic as say demons trate

that patient and control neutrophil funct ion was the same . Monocyt e

chemo tact ic act ivity was no ted to be defic ient in some cas es s tudied .

There was a great deal o f variability no ted in respons es o f normaland patient cells on rep eated tes t ing . S everal factors were identifed

which appeared to ef fec t chemo tact ic respons ivenes s . These included

antib io t ics , smoking , asp ir in , antihi stamines and o ther drugs . There

was a d if ference no ted in ch emo tact ic respons e o f monocytes to s elf or

f ore ign dental p laque . Plaque it sel f was not a chemoattractant .

Lymphocyte trans formation and his tamine release s tudies showed a

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ANNUAL REPORT OF THE LABORATORY OF ORAL MEDHJINE NATIO NAL

INSTI TUTE OF DENTAL RESEARC H

The Laborato ry of Oral Me dic ine is c oncerned wi th the e tiology and

pathogene si s o f di sea se s o f the so ft tissue o f the oral cavity wit

emphas i s on : 1 ) viral infec tions such as herpes s implex virus ; 2 )aphthous ulcers and othe r dermatologic diso rders ; 3 ) exocrine and

endocrine disease s wi th spec ial at tention to disease s o f the sal ivary

glands and pancrea s d iabe te s mellitus ) ; and 4 ) malignant and

premalignant les ions o f the o ral cavi ty . The program is di sease oriente dand highly interdi sc iplinary .

Over the las t ye ar , in depth s tudie s have continued on the pro j ec tsdiscus sed in the 19 7 7-7 8 annual repor t . In addit ion , one new pro j ect was

ini tia ted and one pro j ec t was t e rminated . The new pro j ec t i s c oncerned

with receptors on the pla sma membrane o f cell s . There are two as pec t s o f

this pro j ec t ; the first deal s wi th viral recepto rs and how they inf luence

susceptibili ty to infec t ion ; the second involves al tera t ions o f o ther

t y pe s o f pla sma membrane recepto rs and how they are changed by viral

infec t ions . The te rminated pro j ec t deal t with target ing o r homing o f

b iologically ac tive media tors chemo tac tic fac to r ) t o s pec i fic

s i te s t umors,viral le s ions ) by antibody molecule s . This pro j ec t

was de terminated because o f the departure o f the two principal inves t igato rs .

The progre s s in the Labora tory s ince the las t annual report has been

excellent . We have at las t achieved a crit ical mass o f inves tigato rs

with training and experience in virology ,immunolo gy , patholo gy , e lec t ron

micro scopy,cel l bio logy and molecular biology . Many o f our s tudies on

the e tiology and pathogene si s o f disease require an inte rdis c iplinarv

approach and over the las t year,thi s approach has proved to be part i cularly

frui t ful .

Emphas is on human disease s and clinical material from pa tient s

continue s ; thi s is par ticularly true for the pro j ec t s on inter feron and

diabe te s . This coming fall , a cl inical inves tigato r from Sc o tland

wi th bo th a D . D . S . and M . D . will be j oining our s taf f as a V isi ting

As soc iate . In addi tion,a senior inves tigato r from the Univer si ty of

C alifo rnia,who se cl inical expe r tise i s infe c tious di sea ses , will s pend

his sabbat ical year wi th our group . The only re c rui tment a t the presen t

time i s to f ind a ful l-time , c linical ly—oriented inve s t iga tor to replaceDr . Howe ll Ar chard who will be re tiring thi s fall .

The various serv ice c omponent s o f the Labo ratory continue to func tion

well and thi s i s particular ly the case wi th the Ti ssue C ul ture Unit . A

ma j or e f fort has been made over the las t year to reorgani ze and moderni ze

our de ep free ze r sys tem for s to rage o f viruse s and cell cul ture s . Ef fo rt s

also are be ing made to renovate and moderni ze some o f our s pace so tha t

i t can be mo re e f fe c tively utilized . In the Of fice , a Le xitron typewr ite r

was purchased to more ef ficiently prepare manuscript s .

1 — 7 8 7

The Laboratory continue s to add new technique s and methods to i t s

program . This year we have se t up methods fo r : 1 ) making antibody

producing hybridomas ,2 ) measuring antiviral antibody by micro ti tration ,

3) de tec ting viral and othe r antigens by the peroxidase-anti—peroxida se

method , 4 ) measuring the number o f viral receptors on the sur face o f

cells,5 ) quanti ta ting insulin receptors ,

6 ) enr iching for herpes simplexV iral DNA sequences

,and 7 ) res tric ting HSV DNA by endonuclease s .

A number o f new collaborat ive pro j ec t s have been ini tiated with

inves tigators from NIA ID , NIAMDD , and NC I . In addition,case material is

being sent to us from at lea s t one-hal f dozen universi t ie s throughout the

country .

Since the las t annual repo rt , good progress has been made in severalareas . The s ignificance and implicat ions o f some o f our f indings are

briefly summarized below

1 . Previous s tudies showed that type I I o r immune interferon coul d

be produced by peripheral blood leukocyte s in vitro on an immune—s peci f i c

basis . This sugges ted tha t i t also might be produc ed in vivo in various

autoimmune di sorders . Over the last year , we found immune interferon in

the serum of a high percentage o f pa tient s wi th sys temic lupus erythemato sus ,

rheumatoid arthri ti s , scleroderma and S j ogren 's syndrome . Subdivis ion o f

the patients wi th lupus erythemato sus into ac tive and inac tive disease

showed that approximately 7 l% o f tho se with ac tive di sease had inter feron

in their circulation as compared to only 21% o f tho se with inac tive

di sea se . Moreover,serial serum sample s ob tained over many months showed

a good correlation between interferon ti ters and disease ac tivity .

Thus , i t i s po s s ible tha t the produc t ion o f inter feron may on one hand

c ontribute to immunological aberrations in auto immune di seases , while on

the o ther hand pro tec t the already compromised ho s t from viral infec t ions .

The poss ibili ty that type I I interferon may be found in the circulation

of patients with a variety o f other immunolog ical disorders i s now under

ac tive inves tigation :

2 . New findings have been made in the area o f virus— induced diabetes .

Since the las t annual report,members o f the Laboratory have succeeded in

i solating a virus from a child who died of diabetic ke toac idosi s and

showed that this virus,a variant of C oxsackievirus B4

, could produc e

diabetes in certain inbred s trains o f mice . A varie ty o f fac to rs including

a ri se o f antibody to the virus during the pat ient 's illnes s and the

presence o f viral antigens in the patient 's brain argue agains t the virus

being a laboratory contaminant . Al though cases o f thi s type are a rarity ,

i t support s the hypo thesi s tha t some case s o f juvenile-onse t diabetesmellitus may be triggered by viruse s and backs up the years o f experimentalwork in animal model s .

A second line o f evidence supporting the hypo thesis tha t viruse s can

somet imes produce diabetes comes from his topathologic evaluation o f

pancreas from patient s dying o f overwhelming viral infec tions . The se

studies showed that viruses canHgf e

iiggmgdamage pancreatic be ta cell s .

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NO1 DE 9 2420—01

N AM ES,L A BOR ATO RY AN D I N ST I T UT E AFF I L I AT I ON S, A ND T I T L ES OF PR I NC I PAL I NV E ST I G AT OR S AND ALL OT H E R

PR OF ES S I ON AL PERS ON N EL ENG AG ED ON THE PR O JECT

PI : No tkins , Abner L . Medical Direc tor

COPI : Yoon , Ji—Won Research Microb iolo gis t

Jenson , Al fred B . Surgeon

Onodera , Takashi Vis it ing As soc iateDobe rsen , Michael J . S taf f Fellow

Toniolo , Antonio Vis it ing Fellow

Dr . Marshall Aus t in , NNMC,Bethesda

,Md .

Dr . F redda

[g (b ) HUM AN n ssurs D (c ) N E I T H E R

S UMM AR Y OF W OR K (200 wo rd s or le ss und e rl in e keyword s )

Diabetogenic variant s o f encephalomyocardit is virus,Coxsackievirus B

,and

reovirus have been s tud ied in vitro in beta cell cul tures and in vivo in

animal model sys t ems . One o f these variant s,a Coxsackievirus B4 ,

was

iso lated from the pancreas o f a child who died with acute juvenile— onsetdiabetes (JOD ) . The ro le o f the immuno logic sys tem in JOD also is beingstudied .

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DIS EAS ES O F THE SALI VARY CLANDS AND PAX 3REAS : V IRUS -INDUJED DIABETE S

Juvenile— onse t diabe tes ( JOD ) occurs a t an early age and is

charac teri zed by an acute o nse t , a ppe arance o f i sle t cell antibod y ( EZA ) ,an infil tration o f inflamma to ry c e l ls in and around panc rea tic isl e ts(insuli ti s ) , and a reduc tion in the num be r o f insulin— conta ining be ta

cell s . Be cause o f the abrupt onse t and development o f insul iti s, an

infec tious e tiology has long been s ugge s te d for JOD . Se roe pidemiolog ic

s tud ie s and nume rous case re po rts have shown that a tempo ral re la tionship

exis t s be tween the onse t o f c e r ta i n virus infec t ions and the s ub se quent

d evelopment o f d iabe tes . JOD has been re po rte d to fol low infec tion bymembers o f the C oxsackievirus B g roup ( par ticularly B4 ) within days ,

mumps virus wi thin we eks to years , and congenital rube lla by a number of

year s . Thi s line o f evidence s ugge s t s tha t a number o f viruse s e ither

ac ting alone (C oxsackieviruse s ) or inte rac ting wi th ho s t fac to rs such a s

the immune sys tem (mumps and rubella ) may cause JOD in s usce ptibl eindividual s .

Some viruse s have the capac i ty to infec t and damage human panc rea tic

i sle t ce ll s in vit ro . We have previous ly shown that C oxsackieviruse s B3

and B4 and mumps virus can infec t and deg ranula te human be ta c e ll s in

c ul ture . Al tho ugh these st udie s did no t prove that viruse s c an ac tuall y

infec t and dest roy be ta cells in vivo t o produc e JOD,the y did provide

a sound ba si s fo r the idea tha t viruse s play an ac tive role in JOD .

The best evidence that viruse s cause diabe te s come s from experiments

with an animal model sys tem . Infec tion o f certain s trains o f mice with

the M—variant o f ence phalomyocard it is ( EMS ) vi rus resul t s in the development

o f a diabe te s—like syndrome which clo sely resemble s JOD . Thi s variant o f

EMC infec ts and dest roys murine be ta cell s and produc e s hypoinsul inemia

and hyperglyc e mia . Only c e rtain inbred st rains o f mice develop EMG virus

induced diabe tes which appe a rs to be ge ne t ical ly controlle d at a sing lereces s ive locus .

Mur ine be ta cell s grown in cul ture are suscept ible to the M—variant

o f EMC virus,C oxsack i e virus B3 , C oxsackievirus B4

,and reovirus type 3 .

Al though the M-variant o f EMG is a na turally oc curring diabetogenic virus ,

the C oxsackieviruse s and re oviruse s mainly cause an ac inar c ell pancrea ti tis .

However ,after pa ssage o f re ovirus type 3 and C oxsackievirus B4 thro ugh

murine beta c e ll s grown in cultur e , it was po s sible to obta in var iant s

o f the se viruse s which,whe n inocula t ed in to mic e , infe c te d the be ta

cell s and produced hypo insul inemia ,hype rglycemia and / or abnormal gluco se

tolerance te s t s . The diabe togenic e f fe c t o f re ovirus type 3 c ould be

as ses se d mo s t accura te ly b y pe rform i ng gluco se tole ranc e tes ts whe re a s

the diabetogenic e f fe c t o f EMS and C oxsackievirus B4 wa s bes t a s se s se d

by de te rmining level s o f non fas ting gluco se and insul i n at varying t ime s

after virus infe c tion .

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The ob j ec tives o f thi s research pro j ec t are to determine 1 ) which

viruse s cause diabe tes , and 2 ) the sequence o f event s that occur between

the time o f infec tion and the onse t o f cl inical disease .

STUDIES IN ANIMALS

that the diabetogenic capacity o f the M-variant o f EMC virus i s markedlydimini shed af ter pas sage in mouse kidney cell cul tures . One pa s sage

through animals , however , restored i t s diabetogenic capacity . We sugges ted

that the s tock virus pool was made up o f two populations o f virus ; one

that had a tropism fo r beta cel l s and was diabe togenic , and the o ther

that did not have a tropi sm for be ta cell s and was no t diabe togeni c .

Growth o f virus in the kidney cell cul tures might have favored repl icationo f the nondiabe togenic s train. In vivo

,both be ta-t ropic and nonbe ta

t ropic strains would have an oppo rtunity to replicate as t rue stable

variant s .

To obtain a genetically unifo rm s tock of diabetogenic virus , the M

variant o f EMG virus was grown in secondary mouse embryo cell s ( SME ) and

purified by densi ty gradient centri fuga tion . The purified virus was

pas saged five times in cul ture s enriched in murine be ta c ell s . The virus

was then passaged in mice and harves ted from heart s . By serial plaquepuri fication o f the pa s saged virus , two di s tinc t s table clones were

obtained . When the se clones were tes ted in SJL /J mice , one o f them

(D-clone ) produced hyperg lycemia in up to 1002 o f the animal s . In

contrast , the o ther isolate ( B—clone ) did no t induce diabete s . Virus

ti ters were checked in the i sle t s and heart from mice a t di f ferent time s

af ter infec tion. At two days af ter infec tion , the amount o f infec tiousvirus recovered from the i slet s o f D-clone infec ted mice wa s a t lea s t 1 0

fold higher than recovered from B-clone infec ted mice . Similarly , the

D—clone replicated in the hear t to a grea ter extent than the B—clone .

Mo reover , micro scopic examination o f sec tions o f pancreas from D-cloneinfec ted mice revealed tha t more than 80% o f the i slet s develo ped insuli t i s

( lymphocyt ic infil tration) and necro si s o f beta cell s . In contrast ,

sec tions of pancreas from B-clone infec ted mice showed that about 90 2 o f

the islets appeared no rmal . Us ing immuno fluorescence , about 9 7 2 o f the

i slets f rom D-clone infec ted mic e ( 2 days af ter infec t io n) contained

s pecific viral antigens,while only 132 o f the is let s from B—clone infec ted

mice contained viral antigens . Further studies showed a marked decrea se

in the insul in content o f the panc reas from D-clone infec ted mice ,

beginning a t 2 4 hour s a f ter infec tion . In contras t,infec t ion o f mice

with B—clone resul ted in no significant change in insul in content o f the

pancreas . The se studie s clearly showed that the newly i sola ted diabe togenic

c lone (D-clone) that i s genetical ly s table,di ffered from the non

diabe togenic clone (B—clone ) in i t s capac ity to replicate in the intac t

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dif ferent s tage s o f morphogene sis were observed in insul in—containing

beta cell s,as well as glucagon

—containing alpha cell s and somato sta tin

containing D cell s . The se develo ping V irus par t icles were frequently

a ssociated wi th micro tubules in large cyto plasmic virus fac torie s .

Fro zen sec t ions from the pancreas o f infec ted mice were s ta ined with

f luorescein— labeled anti-reovirus type 1 and rhodamine-labeled antiinsulin

antibody . Reovirus ant igens were found in bo th be ta cell s and noninsul in

c ontaining i sl et cell s . By the peroxidase-anti-peroxidase method us ing

anti-glucagon ant ibody,more than SOZ o f alpha cell s were degranulated a t

days 14 and 2 1 . It i s c oncluded tha t a variant o f widely di sseminated

human virus , reovirus type 1 , can infec t the alpha , be ta and D cell s o f

mouse pancreas . The damage o f the se cel l s al ters the ho st s ' capac ity to

res pond normally to a gluco se load .

monolayer cul tures,but thi s technique has no t been widely used becaus e

o f the dif ficul ty in ob taining large numbers o f cell s and maintaining

them in a f unc tional s ta te . Monolayer cul tures are par ticularly sui tablefo r studying immune phenomena . Fo r the se reasons , we have developed a

func t ional microculture sys tem that would require relatively few c ell s

and make available large number s o f cul tures .

Prel iminary studies reported last year have been confirmed and

extended to includ e new data . The cul tures were prepared in 9 6 wel ltissue cul ture trays from isle t cell s i solated from enz yma tically di srupted

newb orn rat pancreas . The cul tures could be maintained in a func tional

s tate for at lea s t 2 weeks as de fined by their res ponsivene s s to known

modulators o f insul in secretion . Glucose,fo r exampl e , enhanced insul in

release,as did arginine leuc ine and isoleuc ine . The se agent s are known

s timulators o f insul in sec re tion in vivo . As few a s 1 x 10 cell s per

well gave result s that were reproducible within loz .

Experiments are currently underway to study the bio logical significance

o f islet cell ant ibody using this sys tem . The e f fect s o f IC A on isle t

c el l func tion as well as i t s cyto toxic propertie s are being inves tigated .

STUDIES IN HUMANS

Ef forts are being made to determine which viruses may be involved in

human JOD . We are following three approache s . The f irs t approach i s t o

i solate viruse s from the pancreas o f patient s dying wi th acute JOD ,

and then charac teri ze the virus in tissue culture and in animal model

sys tems . The second approach i s to determine which viruse s have a tropi sm

(in vit ro ) for human be ta cell s in cul ture . The third approach i s to

look at the islet s o f pa tients dying wi th fatal viremia and determine if

the se viruses produce cytopathology in islet s .

C oxsackievirus B4 : A heal thy ten—year—old male developed a flu-like

illnes s and within three days a f ter onse t o f s ymptoms,the child was

2 01 DE 00080-06 LOM

admit ted to the hospital with diabe tic ketoacidos is and died seven days

later . Micro sc opic examina tion o f sec tions o f the pancreas revealed

lymphocytic inf il tration o f the is let s o f Langerhans and necro sis o f

insulin—produc ing beta c ell s . Small pieces o f the pancreas were removeda t autopsy and s tored a t Monolayers o f secondary mouse embryo

c ell s , Ve ro cell s and human embryo kidney cell s were inoculated with a

102 homogenate o f the fro zen pancreas . Six days late r , 1 0 to ZOZ o f the

monolayer s showed one or more focal areas o f cytopatho logy . The cell sand media were harve s ted , f ro zen and thawed three t imes

,and then pas saged

twice in secondary mouse embryo cell s . The material obtained af ter thesecond pas sage produced large plaque s on secondary mouse embryo ce llmono layers .

To charac teri ze the virus,sera ob tained from mice tha t had been

infec ted with the human isolate neutrali zed the human isolate and al s o

the mouse-adapted diabetogenic variant o f C oxsackievirus B4 . The seresul t s indica te tha t the human isolate i s clo sely re lated to a diabe togeni c

variant originally derived from C oxsackievirus B4 . To fur ther charac te ri ze

the virus,monolayer s o f secondary mouse embryo cell s grown on cover slips

were infec ted with the human isolate and then s tained wi th FITS — labeled

anti serum to the mouse-adapted variant o f C oxsackievirus B4, C oxsackievirus

B3,and EMC virus . C e ll s s tained with F ITC —labeled antiserum to the

mous e-adapted variant of C oxsackievirus B4 showed brilliant fluorescencein the cytoplasm

,while uninfec ted control cells did no t fluoresce .

Monolayer s infec ted with the human isolate and then s tained with F IBC

labeled anti serum to C oxsackievirus B3 or EMC virus failed to fluo resce .

Evidence that the virus was no t a laboratory contaminant came from

showing : 1 ) a ri se in antibody ti ter to C oxsackievirus B4 during

ho spi tali zation,and 2 ) viral antigens in brain tis sue obtained a t autopsy .

Se rum obtained on the second ho spi tal day failed to neutralize the human

isolate a t a dilution o f while serum obtained six days later

neutralized the iso late a t a dilution o f In contrast , neither

serum ob tained on the second ho spi tal day nor six days later neutrali zedEMC v irus . This resul t indicate s tha t the rise in antibody in the patients

serum was no t a non-S peci fic toxic ef fec t . In order to determine if

there was an ac tive viral infec tion at the time o f autopsy , f ro zen sec tionso f the brain

,hear t and pancreas were s tained with F ITC —labeled ant i

C oxsackievirus B4 antibody . Examination o f brain sec tions revealed

viral ant igens in a smal l number o f i s olated neurons and occas ional ly inendothelial cells l ining small arteries . Out o f 4 0 s ec t ions examined ,

only 7 showed immuno f luores cent— po s i tive cell s . As a contro l , sec t ions

immedia tely before and af ter each sec tion showing immunof luorescentpo si tive cell s were s tained with F IBZ—labeled antiserum t o C oxsackievirus

B3 .These sec tions were all negative . Se c tions o f the heart and ac inar

pancrea s also were negative when s tained with F ITC —labeled anti-C oxsackie

virus B4 antibody . Is let cell s sometimes showed sl ight fluorescence , but

the s taining wa s not s trong enough to be scored as po si tive .

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When a certain inbred s train o f mice (SJL ) was inoculated with the

human isolate,over 502 o f the animal s became hyperglycemic wi th blood

gluco se levels somet imes exceeding 400 mg/ dl . The concentration o f

insul in in pooled serum seven days af ter infec tion was ng /ml ascompared t o ng /ml in serum from uninfec ted animal s . In contrast

,

s ome inbred strains o f mice such as C 5 7BL6 , C BA and Balb/ c mice were no t

hyperglycemic under the same condit ions . These result s indicate that

there were genetically de termined hos t di fferences in the induc tion o f

diabe tes when mice were inoculated with the human isola te .

To see whether the hyperglycemia was secondary to beta cell damage ,

mice ( SJL ) were sacri ficed at dif ferent t imes af ter infec tion and thei r

pancreas was examined by light micro scopy . Inflammation was vis ible in

the islet s o f Langerhans s imilar to that seen in the pat ient 's pancreas .

The cellular columns of the islet s were di ffusely infil trated wi th

mononuclear cell s , the normal archi tecture o f the i slet was disrupted and

f ocal cellular degeneration was observed . Lymphocyt ic infil trates al so

were prominent in a perivascular dis tribution immediately surrounding

s ome islets and in scat tered foci in the acinar tissue . Se c tions o f the

brain were charac teriz ed by an acute meningo-encephalit is with foc i o f

neural degeneration in the cerebral cortex and brains tem . To see whether

his topathological changes in the i slet s o f Langerhans were due to viralinfec tion , sections o f the pancreas from infec ted mice were s tained wi th

F ITC —labeled ant i-C oxsackievirus B4 antibody . At four days a f ter infec t ion,

viral antigens were clearly seen in the cytoplasm of beta cells . Vi ralantigens al so were found in some acinar cell s o f the pancreas and in some

arteries and neurons o f the brainstem. Beginning at about seven days

af ter infection,viral ant igens were dif f icult to detec t .

Finally , cul tures enriched in human beta cell s were inoculated with

the human isolate and the suscept ibility o f the beta cells was de termined

by the double—label immunofluorescent antibody technique . By thi s

technique , human beta cell s prepared from the pancreas o f a 1 7-year-oldmale (HLA type A2 , A3 3 , B1 5 ,

B21 ) were qui te sus ceptible , and 6 0 hours

after infec tion , approximately 37 % of the insulin—containing beta cell sshowed viral antigens .

At present,we do no t know the frequency o f diabetogenic variant s o f

C oxsackieviruses in the human population. However , i t is evident that

diabetes i s not a common sequela o f C oxsackievirus B4 infec tion , sinceantibody t o C oxsackievirus B4 i s pre sent in about hal f the population ,

whereas juvenile diabetes develops in less than percent o f the

population . Moreover , i t i s not known whether the pre sent case i s the

very rare exception in which the patient became diabetic because o f the

pre sence o f a receptor for the virus on his be ta cell s or i f he had a

s pec ific immune deficiency to the virus or because o f o ther fac tors .

Finally , j uvenile diabete s may no t be a single disea se , but several

diseases wi th different cause s . If viruses produce diabetes , they may be

only one o f the causes , and more than one virus or virus group may beinvolved .

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During this study , we found that fixation with Bouin 's solut ion no t

only allowed the demons tration o f IC A , but that antibodies to insulin

(insulin antibody , IA ) , commonly found in insul in treated diabe t ic s

could be detec ted as well . IC A. c ould be detec ted wi th equal sensi tivi ty

on fresh , ace tone-fixed and Bouin 's-fixed pancreas , but no t on formalinfixed

pancreas . Fur thermore , homogenized , fresh , acetone-fixed or Bouin's fixed

pancreas could absorb out ICA. whi1e fo rmalin-f ixed pancreas could no t .

Thus , the antigenic spec ific ity o f ICA on fresh , acetone— fixed and Bouin's

f ixed pancreas was identical . Human IA , on the other hand , could be

detec ted wi th equal sensitivi ty on Bo uin 's-fixed and fo rmal in— fixedpancreas

,but i t could no t be detec ted on fresh or ac e tone fixed pancreas .

The S pecificity o f thi s reac tion wa s confirmed by complete absorpt ion o f

the antibody with insulin . ICA was no t af fec ted by insulin absorpt ion .

Ti tration o f guinea pig anti—proc ine insulin IgG on each o f the

pancreas preparations sugges t s that the lack o f sensit ivity fo r IA seen

with fresh or acetone-fixed pancreas may be due to a decrease in the

amount o f insulin in the sec tion . Staining of the insulin-containing

b eta cell s wi th this anti serum could be demonstrated wi th up to a

dilution on Bouin 's or formal in-fixed pancreas while dilutions up t o

only c ould stain fresh or acetone-f ixed cryo s ta t sec tions . The bes t

explanation is that formal in and Bouin 's solut ion fixe s insul in in s i tu .

Therefore , virtually all insul in contained in the sec tion would be

available for reac tion with IA . However , s ince cryo s tat sec tions are

made o f unfixed pancreas , leaching out o f insul in is l ikely during

sec tioning , ace tone—fixa tion or incubat ion o f fre sh pancreas wit h

anti sera .

Since Bo uin 's— fixed pancreas can de tec t low ti ters o f IA,i t i s

neces sary to absorb all samples with insulin be fore de terminat ions for

ICA . Us ing this technique we have tes ted 2 02 s era for IC A in compari son

with fresh pancreas . Serum samples gave identical resul t s on each tis sue

preparation . The inc idences o f IC A determined in thi s way were : JOD a t

time o f onset ,842 JOD o f les s than one year dura tion ,

JOD o f greater than one year duration , maturity

onset diabetes,02 and controls

,02

An important approach to unders tanding the inter—relationships o f

gene tics , immunologic and o ther fac tors in the e tiology o f JOD is the

careful study of families wi th one or more siblings with this disease .

We have inves tiga ted 3 5 famil ies,31 having one child wi th JOD and four

having two such children .

Inc idence o f IC A in the juvenile-onse t diabe tic s was

Of 7 7 ini tially non—diabe tic s iblings in these familie s (age s 2 1 8

are HLA identical to their diabet ic s iblings . F ive o f these 18

were po sit ive fo r BSA and one o f the se has developed JOD s ince the s tart

o f the study . The inc idence o f IC A in s ibl ings sharing one HLA haplo type

(haploidentical ) wa s and that fo r HLA nonidentical sibl ings

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wa s Oral g luco se tolerance tes t s in the se groups were

no rmal as were HbAIc level s . It is al so interest ing to no te tha t the

inc idence o f BSA in the parents was

Al though thi s s tudy is in i ts preliminary s tages,i t i s becoming

clear tha t IC A i s more prevalent in familie s having one o r more diabe t i c

children than in the general population and therefore sugges ts that IC Amay be more re lated to gene tic fac tor s HLA type s ) or po ss ibly to

environmental fac to rs transmiss ible agent s such as viruse s ) than

to the develo pment o f JOD .,F ollow-up s tudies on the famil ies are in

progres s .

There are over one million insulin-dependent diabetics in the Uni te dState s . This fo rm o f diabe tes is the mos t dif ficult to cont rol and the

short and long term side e ff e c t s are enormous with res pec t to the pa tient 's

health and economic s . C ircums tantial evidence sugges t s that viruses play

s ome role in the ini tiation o f this fo rm of diabetes . Identi fication of

diabe togenic viruses and preparation o f a vaccine might of fer a form o f

preventive medic ine tha t i s no t o therwi se available for geneticallypredis po s ed individual s .

Future Plans

Our future plans fo r research are aimed at : 1 ) identi fying viruse s

in the pancreas o f humans with acute JOD or fatal viremias without JODand de te rmining if they are diabetogenic in animal model sys tems 2 )identifying the proper tie s o f dif ferent clones o f diabe togenic viruse s a t

a cellular leve l (in vi tro ) and at the level o f the intac t organism ( invivo ) which c an ult imately be used to s tudy the molecular basi s o f virus

induced diabete s ; 3 ) identi fying inbred s trains o f animal s that are

susceptible and re si s tant to diabe te s induced by the se viruses with the

goal o f eval ua ting the genet ic and non- genetic fac to rs c ontro lling

susce pt ibility ; and 4 ) ident ifying ant i viral antibod ie s and autoantibodie s

in animal s and humans wi th diabe tes mellitus to determine the ro le o f the

immune response in JOD .

Publ ica tions

1 . Yoon,J . W .

,Onode ra ,

T Jenson ,A . B .

, and No tkins ,A . L . : Vi rus

induced diabe tes mellitus . XI . Replica tion of C oxsackie B3 virus

in human pancrea tic be ta cell cul tures . Diabete s —7 81,1 9 7 8 .

2 . Onode ra ,T Jenson

,A . B Yoon ,

J .W ., and No tkins ,

A . L . : Virus

induced diabe te s melli tus : Reovirus infec tion o f panc rea tic be ta

cell s in mice . S cience —5 31 ,1 97 8 .

3 .

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Ste fan,Y Malais se -Lagae ,

F Yoon , J .W . , Notkins , A . L .,and Orc i

,

L . : Virus-induced diabetes in mice : A quanti tative evalua tion o f

-401 , 19 7 8 .

Omodara ,T Yo on ,

J . W .,Brown , K. S . , and No tkins ,

A . L . : Evidence

for a single locus controll ing suscept ibility to virus-induced

diabetes melli tus . Nature 19 7 8 .

Yoon, J .W . , Onode ra ,

T . , and Notkins ,A . L . : Virus-induced diabete s

mellitus . XV. Be ta cell damage and insulin-dependent hyperglycemia

1 9 7 8 .

Yoon , J .W . : Me thod fo r cul tivation and identif ication o f human

pancreat ic be ta cell s . TCA Manual No . 3 ,-88 8 , 197 8 .

No tkins , A . L .,Yoon , J . W . , Onode ra ,

T . , and Jenson , A . B. : Vi ral

induced diabe tes mellitus : Infect ion o f mice with variant s o f

encephalomyocardi ti s virus,C oxsackievirus B4

, and reovirus type 3 .

Adv . in Exp . Med . Bio l . ( in pres s ) 197 9 .

Yoon ,J . W .

,Aus tin , M . , Onode ra ,

T . ,and No tkins ,

A . L . : Virus

induced diabetes mellitus . XVI . Isolation o f a virus f rom the

pancreas o f a child wi th diabetic ketoacidosis .

—ll7 9 , 19 7 9 .

No tkins ,A . L . : What causes j uvenile diabe te s . Sc ient i fic Am .

( in pres s ) 197 9 .

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ZOI DE00094—06 LOM

LEC ERATIVE LES IONS AND TUMORS : APHTHOUS UUSERS

The ob j ec tive o f this pro j ec t i s to s tudy the etiology and pathogenesi s

of muco sal disease s o f the oral cavity , with the ul t imate goal o f treatment

and prevention. At the present time , emphasi s i s on recurrent aphthous

ulcers . Aphthous ulcers are charac teri zed by recurrent , painful , singleo r mul tiple ul cer s on the mois t , movable oral mucosa .

_

The e tiology o f

this di sease is unknown , however , there i s a s trong familial tendency and

the oral aphthous ul cer s o f ten appear following injury to the mucosa . Inaddi tion

,phys ical and emo tional s tres s , decreased serum level s o f fol i c

acid , iron , and vitamin B1 2 , as well as auto-immune reac tions , have beenimplicated . Microbiological studies have failed thus far to identi fy any

virus or unusual bac terial flora as soc iated wi th the lesions . Except fo r

the s peci f ic replacement therapy in folic ac id , iron , and vi tamin B1 2

deficiency,therapy to date has been direc ted toward palliation o f the

di scomfor t caused by the oral ulcer s .

Several prel iminary report s appeared in the literature in 1949

imp licating local ized mechanical trauma in the e tiology o f o ral aphthous

ulcers . Inves t igators in a study comparing the e f fect s of mechanicalt rauma on the mucosa of aphthous patient s with individual s wi thout oralmuco sal di sease produced oral ulcers in aphthous pa tient s but not the

controls . It has been po s tulated that recurrent oral aphthous ul cerations

are an end resul t common to many e tiological fac tors , provided they are

capable o f initiating suf f ic ient t issue reac tion to produce ul cerationand that they are appl ied periodically to the oral mucous membrane . It

also seems probable tha t aphthous ul cer s may occur as the resul t o f one

o r more precipi tating fac tors superimpo sed upon a muco sa o f l imited

re cuperative or defensive po tential the latter aspec t a ttributed to as

yet unknown local or systemic fac tors .

A pro tocol enti tled The Re lationship o f Mechanical In jury to Oral

Aphthous Ulcer s was ini tiated in 197 7 . The purpo se o f this s tudy i s to

a scertain i f mechanical injury to the o ral mucosa is capable o f induc ing

aphthous ulcers in patients known to have recurrent aphthous ulcers . The

buccal mucosal injury i s produced by inserting and leaving a surgicalsuture in the muco sa fo r 3 days or placing a small por tion o f the muco s a

in a surgical clamp for one second . The se procedures are done after

local anaesthe s ia has been adminis tered in the opera tive area . The

resul ts are compared wi th the findings in a matched contro l group o f

individuals free o f oral ulcerat ive di sease . The s tudy i s ongo ing at the

present time with a to tal of 30 patient s ; 20 aphthous and 10 c ontrol shaving been completed .

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The resul t s o f the s tudy in 3 0 pa tient s are as follows : 1 ) Oral

ulcers developed in 8 (4 02 ) of the 20 aphthous patients a t one or moresi te s o f induced muco sal injury ; 2 ) the average duration o f the

experimentally-produced ul cers was 5 days which compares favorably with

the usua l 7 14 day duration s pan o f s pontaneous oral aphthous ulcers ;3 ) the average to tal si ze and the average maximum pain al so were wi thin

the usual limit s o f s pontaneous oral aphthous ulcer s ; and 4 ) no ulcer s

fo rmed in the 10 c ontrol sub j ec ts,however

,a l t i ssue reac tion

( swelling and rednes s ) was no ted in 3 control sub j ec t s .

The se f indings are significant in the inves tigations into the

e tiology o f aphthous ulcers because a maj ority o f the naturally occurring

o ral aphthous ul cers appear at the si te o f a muco sal injury . Injury to

the oral muco sal may allow entry o f an agent or agents that initia te

ul cer fo rmation . Al ternatively , the oral mucosa o f aphthous patients

re s ponds in an unusual manner to injury,resul ting in an expansion o f

the injury si te . A c ombined immedia te and delayed hype rsensitiviy reac tion

to the penetrat ion or release o f a fore ign antigen i s a di s tinc t pos sibility .

During the pas t several years , we have been inves tiga ting the po s sibleviral e tiology o f recurrent aphthous ulcers

,Be hce t 's syndrome

,S j ogren 's

syndrome and o ther dermatologic disease s by attempting to detec t the

pre sence o f viruse s in s pec imens collec ted from patients with the se

diseases . The emphasi s o f the se s tud ie s in the past has consi sted ofef fort s to demons trate viruse s in vitro , in small laboratory animal s and

in embryonating hen's eggs . During the past two years , we have extended

these inve s tigat ions . Biopsy s pec imens obtained from pat ient s have been

inoculated into seven primate s in an attempt to transmi t a slow , latentor persis tent virus . The se primate s are being moni tored for the develo p

ment o f c linical di sease and fo r abno rmali tie s in clinical and immunologicallabora tory as says . The animals have no t developed clinical di sea se a s o f

ye t and,there fore

,are maintained in order to be observed for late

di sease develo pment .

Future Plans

During the next year,new invest igations into the pa thogenesis of

recurrent aphthous ulce rations will be init iated with part icular emphasi s

on immune defec t s and the e ffec t of various treatment modal ities on the

prec ipi tating fac tors in recurrent aphthous ulcers .

Publ ica tions

1 . Graykowski , B rA . z Aphthous-Behce t 's Syndrome Wo rkshop . Doub le—Bl ind

trial o f te tracycline in recurrent aphthous ulcera tion . J . Oral Path .

—382 ,

2 .

3 .

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Graykowski , E . A . , ar. al . : Appendix I I , Aphthous Stomati ti s-Behce t 's

Syndrome Wo rkshop . Tr eatment o f recurrent aphthous ulcerations . J .

Oral Path .-440 , 197 8 .

Graykowski, E .A . and Ho oks ,J . J . : Re current Aphthous St omati ti s

Behce t 's Syndrome Works hop Summary . J . Am . Dent . As sn .-602 , 197 8 .

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2 01 DE 0012 3-06 LOM

HERPES S IMPLEX VIRUS : LATENCY

Background

Re current disease is the main clinical problem in herpe s s implex

virus (HSV ) infec tion. Af ter the primary infec tion which usually occur s

in childhood , the virus remains latent fo r years in senso ry and autonomic

gangl ia . At irregular intervals , HSV is reac t ivated in gangl ia . Neura lshedding of the reac tivated virus ul timately leads to HSV repl ication in

the skin and a cl inical ly de t ec table recurrent le sion .

Re currences occur in many si te s including the lip and oral cavity ,

the corneal epithelium , the skin , and the genital ia . About oneh third o f

the adul t population suf fers from recurrent l ip lesions (herpe s labialis ) .

Al though les s common , corneal recurrence s (herpes kera ti ti s ) are the mo s t

c ommon nonr traumatic cause o f blindness in young adul t s in the UnitedState s . Bo th lip and eye herpe t ic infec tions are usually caused by type

1 HSV. Thi s virus i s al so the e tiological agent o f one o f the mos t commono f the sporadic encephali t ide s . Permanent neurological sequelae o r death

usually resul t from HSV encephali ti s . A r elated virus s pec ie s , type 2

HSV , causes aseptic meningi ti s and mos t case s of pr imary and recurrent

genital herpe tic infec tions . These genital infec tions cause a great dealo f suf fering in terms o f discomfort

,very f requent recurrences

,and

venereal spread . In addition , newborns who contrac t the virus in thebirth canal develop a generali zed infec t ion which is usually fatal .

Finally , there are se roe pidemiologic al data that suggest that genitalHSV-2 infec tion may play an e t iological role in cervical carc inoma .

Three general strategie s o f anti—HSV therapy can be employed

prevention o f gangl ionic infec tion , trea tment o f the produc tive epithel ialinfection , and treatment a t the latent stage to prevent recurrences .

Previous annual report s have discus sed prevention by prior immunization

and topical drug appl ication . Bo th o f the se treatment s in experimentalanimal s can block the ganglionic infec t ion under cer tain circums tances .

Similarly , some success has been achieved in man wi th drug treatment o f

produc tive HS V infec tion , espec ially encephal itis and kerati tis . In

contrast , very little experimental wo rk has been done to develop the

third s trategy : the prevention o f reac tivation. This s trategy,di scus sed

in las t years annual report,was inves tigated by te sting antibody

,

interferon , and drugs in an in vitro reac tivation model . Al though

reac tivation was prevented by drug s that blocked the HSV DNA polymerase ,

the cont inual pre sence o f drug in the cul ture media was needed the

latent infec tion was no t eradicated ) .

The development o f a rational therapy to prevent reac tivation mus t

wait for a fuller unders tanding o f latency . To this end,our continuing

wo rk in the last year has been mainly concerned wi th the mechanism of

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latency and reac t ivation . F indings in four as pect s o f this work will be

discus sed : ( 1 ) reac tivation o f virus by immunosuppressive agents and

epi thelial irri tant s , ( 2 ) the e f fec t o f ant i-HSV antibody on the produc tive

infec tion and the maintenance o f latency, ( 3 ) the charac teri za tion o f

the latent s tate in molecular terms ,and ( 4 ) the demons tration o f HSV

DNA in brain s tem and cereb ral hemispheres a t the latent stage .

Ma j or F indings

In the experimental sys tem , HSV i s inoculated on the skin sur face . The

virus enters nerve terminals and is transported in axoplasm to the nerve

cell body in sensory ganglia . Then an acute or produc tive ganglionicinfec tion take s place . Af ter about 2 weeks

,the infec tion passe s from

the acute to the latent s tage . During latency,infect ious virus can no

l onger be recovered in cell- free ganglionic homogenate s and viral antigens

canno t be de tec ted by immuno fluore scent techniques . Yet,i t i s clear

that virus or the viral genome is present in ganglia because HSV can once

again be demons tra ted a f ter explantation o f ganglia in tiss ue cul ture .

The se conditions o f a negative homogenate and a po si tive explant cul ture

e s sent ial ly de fine the latent s ta te .

Re ac tivation refers to the process in which the cons traint on HSV

replication i s removed and virus synthesi s re sumes in gangl ia . Reac tivat ion can be detec ted direc tly by recovery o f infec tious virus in the

ganglia or indirec tly by recovery o f HSV at the epithelial sur face . In

our experimental model , reac tivation as detec ted by the homogenate method

doe s no t occur s pontaneously . However , as repor ted in last year 's annual

report , reac tivation c ould be induced by treatment o f latently infec ted

mice with the immunosuppres sive agent s cyclophos phamide and x-irradia tion .

Thi s year , we were al so able to induce react ivation with sys temic

predni sone , but in only 7 2 o f the mice . In the se st udies , mice wer e

t reated wi th 400 mg /kg i. p . twice daily fo r 7 —10 days . Tr eatment was

ini t ia ted about 2 months a fter corneal inoculat ion , and reac tivat ion wa s

dete c ted by recovery o f infec tious virus from cell-free ganglionic

homogenate s af ter 7-10 days o f trea tment . Prednisone in thi s do s e

abolished the response o f s pleen cell s in cul tures to s timulation by

phytohemagglut inin and reduced the spleen weight to about 202 o f control .

All three o f the immunosuppress ive agent s produced a s trong lympholyticef fect in mice . However

,sys temic trea tment o f mice with lysates from

x-irradia ted S pleen cell cul tures failed to provoke reac tivation . Negative

resul t s were al so ob tained with sys temic LPS (bac terial lipopolysaccharide ) ,and antilymphocyt e serum adminis tered i. p . induced reac tivation in only

1 o f 10 0 mice .

During the las t year,we have al so induced reac tivation by epi thel ial

irri tat ion . In these studies mice were inoculated by the lip route . Two

months af ter inoculation ,dry ice was appl ied daily to the lips fo r about

10 s econds . Af te r 7—10 days o f thi s trea tment , reac tivat ion could be

detec ted in about 8 2 o f 7 7 mice from three separate experiment s .

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Reac tivation was detected by recovery o f virus from cell-free homogenate s

o f l ip o r ganglion tissue . Virus or the viral genome could not be detec ted

in lip tis sue of untreated mice either by explanta tion or by a hybridiza tiontechnique for HSV DNA . It seems likely that the lip irri ta tion sets up a

neural signal that in some way trigger s virus repl ica t ion in ganglia .

The nature o f the signal and the mechanism of reac tivation have no t ye t

been es tablished .

Ef fec t o f Antibody on the Product ive and Latent S tage . The se

reac tivat ion studies were t ime consuming since trea tment mus t be delayeduntil the latent stage . To provide a rapid method to sc reen a varie ty

o f agent s ,pas sively immunized mice were used . In these s tudies , mice

were given high—ti tered rabbit anti—HSV s erum 3 , 48 , and 9 6 hour s after

bilateral corneal inoculation with HSV . At the same t ime , experimental

mice were treated with cyc lopho sphamide ) . The control group wa s

inoculated and pas sively immunized , but g iven no trea tment . Two and 4

days after inoculation , ganglia explant s and homogenate s were assayed in

the experimental and the control group .

The e ffec t o f passive immuniza tion is to bypa ss the acute stage o f

the gangl ionic infec t ion without ac tually preventing the infec t ion .

The se resul t s ( i. e . negative homogenate and a po si tive explant cul ture)are the same condi t ions that func t ional ly de fine the latent s tage ( se e

previous sec tion) . The acute stage is bypassed in most ( about 802 ) but

no t all o f the control mice a t bo th 2 and 4 days af ter inoculation . With

cyclopho sphamide treatment,however , the acute stage was bypa s sed in only

212 o f mice two days a f ter inoculation and 132 o f mice four days a f ter

inoculation. Thi s resul t,therefore

,indicate s that cycl opho sphamide

increases the degree o f virus repl ica tion in gangl ia . In contra s t , no

change from control was seen wi th LPS,and only a sl ight change was seen

with lip irri tation (in lip inoculated mic e ) . Additional agents will be

t ested in thi s sys t em to determine if all agent s that induce reac tivation

al so enhance virus recovery in the f ir s t week af ter inoculation o f

passively immunized mice .

To study the e f fec t o f anti-HSV antibody on latency , serum neutral izingantibody was measured various t imes af ter corneal or lip inoculation .

Two groups o f mice were used : pa ss ively immunized mice given rabbi t

ant i—HS V serum i. p . and 144 hours a f ter inoculat ion and controlmice given non-immune serum on the same schedule . In the control mice

the serum antibody ti ters were between 8 and 1 6 NU 7 days a f ter inoculat ion ;1 6-32 NU one month after inoculation ; and 3 2-12 8 NU a t and 4 months

after inoculation; In contras t,ant ibody ti ter s in the pas sively immuni zed

mice were 1 28—2 56 NU 7 days af ter inoculation ; 8-16 NU one month af ter

inoculation ; and 8 NU and 4 months af ter inoculation . Despi te the

serum antibody ti ter o f 8 NU,the infection was s till in the latent

s tage in the pass ively immuni zed mice 4 months af ter inoculat ion (i . e .

negative homogenate , po si tive ganglion explant cul tures ) . Thi s resul t

indicate s that serum neutrali z ing antibody does not play a cri tical role

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2 01 DE 0012 3-06 LOM

preparat ive gel elec tro phore sis were unsucces s ful in enriching recons titution

mixtures o f DNA wi thout a t the same time leading to shearing o f DNA into

small fragment s . This was an unde s irable resul t . It was finally po s s ibleto enrich fo r viral sequences by a combination o f aff inity chromatography

in phenyl-red bisac rylamide ( G~C s pec i f ic ) and rever se-phase chromatography

in RBI-5 . RES-5 f rac tionate s DNA on the basi s o f fragment size ,G 4 3

content,base s tacking and an array o f o ther as yet unknown fac tors . It

is common to obtain a 4—fold enrichment in phenyl-red columns and lO—fold

in RBC -5 al tho ugh the value s vary from batch to batch o f re s in. However ,

the high pressure and high ionic streng th needed to run RES~S has led to

two other problems,viz . fragmenta tion o f the high molecular weight DNA

s pec ies and re—hybridi za tion o f fragment s by tail s made by the res tric tion

enzymes . High—pres sure columns had to b e rede signed el imina ting allf itt ings with pore si ze smaller than 6 0 microns and sub sti tuting them

with hand—made porous polyurethane fi t t ings . Technique s had to be

developed to denature spurious tail— to-tail hybridization without

denaturing the whole o f the DNA fragment .

Thirdly,non— s pec ific hyb ridiza tion to mouse DNA in blo t s and to the

nitrocellulo se support had to be reduced to prac tically zero level s in

o rder to be able to detec t pic ogram quanti tie s o f viral DNA . Af terseveral unsuccess ful tr ial s , it was found tha t inclusion in the hybridi za tion

mixtures o f carrier DNA's from mouse ,

rabbit , dog , cow , sheep , and salmon

s perm would completely el iminate non—s pec i fic hybridi za tion . A graded

serie s o f washings o f the blo ts a t high tempera ture with dec reasing

concentrations o f SSC ~buf fer in the pre sence o f sodium pyro phos phate

W ould eliminate s ticking of the probe to the nitrocellulo se membrane .

“

At this po int , i t i s pos sible to detec t 100 pic ograms o f HSV DNA

f ragmented wi th Ec o RI ( smallest fragment 1 picogram) in a backg round of

50 ug o f carrier DNA . It should be po ss ible to have a de fini te answer to

the integration ques tion very soon , now that most o f the unpredic ted

technical problems have been el iminated .

molecular terms the latent s tage o f HSV infection o f mice , i t i s important

to determine whether expres sion o f the viral genome occurs , and if so , to

what extent .

Published wo rk from thi s Laboratory reported detec tion o f the

virusc oded thymidine kinase ( tk ) enzyme in extrac t s prepared from dorsalroo t ganglia taken from latently infec ted mice up to a maximum of 60 days

po s t HSV inocula tion . In order to extend the se s tudies a highly sensi tive

a ssa

yButil izing very high spec ific radioac tivity , 5— [1é iodode oxy c y tidine

(5—1 IdC ) as enzyme subs tra te was introduced last year .

As discus sed in last year 's annual report,in the presence o f te trahyd ro

uridine phos phorylation o f IdC i s blocked in extrac ts prepared from

uninfec ted cells , but can occur via the viral Tk enzyme in extrac t s

prepared from HS V infec ted cell s . Immune IgG , but not non— immune IgG ,

bloc ks this reac tion in infec ted cell extrac t s .

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During the pas t year , extens ive s tudie s o f Tk ac tivity in mouse

t rigeminal ganglia latently infec ted wi th HSV f ollowing corneal inoculation,

have been undertaken . Gangl ia were as sayed ei ther individually or as

pool s taken at dif ferent times after inoculation up to a maximum of 2 08

days po s t inoculation .

In the cour se o f these studie s,a number o f unforeseen problems were

encountered . F ir s t , the radio-labeled sub s trate (5 12 5IdC ) was found to

be uns table and decompo sed to produce an unidenti fied breakdown produc t,

which though i t d id no t func tion as a sub strate for any enzyme in the

gangl ion extrac t s , did produce high backgrounds in the as say . A puri f ication

system for the sub s trate wa s success fully developed and used to pur ifythe sub s trate about every two weeks . Al so a thin layer chromatography

sys tem , capable o f separating the reac tion produc t ( IdCMP ) from the

impurit y (X) and unreac ted sub s tra te ( IdC ) was used to de termine the

degree o f pho sphorylation o f the sub strate without inter ference from any

breakdown product s .

The second and maj or problem wa s tha t addition o f highly puri fiedIgG (immunoelec t ro phore tically pure ) prepared from immune rabb i t serum

raised against HSV infec ted primary rabbit kidney cell s resul ted in

s t imulation o f the Tk ac tivity in ganglion extrac ts prepared from la tently

infec ted mice and in some case s from uninfec ted mice,but never from

cell s produc t ively infec ted with HS V. Since the source o f the Tk enzyme si s a crude cell extrac t , the as say sys tem is necessarily complex and

numerous control experiment s were under taken to e s tabli sh the reason fo r

the stimulation . The se experiment s sugges ted tha t the stimulation was

no t due to fac tor s"in the IgG preparat ions used

,but was probably due

t o some immunologic cro s s—reac tivity wi th one or more o f the cellular

mi tochondrial Tk i soenzyme s . This problem was largely overcome by usingheat-inac tivated S taphyl ococcus aureus bearing pro te in A a s an immunoadso rbent

for immune complexes fo rmed when ganglion extracts were reac ted wi th

no rmal or immune IgG prior to being assayed fo r Tk ac tivity . Al so ,

addi t ional sera rai sed in rabbit s agains t the highly puri fied HSV Tk

enzyme were ob tained and used in the se s tudies .

The resul t s o f the se s tudies showed that the HS V speci f i c Tk enz yme

c ould only be detec ted during the acute phase o f the infec tion o f mouse

trigeminal or dorsal roo t gangl ia but no t during the la tent phase o f the

infec tion. Exac tly the same resul t was ob tained us ing a modified version

o f the s tandard as say sys tem (using [ 3H ] -Thymidine as sub s tra te ) capabl eo f de tec ting the HSV Tk ac tivity ari s ing from only one in a thousand

produc tively infec ted cell s . The resul t s o f thi s study are in agreement

with da ta reported in last year 's annual report , showing that there i s

a block in transc ript ion o f the HSV genome at the latent s tage o f the

infec t ion .

C urrently,o ther technique s are being developed in the Laboratory to

search fo r HSV antigens o ther than the viral Tk in latently infec ted

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ganglia . The se technique s involve i solating HSV antigens on immuno

adsorbents and analyzing them on denaturing polyacrylamide gel s .

Prel iminary experiment s on latently infec ted ganglia which have been

reac tivated in vitro f o r up to 4 8 hours show that HSV antigens are present

in very smal amount s . This s ugge s t s that i f expres sion o f some HSV

antigens immedia te early polypeptides ) occur s dur ing the latent

phase of the infec tion , their de tec tion would require extremely sensi tive

t echnique s .

has concentrated on HSV infec tion in gangl ia o f the peripheral nervous

system . In the last year , we initiated s tudies o f latency in the central

nervous sys tem (C NS ) .

At 6 days after corneal inoculation , HSV was demonstrated in

homogenates prepared from 18 o f 2 0 brain s tems and 6 o f 2 0 cerebralhemispheres . In the same group a t the latent s tage ( 5

— 8 weeks after

inoculat ion) , homogenates were negative in 2 0 mice . But unlike ganglia,

explants at the latent stage usually failed to yield virus ( po si tiveexplant s in O o f 1 0 brain s tems and 1 o f 1 0 hemis phere s ) . Since the

mortality in these mice was 52 , i t i s clear tha t the animal s survived a

produc tive C NS infec tion . The nega tive explant s may mean that HS V i s

el iminated at the produc tive stage o r that HSV canno t be readily reac tivated

from the C N S by explantat ion technique s .

To settle thi s que st ion , hybridi za tion technique s were used to assay

fo r HSV DNA sequences , bo th at the acute and latent s tage . Since viral

sequences , if a t all present , would be expec ted in a lower relativeabundance than in gangl ia , i t was impor tant to enrich the brain DNA

for viral DNA . The technique employed was aff inity chromatography on

phenyl-red subs tituted bisac rylamide . The phenyl—red l igand b ind s G 4 3

pair s in DNA,that can then be eluted wi th a gradient o f NaC lO4 ; a tighter

binding requires higher pe riodate concentrat ion and i s indicative o f

higher C~C c ontent . . Since HSV has 2 52 higher G 4 3 than mouse DNA , i t

could be separa ted from cell DNA sequences us ing thi s technique . The

complementary dye-sub st i tuted support,malachite-green bisac rylamide , is

s peci fic for A—T rich DNA and was al so used to enrich for viral DNA .

Bo th technique s gave on recons ti tut ion experiment s enrichment fac to rs o f

5-1 0 fold , which were considered suf fic ient for the experiment s to be

attempted .

Thirty animal s were sacrificed at bo th acute and latent phase s o f

the infec tion , and brain s tems and cerebral hemis pheres from the se micewere pooled . DNA was extrac ted and enriched for viral DNA sequences by

the techniques just de scribed . Hybridi zat ions were done us ing high

s pec ific ac tivity 32P-labeled viral DNA . De tec tion o f the hyb rid s was by

hydroxyapati te chromatography . The pool of thir ty acute brain hemispheres

was shown to have approximately viral DNA equivalent s per diplo id

c ell ; in the corresponding pool o f thirty acute brain stems viral

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2 .

3.

6 .

2 01 DE 0012 3-0 6 LOM

Puga,A . ,

Rosenthal, J . D .

, Openshaw ,H .

, and Notkins ,A . L . : Herpe s

s impl ex virus DNA and mRNA sequences in acutely and chronicallyinfec ted trigeminal ganglia of mice . Virol .

-1 11 , 19 7 8 .

Openshaw,H Asher , Wohlenberg , C . R. , Saki zawa

,T . , and

Notkins ,A . L . : Acute and la tent infec tion of sensory gangl ia wi th

herpes s implex virus : Immune contro l and viral reac tivation.

J . Gen . V irol .—2 1 5 , 197 9 .

Wohlenberg , C .,Openshaw ,

H . , and No tkins ,A . L . : In vitro sys tem fo r

studying the ef ficacy o f anti-viral agent s in preventing the reac tivation

—62 7,1 97 9 .

Openshaw,H .

,Puga

,A . ,

and No tkins,A . L . : Herpes simpl ex virus

infec tion in sensory gangl ia : Immune control , latency , and reac tivation .

F ed . Proceed . ( in pre ss ) 19 7 9 .

Openshaw,H .

,Puga ,

A .,and No tkins ,

A. L . : tene y and reac tivation

o f herpes s implex virus in sensory ganglia o f mice . In Virus LymphocyteInterac tions : Impl ications fo r Di sease . Ed ited by Max Pro ff i tt ,

El sevier North-Holland , New York ( in pres s ) 1 97 9 .

N AM E S , L A B OR ATO RY AN D I N ST I T UT E AF F I L I AT I ON S , AN D T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND AL L OT H E RPR OF E S S I ON AL P ER S ON N EL ENG AG ED ON THE PR O JECT

PI : Jenson , Al f red B . Surgeon

COPI : Puga—Carras co , Alvaro Sr . S taf f F ellowNo tkins , Abner L . Medical Director

Laboratory o f Oral Medicine

HUM AN T I S SU E S [j (c ) N E I T H ER

SUMM AR Y or W OR K (2 00 word s or less und e rl in e keywo rd s )

o f human papilloma virus (HPV ) by an antiserum which is cro ss— react ive with an

antigen present in al l pap il l omaviruses . Fluorescent antibody (FA) test s are

being done on fro zen sect ions o f wart tis sue s and peroxidase antiperox1 dase

(PAP ) tes t s are being done on sect ions o f formalin— f ixed ti ssue .

2 01 DE 00200-03 LOM

IHC ERATIVE LES IONS AND TUMORS : PAPILLOMAS OF THE ORAL <3 AV ITY

The papillomaviruses cause cutaneous and muc osal papillomas in man

and animal s . The human papillomavirus (HPV ) is the only known virus tha tunequivocally cause s neoplasia in man . Al though mos t o f the neoplasms

known to be induced by HP V appear to be benign and many s pontaneous ly

disappear , occasional oral and cutaneous papillomas will undergo mal ignanttrans formation into squamous cell carc inomas . During the las t few years

a t leas t five dif ferent HPV geno types (HPV—l , HPV BF V -5 ) have been

identi fied in cutaneous papilloma s . The se geno type s are no t immunolog icallyc ro ss-reac tive and can be distingui shed from one ano ther by immunologic

te st s as well as by DNA homology s tudie s . Recent s tudies , using te st s to

d istingui sh between the HPV geno type s,sugges t that each type of HPV

appears to be the causat ive agent o f s pec if ic clinical type s o f papillomas .

A g reater varie ty o f cl inical types o f papillomas occur in the o ral cavity

(verruca vulgaris,verruca plana , focal epithelial hyperplasia ( FEH ) ,

condyl oma ac c uminatum , oral papillomas and laryngeal papillomas ) than

anywhere el se in the body . It i s highly probable that some o f the s e

papillomas are caused by HPV o ther than HPV type s 1-5 .

The ob j ec tives o f this research s tudy are to ob tain representative

sample s o f cutaneous and oral cavity papilloma s,de termine which les ions

have an HPV e tiology,and if they do

,identi fy the type s o f HPV .

We have collec ted FEH f rom native Americans in the southwes tern

United States , laryngeal papillomas from o tolaryngology clinic s speciali z ing

in this disease , and o ther oral cavity papillomas from dentis t s in this

area . We also ob tained cutaneous papillomas f rom Univer si ty wart virus

c linic s , podia trist s,and dermatologist s . Mo s t o f the papillomas that we

are working with have been eas ily identi fied clinical ly and the diagnos i s

c onfirmed pathologically . Each fresh spec imen has been divided up for

elec tron microscopy , immunolo gy,and molecular virology s tudies for

identi fication and charac teriza tion o f HPV .

All o f the cutaneous papillomas in our study were either verruc a

vulgaris or verruca plantaris . When examined wi th the elec tron micro sc ope ,

approximately one—half o f these wart s contain HPV part icle s . We de termined

the type o f HPV in many o f the se virus—po si tive lesions by ei ther

immunologic studies us ing speci fic antisera agains t HPV —l or molecularvirologic s tudies on purified HPV . Almost all of the plantar warts

contained HPV —l and mo s t o f the common warts contained HPV -2 . We then

turned our attention to the oral cavity papillomas . Only one o f the oral

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2 01 DE 00200-03 LOMFuture S tudies

We have jus t s tarted screening large numbers o f fo rmalin—fixed,

paraf fin-embedded oral cavity papillomas for the presence o f HPV . We are

us ing antiserum direc ted agains t the common papillomavirus antigen,using

the PAP technique . Af ter identifying the le sions that mo s t o f ten contain

s ignificant amounts o f HPV , we can then concentrate on collec ting these

papillomas from various clinic s for the purpo se o f virus isolat ion. HPV

can then be isolated from them and the HPV DNA can be cloned in bacterialplasmids . Large quanti tie s o f HPV DNA can then be ob tained from oral

cavity lesions fo r molecular virologic studies ( endonuclease restric tionstudie s and molecular hybridi zation s tudie s ) .

Publ ications

Jenson ,A . H. ,

Op enshaw,H .

,Ho oks ,

J . J ., Puga

,A .

, and No tkins ,A . L . :

Herpes and o ther virus-induced oral di sease s . In C urrent Advance s in

Oral Biology,edited by Dr . H .C . Slavkin ( in press ) 19 7 9 .

..D

LN

00

N AM ES , L A B OR ATO RY AN D I N ST I T U T E AFF I L I AT I ON S , AN D T I TL ES OF PR I NC I PAL I NV EST I G AT OR S AND AL L OTH ERPR OF E S S I ON AL PER S ON N EL ENG AG ED ON THE PR O JECT

P I : Hooks , John J . Research Microb iologis tCOP I : Hernandez , Mercedes Vis it ing As so ciate

Cha , Chang—Yong Vis iting FellowNo tkins , Abner L . Medical Director

Yoon , Ji—Won Research Microb iolo gis t

Decker , John L .

rmine the role o f viruses andlogic and immunopathologic proces ses .

immune inter feron could be produced by peripheral blood

on an immune specif ic bas is sugges ted that it might be

various auto immune di sorders . We found immune interferon in

tients with act ive immunologic diseases , such as sys temic lupus

rheuma to id arthrit is , s cleroderma and Sj ogren's syndrome .

urthe rmore , the presence o f interferon was correlated with clinical disease

c tivity .

t is po s s ib le that the product ion o f interferon may contribute to immunologicbe rrations in autoimmune diseases and al so pro tect the already compromised

os t from viral infec tions .

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ZO1 DE 00219-03 LOM

C ELL-MEDIATED IMMUNE MEZHANISMS AND INTERFERON

Thi s research pro j ec t i s divided into three component s : 1 ) the roleo f viruse s and inter feron in immunological proces ses , 2 ) chemo tac t ic and

mitogenic antibodie s,and 3 ) hyb ridomas and monoc lonal antibodies to

viral antigens . Each pro j ect will be discus sed separat ely .

INTERFERON

Interferon was f irs t recogni zed as a soluble antiviral glycopro te ininduced by viruse s . Re cently , i t wa s shown that there are at leas t two

types o f inter feron . Type I,virus-induced inter feron is produced by

both non—lymphoid and lymphoid cell s in response to viruse s . Type I I o rimmune interferon is a mediator o f cellular immunity , a lymphokine and i s

produc ed by sensiti zed lymphocytes in response to s peci fic antigens . In

addi tion to their antiviral ac t ivity , both type s o f inter feron can af fec t

antibody produc tion and cell-mediated immunity .

The ob j ec tive o f thi s re search pro j ec t i s to de termine the role o f

viruse s and inter feron in immunologic and immunopa tholog ic processe s . In

the last two annual report s , we de scribed our exper imental data on the

role o f viruse s and interferon in immediate hypersensi tivi ty ( allerg ic )reac tions .

Hi s tamine and o ther mediators o f anaphylaxis are released when IgE

on the membrane o f human peri pheral blo od basophil s interac t s with speci fic

allergens ( e . g . ragweed antigen ) . Thi s release o f hi stamine from human

basophils by allergens has proved to be a good in vitro model for immediate

hypersensitivity ( allergic ) reac tions . Us ing thi s model , we found that

incuba tion o f human leukocytes with certain viruse s be fore challenge wi th

ragweed antigen enhanced the release o f his tamine . Furthe r . s tudie s showed

that thi s enhancement o f hi s tamine release was in fac t due to inter feron

induced by viruses . Moreover,an induc tion period and new RNA synthe sis

was required for interferon to exer t i t s ef fec t .

Interferon al so can be induced by s timulation o f leukocyte s from animmune ho s t by spec i f ic antigens . In addi tion to the relea se o f inter feron

,

the st imulation o f immune leukocytes by spec i fic antigens resul ts in the

release o f a variety o f o ther biologically ac tive mediator s (l ymphokine s ) .

La st year , we showed that immune— spec ific s timulation o f leukocytes

resul t s in the release o f soluble media tor s tha t are capable o f enhancingIgE

-mediated his tamine release . Thi s data sugges ted to us that inter feron

al so might be produced in pa tient s with di sease s involving an immune

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ZO1 DE 00219—03 LOM

Antibody to double stranded DNA and low level s o f the 3rd component

o f complement in the sera are both recognized laborato ry measures o f di seaseac t ivity o f systemic lupus erythemato sus . Our studies showed that cl inicald isease ac tivity wa s correlated wi th inter feron ac tivity and wi th antibodyt o DNA and low sermm <3 3 level s . The s triking correlat ion with di sea se

ac tivity , in fac t , raise s the po ssibil ity tha t interferon level s may be

a be tter index o f such ac t ivity than the laboratory measure s now in use .

C harac terizat ion o f interferon . The antiviral ac tivity in the

patient s sera was shown to be interf eron . The serum sample s inhibi ted

replication o f vesicular stomati ti s virus (V SV ) on human cell lines but

no t on mouse cell s . Moreover , the serum samples were no t toxic for

human cell s ; an induc tion period wa s required for development o f the

antiviral s ta te , incubation o f serum samples with V SV did no t resul t in

viral neutral ization , and the antiviral ac tivity was des troyed by trypsin .

Since immune interferon is inac tivated by trea tment with ac id , whereas

s tandard inte r feron is resi stant to such treatment , the sera were te sted

with acid . Since ac id trea tment des troyed the antiviral ac tivity o f the

pa tient 's sera , we conclude that the inter feron was immune interferon.

Our studies show that immune interferon is produced in patient s

with immunologically ac tive di sea se . The role o f inter feron in the se

immunological diseases i s s t ill conj ec tural . However , i t i s known that

interferon may al ter or modulate immune res ponse s . It is po s s ible that

immunolog ical aberrations assoc ia ted wi th sys temic lupus erythemato sus ,

such as increased antibody level s , autoantibodies , immune complexes,

depressed cell-media ted immune responses and kidney disease may in part

be mediated by circula ting immune interferon .

C l inical disease ac tivity in pa tient s with sys temic lupus erythema

tosus follows a course o f exacerbations and remis sion . Unfortunately ,

the laboratory measures o f disease ac tivity do no t always correlate with

cl inical di sea se . It i s therefore some time s dif ficul t to carefullyevaluate the e fficacy o f experimental drug treatment s in these pa tients .

Our findings s ugge s t that inter feron level s may be a be t ter index o f

disease ac tivity than the laboratory measures now in use .

Interferon is ef fec tive as an antiviral agent and de fends the ho s t

against viral infec tions . It was recently repo rted that herpe s-zo ster

virus infections occur les s frequently in patients with ac tive systemic

lupus erythematosus than in tho se with inac tive disease . Thi s ob servation

might be explained by the pre sence o f interferon in the blood o f tho se withac tive disease . Thus

,produc tion o f interferon during the ho st 's immunologic

res ponses to one di sease may pro tec t the ho st from contrac t ing a second (e . g .

viral ) disease .

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2 01 DE 00219-03 LOM

Future Plans

The studies reviewed in thi s repo rt provide insight into the po ss iblemechani sms involved in auto immune di sea ses which are charac teri zed byaberra tions in immunoregulation. Over the next year , the se s tudie s willbe continued and expanded . Inter feron in the sera o f lupus pa tient s

will be further charac teri zed and it s c ontinued presence in patient sera

for prolonged periods o f t ime will be doc umented . We al so plan to evaluatethe ro le o f virus infec tions or inter feron produc tion in the exacerbation

o f di sease ac t ivity in pa tients with sys temic lupus erythematosus.

Our s tudie s raise the possibility that type I I interferon may be

pre sent in the peri pheral blood o f pa tients with a varie ty o f otherimmunologic di so rder s . We are presently investigating thi s po ss ibil ityin di sea ses such as mul tiple sclero sis

,Wegener

's granuloma to si s,

vascul i ti s and sarco ido si s .

C HEMOTAC TTC AND MITOGENHS ANTIBODIES

Background

Mo use epidermal growth fac tor (mEG F ) i s a small polypeptide (molecularweight puri fied from the s ubmaxillary gland o f adul t male mice .

Thi s fac tor , at very low concentration ,st imulate s epi thel ial growth in

in vivo and in organ cul ture and i s mi togenic for many ec toderm and

endoderm— derived cell s .

Developmental biolog i st s c oncerned wi th in vivo control o f cell

proli feration and- di f ferentiat ion , and pathologi sts s tudying wound—heal inghave been very interes ted in the role o f EG F in the pro li feration o f

dif ferent cell type s involved in the se proces ses . As discus sed la s t

year , the coupl ing of biologically ac tive molecules chemotac tic

fac to r s ) to immunoglobulins , a highly spe c ific_

c arrie r ,could repre sent

a po tent tool fo r"targe ting the se molecules to s pec i fic si te s in vivo .

C hroma tog raphically puri fied antibody ( IgG ) t o herpe s simplex

virus or sheep red blood cells was coupled to mouse epidermal growth

fac to r (mEG F ) in the presence o f trace amount s o f iodinated mEG F ,

using gluteraldehyde as the coupl ing agent . Samples were dialyzedand chromatographed on a Se phac ryl S— 2 00 c olumn . The 12 5 -mEG F appeared

in the same peak as the Ig G . This demons trate s that mEG F was coupled t o

the IgG molecule . The coupled material then was assayed for it s

ability to st imulate DNA synthe si s o f human foreskin fibroblas t s . Bo th

preparations coupled to mEG F s t imulated DNA synthe si s . The control s ,IgG

alone and Ig G trea ted with gluteraldehyde , were devoid o f s timulatory

ac tivity .

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2 01 DE 00219-03 LOM

To see i f the antibody-b inding si te o f the IgG molecule s remained

intac t after coupl ing wi th mEG F , neutral iza tion test s for/ HSV and

hemagglutination te st s fo r SRBC were per formed . In bo th cases , the Ig G

r etained i ts capaci ty to b ind to antigen .

The coupling o f EG F to spec i fic immunoglobul ins may make it po ssibleto target mitogenic molecule s to s peci fic in vivo si tes . If succes s ful ,

thi s may aid in the resto ration o f damaged ti ssue as in wound—heal ingwhere mas sive proli feration o f epidermal and fibroblas t cell s i s required .

Future S tudie s

In the future , we hope to determine whe ther immune complexes compo sed

o f ECF coupled to antibody (mi togenic antibody ) and bound to ant igen are

capable o f s timulating DNA synthe s i s and whether o ther growth s timulatingfac tors can be coupled to immunoglobul ins .

HYBRIDOMAS

Background

By conventional immuniza tion methods , i t has been diff i cul t to

ob tain monoclonal antibody to s peci fic antigenic de terminant s on c omplexantigens . Re cently developed technique s have made it po ss ible to

produce antibody o f re s tric ted s peci fici t y (monoclonal ) to cer tain

antigens . Fo r example ,P3 x 6 3 Ag8 mye loma cell s [which are resis tant

t o 8-azaguanine due to de fic iency o f hypoxanthine phos phoribo sylt rans ferase and canno t survive in the media containing hypoxanthine ,aminop terine and thymidine (HAT ) ] i s hybridi zed to primed spleen cell sfrom animal s immunized wi th spec ific antigens . The se hyb rid cell scan survive in HAT media , they proli ferate continuously , and secre te

large amounts o f homogeneous antibody to specific antigens .

Over the last eight months,we have established the hyb ridoma

technique in our Laboratory to ob tain monoclonal antibodie s agains t

different antigenic determinant s on viruse s such as herpe s s implex ,

C oxsackievirus B4, encephalomyocardi ti s and reovirus . In our experiment s

,

Ba lb/C mice were immunized intraperi toneally with increasing do se s o f

virus . On the fi fth day,after the las t intravenous boo s te r , s pleen

cell s were prepared from the sacrificed mice and fused with P 3 x 6 3 Ag8

me yloma cell s , using polye thlene glycol (PEG ) as the fus ing agent . The

hyb rids were grown in HAT media fo r 1-2 weeks and then neutraliz ingantibody levels were checked f rom dif ferent well s o f the hybrid cul tures .

About 2 02 o f the tested samples produced neutralizing antibody . Well s

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Recep tors , Membranes and Diseases

N AM ES,LA BOR A TORY AN D I N ST I TUT E AF F I L I A T I ON S , ANO T I TL ES OF PR I NC I PAL I NV EST I G ATOR S AND ALL OTH ER

PROF E S S I ON AL PER SON N EL ENG AG ED ON THE PRO J ECT

Mccl into ck Patrick R . S taf f Fellow

COPI : Shimizu , F umio Vis iting FellowNo tkins , Abner L . Medical Director

Dr . J .D . Longs tre th , LMI , NIAID

Laboratory o f Oral Medicine

SUMMAR Y or W OR K (200 word s or less und e rl in e keyword s )Cell surface receptors for viruses and po lypep tide hormones were s tudied in

cells o f murine and human origin . The human membrane receptors for

Encephalomyocardit is virus were found to be quantitat ively and qualitat ively

different from those on murine cell membranes , although cell s o f bo th species

are sus cep t ib le . Ano ther clas s o f membrane recep tors , those for the po ly

pep tide hormone ins ul in,was s tudied following infec tion with Herpes S imp lex

herpes s implex and ves icular s tomat it is viruses but no t after infect ion with

measles and Sinbis viruses .

PI‘IS-6040

(Rev.

2 01 DE 00255-01 LOM

REJEPTORS, MEMBRANES AND DISEASE

Background

The sur faces o f cell s are known to po ssess receptors which determine

i f and how tho se cells will respond to viruses,ho rmones ,

and o ther cell s.

In recent years , the relationship between cellular receptors and disease

ha s become increas ingly evident . Mo s t viruses mus t at tach to a cellularreceptor in order to infec t a target cell

,and the absence o f an appropriate

recep tor can render a cell re sis tant to tho se viruses . Antibody to

hormone and neuro transmitter recepto rs can produce serious disease as in

extreme insulin re sis tance , Graves disease and myasthenia gravis . Obe si ty

can lead to decreased insul in reception and a mild to severe insul inresis tance .

In recent years , it has become evident tha t many disease s are

as soc iated with s peci f ic hi s tocompatibility antigens . The HLA (human )and H-2 (mouse ) ma j or histocompatibili ty loc i govern the expression of

the s tronge s t and perhaps the mos t important o f the cell surface antigens .

Insul in defic iency , diabetes mellitus , mul tiple s clerosis,and a ho st o f

rheumatological disorders have been found as sociated with increased

representation o f certain HLA haplotypes . It i s no t unreasonable to

cons ider tha t an apparently minor change in one or more cell sur face

c omponents c ould lead to al tered cellular func tion and eventually to

mul t i—sys temic disea se .

We have been inves tigating the relationship between receptors and

di sease from several approache s . Fir s t , since the susceptibility o f a

g iven cell to a virus largely depends upon the presence and charac teris tic s

o f a cellular receptor for that virus , we have been comparing cell s o f

di ffering susceptib ili tie s to encephalomyocarditi s virus (EMC ) withrespec t to the kinet ic s o f receptor binding . Second , previous work from

the Laboratory o f Oral Medic ine has demons trated that a single virus can

produce dramatically dif ferent di sease s in dif ferent mouse s trains . We

have been attempt ing to determine the genetic basi s o f differentialsusceptibility by studying the segrega tion o f susceptibility and re si s tance

in cro sse s o f sus cept ible and res i stant s trains o f mice . Third , some

viruses are known t o modi fy the plasma membrane o f their ho s t cell s . In

some cases,viral antigens o r new ho st antigens are displayed , while in

others,normally present component s are decreased . There fore , we have

b een using a selec ted sur face cell receptor , the one for insulin , as a

probe of pos sible cell sur face changes in viral infec t ions .

o f EMG virus with it s receptors,we made the as s umption that the behaviour

o f EMG would be similar to tha t o f o ther picornaviruses . In fac t ,EMC

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Z OL DE 002 55 6 L 0M

virus proved to be qui te di f ferent in it s b inding kinet ic s and opt imum

condit ions for attachment . In these studies , the cell s used were

secondary mouse embryo cell s ( SME ) , mouse L-92 9 fibroblas t s , mouse

e rythroblastoid cell s ( FTC ) , HeLa cell s , and human and mouse erythrocytes .

The virus used was plaque-puri fied from hear t—pas saged M-variant EMC , and

was designated EMC-108D .

When we initially attempted to measure the binding of EMC to SME

cell s o r L-92 9 c e11 s_

at ZO°G and 3 7 23 , no at tachment was found .

Surprisingly,when the binding reac tion was attempted at 0 23 , the virus

rapidly attached_ to the . c e ll s . The binding was spec ific as judged by the

failure o f mouse cell s to attach poliovirus of inac tivated EMC . It was

al so found that EMB would at tach t o HeLa cell s 5 to 1 0 t imes more rapidlythan to cells of mur ine o rigin . The attachment o f [ 3H ] l euc ine 1abeled EMC

t o HeLa cells was completely blocked by pre-incubation with a 100-fol dexcess o f unlabeled virus .

Others have reported that EMC attache s to and agglutinates human

erythrocyte s . When bo th human and murine erythrocytes were tes ted in our

assay , the human cell s rapidly adsorbed the 3H-labeled virus ; however , no

binding o f radioac tive virus to the murine erythrocytes was ob served .

When these experiment s were per fo rmed using plaque assays to monitor the

number o f infec tious unit s , it was found that bo th HeLa cell s and FTC

a ttached and eclipsed the virus at 0°

C . In contrast , human erythrocytes

at tached virus but did no t eclipse i t , and murine erythrocytes did nei ther .

The binding of EMC to murine cell s and to HeLa cell s proved to be

highly temperature—dependent . At temperatures above 10 23 , the b inding t o

HeLa cell s i s apprec iably sl ower , and the binding to L—9 2 9 c ell s i s almos tcompletely inhibited . All o f the available data indicate tha t HeLa cell shave more receptors per cell fo r EMC and that the se receptors may have

a higher af f inity for the virus . Mouse cell s on the o ther hand,appear

to have fewer receptors per cell , and once bound there i s a greater

tendency for the virus to elute from the murine cell s . Furthermore ,the

rate o f elution from murine cell s i s increased wi th increasing temperature

to a greater extent than the ra te o f elution from HeLa cell s . We are in

the process o f determining whe ther the difference in the number o f receptors

per cell can completely account for the dif ference in the kine t ic s o f

a ttachment o f EMG to HeLa cell s and murine cell s,or whe ther the thermo

dynamic proper ties o f the receptors are truly dif ferent .

Genet ic s o f re si s tance to virus infec t ion . Earl ier wo rk in the LOM

and that SJL /J’

and DBA/ ZJ mice were highly susceptible . Furthermore , i t

was shown that resi s tance was mo s t likely inheri ted as an auto somaldominant gene . We have recently begun a series o f experiment s designed

to es tablish linkage o f thi s gene with known enzymatic and antigenic

polymorphisms . Us ing mice from the ( SJL /J x C 5 7BL / 6J ) F1 x SJL /J backc ro ss

4 - 3 2 8

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2 01 DE 002 55—01 LOM

been charac terized adul t obe si ty and insul in re si s tanc e ) . However,

we are still lef t wi th a large deg ree o f igno rance concerning the role o f

the cell surface in de termining susceptib ility to di sea se . By the

appropriate s tudy o f selec ted membrane receptors , the relationship o f

receptor func tion and/ or dys func tion to di sease may be illuminated .

C ourse o f Future S tudie s

Our immedia te plans are to charac terize the human and murine receptors

for picornaviruses wi th res pec t to their physical—chemical proper tie s and

how these properties are related to dif ferences in suscept ibil ity in the

cell tis sues and ind ividuals. At present , we are at tempt ing to as say fo r

picornavirus receptors in par tially puri fied membrane frac tions o f t is sue

from which s ingle cell preparat ions are no t prac t ical . Since tiss ue

s usceptibili tie s appear to dif fer wi thin and among mouse strains , we hope

to be able to de f ine the reasons for the se tiss ue tro pi sms . With a

combination o f b iochemical , biolog ical and genetic approache s , the nature

o f the receptors , the degree to which dif ferent viruses share receptors,

and whether EMC recepto rs have o ther cellular func tions will be s tudied .

The study o f the influence o f viral infec tion upon sur face receptor s

will focus on defining the mechanism of the reduc tion in insul in recepto r

number by HSV-l and VS V infec tion . In addi tion,we will at tempt to

determine whether o ther receptors such as tho se fo r acetyl choline , thyroid

s t imulating hormone , and growth hormone are af fec ted by viral infec tion

in vitro .

ANNUAL REPORT OF THENEUROBIOLOGY AND ANESTHES IOLOGY BRANCHNATIONAL INSTITUTE OF DENTAL RE SEARCH

The Neurobio logy and Anes thes iology Branch is concerned with the

elucidat ion o f bas ic mechanisms of oral— facial sensat ion with par t icular

emphas is on pain sensat ion , and with the as ses sment and measurement of

exp er imental and cl inical pain in humans . Fundamental resear ch on

per ipheral and central nervous sys tem mechanisms of oral-facial sen

sa t ion i s conducted by the Neural Mechanisms S ect ion . The pr imary

obj ec t ive o f this res earch group is to correl ate behavioral respons es to

noxious and non—noxious s t imul i with neuronal funct ion at various

level s of the tr igeminal sys tem . A mul t id is cip l inary approach is

emp loyed and includes the fo llowing res earch act ivities : ( 1) electro

phy sio log ical s tudies of the response of tr igeminal brain s tem and

sp inal cord neurons t o noxious and inno cuous thermal and mechanicals t imul i ; (2 ) behavioral and pharmaco log ical s tudies involving pain and

t emperature d is cr iminat ion in monkeys trained to es cape po t ent ially

noxious or noxious s t imul i and to det ect non—noxious s t imul i app lied to

the face ; (3 ) correlative behavioral and neural s tudies to determine the

role of d if f erent peripheral and central neural populat ions in pain and

temperature discr iminat ion ; and (4 ) psychophys ical s tud ies on the

measurement and as s es sment of exp erimental and cl inical pain .

Fundamental s tudies on neuronal s tructure and funct ion are carr ied

out by the S ec t ion on Neuro cy to logy and Exp er imental Anatomy . This

program is concerned primarily with neuro cy tologic al s tudies of the

po s tna tal matur at ion and adul t organizat ion of the brain s tem trigeminal

nucleus in ca t and monkey,utiliz ing l igh t and electron micros copy and

var ious tracer t echniques . Correlat ive s tudies on neuronal s tructur e

and func t ion ar e carried out in conj unct ion with inves t igators in the

Neural Mechanisms Sec t ion . The Anes thes io logy Sect ion conduct s cl inicalres ear ch on the us e of intravenous s edat ive and anes thet ic techniques to

control anxiety and apprehens ion as so ciated with dental pro cedures .

Pr esent a ct ivit ies include phys io log ical , pharmacolog ical and psycho

log ical s tudies of the eff ects of intravenous sedative drugs dur ing and

a f ter oral surg ery pro cedures .

Al though the programs of the Branch are divided into thes e three

S ec tions,there is cons iderab le int eract ions between resear ch group s .

Correlat ive s tudies on neuronal s tructure and funct ion are being carr ied

out to elucidate in deta il the cir cui try of the upper layers of the

dor sa l horn of the medulla and S p inal cord and their relat ionship to

pain me chanismsu S tudies of chronic pain in humans involve the us e o f

behav ioral and p sychophys ical t echniques that also have been emp loyed in

correlat ive animal behavioral and phys iolog ical s tudies . The eff ects of

intravenous s edat ive drugs ar e evaluated ut iliz ing the improved psycho

phys ical as ses sment metho ds developed by the pain measurement group .

Final ly,our pr evious animal res ear ch has provided the concep tual

framework f or pr esent s tudies on endogenous pain— suppres s ing mechanisms .

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In this las t year,cons iderab le progress has been made in develop ing

a program for the sensory evaluat ion of chronic pain pat ients . In

co llaborat ion wi th lo cal consultants , we now have a mul tidis cip linaryteam with expertis e in neurology , neurosurgery , anes thes io logy , psychiatry ,

p sychology,pharmacology and oral surg ery . This group evaluates patients

admitted to the Clinical Center f or a 2—3 week period . This detailed

experimental and cl inical work—up f orms the baseline agains t which the

ef f icacy of new pain control methods is evaluated .

Af ter 42 years of government s ervice , ov er 20 of them at the NIDR ,

Dr . Edward Dris coll,Chief of the Anes thes io logy Sect ion retired . Dr .

Dr is coll was one of the p ioneer researchers in the f ield of dentalanes thes iology and his res earch initiat ive and leadership have been

critical for the development of the clinical program of the Branch . The

Anes thes iology S ect ion under his leadership has conducted unique s tudies

on the us e of dif f erent combinat ions of pharmaco logical agents useful in

the relief of anxiety and apprehens ion ass ociated with dental pro cedures .

Inves tigators in the Branch par t icipated in a Consensus DevelopmentConf erence on Pain

,Dis comf or t and Humanitarian Care held at the NIH in

February . This conf erence was held under the auspices o f the Inter

agency Committee oh New Therap ies f or Pain and Dis comfor t , compos ed of

s cient is ts and phys ic ians f rom the NIH and o ther f ederal government

agencies . A maj or goal of this group is to encourage res earch on the

mechanisms and appropriate treatment of s evere pain .

Other inves tigators in the Branch part icipated in a workshop at

Cold Spring Harbor Laboratory on the neurobio logy of pain . Maj or

emphas is here was on the development of new immunocy to chemical tech

n iques f or characteriz ing neuronal pathways involved in pain mechanisms .

Finally , members of the Branch were invited speakers at sympos ia pre

sented at the So ciety for Neuro s cience , the American Pain So ciety,and

the Ass o ciat ion f or Research on Nervous and Mental Diseas es .

Anatomical S tudies

Thes e s tudies are aimed at del ineating the synap tic cir cui try of

the caudal end of the trigeminal brain s tem nuclear complex and i ts rolein the pro ces s ing of pain and temperature information . The caudal and

of this nu c leus e c alled nucleus caudalis , or the dorsal horn of the

medulla , is laminated and cons is ts of an outer marginal layer (layera middle subs tant ia ge latinosal layer (layers 11a and IIb ) , and an innermagno cellular layer .

Recently we have shown that the superf icial layers of trigeminalnucleu s caudalis are organized s imilarly to the S pinal cord dor sal horn .

In light of this f inding , we have now pro ceeded in bo th the trigeminalsys tem and the S pinal cord to elucidate the neural circuitry involved in

sp inal and brain s tem pain mechanisms . The obj ec tives of thes e s tudies

during the pas t year were twofold . Fir s t,small calib er primary axons

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Other S tudies have used autoradiographic t echniques at light and

el e c tronmic ro s c ope level s to characterize the morphology of s ero tonin

containing and norep inephrine-containing axons in the medullary dorsal

horn . Electron micro s cop ic analys is of axonal endings labeled wi th

tr itiated sero tonin demons trates two categor ies of morpholog icallyd is tinguishable ending s : dome—shaped endings which form sing le synap ses

and s ca110 p ed endings which f orm mult ip le synap ses . These endings

synap se p rimarily on small cal iber dendritic shaf ts and sp ines . Dome

shaped endings were mos t common and three dif f erent types were found in

layers I and/ or I I . One typ e of s ca110 p ed ending was present in layer I

while layer 1 1 1 contained thr ee types . Dome— shap ed and s calloped-shapednorep inephrine ending s also are present in these layers . They can be

fur ther subdivided into f ive types . The presence of these s ero tonergic

and noradrenerg ic endings in layers I and I I sugg es t that they have

acces s to bo th the proj ection neurons in layer I and the interneurons in

the SC or layer I I . I t also has been obs erved that bo th typ es of

endings synap se on the same dendr ite,sugges ting that bo th monaminergic

sys tems p lay a ro le in the modulat ion of the output of s ing le neurons .

Correlat ive Anatomical and Phy s io logical S tud ies

Previous anatomical s tudies in our laboratory have provided a

detailed analys is of the morpho logy of neurons in the upper layers of

the dorsal horn tha t presumably par t icipate in pain mechanisms . However , the funct ion of the various types of proj ection neurons and

interneurons ident if ied in these anatomical s tudies is only poorlyunders tood . In recent s tudies we have at temp ted to correlate s tructure

and funct ion us ing an intracellular lab el ing technique . Neurons in

layers I and II have been funct ionally characterized and then intra

cellularly s tained with hor seradish peroxidas e .

Our result s conf irm that there are two common morpho log ical typ es

of SC neurons , s talked cell s and is let cell s . The s talked cel l 's

phys iolog ical responses are of two general types . No cicep t ive-specif icneurons respond only to noxious s t imul i . Wide— dynamic—range neurons

respond to bo th innocuous mechanical and to noxious s timul i . Thes e

responses are very s imilar to tho se of proj ec t ion neurons in layer 1 .

This f inding suppor ts the hypo thes is tha t s talked cell s are excitatory

interneurons that relay primary af f erent input to layer I neurons .

There app ear to be two types of is let cell s . I s let cells in the

superf icial par t of SG (layer I la ) have dendrit ic arbors with ros tro

caudal expanses of 400— 900 pm . Nearly all of the dendrites are conrained within layer I la . Thes e cell s have ei ther nocicep tive—spec if ic

or wide—dynamic-range responses . I slet cells with dendrites conf ined to

the deep part of SC (layer IIb ) have ros tro caudal extens ions of 1 200

1400 um . Their phys iolog ical resp onses indicate that they are innervatedonly by low thr eshold mechanorecep tive primary af f erent s . These dif f erenc e s between layer Ila and I Ib islet cells sugges t that each type may

b e involved in separate inhib itory pro ces s es .

This s tudy should provide long needed knowledg e on the role of the

sub s tant ia g elatinosa (layers Ila and IIb of the dor sal horn) in pain

mechanisms as well as adding to our unders tanding of the funct ion of

shor t interneuronal c ir cuits in the brain .

Phy s io logic al and Behavioral S tud ies

In previous s tud ies we det ermined the peripheral nerve f iber groups

resp ons ible for the ab il i ty of the monkey to detect inno cuous thermal

s t imuli and to es cape noxious thermal s timul i app l ied to the face

These co rrelated b ehavioral and neurophys io log ical s tudies fo rmed the

foundat ion for present and future s tudies of central nervous sys tem

co ding of pa in and temperature sensa tion .

Where do thes e dif f erent peripheral f iber populat ions proj ect in

the central nervous sys tem and what are the proper t ies of central

neurons invo lved in the monkey 's ab il ity to detect non-noxious and

noxious thermal s timul i . We began to answer thes e ques tions by examining

the re spons e proper ties of neurons in tr igeminal nucl eus caudal is in the

brain s tem in anes thet ized monkeys . Many of these neurons are part of a

pathway that proj ec ts d irectly to the thalamus and is exclus ive of o ther

component s of t h e trigeminal brain s tem nucleus . This analys is revealed

two general clas s es of tr igemino thalamic neurons that convey informat ion

related to pain . Current s tudies extend these observat ions to the

awake , behaving monkey . These s tud ies allow detailed analyses of how

the brain codes various proper t ies of no cicep t ive input . In contras t to

the relat ively s ta t ic and pas s ive input-outpu t relat ionsh ip s att ending

exp eriment s on anes thet ized preparations , s tudies of awake b ehavingmonkeys provide the oppor tunity to exam ine variables such as att ent ion ,

r espons e appropr iatenes s,expec tation and s t imulus salience , and the

role they play in no cicep tion .

Monkeys were t rained to detect the terminat ion of inno cuous heat

s t imul i and the onset of noxious heat s timul i (45 -49 C ) . In a s econd

task,the same monkeys detected the onset of a l ight s t imulus while

b ehav iorally nonrelevant thermal S t imul i were presented . Neuronalact ivity in the dorsal horn o f the medulla has b een correlated with a

number of b ehavio ral event s such as panel pres s , temp eratur e ons et and

terminat ion,and panel releas e . Two clas ses of neurons were s tudied .

No cicep t ive neurons exhibit ed respons es correlated with the onset and

terminat ion o f thermal s t imul i . In bo th behavioral tasks , thresholdso f ten were b elow 45

°C ,

and s t imulus-response functions were po s it ively

accel erat ing in 'the noxious hea t rang e . However , the sens it ivi ty o f

these neurons was cont ingent upon the behavioral s ituat ion . Responsesto behaviorally non-rel evant thermal s timul i pres ented during the second

task (l ight task) were reduced in magnitude as compared to s t imul ipres ented dur ing the thermal task . Increas ing vigilance demands by

reducing the intens ity of the l ight fur ther reduced the amp litude of

heat—evoked neuronal act ivi ty . Finally , a warming s ignal predict ing the

onset of a noxious heat s t imulus ( 470or 49

°C ) decreased heat—evoked

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ac tivity dur ing the thermal task , as compared to uns ignaled tr ials . Asecond clas s of neuron exhibi ted maximum ac t ivi ty dur ing the execut ion

o f taskr related behavior s . Su ch taskh related neurons exhib ited a main

tained dis charg e during the panel pres s p eriod . Dis charge rate was

indep endent of s timulu s intens ity or modality , s ince the maintained

d is charge was present in both tasks and o ccurred in the ab sence of

thermal s timulation . A bur s t of neuronal act ivi ty o ccurred in bo th

tasks pr ior to panel releas e . The bur s t was ab sent if the monkey failedto detect the cue or rel eased inappropr iately . Burs t magnitude was

indep endent of the intens ity of the relevant cue and i ts latency was

more highly correlated with panel release than with s timulus terminat ion .

The bur s t dis charg e o ccurred on exp erime nt er— init iated trials in which

monkey hand movements were no t pres ent . In addit ion,the bur s t dis charge

could be temporally disso ciated from lip movement s made in preparat ion

of liquid reinf or cement . Thus , taskr rela ted responses at panel pres s

and panel release were indep endent of phys ical characteris tic s of the

s t imulus and could b e dis so ciated from mo tor p reparatory behav ior

No cicep t ive neurons of ten exhib ited taskr rela ted respons es but of les smagnitude than their resp onses to noxious thermal s t imulat ion . These

data sugges t that no cicep t ive neurons provide informat ion about the

intens ity of noxious heat s timul i and can b e mo dulated by behavioralcont ingencies . These funct ional prop er t ies demons trate the impor tance

of mo dulatory inf luences on medullary dor sal horn neurons in aler tbehaving animals . F inally , the taskr related responses of thes e neurons

indicate that var iables such as S timulus relevance,at tent ional process es ,

response appropriatenes s , and reinf or cement cont ingencies al ter neuronalact ivity at the brain s tem level . Such respons es app ear to be cr itical

in s ignall ing the chain of neural events fr om s timulus per cep t ion to

mo tor response tha t are neces sary f or the execut ion of goal direc ted

behaviors .

We have employed the same behavioral thermal task in pharmacolog ical

s tudies in awake monkey to as ses s the relat ive ef f ect s of morphine on

the sensory dis cr iminat ive and af f ec t ive dimens ions of respons es to

noxious thermal s timul i . Our resul ts indicate that morphine at tenuates

the monkey 's ab il ity to dis cr iminate noxious and inno cuous thermal

s t imul i indep endent of it s ef f ec t on the aver s ive qual ity of the s timulus .

This result parallel s resul ts in human exper iment s descr ibed b elow °

nar co t ic analges ic drug s app ear to al ter the sensory dis cr iminat ive

aspec t of the pain exp erience apar t f rom their ef f ect on it s aff ect ive

component . The resul t s in the animal s tudies al so demons trate that our

behavioral model may be useful in the evaluat ion of new pain contro lagents .

Human P sychophy s ical and C linical S tudies

The cl inical program of the branch was es tabl ished to take advantage

of new knowledg e gained in the laboratory and to apply it to the cl inical

s ituat ion . This program has been expanded this year to include new

s tudies of chronic oral—f acial pain condit ions .

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Our s tud ies on the ef fect ivenes s of intravenous s edat ion t echniques

in the pas t on dental pat ients have concentrated on amnes ia ef f ect s and

recovery of s ensory and motor funct ions . In the pas t year , our eff or ts

have been towards an evaluat ion of phys iolog ical resp onses , such as

cardiac funct ion , b lood pres sure , and oxygen sa turat ion . The phys io

log ical respons es are mon itored non-invas ively to avoid introducing

fur ther anxiety and morb idity which may be as so ciated with invas ive

techniques . Our resul ts indicate that drug comb inations that resul t in

greater degrees of s edation are accomp anied by measurab le impa irment in

cardiovas cular and resp iratory hemo s tas is . Thes e comb inat ions have no

clear advantag e to the pat ient in terms of anxiety-rel ief or amnes ia .

Our future goal is to con t inue to evaluate phys io log ical and p sycho log icalfunct ion in this exp erimental model in order to ident ify pharmacolog icalas well as non-pharma co lo gical te chniques that produce anxiety rel ief

and a reduct ion in pa t ient app rehens ion with a minimal po tent ial for

cl inical toxic ity .

er 1 , 197 8 to S ep tember 30 , 197 9 CT 0060 102

N AM ES , L A BOR A T O RY AN D I N ST I T UT E AFF I L I AT I ON S AN D T I TL E8 OF PR I NC I PAL INVE GPROF

ES§JONAL PERSO

Q§ EL ENG AG ED ON TH

eiPROJECT

STl ATORS AND ALL OT H E R

i onne , Raymon A . S taf f Fellow NIDR

Dr is co ll,Edward J . Chie f

,Anesthe sio logy Sec NIDR

OTHER : Amato , Peggy R . Cl inical Nur se NIDR

But ler , Donald P . Oral Surg-Anesthe s io lo gis t NIDR

Clark,Barbara Ann C linical Nur se

Streko ,Thomas S taf f Dent ist NIDR

Swee t,James B . Sr . Dental Surgeon

Neurobio logy and Anesthe s io logy Branch

Anes the s iology Sect ion

NIDR ,NIH

,B ethe sda , Maryland 20205

I] (b ) H UM AN T I S SU ES [ 1 (c ) N E I TH ER

SUMM AR Y OF W OR K (2 00 wo rd s or le ss un d e rl in e keyword s )The purpo se o f this proj ect is to sys tematically as ses s the phys iologicalres

ponses to dru

gcomb inations u sed to alleviate anxie ty in dental out

pat ient s

.Cardiovascular and resp iratory funct ions are monitored non

invas ively by means o f an impedance cardiograph , earp ie ce oximeter andautomat ic b lood pres sure recorder . Resul t s o f the proj ect ind icate that

thec ombinationr o f diaze

pam- fentanyl and me thohexitol cause s a d ecrea se

in s troke volume which is ac companied by a ref lex tachycard ia . This

comb inat ion also result s in a decrease 19 arterial oxygen saturat ion .

The use o f diaz epam alone and d iazepam plus f entanyl does no t result in

any detectab le cardiovascular or resp iratory eff ect s . The methodology

emp loyed in thi s s tudy will b e used to as ses s o ther intravenous ,

inhala t ional,and oral agent s which can be useful for alleviating pain

and anxiety in dental outpat ient s .

PmS-604O

(Rev. 10—76 )

Z01 DE 00 132 — 05 NA

Ob j ect ives : The purpo se of the proj ect is to as ses s the phys io logicalrespons e to drug combinat ions commonly us ed in dental outpat ient s to

rel ieve anxiety . These ag ents are used in mo derately to highly anxious

pat ient s undergoing rout ine dental procedures and in pat ients undergo ing

extens ive pro cedur es such as the removal of third molar s and periodontalsurg ery . Desp ite their relat ively widespread use in cl inical pract ice ,

no systemat ic as s es sment ha s been made of the various agent s and com

binations . By comb ining the resul ts of this as sessment with the result so f previous s tudies which quantif ied the var ious parameters of therap eut ic

ef f icacy such a s amnes ia , s edat ion and anxie ty rel ief , it i s po s s ible to

quantify the relat ionship between ef f icacy and cl inical toxici ty , i . e . ,

the therapeut ic ratio . This information provides an obj e ct ive , rat ionalbas is for s elect ing the op t imal drug or combinat ion for the var ious

therapeutic s ituat ions in which they are used . In addi tion , the therap eutic

rat io provides an obj ec tive metho d for as ses s ing new agent s propo sed for

use in alleviat ing anxie ty in dental outpat ient s .

Metho ds Employed : Pot entia l pat ient s , 18 year s or older of either

s ex , are screened by member s of the NIDR Dental C linic to verify the

need for extract ion o f thir d mo lar teeth , the surgical procedures elect edfor use in this s tudy . Af ter val idat ion of this surg ica l need , a comp letehis tory and physical examination ( including appropr iate laboratorys tudies ) i s p erformed by qualif ied NIDR S taf f .

The response to four dif f erent drug comb inat ions and to placebo ar e

s tudied . Pat ient s are randomly allo cated to the var ious treatment

group s . Thes e treatment s are : 1) d iazepam 15 mg , me thohexitol 10 to 40

mg ; 2 ) diaz epam mg , f entanyl mg and me thohexito l 10 to 40 mg ;3 ) f entanyl mg /kg , diaz epam mg /kg ; 4 ) diazepam to clinicalendpo int ; 5 ) saline p lacebo . All drugs are adminis tered intravenous lyand all pat ients receive lo cal anes thes ia . A within subj ect de s ign is

used to compare treatment # 1 to treatment # 2 and to compare treatment #4

to p lacebo . All group s are compared to each o ther and to p lacebo by

appropr iate between subj ect s analysis .

The phys iolog ical response is monitored non— invas ively to avo id

introducing fur ther anxiety and morb idity which can be as so c iated with

invas ive technique s . Oxygen saturat ion is recorded via an earp iece

oximeter , blood pres sure via an automat ic blood pres sure device and

heart rate , s troke vo lume and cardiac output with an impedance cardio

graph . Preopera t ive ba sel ine reading s are taken every 5 minutes , intra

op erative reading s ar e taken every minute and po s t-O perat ive readings

every 5 minutes . This permits comparison of maximum drug ef fect s to

ba seline values and comparison of maximal eff ect s of the d iff erent

treatments . The t ime o f recovery to normal ba seline is al so monitored .

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ZO1 DE 00132—05 NA

Propo sed Cour se : F uture s tudies are aimed at as ses s ing o ther

modali ties for pain and anxiety control in dental outpat ient s . The se

include oral premedicat ion with non-s tero idal ant i-inflammatory analg es icssuch a s flurbiprof en , and the use o f long-act ing local anes the tics such

as bup ivacaine and etido caine . In addit ion , the ef f icacy of intravenous

d iazepam p lus nitrous oxide will be compared t o diazepam alone . The

overal l goal of the se three proj ect s will be to develop methods for pain

and anxie ty contro l in dental ou tpat ient s tha t do no t require special

tra ining or result in prolonged per io ds for recovery or alterat ion in

physio log ical home os tas tis .

2 . Publicat ions

G e lfman , S . S . , Gracely , R .H . , Dris coll , E . J . , Wirdz ek , P . R . , Butler ,

D . P . and Sweet , J . B . : C ompari son o f recovery test s af ter intravenous

s edat ion with diazepam—me thohexitol and dia z epame me thohexitol and

f entanyl . J . Oral Surg . ,-39 7 , 19 79 .

Dr iscoll , E . J . and G elfman , S . S . : Thrombophleb it is : I ts Inc idence

and Natural History . J . Oral 3 7 : In Press , 19 7 9 .

U 3 4 2

1, 19 78 to S ep tember 30 , 19 79

c and Brain S timulation Analg es ia on HumaPain

N AM ES , L A BOR ATORY AN D I N ST I T UT E AFF I L I AT I ON S , A ND T I T L ES OF PR I NC I PAL I NV E ST I G AT OR S AND ALL OT H ERPR OF E S S I ON AL PER S ON N EL EN G AG ED ON THE PR O JECT

P I Gracely , Ri chard H . Research P sycholog is t NIDR NAOTHER : Dubner , Ronald Chief , NAB NIDR NA

Dionne , Raymond A . S taf f Fellow NIDR NAHo f f er t , Marv in J . S enior S taf f Fellow NIDR NAAma to , Peggy R . Cl inical Nur se NIDR NALee s , David E . Deputy Chie f

SUMM AR Y OF W OR K (200 wo rd s or less und e rlin e keyword s )The purpo se s of the s tudy are ( 1 ) As ses s the ef fect ivenes s of chronic electri cal

st imulat ion of midbrain s ites for the relief of chronic pain in humans ; ( 2 )

compare the se to chronic electrical s t imulat ion of midbrain s ites ; (3 ) Validate

experimental models pf pain and the ir po t ential diagnos ti c use in chronic pain

emo tional wel l be ing o f the se pat ient s . Part icipants in this s tudy will be ( 1 )chronic p ain pat ient s receiving surgically implanted s t imulating electro de s for

pain contro l ; (2 ) chronic pain pa tient s maintained on tradi t ional narco t ic

analges ics who will not receive imp lanted s timulat ing elect rodes ; and (3 )healthy normal vo lunteer s . The eff ect s of chronic brain stimulat ion in surg icalp at ient s will b e compared to the eff ect s of narco tics previously adminis t ered to

pa tients and to eff ect s of nar cot ic regimes in nonsurgical chronic pain patient s .

In addi tion,the ef fe ct s of narcot ics on p ercep tual and neural mechanisms of

(Re v. 10-76 )

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1 . Proj ec t De s cr ipt ion

Ob j ect ives : Thes e s tudies will as s es s both the mechanisms and

relative ef f icacy of brain s timulation and narco t ic analg esics for the

contro l of chronic , intractab le human pain . S timulat ion of midbrain

s ites is a recent analg es ic technique perf ormed by a small number of

neuro surgeons . The present s tudies addres s several is sues p ertient to

the us e of this method . How do es brain s timulat ion compare to con

ventional nar co t ic adminis trat ion in terms of : ( 1) magnitude of\anal

gedia , (2 ) mechanisms of analges ia , (3 ) adver se s ide ef f ect s , and (4 )to -e ran c e .

I Me thods Employed : Pat ients scheduled to receive imp lant s are

admLtt ed to the NIH Clinical Center before and af ter surgery for ex

t ern ive t es ting including neurolog ical workup s , p sychiatr ic and p sycho

logical evaluat ion,appropr ia te laboratory tes ts , clinical pain ques

tic naire s , responses to noxious too th pulp and heat s timulat ion , and

tes ts of cognit ive and p sychomotor funct ioning . Mo s t pat ient s are

ass .s sed before and af ter adminis trat ion of analg es ics , p lacebo s ,

nar o t ic antag onis ts or brain s timulat ion . Pat ients no t receiving

impiant ed electrodes and normal vo lunteer s are as s es s ed in a similarmanger .

I Maj or Findings : Eight pat ients have been asses s ed to date . Five

hav received electrodes , one was rej ected and two are s cheduled f or

imp ana tion . Of the f ive receiving electro des , three had lit tle pain

rel af within one week of surgery and the electro des wer e removed . Two

of nese had required larg e do ses o f narco t ic medicat ion for pain rel iefand Ilad developed cons iderable tolerance to these medicat ions

, 15 mg

morphine did not alter appreciably ei ther exp erimental or clinical pain

mea shre s .

'

The third elec trode removal was from a p atient with atypical

facial pain who was no t nar co t ic tolerant . Adminis tration of morphine ,

howeLer , did no t relieve her cl inical pain .

Iof the two pat ient s wi th electrodes implanted for more than one

week pos tsurg ery , one had satis factory pain rel ief for 7 months before

the le c trode was removed because of complaint s about s calp irr i tat ion

over Ithe electrode s ite . The other has had the electrode for a shor t'

timekand has no t been reevaluated at NIH .

The es ent result s provide addit iona l evidence that the analg es ia

p rod ed by brain s timulat ion is cro s s- tolerant t o analges ia produced by

narc t ic adminis trat ion and that brain s timulation canno t b e ut ilized in

narc -ic tolerant pat ient s without a t l eas t a suitab le weaning from

narc pic medication . The negat ive result s with atyp ical facial painsugge ft that this syndrome may no t re spond to this pro cedure . The lackof pai n relief by conventional narcot ic analg es ic s in this pat ientsuppoft s the hypo thes is that re sp onsitivity to narcot ic analg es ic s may

predi t the ef f icacy of brain s timulat ion analges ia .

[P 3 4 4

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O ctober 1, 19 78 to Sep tember 3 0 , 197 9

N AM ES , LAB OR ATO RY AND I N ST I T UT E AFF I L I AT I ON S , A ND T I T L ES OF PR I NC I PAL I NV E ST I G ATOR S AN D ALL OTH E RPR OF E S S I ON AL PER S ON N EL ENG AG ED ON THE PRO JECT

Brown,F rederick J . Electronic Eng ineer (Instru) NIDR

Medlin , Terry P . Supv . Computer Specialist

Neurob io logy and Anes thesio lo gy Branch

Neural Mechanisms Sect ion

SUMM AR Y OF W OR K (2 00 wo rd s or le ss und e rlin e keywo rd s )

This work invo lves the development of sui tab le elec tronic and elec tromechanical instrumentat ion to be used in neurophys io log ical , phy siologic a

and behavioral research . I t involves the adap tation and interfacing of

the se and o ther instruments to a labo ratory or mult ipurpo se computer

installation .

PHS—604O

2 0 1 DE 0003 1- 11 NA

1 . Proj ect Des cript ion

a ) A DEC ll / 34 computer has been inter faced to equipment in one

laboratory through an INTEL SBC— 80 / lO computer . The ll / 34 interact s

with scient ist s in the laboratory while con trol ling a behavioralexp er iment on monkeys . Co llec t ion of the behavioral data and control o f

the equipment is aided by the SBC Neurophys iological data from

the monkey are presently recorded on analog magnetic tape for later

c omputer pro ces s ing .

The ll / 34 c omputer.has also been interf aced to a data entry facility .

Here nerve cell and EMG data previous ly recorded on analog magnetic tape

are enter ed into the ll / 34 through an INTEL MOS-8 5 micro computer . The

MOS—85 allows accurate timing o f the neurophys iolog ical and behavioral

events while leav ing the ll/ 34 relatively free to pro ces s data or run

o ther exper iments .

b ) A pair of high gain,high impedance , low no ise , dif ferential

amp li f iers have been de signed and built to faci lita te the recording of

electr ical s ignals from awake monkeys . The amp lif ier s are used to

monitor s ingle cell act ivity of neurons in the brain or EMC muscle

act ivity .

2 . Pub lications

None

O c tober 1 , 1 978 to S ep tember 30 , 19 79

N AM ES,L ABOR ATO RY ANO I N ST I TU T E AFF I L I A T I ON S , AN D T I TL E S OF PRINCIPAL J NVESTIGATORS AND ALL OT H ER

PR OF E S S I ON AL P ER S ON N EL ENG AG ED ON THE PR O J ECT

P I Dubner , Ronald Chief , NAB

Selt z er , Z eev Vis it ing F ellowYoko ta

, Toshika tsu Vis i t ing S c ient is t

Neural Mechanisms Sect ion

D (b ) HU M AN T I S S U E S g] (c ) N E I T HE R

SUMMAR Y OF WOR K (2 00 wo rd s or le ss und e rl in e ke yword s )Under barbiturate anes thes ia , microelec trode reco rdings from the tr igeminal

ganglion o f monkey are perf ormed and electr ical act ivity is recorded and

s tored on magne tic tape . Thermal and noxious heat s t imuli are delivered

with a prec isely-controlled the rmode which has a temperature range o f 2 0

0C

to 60 C and can produce a temperature change of 10°C at the the rmode— skin

j unct ion within one second . Data are analyz ed with the aid of an on-line

real— time computer sys tem . In related experiment s , intracellular recordings

are made from primary aff er ent neurons in trigeminal do r sal roo t ganglia ,

dor sal roo t f ibers , or the sp inal trigeminal tract , in response to

inno cuous and noxious thermal and mechanical s timuli . Af t er functional

charact er izat ion , hor seradish peroxidas e is iontophor e sed into the cell

or f iber . This intracellular label is then lo cated in his to log ical

mat erial and the central terminal proj ect ions of each axon determined .

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2 0 1 DE 00040— 10 NA

them with p sychophy sical measurements us ing s imilar parameter s , we can

as certain the ir role in pain sensat ion . Transformat ion or proc ess ing of

act ivity from each nocicep tive c las s then can be evaluated at dif f erent

level s of the neuraxis . In addit ion , the animal behavioral model which

has been developed in this laborat ory , in comb ina tion with electrophysio log ical studies , should be useful in the development and tes t ing

o f new and improved method s o f pain contro l .

Propo sed Course : We have init iated s tudie s which Co rrelate func

tional propert ies of primary aff erent s with the morphology and lo ca t ion

o f their terminal axonal arbor s in the brain stem , util iz ing intra

cellular tracer technique s .

2 . Pub licat ions

Dubne r , R . , Se ssle , B . J . and S torey , A . T . z The Neural Bas is of

Oral and Facial F unct ion . Plenum Press , New York , 1 978 , 483 pp .

Oct ober 1, 19 78 to S ep tember 30 , 197 9 CT 0060 10 1

N AM ES , LA B OR ATO RY AN D I N ST I TUT E AF F I L I AT I ON S , AN D T I T L E S OF PR I NC I PAL I NV EST I G ATOR S AND ALL OT H E RPROF E S S I ON AL P ER S ON N EL ENG AG ED ON THE PRO JECT

P I Gr acely , Richard H .

OTHER : D ionne , Raymond A .

Dubner , RonaldSweet , James B .

Hef t , Marc W .

S treko , Thoma s

Wol ske e , Patric ia J .

Research P sycho log ist

S taf f FellowChief , NAB

S r Dental Surg eon

Clinical As soc (Dentistry )S taf f Dent ist

Psycho logist

Neurob io logy and Anesthe s io logy Branch

Neural Mechanisms Sec t ion

NIDR

NIDR

NIDR

NIDR

NIDR

NIDR

NIDR

The obj ec t ive s o f thi s proj ect are ( 1) to asse ss psychophysic al m

methods

o f experimental pa n measurement , i . e . , magnitude est imat ion , category

scal ing ,and cro s s—modal ity mat ching . Pain will be exp erimentally induced

(2 ) to as se s s c linical pain measures , such as pain quest ionnaires and

s ensory mat ching methods,in a dental set ting ; (3 ) to det ermine the val id ity

o f experimental pain mo del s by compar ison of experimental and clinicalpain response s ; and (4 ) to evaluate known pharmaco logical and non

PHS-6040

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1 . Pro j ec t D escription

Ob j ect ives : The purpo se o f these s tudie s i s to develop p sycho

physical measurement techniques for experimental and clinical pain , and

to use validated techniques to evaluate pharmacolog ical and non-phar

ma c ologic al pain contro l ag ent s . As ses sment techniques mus t at temp t to

independently measure the sensory and mo tivat ional dimens ions of the

pain experience . The sensory dimension i s generally as sumed to be

associated with the di scriminatory aspect s o f pain . The mo t ivational or

unp leasantnes s dimension is generally a ssumed to be a complex percep tual

cogni tive component inf luenced by p sycho log ical f ac tor s . There i s

evidence that the se two factors may be dist inguished at a phys io log icallevel and evidence which sugges ts that pain control agent s can ac t

dif f erent ially on the se two pain component s .

s timulation o f skin and teeth , by hea t appl ied to skin and by noxious

co ld s timulat ion of expo sed dentin . The sensory intens ity and unp leasant

nes s as so ciated with the se s t imul i are as ses sed by verbal de scr ip tor

s ca les and cros s-modali ty mat ching techniques .

'

Clinical p ain is being

as ses sed by verbal s cal ing , by sensory ma t che s to experimental pain

s timuli , and by ques t ionnaire . The verbal pain des cr ip tor s have been

obj ec tively and reliab ly quantif ied in previous s tudies

Maj or Findings : The rel iab le , obj ect ive and val id verbal des crip tor

s cales developed in this proj ect have been used to as se ss po s tsurgical

pain following third molar extract ions in nonseda ted patient s . Pain was

a s ses sed before and af ter the intravenous adminis trat ion of either a

narco t ic antagonis t (naloxone) or a placebo , or af t er no treatment .

Verbal descrip tor que s tionnaires showed that po st surgical pain fol lowingp lacebo was s ignif icant ly reduced in compar ison to the pain exp erienced

af ter either na loxone or no trea tment . Commonly used visual analo g pain

s cales showed f ew signif icant ef fect s . The experiment s conc erned with

the a sses sment of nar co t ic and brain s t imulation produced analges ia in

c hronic pain patient s are des cribed in a new NIRP ,

"Ef fec t s of Narco t icand Brain S t imulat ion Analge s ia on Human Chronic and Exp erimental Pain".

An addit ional s tudy used verbal des crip tor mea sures and exp erimentalpain matching technique s to a sse s s pain in pat ient s suf f ering from

chronic oro— f ac ial pain syndromes . A correlat ional analy sis'

showed that

s elect ed exp erimental measures corr elated with cl inical measures of

mus cle tendernes s in pat ient s with Myof as c ial Pain Dy sfunct ion Syndrome .

The exp er imental measures also showed that these pa t ients can s caletheir clinical pain as readily and reliab ly as pain p roduced by experimentaltooth pulp s timulat ion and that they can reliably mat ch the sensory

intens ity o f the se quali tat ively diff erent pains .

Re sult s o f addit ional s tudie s of Myo fasc ial Pain Dys funct ion syndrome

are presented in two addit ional NIRP s ,S ensations Produced by Too th

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detec t changes in endorphin levels produced by the anxiety , pain and

trauma asso ciat ed with oral surgery . Addit ional s tudies also willdetermine the role o f endorphin relea se on placebo respons ivity . The

sens itivity of the verbal de scr ip tor ques t ionnaire used in the exp eri

ments will be op timized by standard mul t ivaria te techniques .

The s tudies o f chronic oro—f acial pain syndromes will cont inue with

a long itudinal analysis o f the eff ect ivenes s of conservat ive treatment

techniques fo r Myofas cial Pain Dysfunct ion Syndrome . These patient s

will al so rate the sensory intens ity or unp leasan tness of exper imentaltooth pulp s t imuli before and af ter the adminis trat ion of either fentanylor placebo to determine if the dif f erential resul t s ob served previouslywith no rmal volunteer s also o ccur with pat ient s suf f ering from chroni c

pain .

The s tudies us ing noxious thermal s timulat ion will cont inue with

the development o f new behaviora l and p sychophysical paradigms tha t will

ass ess the eff icacy and mechanisms of narco t ic analg esia . S tudies us ing

chronic pain pat ient s will provide informat ion about neural mechanisms

of narco t ic t o lerance .

2 . Publ icat ions

Gracely , R .H . , Dubner , R . and Mc G rath ,P . A . : Nar co tic Analgesia

Fentanyl Reduce s the Intens ity but not the Unpleasantnes s of PainfulToo th Pulp S t imulat ion . Sc ience ,

— 12 63 , 19 79 .

Gracely , R . E . : P sychophys ical Asses sment of Human Pain . In press

in : Bonic a , J . J Liebeskind , J . C . and Albe-Fes sard , D . , Advances in

Pain Resear ch and Therapy , Vo l . 3 , Raven Pres s , New York .

Gracely, R . H . : Pain Measurement in Man . In :

‘

Ng . , Pain , Discomf ort

and Humanitarian Care . Pro ceeding s o f NIH Sympo s ium , F e b . 197 9 , in

press .

LI

O ctober 1 , 19 78 to S ep tember 30 , 19 7 9

nothalamic Neurons Resp ons ive to Innocuous and No

N AM ES , LA B OR AT O RY AND I N ST I T UT E AFF I L I AT I ON S , AN D T I TL E S OF PR I NC I PAL I NV E ST I G ATOR S AN D ALL OTHE RPR OF E S S I ON AL P ER S ON N EL EN G AG ED ON THE PR O J ECT

P . I . : Ho f fman , Donna Po s tdo c toral FellowOTHER Dubner , Ronald Chief , NAB

Ziriax , John M . P sycho logis tMueller , Roy Psycho log is t

Neurob iology and Anes thes io logy Branch

1 2 0205

[3 (b ) HUM AN T I S S U E S Dfl c ) N E I T H E R

SUMM AR Y OF M0RI< (200 wo rd s‘

or le ss und e rlin e keywo rd s )This p roj ect s tudi ed the responses of tr igeminal nucleus caudal i s neurons to

noxious and inno cuous mechanical and thermal s timuli in awake behaving

rhe sus monkeys . Monkey s were trained to detect the terminat ion o f inno cuous

heat s timul i and the onse t of noxious heat s t imul i . In a second task , the

same monkeys detect ed the onset of a light s timulus while behaviorallynonrelevant thermal s t imul i were presented . No cicep tive neurons exhibi ted

thermal thr esho lds of 450C or lower and monot onic s t imulus— response funct ions

t o noxious s t imul i presented during bo th ta sks . Responses to thermal s timul ipresented during the light task were reduced in magnitude when compared to

responses during the thermal task . A second clas s of neuron exhibi t ed it s

maximum act ivity dur ing the execut ion o f task—related behaviors . This

act ivity was independent of s t imulus charact eri s t ics and could be sep arated

from moto r act ivity . No c icep t ive neurons als o exhib ited task-relat edact ivity

,but i t was of les ser magnitude than the responses to noxious

thermal s timul i . The task— related responses of both clas ses of neurons

may be as so cia ted with at tent ional pro ces ses involved in organiz ing goaldir ected behavio rs .

PH8-6040

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2 0 1 DE 00 15 1—05 NA

1 . Proj ect Descrip tion

Ob j ect ives : Previous work in thi s proj ect employed ane sthe ti zed

p reparat ions to examine the response proper t ie s o f nucleus caudali s and

subj acent reticular f ormat ion neurons that are par t o f the trigeminal

nuclear sys tem invo lved in no cicep t ive response s by rhe sus monkeys . The

present s tudies represent an effor t to evaluat e the contribution of

general anes thes ia to the result s of earlier work . Mor eover they allowdetailed analyse s of the dynami c proper tie s of the enco ding of noc icep t ive

input . In contras t to the relat ively s tat ic and passive input-output

relat ionship s at t ending experiment s on anes the t ized preparat ions,

s tudies of behaving monkeys provide the oppor tuni ty to examine the

interactive proper ties of somato sensory sys tems . Variab les such as

att ent ion , response appropria tenes s , expecta tion and st imulus salienc ecan be s tudied , and their ro le in no cicep t ion evaluated .

Methods Employed : In the phys io log ical s ingle unit recording

experiment s , a micro electrode is s tereo taxically introduced into the

brains team tr igemina l nucleus caudali s through a sea led chamber chron

ic ally f ixed to the monkey 's skull . During the experiment the monkeys

are temporarily res trained . O therwise the animals are kep t unre s trained

in their home cag es .

In one behavioral task currently being used and repor ted on p re

vious ly , monkeys are trained to detect the t erminat ion o f inno cuous hea t

pulses delivered via a f e edbac kr c ontrol led contac t the rmode ( 1 cm diam )app lied to the upp er hairy lip . Adap ted skin temperature i s 35

°C . A

panel i s illuminated to s ignal the beginning o f each trial . Monkeys

depress the lighted panel to ini t iate innocuous heat pulses (3 7

0

, 390

,

4 1 43 C ) o f randomly varying durat ions ( 2-8 sec ) . Panel re leases o f

l ess than 2 s ec (reac tion times or RT s ) af ter the terminat ion o f the se

s timuli are rewarded by water cc ) . Addit ional heat pul se s of a

cons tant dura tion (2 0 sec) ar e presented quasi-randomly in which the

temperature increase reaches the noxious range (45 470

,

Release of the panel at the terminat ion o f noxious s t imuli is no t

r ewarded by water . However,early relea se s terminate heat pulse s on

these trials , thereby allowing the monkeys t o es cape noxious heat

s t imul i . Thus the ta sk as se s ses RTs indicat ing the detec tion o f the

termination of innocuous hea t s t imuli or the di scr iminat ion of the onse t

o f higher s timulus intens i ties shif t ing into the noxious range .

A second behavioral paradigm has been introduced to addres s the

ro le of att ent ional pro ces ses as well as the ef fect s o f requiring the

monkey to respond pr imarily to visual rather than the rmal s t imuli . In

this ta sk the water re s tr ic ted monkey is trained to detect the onse t o f

l ight s timul i . Correct detec tion is rewarded by water cc ) . Con

s tant dura tion 3 ) heat pulse s are presented at quas i— random in

t e rval s bo th during and between trials . Response s by the monkey do no t

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Z 0 1 DE 00 15 1—05 NA

contingencies , and (3 ) the ac t ivity of bo th nocicep tive and task— related

neurons may be influenced by at tent ional pro ces ses .

The se s tudies have succes sfully identif ied no c icep tive neurons in

trigeminal nucleus caudali s — that i s , neurons which could transmit

informat ion about noxious s timuli and thus po s s ibly par ticipate in the

percep tion of and response s to oral— facial pain . S ince these data were

co llected in awake , behaving animals , our observa tions ext end and modify

previous work in acute , anes the tized preparat ions . No tab ly , certain

clas ses of no cicep tor s des cribed for acute preparat ions respond s imilarlywhen tes ted with s timul i pas s ively administer ed t o awake monkeys .

However , when the monkey perf orms the task , neurons des cribed as potential

no cicep tors in anes thet ized preparat ions are clas sif ied as task-relatedor no cicept ive neurons in awake behaving monkeys . Thus descrip t ions of

the func t ional prop er t ies of po t ent ial noci cep t ive neurons in anes the t ized

prepara t ions are incomp le te . The se s tudie s have also shown that the

responses of nocicep tive neurons can be mo dif ied by environmental and

behavioral contingencies . Thes e data s trongly sugg es t that the neuralrepresentation of oral-fac ial nocicep tive input can be modif ied at the

ear lie s t s tage of central nervous sys tem integrat ion . In addi t ion , this

work could sugg es t non—pharmaco lo g ical approaches to the contro l of

dental pain . Finally , this work has shown promise of providing valuab le

ins ights into the roles of such variables as at tent ion , motivation and

exp ec tancy in the neural representation of somato sensory st imuli .

Propo sed Cour se : F uture experiments inc lude the following : 1)antidromical ly act ivat ing neurons from chronically imp lanted thalamic

s t imulat ing elec tro des ; 2 ) shift ing the tempera ture values which dis

tinguish reinfor ced from non—re inforc edOtrial s . Shif ting the present

430C vs 45

0C compar ison to a 45

0C v s 47

0C comparison , fo r example , could

inf luence st imulus— re sponse func t ions and thresholds ob served for

noci cep tive neurons . 3 ) intro ducing a new behavioral task des igned to

maximize the sensory— d is criminat ive funct ioning of monkeys . This task

will require monkeys to att end to s timulus intens ity in order to cho se

the grea ter of two s t imuli which are clo se in magnitude . This new task

should maximiz e the responses of nucleus caudalis neurons to noxious and

inno cuous thermal s timuli and wil l a llow us with greater reliabili ty to

imp licate cer tain clas ses o f nucleus caudalis neurons in pain di scrim

ination .

2 . Publications

Dubner , R . and Hayes , R . L . : Pain mechanisms in the trigeminal

system . In : Mechanisms o f Pain and Analges ic Compound s , edit ed by R . F .

Beer s and E . G . Bas se t t,Raven Pre s s , New Yo rk , 19 7 9 , pp . 157— 16 9 .

Dubner, R . :

‘

P eripheral and central mechanisms o f pain . NIHSymp o s ium , in pres s .

1I ~ 7H§ 8

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Hayes , R . L . , Price , D . D . and Dubne r , R . : Behavioral and p hy s iolog icals tudies o f sensory coding and modulat ion o f trigeminal nocicep t ive

input . In : Advances in Pain Research 2 2d The rapy , 231 . 3 , edit ed by

J . J . Bonic a e t al , Raven Pres s , New York , 197 9 , pp . 2 19-2 43 .

Dubner , R . and Pric e , D . D . : Mechanisms of oral- facial pain . In

Oral Phys io logy and O cclus ion , ed ited by J .H . Perryman , Pergamon Pres s ,New Yo rk , 19 78 , pp . 193—2 05 .

Dubner , R . , Hayes , R . L . and Ho f fman , D . S . : Neural and behavioralcorrelate s o f pain in the trigeminal syst em . In : Pain , edi ted by

J . J . Bonic a , Raven Press , New York , 19 79 , pp . 63—72 .

N AM ES,LA B OR AT ORY AN D I N ST I T U T E AF F I L I AT I ON S , AN D T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H E R

PR OF E S S I ON AL PER S ON N EL ENG AG ED ON THE PR O J ECT

P I : Hayes,Ronald L . S taf f Fellow

Dubner ,Ronald Chief , NAB

Neurobio logy and Anes the s io lo gy Branch

Neural Mechanisms Sect ion

SUMM AR Y OF W OR K (2 00 wo rd s or le s s und e rl in e keywords )The maj or concern o f this proj ect was two fo ld : l ) to d evelop a primate ,

i s primar ily,if no t to tally , dependent upon the sensory ( intens ity )

dimension of pain while indep endent o f the af fect ive dimens ion of pain ;2 ) to tes t the hypo thes is that nar co tic analg es ic s (viz , morphine ) can

suppres s the sensory ( intens ity ) dimension o f noxious heat st imul i . Acoro llary of thi s hypo the s is that a lt er ing the affe ct ive stat e by minor

tranqui lizer s,l ike diazepam ,

is no t suff ic ient to suppres s the sensory

( intens ity) d imension of pain was al so tes t ed . Techniques emp loyed in

this s tudy were app l icat ion o f thermal heat via a contac t the rmode , us ing

latencies as a behavioral index of percep tual sens it ivity , i .m . inj ect ions

o f drug s , and real-time computer contro l o f the exp er imental se s sions . The

obj ect ives o f this s tudy were to develop a more comprehensive behavioralmodel o f pain in animals (primates ) and to broaden our unders tanding of

the analges ic act ion of narco ti c s .

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By showing that morphine can at tenuat e the sensory dimens ion it provides

a rationale for develop ing new metho ds o f analges ia which fo cus upon

s ensory at tenua tion rather than influencing the aff ec tive dimension o f

pain .

Propos ed Cour se : This animal model presently i s being used in

comb ined behavioral and neurophys io logical studies o f neural activi ty in

the tr igeminal brain s tem nucleus . The resul t s of the morphine-diaz epam

s tudy presently are being pr epared for publi cat ion .

2 . Publ icat ions

Hayes , R . L . , Bennet t , G . J . , Newlon , P . G . and Mayer , D . J . : Behavioraland phys io log ical studies o f non— narcot ic analges ia in the ra t elici tedby cer tain environmental s timuli . Brain Resear ch , 15 5 z 69— 9 0 , 19 78 .

Hayes , R . L . , Pr ice , D . D . , Bennett , G . J . , Wilcox , C . L . and Mayer ,

D . J . : Dif f erent ial ef fect s of sp inal cord les ions on narco t ic and non

nar co t ic suppres s ion o f no cicep tive reflexes : fur ther evidence for the

phy sio log ic mul t iplicity o f pain modulat ion . Brain Research , l55 : 91

101 , 1 9 78 .

Hayes , R . L . , Price , D . D . , Ruda , M . A . and Dubne r , R . : Suppres s ion of

no cicep tive responses in the primate by electrical s timulation o f the

brain on morphine administrat ion : behavioral and electrophysio log ical

compari sons . Bra in Research, l6 7 z 4l7-421 , 1 9 7 9 .

N AM E S , LA B OR ATO RY ANO I N ST I TUTE AFF I L I AT I ON S , AN D T I T L E S OF PR I NC I PAL I NV EST I G ATOR S AND OT H E RPR OF E S S I ON AL P ERSON N EL ENG AG ED ON THE PR O JECT

Mc G rath , Patr icia A . Bio log is t NIDR

Gracely , Richard H . Research Psycho log is t NIDR

Heft , Marc W . Clinical As so c (Dent ) NIDR

Dubner , Ronald Chief, NIDR

Neural Mec hanisms S ect ion

D (b ) H UM AN T I S SU E S C] (e ) N E I T H E R

SUMM AR Y OF MOR K (200 word s or les s un d e rlin e keywo rd s )The obj ec t ive o f this proj ect i s to de termine the nature of sensat ions

are evoked when low intensi ty elect ric current is app lied to human teeth .

tradi tionally exclusive pain Sys tem,these sensa t ions were s tudied bo th

p sycholog ically and phys io log ically : 1) the minimum level s of curr ent

neces sary to produce non—pa in and pain sensat ions were determined for

diff er ent f requencies of s timulating current ; 2 ) the intens i ties o f

s ensat ions from detec tion thresho ld to pain thresho ld were s caled by

act ivity o f the mas seter inhibi to ry perio d was recorded dur ing to o th pulp

s timulation at bo th non—pain and pain current s ; 4 ) the eff ect of a narco t ic

on s ensat ions produced by too th pulp s timulat ion and on the mas seter

inhib i tory period was evaluat ed ; and 5 ) the ef f ect s o f a condi tioningelectr ical s timulus , appl ied to the too th

,on sensat ion and on mas se teric

inhibi tory p eriod were determined .

PHS-6040

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Ob j ect ives : The purpo se of these s tud ies is to inves tigat e sen

sat ions evoked by electrical too th pulp s timulat ion . The too th has been

as sumed to be an exclus ive pain or no cicep t ive sys tem , and thereby a

unique mo del for the s tudy of pain , pain pathways , and contro l agent s .

However,a wide var iety of non—pain sensat ions ( such as warmth , t ingling ,

and pr es sure) is experienced when low intens ity electric current is

app lied to human teeth . The exis tence of non-pain sensa t ions , in

addition to pain sensations,may indicate the presence o f a sensory

sys tem dis tinct from a pain sys tem , or these non-pain sensat ions may

simp ly be a pares thes ia and re sult from near thresho ld s timulat ion of an

exclus ive pain sys tem . If there are 2 dis tinct sensory sys t ems , there

may be consis tent dif f erences be tween the levels of curr ent suff icient

t o produce sensa t ion and pain-assuming diff erent thresho lds for non— pain

and pain nerve f iber s . Or,the 2 sensory sys tems may diff er in ano ther

neural prop er ty,t emporal summat ion . Temporal summation can be s tud ied

by varying the frequency of s timulation ( that is , the number of pul se swithin a s t imulus train ) and no t ing whether there is a uniform or

diff erent ial ef f ec t on non— pain and pain sensat ions . If all sensat ions

produced by tooth pulp s timulation have similar thre sho ld and summation

prop er t ies , it is probable tha t all sensa tions result from s timulat ionof one sensory sys tem .

Ano ther means of s tudying po s s ible dif f erences in the sensory

innervat ion of the too th pulp is moni toring the ma sse ter‘

inhibitory

per io d , inhib ition of mas seter act ivity during subs tained contract ion

that o ccur s af ter the too th pulp is s timulated . This inhib ition may

provide phys io log ical correlates for the non-pain and pain sensationsproduced by too th pulp s timulat ion . Inhibitory .pe riods produced by

current s tha t produce def ini te non—pain sensa tions may dif f er from tho se

caused by current s that produce pain sensat ion . In order to as ses s the

value o f these inhib i tory periods as phys io log ica l corr elates of sen

sat ion : 1) the rel iab ili ty and s tab ili ty of EM recordings of ma s seter

activi ty dur ing t oo th pulp s timulat ion at non-pain and pain currents are

determined ; 2 ) the correlat ion between sensat ion experienced (non-pain

or pain) and the inhib i tory period is determined ; 3 ) the eff ect s of

narco t ic ( f entanyl ) on sensat ions experienced and on the inhib itory

p eriod are evaluated ; and 4 ) the eff ec t s of a peripheral electricalconditioning s timulus on sensat ion and mas seter ic inhib i tory p eriod are

determined .

Methods Employed : Non—pain and pain sensat ions were produced by

electr ical st imulat ion ( 1 sec trains of monopo lar , monophas ic , ca thodal ,

1 msec durat ion cons tant current pul ses ) del ivered to the lab ial and the

incisal edge of upp er'

c entral incisor s . Frequency of stimulat ion rangedf rom 5 to 500 HZ .

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The thresho ld for the mas seteric inhib itory period co incides

approximately 2 nA ) with an individual 's sensory thre sho ld . The type

o f inhib it ion is related to the s treng th of the s t imulat ing

current,no t to a sp ecif ic sensation . Mas seter ic inhib ition and sen

sa tion are two somewhat independent responses to s t imulus intens ity .

The indep endence of mas seteric inhib ition and sensation was demon

s trat ed cl early by the invariance of the mas se teric inhib itory per iod by

the narco t ic alt eration of pain sensation . The adminis tration o f

f entanyl ug /kg IV ) increased s ignif icantly sensory and pain thresho lds ,

but did no t af f ect the mas seter ic inhib itory p eriod s . The mas seteric

inhibitory period evoked by a par ticular s t imulus current was the same

before and af ter f entanyl , desp ite the changes in sensat ion produced by

the f entanyl .

This di sso cia tion be tween sensat ion and inhib i tion is also shown by

the app lication of an elec tr ical condi tioning s t imulus to the to oth .

The condi tioning s timulus consis ted of a train of 30 m sec pulses ,

del iver ed at 2 s ec int ervals to the central inci sor . The s timuluscurr ent was cho sen at the highes t value tha t was to lerab le to the

subj ect ; usually , but no t always , this current produced pain . The

appl icat ion o f the elec tr ical condi tioning s t imulus increa sed sensory

and pain thresho lds , withou t any accompanying changes in the mas seter ic

inhib itory period . S ensation is reduced in the adj acent non— condi tioned

too th as well as the cond itioned too th indicat ing the act iva tion of a

descending pain suppres s ing sys tem . S ince the pain suppress ion , produced

in par t elec trical condit ioning , is revers ed by the administrat ion of an

op ia te— antagonis t , naloxone ( 10 mg IV) an op iate— like sys tem is in par t

invo lved .

Prel iminary inves tigat ions indicate that the time course for pain

suppres sion on the condit ioned central incisor is diff erent from that on

the non— condit ioned lateral incisor . Quanti tative s tudies of the

diff erent ial ef fects o f electr ical condit ioning are now in progres s .

Signif ic anc e _pp Biomedical Research apv rogram p f _

the Ins t i tute

In recent years the tooth pulp has replaced the cornea (now known to

have therma l and pres sure innervat ion ) as an exclus ive no cicep t ive

sys tem . Consequent ly much res ear ch has fo cused on producing pain in the

too th by mechanical , chemical , and elec tr ical s timulation in order to

inves tiga te pure exper imental pain and various methods o f contro ll ingit -drug s , hypnos is

,e lec troanalges ia and acupuncture .

The present result s show there i s also a wide range of non-pain

sensations produced when the too th pulp is s timulated electr ica lly .

Subj ect s are able to scal e the intensi ties of these non— pain sensa tions ,

in addit ion to the pain sensa tions usually as so ciated with too th pulp

s timulat ion . Their scales or rat ing s may be used to as ses s the eff ect s

o f various pain control metho ds and to de termine if they act uniformlyor dif f erent ially on non—pain and pain sensat ions .

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Elec tromyographic moni toring of mas se ter inhibi tory ac tivity

( f o llowing chin tap ) has been used as a diagno s tic too l for as ses s ingtemporomandibular j o int dysfunc t ion (TMJ ) . This inhibi tory p eriod

( f o llowing too th pulp s timulation) has b een used al so as a nocicep tive

ref lex,a phys iolog ical correlate f or pain . However , the present

resul t s show that the inhibi tory period is no t correlated with pain

sensation . The inh ib it ion is as so cia ted with the intensi ty of the

s timulation , no t wi th sensat ion . The inhib i tory perio d may b e used as

an index of the current app lied to an un—ane s the siz ed too th ( in an

ane s the siz ed too th , there is no sensat ion and no inhib i tory period ) , but

no t as a reliab le index of pain . This f inding , conf irmed by the reduc t ion

of pain sensat ion by a narco t ic and by an electr ical condi tionings timulus wi thout any chang e in the mas se teric inhib i tory period , dis

qual if ies the use of the mas seteric inhibi tory p eriod as a nocicep tive

ref lex .

Propo sed Course : Inhib i t ion of pain sensation in a too th (produced

ei ther by a current app lied to the too th i ts elf or to an adj acent too th )will be fur ther evaluated . The succes s of some of the elec troanalges iacurrently us ed to relieve pain in dentis try is based in par t on this

inhib i tion princip le . Yet , the specif ics of the lateral sensory in

hibition pro ces s -a generalized phenomenon common to o ther sensory

sys tems — are no t de termined for the too th . Future res earch will fo cus

on elucidat ing this inhibi t ion pro cess in the too th . By concurrent EMG

monitoring of mass e ter ref lex act ivi ty , i t will be po s s ib le to as sess

the extent to which the sensory inhib i tion i s peripheral or central .

2 . Publicat ions

None

October 1 , 19 78 to S ep tember 30 , 197 9

N AM E S,LA BOR ATO RY AN D I N ST I T U T E AF F I L I AT I ON S , AND T I TL ES OF PR I NC I PAL I NV E ST I G ATOR S AND ALL OTH E R

PR OF E S S I ON AL PER SONN EL ENG AG ED ON THE PR O JECT

Mc Grath ,Patr icia A . B io log is t NIDR

OTHER : Gracely,Richard H . Research P sycho log is t NIDR

Dubne r ,Ronald Chief , NAB NIDR

Hef t,Marc W . C linical As soc (Dent ) NIDR

Neural Mechanisms Sect ion

D (b ) HU M AN T I S SU E S a (c ) N E I T HE R

SUMM AR Y OF W OR K (2 00 word s or les s un d e rlin e keyword s )

The obj ectives of this proj ect are : to es tab lish sensory and pain

thresho lds to electrical tooth pulp s t imulat ion in myo fascial paindysfunct ion (MPD ) pat ients and to relate sub— sensory , s ensory and pain

in MPD pat ient s,by means of electromyographic (EMG ) recording s . In

bo th the se cases compari son be tween MF D pat ient s to normal subj ect s willenable a be tt er under s tanding pf the MPD sypdrome pain and dysfunc tion

of the inhibitory perio d .

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act ing narcot ic , f entanyl , ug / kg IV ) or sal ine , in a doub le b lind

paradigm . Their EMG recordings o ccas ionally ( 2 5 2 ) showed a tendency

towards group ing .

The inhibi tory period has been found to be s ignif icantly longer in MF D

patients . This has been at tributed t o musc le spasm and a peripheral

mechani sm of pro longed inhib itory di scharge from Golg i t endon organs and

muscle sp indles . A central mo torneuron poo l mechanism could no t ,

however , be excluded . The pr esent s tudy demons trates a normal ext ero

c ep tive inhib itory p eriod in MF D pa tient s which sugges ts that the

di s turbance i s no t in the central mo torneuron poo l . The etio log icalconcep t as so cia ted with a dis turbed extero cep tive f eedback due to a"pathogeni c" o cclus ion s eems unlikely in l ight of the se new f inding s .

This will have fur ther implications in the trea tment of the syndrome .

Propo sed Cour se : This s tudy has been comple ted and a manuscrip t i s

being wri tten for pub li cation .

2 . Pub li cations

None

Oc tober 1,19 78 to S ep tember 30 , 19 7 9

N AM ES , L A BO R ATORY AN D I N ST I T UT E AFF I L I AT I ON S , AN D T I T L ES OF PR I NC I PAL I NV EST I G ATO R S AN D ALL OTH E RPR OF E S S I ON AL P ER S ON N EL ENG AG ED ON THE PRO JECT

PI Bennett , Gary J . Pos tdo ctoral FellowOTHERS : Abde lmoumene , Mohammed Vis i ting S cient i s t

Ho f f er t , Marvin J . S enior S taf f FellowDubner , Ronald Chief , NAB NIDR

S UMM AR Y or WO R K (2 00 word s or less un d e rl in e ke yword s )

Neurons in the sp inal cord dor sal horn o f monkeys and cats , e sp ecially

tionally charac ter i zed and intracellularly s tained with horseradi sh

p erox idas e . The dendri t ic arbors and axonal extens ions of these func

tionally ident if ied neurons have be en examined a t the light micro s cop e

level . Wide dynamic range neurons , respons ive to innocuous s t imuli but

ac tiva ted maximally by noxious s t imuli ar e found in layers I-V of the

dor sal horn and have more extens ive arbor s than neurons in the same

layer s act ivated only by noxious s t imuli (no cicept ive— specif ic ) . The

cy tomorpho log ical analysis o f these func t ionally characteri zed neurons

will so on be extended to the level o f re so lut ion of the electron micro s cope .

PHS-604O

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1 . Proj ect Des cr ip tion

Ob j ectives : The small interneurons of the sub s tan tia gelatinosa

( SG ,Rexed 's lamina I I ) are believed to be involved in the integration

and modulat ion of somatos ensory primary af f erent input . These cell s may

b e part icularly invo lved in pain s ensation . Extrac ellular '

e l e c trophy si

log ical analyses of SC neurons have recently app eared . The extracellularmethod

,however

,does not allow one to unamb iguous ly identify SG neurons ,

s ince this area al so contains the ap ical dendri tes of neurons who se

perikarya lie beneath the SG . Moreover , the extracellular me thod can

no t ident ify which of the several morpholog ical typ es of SC neurons the

physiolog ical recordings emanate from .

We have physio log ically character i zed individual SG neurons and

then intracellularly inj ec ted the same neurons with the enzyme , horse

radish p eroxidase (HRP ) . This enzyme spreads throughout the cell 'sinter ior and , when expo sed to cer tain his to chemical reag ents turns

b lack ; thus visual i zing the cell and i ts pro ces ses . This technique

obviously allows the neuron in ques tion to be preci sely lo cated .

Impor tantly , the technique also reveals the neurons morphology . Because

the HRP react ion product is elec tron opaque , the morpho logical charac

t e ri z ation can be extended to the ultras tructural level . Using this

methodo logy , we have begun a detailed examinat ion of the phys iology and

morphology o f the somato sensory neural cir cui try in the SG .

Methods Employed : The lumbo sacral spinal cords of anes the ti zed

adul t cat s are prepared for electr ophys io log ical recording in the usualmanner . We use glass microp ipett es with tip diameter s of about um .

Because thes e relatively large tip s yield poor intracellular records,we

physiolog ically character i ze the neuron b efore pene trat ion . The cell 'sresponses to a bat t ery of natural s timuli ( touch , hair , movement ,

p ressure , p inch , noxious heat,et c ) , and to percu taneous electrical

s timulat ion of the cell 's recep tive f ield are determined . The per

cutaneous elec trical s timulus allows us to es timate the conduct ion

velo ci ties of the cell 's input s . Af ter phys io log ically charact eri z ing

the neuron , an attemp t is made to introduce the microp ipet te into the

neuron 's interior . Succes s ful penetrat ions are carefully checked in

order to be cer tain that the impaled neuron is the same as the one

charact er ized . HRP is iontophore se d into the cell for the durat ion of

the impalement ( 1— 15 min) . At the conclusion of the experiment , the

animal is perfused with f ixat ive and the sp inal cord removed . The cord

is sect ioned p arasagittally into 150 um thick sec tions . The tis sue is

then incubated in COCL and the HRP react ion product is develop edby incubating with diaminoben z idine and hydrog en p eroxide . The develop edsect ions are cleared wi th g lycerin and examined with the light micros cope .

Camera lucida drawing s X) are prepared for well s tained examp les .

The t is sue is then removed from the glycerin and pro ces s ed for electron

micro s copy in the usual f ashion .

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The neuronal circui try tha t sub serves the sensation o f pain is only

d imly elucidated . The des ign o f ra t ional therap ies for the ameliorationof neuropatho log ical syndromes tha t present dis turbed pain sensation . as

their mo s t salient symptom requires an accurat e under s tanding o f the

neural mechanisms tha t mediate normal pain sensation . This proj ec t aims

toward an unamb iguous and detailed elucidation of the phys io logy and

morpho logy of the neuronal cir cui try of the SG .

Propo sed Cour se : We intend to continue to examine phys iolog icallycharac teriz ed neurons with bo th the light and elec tron micro scop es . In

addi t ion to neurons in the SG , we will extend our analys is to the

neurons immedia tely benea th the SG in the nucleus proprius . Moreover ,

we hope to be ab le to emp loy immunohis to chemical t echnique s in order to

ident ify the neuro chemis try of the HRP f illed neurons and their con

ne c tions .

2 . Publications

Bennet t, G . J . , Hayashi

,H . , Abde lmoumene , M . and Dubne r , R

Physio log ical p roper t ies of s talked cell s of the subs tant ia gela tinosa

intracellular ly s tained with horseradi sh peroxidase . Brain Re sear ch ,

’

— 2 89 , 19 7 9 .

Price , D . , Hayashi , H . , Dubner , R . and Ruda , M . A . : Func tionalrelationship s be tween neurons of the marg inal and sub s tan tia gelatino salayer s of the prima te dorsal horn . J . in pre s s .

N AM ES , L A B OR ATO RY AND I N ST I T UT E AFF I L I A T I ON S,AN D T I T L ES OF PR I NC I PAL I NV EST I G ATOR S AN D ALL OTH ER

PROF E S S I ON AL PER S ON N EL ENG AG ED ON THE PR O JECT

P I Gobel , S tephen Chie f , NEA S ect ion

Co invest igator s Fal ls , William Senior S taf f F e llowArvidsson ,

Jan Vis it ing F ell owB rown , Emma B io . Lab . Tech . (Ele . M1 c

Al len , Barbara M . B io logis t

r oo t o f the trigeminal nerve and the morpho logy o f the neurons which

comprise the ma in sensory and sp inal tr igeminal nucle i . The se s tudie s

emp loy electron micro s copy ,the Golgi method and the use of intraneuronal

markers such as hor seradi sh peroxidase . The obj ec t s o f the se s tudies

are to delineate tr igeminal pain—temperature pathways and to broaden

our unders tanding of o ral-fac ial sensat ion .

PHS-6040

2 0 1 DE 0002 0-14 NA

1 . Proj e ct Des cript ion

Obj ect ives : The obj ect ives o f our anatomi cal s tud ie s during the

pas t year were two fo ld . Firs t , to f ill small caliber primary axons

whi ch respond to noxious s t imuli with the protein marker horseradish

peroxid ase (HRP ) . This marker can be visualized bo th at the l ight and

electron mo c roscopic al levels and provide s an oppor tunity to de termine

the laminar dis tribut ion o f these axons within the sub stant ia gelat ino sa

(SC ) and charac terize the ir terminal axonal arboriz at ions . Se cond , to

examine the synap t ic connect ions o f the two maj or interneurons of the SG

whi ch have been impaled with a microp ipet te had their primary inputs

charac ter ized and subsequently f illed with HRP .

Me thod s Employed : Detailed camera lucida drawings were made of

HRP— f illed neurons at X using the l ight micro s cope which dep icted

their axonal and dendrit ic morpho logy , branching pa t tern and laminard is tr ibut ion . Af ter comp let ion of the drawings , the sect ions containingthe HRP-f illed neurons were proc es sed for elec tron microscopy in order

to study the ir f ine s tructural charac ter ist ics and the ir synap t ic con

ne c tions .

Maj or Find ings : The SC , which cap s the dorsal horn in the medullaand sp inal cord , is a maj or terminat ion s ite of primary tr igeminal and

sp inal neurons respect ively whi ch respond to noxious s timul i . Individualsmall cal iber um ) myel inated primary axons enter layer s 1 1a and

I Ib where they give rise to as many as f ive f ine um) axonals trands which are extended for s everal hundred pm in the ro s tro caudalaxis o f the se layer s . Each o f the se strands be ar numerous bead— l ikeaxonal endings which l ie in the center o f the layer 11a and I Ib glomeruliwhere they synap se on two d iff erent kinds o f dendr ites , i . a .

, tho se

whi ch contain synap tic ves icles and are sour ces of as well as rec eiver s

o f synap t ic input s ( type 2 dendr ites ) , and tho se without synap t ic

vesicles which only receive synap t ic input (type 1 dendr ite s ) . The se

f ind ings conf irm par t of the hypo thes is which aro se from our too th pulpext irpation s tudies whi ch s tated that layer I I is a maj or terminat ion

s ite o f pr imary neurons with small myel inated axons while layer I is a

maj or terminat ion s i te o f pr imary neurons with unmyelinated axons .

The axonal endings o f the pr imary axonal s trands in layer II synapse

on the dendr ites o f two maj or kinds o f interneurons,i . a . ,

neurons whose

axons arborize wi thin SC and do no t pro j e c t to higher level s o f the

neuraxis . In ear lier EM analyse s , we have been able to thoroughlycharacter ize the kinds o f dendr ites which receive synap t ic input fr om

primary endings in layer II . However,it had no t been po s s ible to

determine which kind s o f dendr ites belong to which of the many celltyp es that make up the-neurop il o f SC largely be cause all o f the dendr ite s

are about the same s i ze and shape . During thi s pas t year , our colleaguesin the Neur al Mechanisms Sect ion have succeeded in iontophore t ically

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The rat ionale for u t il iz ing orofacial pain in diagno s ing dental patho logy,

for providing anes thes ia for dental pro cedures and for unders tanding the

role of pain in ref lex movement of the mus culatur e of the head and neck

is based on our knowledge of orofacial pain pat hway s . Our knowledge of

much of these neural pathways today is fragmentary . Our neuroanatomicals tudies are aimed a t es tablishing a mor e def init ive circui t diagram of

trigeminal pain pathways .

The tr igeminal nerve is involved in a ho s t o f chronic pain s tates

which include tr igeminal neuralgia ( t ic doulour eux) , glo s so dynia (burningtongue) and o ther facial neuralgias . Exp lanat ions of these pain s ta tes

u sually involve mechanisms related to pathology of the peripheral nerve .

However many of the symp toms of t ic doulour eux , for examp le,canno t b e

explained without cons idering synap t ic cir cui try in the central nervous

sys tem . For example , why are only the maxillary and mandibular divis ions

involved i. e . , only tho se d ivis ions supplying the teeth? Why do the

mo s t innocuous s timuli trigger the pain ep isode ? I s the lo ss of teeth

s omehow involved in a d isrup t ion of synap tic connect ions in the spinaltr igeminal nucleus ?

Recent technical advances have made it po s s ib le to select ivelys tudy individual neurons in trigeminal pain pathways at the l ight and

elect ron micro scop ical level s . Data from such s tudie s will provide more

detailed informat ion about the neural circui try about trigeminal pain

pathways and will permit us to des ign more cr itical exp eriments to

approach the above a s well as other que stions concerning chronic pain

s tat es in the orofacial region .

Propo sed Cour se : Dur ing the coming year we will u se our ab ility to

select ively mark the terminal axonal arbors o f pr imary neurons with HRP

in order to cr it ically examine their synap tic connect ions in the three

layer s of the SG . This approach is superior to cut t ing the trigeminal

roo t and looking fo r degenerat ing endings be cause diff erent kinds of

primary neurons degenerat ed at di f ferent rat es and require many ex

pe riment s . In addit ion it will provide an oppor tunity to examine the

intact primary ending rather than degenerat ing , shriveled , fragmenting

endings as seen with degenerat ion techniques .

We wil l also reexamine the terminal dis tribut ion o f primary neurons

which innervate too th pulp s us ing several new approaches . HRP and

f errit in will be conj ugated to a variety of dif ferent p lant lect ins such

as wheat germ agglut inin in at temp t s to specif ically mark the axonalendings of these neurons . S ince lect ins may be transpo rted orthograde ly

in primary neurons in great er amount s than HRP it may be po s sib le to

obtain a much more accurat e p icture o f the dis tribution o f these neurons

than ever before po s s ible . In addi tion we will continue our study of

the trans synap t ic deg enerat ive changes in SC neurons fo llowing pulpotomie s .

Finally , we will cont inue our analyses of the synap tic connect ions

of the neurons of SG . The EM analyses of the synap tic connect ions of

4 — 3 7 8

2 0 1 DE 0002 0- 14 NA

t he se neurons should add grea tly to our under s tanding of how primary

inputs are proces sed in the SG .

2 . Publicat ions '

Gobel , S . : Neural c ircui try in the sub s tant ia gelatino sa o f

Ro lando : Anatomical Ins ight s . In : Advances in Pain Research and

Therapy , Vol . 3 , edi t ed by J . J . Bonic a . Raven Pres s , 19 7 9 , in pres s .

Gobe l , S . and W .M . Fal ls : Anatomical ob servat ions of hor seradish

p eroxidase f illed terminal primary axonal arborizat ions in layer II of

the subs tantia gelatino sa o f Rolando . Brain Res .,19 7 9 , in pres s .

O ctober 1 , 1978 to Sep tember 30 , 197 9

N AM E S,LA BOR ATO RY ANO I N ST I T U T E AF F I L I AT I ON S , AN D T I TL E S OF PR I NC I PAL I NV EST I G AT OR S AND ALL OTH E R

PR OF E S S I ON AL P ER S ON N EL ENG AG ED ON THE PR O JECT

P I Falls,Will iam M . S enior S taff Fellow

OTHER : Gobe l,S tephen Chief , NEA Sect ion

Neurobio logy and Anes the s io logy Branch

SUMM ARY OF WOR K (200 wo rd s or le s s un d e rlin e keywo rd s )The se studies examine the pos tnatal development o f neurons in tr igeminal

pain pathways in the dorsal horn of the medul la ( tr igeminal nucleuscaudalis ) with Go lgi and electron micro sc opic techniques . They compare

in newborn ki ttens the maturity of the two maj or interneurons in the

adult counterpart s . They fo llow the developmental sequenc es in the

formation of the dendrit ic and axonal arbor s of bo th stalked cells and

the i slet cell s and examine how these interneurons el iminate exce s s

dendri tes as well as how they provide addit ional plasma membrane for

their elongating dendrites and axons . The se studies al so determine which

components of the synap ti c circuitry in trigeminal pain pathways are

present at birth and which develop po stnatally .

4 — 3 8 0

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October 1 , 197 8 to S ep t ember 30 , 19 7 9

Caudalis

N AM ES,L A BOR ATORY AN D I N ST I T UT E AFF I L I AT I ON S , ANO T I T L ES OF PR I NC I PAL I NV EST I G AT OR S AND ALL OT H ER

PROF E S S I ON AL PER S ON N EL ENG AG ED ON THE PRO J ECT

P I Ruda,Maryann T . S taf f Fellow NIDR

OTHER : Gobel,S t ephen Chief , NEA S ect ion NIDR

Neurob io logy and Anes thes io logy Branch

Neuro cy to logy and Experimental Anatomy S ection

NIDR ,NIH

,B ethesda , Maryland 20205

[3 (b ) HU M AN T I S S U ES g] (c ) N E I TH ER

S UMM AR Y OF W OR K (200 word s or les s und e rlin e keyword s )

This s tudy at temp ts to pharmacologically charact eri ze axonal endings in

the dorsal horn of the medulla (DHM) . The methods employed include

light and el ec tron micros copic autoradiographic lo calization labeledaxonal endings in the DHM fo llowing app licat ion of trit iat ed H ] neuro

transmitt ers onto the DHM and immunohis to chemical lo cali zation o f spec if ic

populations of axonal endings . The neuro transmitters being inves tigat ed

purpos e of these s tudies is to lo cate and morpholog ically charact eri ze

pharmacolog ically dis tinct axonal endings in the DHM which might be

ZOl DE 00 207—0 3 NA

Ob j ectives : The purpos e o f thes e experiments is twof old : l ) to

character ize the ul tras truc tural morpho logy of pharmacologically ident if ied

axonal endings in the sub s tantia gelatino sa (SG ) of the dors al horn of

the medulla (DHM) ; 2 ) to develop a pharmaco logically charac teri zed

circui t diagram of po tential synap tic interactions in the DHM . Ourinitial s tudies f o cus ed on the monoamines s ero tonin (SHT) and norepine

phr ine (NE ). The data on 5HT terminals are in pres s while those on NEterminals are pres ently b eing analy zed and prepared f or pub licat ion .

Having morpholog ically identif ied the monoaminergic terminals in SG , we

are pres ently inves tiga ting raphe magnus and lo cus coeruleus aff erents

to SC in an at temp t to correlate the morphology and synap tic inter

actions of the monoaminergic terminals we have identif ied , with their

nuclear origin . This typ e o f correlation is important in that i t

des crib es p o tential neural cir cui ts through which s timulation produced

analges ia can act . Other exp eriments which are in their ini tial s tages

will examine dif f erent pep tiderg ic axonal endings in SG .

Methods Employed : Pharmaco logical charac teri zation of cell bodies

and axonal endings in our s tudies invo lve three dif f erent techniques .

The f irs t technique ut ili zed in s tudying monoamines invo lved ligh t and

electron micros cop ic autoradiography . The approach relies on the

ab ili ty of axonal endings which normally utili ze a specif ic neurotrans

mit ter , to take up this same neuro transmi tter when it is exogenous ly

app lied in a radioact ive form . In our experiments , ei ther H ] SHT or

H ] NE was top ically app lied onto the DHM . Following fixation to b ind

the monoamines , the DEM was proces s ed f or autoradiography . Slidescarrying l u or EM sections were coated with liquid nuclear emuls ion ,

exposed in the dark for s everal weeks to months and sub s equently treated

with a pho tographic develop er . This pro ces s results in the depos i tion

o f s ilver grains over areas containing the H ] neuro transmi tter3nd

thus lo cal ize S p ecif ic axonal endings . In o ther experiments an H ]amino acid was inj ected into nucleus raphe magnus and the locus coeruleusand anterograde transport of the radioactive lab el to SG was used to

mark the axonal endings .

A s e cond technique utili zed in our experiments involved the s ero tonin

neuro toxin , dihydroxy tryp tamine DHT) . This neuro toxin des troys

5HT terminals , resul ting in morphological changes indicat ive of de

generation . Thes e changgs can b e seen at the EM level . Experiments in

which b o th DHT and H ] NE are us ed , provide two dis tinguishab le EM

markers s o that interactions of b o th monoamines can b e compared in the

same experiment .

A third method emp loys antib odies rais ed to a specif ic neurotrans

mitter to immunohis to chemically lo calize p ep tide neuro transmit ters at

l ight and EM level s .

Z0 1 DE 00 20 7-0 3 NA

Maj or F indings : Light micros cop ic analysis of the dis tr ibution of

silver3grains in l

3s ect ions of the DHM demons trates the pres ence of

b o th H ] SHT and H ] NE in layers I and II . S ilver grains oc cur bo th

as individual. grains and as aggregates compos ed of many individua

grain In comparing the dens i ty of s ilver grains in SC in the H ] SHT

and H ] NE exp eriments,there is a greater amount of o th individual

ggains and gra aggre gate s . f ollowing app lication of H ] SHT . NeitherH ] SHT nor NE labeled neuronal cell b odies have b een found in the

DHM .

Electron micros cop ic analysis of axonal endings lab eled Wi th [3H ]

SHT demons trates two categories o f morphologically dis tinguishable

endings : dome— shaped endings which f orm a s ingle synaps e and s callopedendings which form mul tip le synap ses . Each category can b e furthe

gdivided into s everal types based on morphological cri ter ia . The . [ H ]SHT labeled endings synaps e primarily on small Calib er dendritic shaf ts

and sp ine s , with the dome— shap ed endings f orming b o th symmetrical and

asymmetrical synaps es and the s callop ed endings forming only symmetricalsynaps es . Dome— shap ed endings were mos t common and two typ es were found

in layer I and II while a third type was found only in layer II . LayerI contained a s ingle type of s callop ed ending While layer II contained

three typ es of s callop ed endings .

The pres ence of s ero tonergic endings in layer I sugges ts that someof thes e endings synaps e on the dendri tes o f layer I proj ection neurons

where they may inhib i t the ou tput of the proj ection neuron direc tly .

S ero tonergic endings in layer II may modulate the ac tivi ty of layer II

interneurons by synaps ing directly on thes e interneurons . The inter

neurons ih layer I I may funct ion by mediat ing the trans f er of inputsfrom primary endings in thes e layers to layer I proj ection neurons .

Dome- shap ed and s callop ed shap ed [3H ] NE labeled endings are also

p res ent in the sub s tantia gelatinosa . These categories of'endings can

b e fur ther divided into f ive types b ased on morphological cri teria .

Three types of dome-shap ed endings are found in layers I and II and are

dis tinguished by the pres ence o f ei ther p leomorphic , small oval or largeoval agranular ves ic les . They form mainly asymmetrical synap ses on

dendri tic spines and shaf ts al though some symmetrical synap s es and

occas ional axosomatic synapses are f ougd . In the exp eriment combining

the s ero tonin— neuro toxin DHT and H ] and H ] NE up take,s everal

examp les of the samgdendrit e receiving synaps es from bo th a degenerat ing

SHT ending and an H ] NE lab eled ending were f ound . One type of

s callop ed ending is found in bo th layers I and II while a s econd type is

f ound only in the deeper par t of layer I I . The s calloped endings fo rm

asymmetrical synap s es on dendritic shaf ts and spines .

The presence o f [3H ] NE lab eled axonal endings in layers I and II

o f the DHM sugges ts that NE aff erents have acces s to bo th the proj ection

neurons in layer I and the interneurons in layer II . The ob servation of

b o th SHT and NE endings synap s ing on the same dendri te further sugges ts

[I- 3 8 LI

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ZO1 DE 00207-03 NA

2 . Publications

Ruda , M .A . and Gobel , S tephen : Ul tras tructural Characgeriz ation of

Axonal Endings in the Sub s tantia Gelat inosa which take up H ] S ero tonin .

Brain Resear ch , in pr ess .

Oc t ober 1 , 19 78 to S ep tember 30 , 19 7 9

N AM ES , L A BOR AT O RY ANO I N ST I TUTE AF F I L I AT I ON S , A ND T I TL ES OF PR I NC I PAL I NV E ST I G ATOR S AN D ALL OT H ERPR OF E S S I ON AL P ER S ON N EL EN G AG ED ON THE PRO JECT

Ho ckfie ld, Susan J . Bio log is t NIDR

Gobel , Stephen Chie f , NEA S ect ion NIDR

S UM M A R Y OF WOR K (200 wo rd s or le ss und e rlin e keyword s )This p roj ec t examines the neural c ircuit s invo lved in the appreciat ion

o f oral-f acial sensa t ions , par t icular ly the circuit s mediat ing pain and

temperature sensations . Incoming sensory informat ion may b e mo dif ied by

neuronal activi ty in many areas of the nervous sys tem b efore be ing relayedto higher brain center s . Neuron marking technique s , in which a foreign

carried back to their neuronal cell bo die s , accurat ely demons trat e which

neurons par t icipate in a given neural circui t . Previous experiments in

thi s s tudy identif ied the neurons that relay incoming oral- facial sen

sa tions t o higher brain centers . Exp eriments conducted dur ing the pas t

year have ident ified the neurons . that can modify incoming sensoryinforma t ion and that may , in par t , mediate analgesic and hyperalges ic

s tates .

PHS-6040

(Re v. 10-76 )

2 0 1 DE 00248—02 NA

1 . Pro j ec t Description

Obj ectives : The obj ect ive of thi s s tudy is to identify the neurons

in the central nervous sys tem that proj ect to the dorsal horn of the

caudal medulla and may,in par t , modify incoming sensory informat ion

f rom the face . Three general lo cations of these neurons will be analyzed

( 1) wi thin the main senso ry and sp inal trigeminal nucl ei ; (2 ) in areas

o f the brains tem out s ide o f these trigeminal nuclei ; and ( 3 ) in the

cervical sp inal cord .

Methods Employed : Horseradish peroxidase (HRP ) , a pro tein of about

molecular weight was micro inj ected into nucleus caudalis . This

p ro tein is p icked up p ino cyto tically by axonal ending s and transpor ted

retrogradely through the axon to the cell body of origin where i t is

s eques tered in lyso somes . Af ter the appropriate his to chemical reaction ,

cells of orig in which send their axons into nucleus caudalis can b e

clearly visuali zed in the brains tem .

Maj or Findings : The ini tial experiments of this proj ect identif ied

three lo cations of trigemino thalamic proj ec tion neurons in and adj acent

to nucleus caudalis . These are : layer 1 , layer V , and an area j us t

ventral to nucleus caudalis which represents the trigeminal component o f

the lateral cervical nucleus . The s imilari ties be tween the morpho logyo f nucleus caudalis and the sp inal cord dor sal horn , and b e tween the

locat ions of trigemino thalamic neurons in nucleus caudalis and of sp ino

thalamic neurons in the sp inal cord dorsal ho rn , has led-us to regard

nucleus caudalis as the medullary continuation of the head of the dorsal

horn o f the spinal cord . I t receives trigeminal sensory input in the

same way that the dorsal horn of the sp inal cord receives sensory input

for the res t of the body .

In the next s eries of experiments , by inj ecting hor seradish p eroxidase

into the dorsal ho rn o f the caudal medulla , we have identif ied the

sources of input to the medullary dorsal horn which are invo lved in

modifying the output f rom this nucleus to higher brain centers . Neuronsin many lo cat ions in the sp inal cord and brains tem send axons to the

medullary dorsal horn . In the sp inal cord , neurons that proj ec t to the

caudal medulla are f ound predominantly in layer s I , III , IV , V and VII .

Contrary to clas s ical des crip tions , they are no t f ound in layer II . In

the caudal medulla , neurons in layer s I , IV , V and VII proj ec t to the

oppos i te medullary dorsal horn . Neurons in the more ro s tral trigeminals ensory nuclei and the areas bordering them also send proj ections into

the dorsal horn of the caudal medulla . The main sensory nucleus and the

subnuclei of the sp inal trigeminal nucleus can be characteri zed by the

dens i ty and morpho logy of their neurons that proj ec t to the caudalmedulla.

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Repor t of the Clinical Inves t igations Branch

Na tional Ins t itu te of Dental Re searchSummary S tatement FY 197 9

The Cl inical Inves tigations Branch is concerned with the s tudy o f

f ac tors inf luencing diagno s is of oral disorder s from theoret ical to

des crip tive analyses of known func tional relationship s .

The Branch is comprised of two sec tions : the Oral and PharyngealDevelopment S ec t ion (OPD ) under Dr . James Bo sma , and the Diagnos tic

Methodology S e c t ion (DMS ) head ed by Dr . Richard L . Webber . These sec tions

work largely independently . OPD emphasi zes the s tudy of form , developmentand func tion of oral and pharyng eal tissues through de tail ed anatomical

descr ip tion and phys iological analyses coupled wi th psycho—phys ical

measurements . DMS approaches the s tudy o f oral disorders with more of

a sys tems or ientation which is grounded in image process ing and infor

mation theory . Together the sec tions complement each o ther by providing

a mul tidis cip linary approach to diagno s t ic problems of mutual interes t .

Re cent ac tivit ies in OPD have involved the s tudy of sensory ,

mo tor and salivary secretory mechanisms . Sensory s tud ies make use o f

p sycho-phys ical tes ting pro cedures to de termine de tec tion and recogni tion

thresholds and to quantify supra threshold percep tion of sensory s timul i .

Of par t icular interes t are proces s es und erlying the percep tion o f sal t

and sweet tas te among normal s and individuals having sensory impairment .

S tud ies of mo tor func tion o f the oral—pharyng eal complex have focused

upon the pharynx and larynx as related to swallow , sp eech , and airway

maintenance . Rad io log ic data provide the sub s tance for a book ,

Radiog paphy o f the Pharynx and Larynx , which is to be publ ished by

Spr ing er—Verlag .

S tud ies o f normal and dis ease-related sal ivary func tions are al sounder way to determine the relationship be tween sp ec if ic salivary

cons t ituent s (pro teins , sp ecific en z yme s ) .and func tional def ic it .

Diseases o f inter es t include Sj ogren's syndrome , aphthous s tomatitis ,

and pancrea tic disorder s .

Two long—range anatomical proj ec ts are nearing comple tion which

provide the sub s tance for two ref erence books . One , Po s tnatal

Bo sma has b een cri tically reviewed and is being cons idered for publica t ionby the Na tional Library of Med icine . The o ther , Anatomy of the Infant

Head,by J . F . Bo sma has progres sed to a po int where its illus trations

are es s entially f inished .

DMS ac tivities are balanced be tween the inves tigation of new bases

f or quanti tat ive ly clas s ifying info rmation of diagnos tic interes t and

the s tudy of al ternative technology via computer s imulation and development of pro to type sys tems .

Re cent work has centered on application of the symmetric—axis

g eometry pioneered by Blum to the des cr ip tion of growth and development'

of the human mandibl e . This mathema tical tool has been used to describ e

mandibular shape in a way which unequivocally demons trates a ser ies of

invariant angular re lationship s . These invariants appear to be independent

of the age of the individual s tudied and may be sp ec ies specif ic . They

also are independent of the coord inate space used to descr ibe the shape .

O ther s tudies deal explicitly with prob lems assoc iated with the '

demons tra tion of impaired performance of explici tly def ined diagno s t ic

tasks from various d if ferent x—ray sys tems inc luding a second-genera tion

CAT machine and various pro to typ e dental x-ray sys tems . In add ition

to me thc dologic al cons iderations these s tudies provide rational bases .

for tailoring the information— carrying capaci ty o f the sys tem to the

amount of inf ormation required for reliable performance of sp ec if ic

diagnos tic tasks .

Hardware d evelopment is done large ly in co llaboration wi th o ther

Federal agencies . This includes technolog ical evaluation and the

deve lopment of pro to type sys tems . The applica tion of broad-sp ec trum

rod—anode x—ray sour ces coupled to various high- sp eed imag e—de tec tor s

is of p ar ticular interes t at this time , s ince iso top ic intra—oralsources have b een shown to be less than ideal for many fluoros cop icapplications . The use of a f eed— back sys tem to determine appropriate

proj ec tion geometry is also being cons idered in this context in order to '

f ac ilitate the de tec tion of les ion— changes from one examination to the

next .

This has b een a year of s ignif icant chang e wi thin DMS wi th regard

to resources and personnel . The entir e labora tory fac il i ty was relo catedinto larg er quar ters and key programming per sonnel including one senior

inves tiga tor , Dr . Roger Nagel , trans f erred to a dif f er ent program at the

Nat ional Bureau of S tandards . This move parallels an evolutionary trend

wi thin DMS , toward more sp ecif ic sc ientif ic app lications research now

that the bas ic technolog ical capab ili ties of the labc ratory have been

e s tabl ished . Dr . Nag e l 's rep lacement as a Senior S taf f Fe llow is

Dr . Waldemar de Rij k , a phy sic ist / dentis t with expl ici t research interes t

in diagno s tic sys tems .

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2 01 DE 00065-08 CI

I . Obj ec tives

The purpo se of this proj ec t is to integra te theore tical and

prac tical information currently availab le into pro to type dental radio

graphic sys tems which can be op timized for spec if ic diagnos t ic tasks .

Emphas is is direc ted toward improving ef f ic iency by reducing do se and

turn—around time for appl icat ions requir ing les s than maximum infor

mation capacity . Ano ther obj ec tive is the ab il i ty to reproduc ably

al ign the radiographic source and de tec tor relat ive to the tis sues o f

interes t to facili tate longitud inal analys is o f des truc tive les ions .

II . Metho ds

Promis ing x— ray sources and imag e— de tec tor conf igurations are

being developed and evaluated in col labora tion with o ther Governmentagenc ies and pr ivate interes ts . The id ea is to more ra tional ly relate

sys tem elements to the diagnos t ic task to be accomplished .

Previous ef for t s included cons ideration of seal ed sources

produced from 1 25 1 to provide x rays from ins ide the mouth as or iginallydescr ib ed by Henrikson . A ser ies of in vitro experiment s with sources

of this type were under taken with model sys tems to de termine the ef f ec t

o f source s ize , intens ity , and proj ec tion geometry on images o f clinicalinteres t . Sp ec tral charac ter is t ics were analyzed by computer simulationto determine impac t on s ignal— to-noise ratio and exposure modulat ion .

Ano ther inves t igation involves analys is of various film/ s c re en

comb inations which have been sensime tric ally class if ied in terms o f

number of nois e-equivalent quanta required to produce an image of

comparable qual i ty . Thes e de tec tor s are being used to record in vitro

images from dental phantoms undergo ing endodontic ins trumentation in

an at temp t to determine the minimum rad iographic information capac ity

required to rel iably perform such procedures in a rout ine fashion .

Various exper ts including technically knowledgeable med icalphys ic is t s , radio—nucl id e eng ineers , and transducer technologis ts have

been consul ted as a prelude to sp ec if ic ef for ts direc ted towardop timization of a prac tical f luoro s cop ic sys tem .

Ef for ts toward geometric op timization have evolved from dis cuss ions

on the app lication o f f eed—back control theory to op timization o f

expo sure geometry which were elicited at a workshop held May l6 , . l9 7 8

at the Univer s ity o f Connec ticut . Effor ts are curr ently underway to

explore the technical f eas ib ili ty of feed—back and / or mul tip le proj ec tion

sys tems via a sui table reques t for an extramural research contrac t or

interagency agreement .

ZO1 DE 00065-08 CI

I I I . Maj or F inding s

Extens ive measurements and computations us ing I as a sealedpo int-sour ce o f x radiation for fluoro scop ic purpo ses have shown that

the f ini te hal f-lif e (56 days ) coup led with the fall-of f in output

as so c ia ted with coll imations suitable to yield a reasonable ef fec tive"fo cal spo t

"pr eclude image-qual i ty suf f icient for all but a few

clinical appl ica tions irrespec tive of the gain of the de tec tor . This

conclus ion al so fol lows from computations b ased on the use of an

extended source having output fo cused by a suitable coll imator . Even

if ac tivit ies of 300 milli cur ies could be rout inely produced in a

sui table packag e f ree from shor t-lived high— energy contaminants,

shield ing and handl ing such an intense source would be problema tical .

Computer s imulations us ing a non-linear at tenuat ion model sugges t

that the sp ec tra ass oc ia ted with 1 251 and 153G d while op timal for

cer tain tasks are no t g enerally well suited to fluoro scop ic appl icat ionseven when an allowance is made for op timal spec tral sens it ivity o f

the detec tor . This is part icularly true for 153Cd which in add it ion

to energy in the op timal range ( 24 45 kev fo r caries detec tion— tasks )al so produces a s ignif icant high— energy peak near 100 kev. More

convent ional (broad— spec trum) sources were shown to be les s sensi t ive

to the normal range of at tenuations encountered in tis sues o f dental

interes t .

As an al ternative modif ied rod-anode sources are being cons idered

which are po tential ly saf er and more fl exib le in this regard . These

devices al so exibit a po tential for miniaturizat ion made po ss ible by

high-sp eed detec tion technology , which minimizes the power—handl ing

requirement o f the source .

Previous work has demons trated that an es t imated reduction in

s ignal-to—no is e power ra tio of 30% does no t s ignif icantly influencethe d etectab il ity o f induced incip ient enamel les ions , and preliminaryf ind ings sugges t that the same will ho ld for o ther diagnos t ic tasks

o f dental interes t,such as the determination o f the height of int er

s ep tal bone relative to the cemento— enamel j unc tion of adj acent tee th .

Hence the use o f fas ter de tec tors permits cons iderable latitude in

automation o f beam-angulat ion via an on— line f eed-back control sys tem

or through the use o f mul t iple expo sures with no increase in do se to

the pat ient relat ive to the s tatus quo .

IV . S ignif icance

Taken en mas s e these f ind ings sugges t that the ul timate des ign of

a prac t ical dental f luoros cope will prob ably no t include an iso top ic

source.

A suitable minia tur ized rod— anode type intra—oral x-ray tube

provid es a promis ing al ternative which is currently being inves tigated

for this appl ication .

LI 3 0 5

ZO1 DE 00065-08 C I

The use of an automated sys tem for reproducing proj ec tion geometry

from'

one x—ray examination to the next would permit relatively smallchanges in tissues to be accurately and conveniently determined . This

is par ticularly impor tant for inter cep t ive treatment o f largely irre

versible , deg enerative proces s es such as caries and periodontal d isease .

V . Propo sed Cour se

Future ef for t s will continue to'

emphasiz e the mul tid is cip l inaryapproach des cr ibed above . Collaborative ef for t s will b e coordinated

in ways which op timize the resour ces conveniently available from the

many sources and agencies having interes ts in this area.

Publ ica tions

Lieberman , J . E . and Webber , R . L . : C linical evaluation o f pro to typ e

intraoral source x-ray sys tem . Oral Surg .,

Oral Med .,Oral Path .

46 : No . 2 , 318 3 27 , 19 78 .

Yin,L . I . , Trombka , J . I., S el t zer , S . M . , Webber , R . L . , Rennie , J

and F air , M . R . : The lixisc ope . SPIE Conf . Pro c . 14 3 : 106 11 3 ,

197 8 .

Webber , R . L . , and Nagel , R . N . : Pro ceed ings from Workshop on Feedback

Contro l of Exposure Geometry in Dental Rad iography . (In Pres s )

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2 01 DE 00158-05 CI

I . Obj ec tives

Charac ter ization of the shape of b iolog ical s truc tures which

grow in predic table ways is dif f icul t us ing conventional techniques

s ince mo s t common mathematical tool s are based on Eucl id ian geometry

which does no t take into account developmental redundancy . Attemp tsa t sys temat ic des cr ip tion are o f ten compl icated by coord inate

dep endent transf ormations which yield dif f erent resul ts depending

on the arb i trary selec tion of f iducial references . A coordinate

independent trans formation or ig inally descr ibed by Blum permits the

shape o f clos ed forms which grow incrementally in time to be e f fic

iently des cr ib ed by means of a central , as opposed to per ipheralpat tern called the symmetric-axis . The resul t is a new geometry which

cap italizes on the redundancy of l inarar growth proces ses and therefore

provides an ef f icient way to des cr ibe shapes which are of interes t in

s tud ies of biolog ical growth and development . The purpose o f this

inves t igat ion is to continue to explore the utility o f this new

geometry in the des cr ip tion o f b iolog ical shape , and to use it as a

tool to s tudy f ac tors cons training growth and development in controlled

b io logical experiments .

II . Methods

Any clo sed continuous plane curve can be expres sed as an

isomorphic trans form which relates all po ints to a unique central

descr ip tion called the symmetr ic—axis . This axis has two components ;

The lo cus o f center points of maximal ly ins cr ibed

disc s , call ed the axis points , and the ordered

collec tion of dis c radii , called the rad ius func tion .

Algor ithms have been implemented which permit a digi tal computer

to trans form a g iven two— dimens ional f igure into i ts unique symmetric

axis representa tion and conversely recons truc t it from its symmetric

axis descr ip tion in a rever s ib le fashion . Addi tional algor ithms have

b een cons truc ted to analyze the symmetr ic axis components and produce

a set o f shape descr ip tors . These descr ip tor s provide a rich

environment based on the symmetric axis which permits quanti tative

analys is o f the b iolog ical shape under s tudy . By compar ing these

shape descr ip tors ob tained from two dimensional proj ec tions of

b io lo gical s truc tures recorded at var ious s tages o f development in

man and homologous spec ies it is poss ib le to ident ify and analyzepro ces s es underlying biolog ical growth and development .

11 1 . Maj or F inding s

An inves tigation of a varie ty o f lateral cephalometric proj ec tions

of human mandibl es including normals abs trac ted from the Broadbent-Bol ton

serie s . from age 1 through adul t,a long itudinal ser ies ob tained from

two dif f er ent patients af f lic ted with severe untreated mandibulo facial

14 - 3 9 8

2 01 DE 00158-05 CI

dyso s to s is (Trea ture— Coll ins syndrome ) , and an isolated example o f

a Polynes ian anomaly known as a rocker— j aw has conclus ively demons trated the pres ence o f an invar iant angle be tween branches of the

symmetric—axis determined by the j unc tion of thr ee axis— segments near

the mand ibular foramen . This f ind ing has now been indep endently con

f irmed by ano ther inves t igator on d iff erent data us ing a dif f erent

symmetr ic-axis algor ithm thus provid ing a reasonably secure bas is forg enerali za tion .

Preliminary f inding s based on a cro ss— s ec tional analys is of

lateral proj ec tions of rat mandibles sugges t that a similar invariance

may al so o ccur in this animal al though the s izes o f the invariant anglesare likely to be dif ferent from tho se seen in humans . O ther preliminary

data derived from a s tudy df homologous radius func tions asso c iated

wi th symmetric— axes near the angle of the mandib le ( gonial region )sugg es t that the curves de termined by the upper and lower boundaries

o f the mand ible in this reg ion are relatively parallel .

New methods for analyzing shap e from symmetr ic—axis descrip tors

have been devised which permit meaning ful subd ivis ion of shape into

component element s which can b e s tud ied ind ividually . As descr ibed

las t year thes e include methods for s tudying d if f erential growth—ra te

within a compo s i te s truc ture . O ther promis ing schemes involve cannon

ical des cr ip tion of deformations caused by encroachment or flexure,

wherein ar ea and per iphery o f the proj ec tion o f interes t are preserved .

IV . S ignif icance

The discovery of a prec isely def ined morpholog ical invariant

proper ty which follows direc tly from symmetr ic—axis descr ip tion of

homologous proj ec tions o f the human mand ib le provides a sol id j us t i

fic ation for future appl ication of this new analyt ical tool . The fac t

tha t this invariant pat tern was dis cover ed on the f ir s t appl ication of

the method to developmental data which can b e meaningfully express ed

as a two— dimens ional proj ec tion sugges ts that o ther invar iant proper ties

are likely to emerg e from comparab le analys is o f o ther b iolog ical

s truc tures charac ter ized by s imilar growth dynamics . C er tainly the fac t

tha t a rela tively invariant angle ex is t s in the j aw impl ies that cer tain

aspec ts of mandibular growth and development can be rel iably predic ted .

This neces sarily reduces the number of pos s ib le al ternatives that an

Or thodontis t mus t cons ider when taking into account the ro le o f man

dibular growth in his trea tment p lan .

V . Propo s ed cour se

Ef for ts will be extended to determine fac tors underlying the

ob served angular invariance with emphas is on experiments des igned to

dis cover cond it ions for which it does no t ho ld . O ther b iolog ical

.Zol DE 00158-05 CI

s tructures wil l be s tudied to determine the extent to which it may apply .

to o ther areas and / or s tructures . To this end a more ef f ic ient algorithmdeveloped by Books te in at The Center for Human Growth and Development

,

The Univer s i ty o f Michigan will be implemented on a mini—computer to

fac ilitate . app1ic ation of this methodology in our laboratory .

Publica tions

Blum , H . and Nagel , R . N . : A Symme tric Axis Bas is for Obj ec t Recognit ionand Des cr ip tion . Pat tern Recognit ion (19 7 9 )

Webber , R . L . , and B lum , H . : Angul ar Invar iants in Develop ing HumanMandibles . Science (In Pres s )

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2 01 DE 00211—03 01

Proj ec t Des cr ip tion

1 . Obj ec tives

This proj ec t is concerned with further s tudy o f fac tors underlyingd iagno s tic per formance ob tainable from images with emphas is on the

ef f ec t of mod e of d isp lay on p erformance by human interpreters . Previous

work. has shown that image proces s ing can improve d iagno s tic performance

ob tainable from dental radiographs for specif ic tasks no t limited by

the inf orma t ion capaci ty of the sy s tem . Thi s work al so demons trated

that mono cular cues to dep th can be meaningfully used to augment per

c ep tibility of complex images o f diagno s tic interes t .

Curr ent obj ec tives ar e based on log ical extensions of these f indings

with emphas is on pe rformance tes t ing o f promis ing enhancement s chemes .

II . Me thods

The enhancement schemes descr ibed las t year are being ref ined and

made available for computer impl ementa ton in ways which fac ili tate formalevaluation of their impac t on diagno s tic performance by human observers .

This involves develop ing methods for automating image transformation

and d isplay as well as es tab lishing research parad igms which facil itate

unb iased acquis i tion and s tatis tical analys is of per fo rmance d ata

obtainab le from human ob server s .

So f t ware has been wr it ten which permit s mul tip le disp lays to be

viewed and manipulated simul taneously on a s ingle video monitor . Other

programs permit wel l— def ined "l es ions to be induced analy tically via

computer s imulation so tha t perf ormance can be based on expl ic it measures

o f diagno s tic ut ility .

One such program permits an ac tual dental rad iograph to be used

to cr ea te a c l inically meaningful at tenuat ion model of the te e th and

related so f t tis sues . The output o f this program is a grey— level

look-up table which permits a radiograph taken under one se t o f cond it ions

(source spec trum,f il tration , detec tor s ens it ivity , detec tor response

character is tic ) to be mapped into the simulated resul t of expo sing

the same tis sues under ano ther set of user-selec ted condit ions with the

s ame expo sure geome try . This program thus pe rmits real is tic s imula tion

of ideal x—ray sys tems as well as providing for enhancement o f tissues

having known x— ray at tenuation charac ter is tic s .

S tatis tical parameter s us ed to evaluate diagno s t ic performance

include measures of s ignal de tec tab ili ty (ROC analysis ) and transin

formation as well as a tally o f diagno st ic error s . A communications

l ink has been es tabl ished which permits CAT scan data to be ac ces sed

dir ec tly on a miniv c omputer so tha t enhancement stud ies can'

be performed

with no lo s s of d iagno s t ic information from the source .

2 01 DE 00211-03 C I

111 . Maj or F inding s

Extens ive analysis of the detec tab il ity of induced dental les ions

us ing the p seudo-so l id enhancement technique sugges t tha t the selec tion

of proper ang le for il lumination relative to the s imulated contour is

cr i tical .-Ev en a relatively small change in angulation can have a

tremendous ef fec t on the s i ze , number and conf iguration o f resul t ingshadows . This po ses f ew concep tual problems if the angle of illuminationis manipulated in an on-line fashion . However

,the extens ive computations

involved pr e c ludes this for a prac tical sys tem at the current s tate o f

the ar t . What appear s to be needed is a cue to dep th which is les ssens i tive to illumination geome try , so that les s searching is required

b efore a meaningful conclusion can be reached .

Theoretical analysi s of fac tor s kn own to influence the amount of

inf ormation available from CAT scans made on se cond— generat ion machines

sugges t s that v isual interpre tab il ity may be enhanced by a suitab le

po s i tion— invariant spa tial-frequency f il ter coup led wi th a context

d ep endent , contras t enhancement technique (his to gram equal ization ) .

Pr el iminary ob servations based on the us e of such a scheme are encourag ing

but requir e conf irmation by s tatis t ical analyse s o f the performance

measures ci ted above .

Application o f the s imulation program to model x— ray sys tems having

a dete c tor charac ter is t ic with a unif orm ( linear ) contras t gain (gamma )charac t er is tic has produced r esul ts which sugges t tha t an ideali zed

d etec tor W i th these charact er is tic s might be us eful in dental radio

graphy . As s imulated , a les ion charac ter ized by a f ixed d iff erence in

a t tenuation would exhib i t the same contras t irre sp ec tive c f the ab so lute

a t tenuation o f the ti s sues involved . Hence , under these condit ions a

compara tively small carious les ion would l ikely be as easy to see in a

molar as in an incisor .

IV . Signif icance

For the mo s t par t it is too early to determine the ul timates ignif icance of this proj ec t s ince mo s t of our ef for ts to date have

b een pr eparatory in nature . Only recently have we ac tually undertaken

p ilo t exp er iment s des igned to measure diagno s tic performance ob tainab lefrom enhanced images . However , the me thodology is well es tab l ished

and has yield ed promis ing f inding s par ticularly with regard to diagnos tic

tasks o f dental inter e s t . The theor etical rationale underly ing this

proj ec t is ba sed on sys tematic ass essment of image pro cess ing within

the context of a compl ete diagno s tic sy s tem . Hence , the number o f

per tinent al ternatives to be exp lored are reduced a pr ior i to a manage

abl e number thus increas ing the likelihood of meaningful resul ts .

V . Propo sed cour se

F uther ac tivity . will emphas ize formal evaluat ion of selec ted

enhancement techniques within the context of sp ec if ic diagno s t ic

tasks.

I t wil l includ e an ef for t to develop a generali zed dif fuse

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reflec tance model to be used in assoc iation with the pseudo-so l id

transformation to facilita te contour percep tion which is les ss ens it ive to illuminant geometry than the proj ec tion o f hard shadows .

Theoretical analyses o f ideal x-ray sys tems will continue with emphas is

on the p ercep tual impact o f ideal detector s .

Publications ;

Webber , R . L . , and Nagel , R . N . ; Image prepro ces sing as an aid to visualinterpreta tion . IADMF R Proceed ings (1978 ) (In pres s )

Webber ,R . L . e t al . z Thr ee—Dimens ional Enhancement o f Two

Webb er,R . L . : On Model ing Dental Rad iographic Syst ems . Proceedings

o f the Bureau o f Rad io logical Heal th Sympo s ium on B io logical Ef fec ts

and Do s imetry o f Ioniz ing Rad iation . (In Pres s )

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201 DE OOO48—O8 CI

I . Obj ect i ves

To prov i de a base of des cri pt i ve anatomy of the human i nfan t and

c h i ld for s tud i e s of normal developmen t and for def i n i t i on of malformat i on s .

II . Methods

Rout i nes of d i s sec t i on and i llus trat i on wh i c h are s tandard i n thef i eld of anatomy , supplemen ted by spec i ali zed rad i ography .

III. Major F i n d i ngs

Publi cat i on of book :'

The Cran i um of the Newborn Human : An Atlas

i n the coronal plane , l7 levels i nthe tran s verse plane and 25 levels i n the sagi t tal plane .

Preparat i on of book : The Head of the Newborn In fan t . T h i s general

manus cri pt , wi t h add i t i onal i llus trat i on s , to total of approx imately240 , and corre spon d i ng enlargemen t of tex t . Its i llus trat i on s are

es sen t i ally completed . Its cri t i que i s s imultaneous ly i n progres s .

IV . S i gn i fi can ce

The mouth , nose , phary nx and larynx of the i n fan t d i ffer from t heseareas i n mature man. Th ese d i fferen ces are i n gros s form and proporti onas well as i n detai led anatomy . The des cri pt i on s afforded by th i s

proj ec t are bas i c to def i n i t i on and un ders tand i ng of pos tnatal developmen t ;Suc h i n format i on i s requi s i te for des cri pt i on of abnormali t i e s of developmen t ,

i n clud i ng t hose ori gi nat i ng i n the peri pheral s truc tures and t hose wh i c h

evolve s econ dari ly i n t he se s truc ture s as effects of cen tral neurologi cald i sease .

Suc h i n format i on i s also pert i nen t to des cri pt i on of a vari ety of

d i seases and d i sorders wh i c h are acqui red i n i n fan cy . As example , the'

evaluat i on of mas ses , i n flammatory or neoplas t i c , mus t be i n pertinenceto the normal anatomy of the adj acen t area i n the i n fan t . The normat i ve ~

rad i ograph s of The Cran i um of the Newborn Human have already proven to

V . Propos ed Cours e

Publi cat i on Prospec tus of a book , The Head of the Human In fan t , has

been submi t ted to a publi s her , Spri nger-Verlag ,

Publi cat i on s :

P i erce , R. H. , Hai nen , M.w. and Bosma , J . E .

The Cran i um of the Newborn In fan t : An Atlas of Tomography

q - a oe

N AM E S , LA BOR ATO RY AN D I N ST I TUT E AFF I L I AT I ON S , AND T I TL ES OF PR I NC I PAL I NV E ST I G ATOR S AN D ALL OTH ERPR OF E S S I ON AL PER S ON N EL ENG AG ED ON THE PR O JECT

Weiffenbach, Jame s M . Re searc h Psyc hologi s t CI NIDRCOPI : NoneOTHER : Cowart , Beverly Psyc holog i s t

ri c s , Nat i onal Naval Med i cal Cen ter , Bet he s da , MD

[ 1 (c ) N E I TH E R

S UMM AR Y OF W OR K (200 wo rd s or les s un d e rl in e keyword s )

The d i s crete eli c i ted lateral tongue movemen t of the human newbornf i rs t des cri bed un der th i s proj e c t con t i nues to be used i n psy c hophy s i cals tud i e s of oral sen sat i on . The tran s verse tongue reflex des cri bed

prev i ous ly can be eli c i ted by separate 5 mi croli ter drops of flui d .

The reflex adapts to repeated flui d el i c i tat i on s . In earli er s tud i es ,

the re spon se returned when glucose i n suprat hre s hold con cen trat i on was

added to the s t imulus flui d after adaptat i on to water . T h i s experimen talsequen ce t hus i n d i cate s tas te d i s crimi nat i on . Current s tud i es addres sthe ques t i on of sen s i t i v i ty to the salty tas te and i ts relat i on to

se n s i t i v i ty to sweet .

LI— A O 7

ZOl DE OOl24-O6 CI B

Proj ec t De s cri pt i on

I . Object i ves

T h i s proj ect explores the oral sen sory competen ce of the newbornhuman i n fan t. A novel reflex tec h n i que has been. developed as part of

th i s proj ect . It i s equally well sui ted to measuri ng the local s en s i t i v i tyof the tongue to touc h and to tas te s t imulat i on . In the begi n n i ng ,

proj ec t i n ve s t i gat i on s were primari ly con cerned wi t h developmen talc hange s i n touc h sen s i t i v i ty . Progre s s i vely greater emphas i s i s now

be i ng placed on s tud i e s of the sen sory mec han i sms underly i ng neonataltas te experi en ce . S i n ce i n fan ts are sen s i t i ve to tas te at b i rth i t i s

pos s i ble , as has been con ten ded on the bas i s of an imal s tud i es , t hatearly tas te experi en ce s hape s later food preferences and d i etary hab i t s .

II. Methods

An eas i ly observed reflex d i splacemen t of the tip of the newborn's

tongue toward the s i de of s t imulat i on serves as the bas i s for as se s s i ngthe sen s i t i v i ty of the tongue to bot h touc h and tas te . For touc hs t imulat i on , the frequen ce of re spon se oc currence i s a fun c t ion of

s t imulus i n ten s i ty . An analogous relat i on of s trengt h of tas te s t imulat i on

(con centrat i on of tas tant i n solut i on ) to frequency of respon se i sobtai ned prov i d i ng t hat re spon d i ng to the solven t alone has been f i rs treduced by repeated s t imulat i on . Postadaptation i ncremen ts i n respond i ngabove that foun d wi t h con t i nued water s t imulat i on have been demon s tratedfor both glucose and sod i um c hlori de te s t solut i on s .

III. Maj or F i n d i ngs

A procedure i n wh i c h eac h s ubj ec t i s exposed .to bot h control and

te s t con d i t i on s fai led to produce the expected results . Re spon se to

sali ne af ter sucrose adaptat i on y i elded on ly margi nally h i gher re spond i ngthan t hat eli c i t i ng by con t i nued s ucrose s t imulat i on . Sucrose tes t safter sali ne adaptat i on eli c i ted re spon ses s imi lar to con t i nued sali nes t imulat i on . T he se results appear to reflect i nterferen ce of the f i rs ttri al wi t h the secon d s i n ce the f i rs t tri al data s how the expec tedbetween subj ec ts effec t (sugar after salt salt after salt and saltafter sugar sugar after sugar) . Earl i er s tud i es i n wh i c h subj ect srece i ved the same sequen ce of adapt i ng and tes t flui d s on tri als one and

two fai led to s how more than mi n imal i n terferen ce .

IV . S i gn i f i can ce

Stud i es of neonatal tas te i n the human , and of the normal developmen tof tas te i n i n fan cy and c h i ld hood wi ll make an importan t con tri but i on to

un ders tand i ng the developmen t of t hose de s truc t i ve food i n take pattern sthat have been impli cated i n the et i ology of cari e s . In ve s t i gat i on s of

the impact of normal"vari at i on i n the pattern of early tas te experi en ce

on later food preferen ce s s hould lead to the des i gn of effec t i ve i n terven t i ons trategi es .

u- uoa

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SUMM AR Y OF WOR K (200 word s or le s s un d e rlin e keywo rd s )

A PHS Gran t Appli cat i on has been submi tted to the Nat i onal Li braryof Med i c i ne for subven t i on of publi cat i on of the book , Pos tnatalDevelopmen t of the Rat S kull .

In Part l of the book , the cran i um and man d i ble of the rat are demon s tratedby drawi ngs and photograph s at suc ces s i ve pos t natal ages . In Part 2 ,developmen t of the cran i um and man d i ble are demons trated by ali zari nv i tal s tai n i ng and photograph s of sect i on s . T h i s comb i nat i on of met hod safford s compre hensi ve demon s trat i on of the development of i n d i v i dualbones , of s keletal compos i tes and of the s kull as a whole .

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Projec t De s cri pt i on

I . Obj ec t i ves

Normat i ve demon s trat i on of pos tnatal development of the i nd i v i dualbones and teet h and of the cran i al compos i te and man d i ble i n the rat .

II . Met hods

Of Part l . Rat s kulls of day s 1 , l5 and 60 were d i s sec ted after

papainiz ation. The i n d i v i dual bone s were t hen photographed and drawn .

Of Part 2 . Rat s at 8 pos tnatal ages were i nj ec ted alternately wi t hAl i zari n Red S or Al i zari n Blue BB and sacri f i ced at i n tervals afterlas t i nj ec t i on . Defleshed cran i a and man d i bles were moun ted i n Bi oplas tand sawn i n sect i on s . Pattern s of development i n separate areas of eac hbone and toot h and of de velopmen t of compos i te s of related bones and

teet h were worked out by compari son s tudy of s pec imen s s tai ned on vari ouscalenders .

III . Maj or F i n d i ngs

Normat i ve i llus trat i on s are prepared i n the cran i um and i t s bonesand of the man d i ble . T hese con s t i tute the bas i s of a textual de s cri pt i onof the pos t natal developmen t of the rat s kull on an i n d i v i dual bone and

a regi onal bas i s .

The pattern s of appos i t i on and resorpt i on at the margi n and i n the

i n teri or of the vari ous port i on s of i n d i v i dual bones and teet h have beendefi ned and furt her i llus trated by s c hemat i c s . The s tudy materi el of

Part 2 as prepared for publi cat i on , comb i nes color photograph s ( i n l20

proj ec t i on s l i de s ) wi t h trac i ngs of the correspon d i ng anatomi caltranssections . Eac h of t hese atlas un i t s i n cludes a proj ect i on s li de ,

deri ved i llus trat i on s and textual commen tary .

T h i s volume also i n clude s a rev i ew of v i tal mark i ng of s keleton byali zari n and other s tai n s wh i c h are c hemi cally i ncorporated i n to os s i fy i ngt i s sue s .

IV . S i gn i f i cance

T h i s i s the f i rs t comprehen s i ve de s cri pt i on of s kull developmen t of

a laboratory mammal i n t h i s ex ten t of age range , i n t h i s i n clus i on of

en t i re s kull and all teeth , and i n t h i s exten t of detai l . The

de s cri pt i on s and as soc i ated i n terpretat i on s are s i gn i f i can t contri but i on sto i n format i on and unders tan d i ng of normal mammali an developmen t . And

they are of normat i ve value i n def i n i t i on of vari at i on s of rat s kull and

toot h developmen t .

ZOI DE OOl8l-03 CI

V . Proposed Course

A PHS Gran t Appli cat i on , I-ROl LM03464-0l , has been submi tted to

the Nat i onal Li brary of Me d i c i ne for subven t i on of publi cat i on and salesd i s tri but i on of t h i s book by the Un i vers i ty of M i c h i gan Pres s .

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ZOI DE 00182-04 CI

1 . Obj ec t i ves

The s tudy of c li n i cal vari at i on s wh i c h are as soc i ated wi t hmalformations. of i n flammatory d i sease , or of surgi cal mod i f i cat i on sof oral and phary ngeal s truc ture and form, or wi t h peri pheral sen sory or

motor impai rmen ts , or wi th abnormal i t i e s of the repre sen tat i on s of the

oral and phary ngeal performan ces i n the brai n .

T he se s tud i es are des i gned to defi ne anatomi cal abnormali t i e s and

t he i r phy s i ologi cal correlates . And, for the purpose of dev i s i ng and

apply i ng t herapi es for d i sab i li t i e s of feed i ng and of speec h and ot herre spi ratory func t i on s .

II . Met hods

Standard procedures of cinephotography and c i nerad i ography , as well

as acous t i cal and other phy s i ologi cal record i ngs are employed . The

regi onal performan ce s of feed i ng , speec h and ot her re spi ratory act i on sare de s cri bed i n i n fan ts , c h i ldren and adults . Selected pat i en t s hav i ngsen sory d i sorders of the oral and phary ngeal regi on s are also evaluatedby sen sory tes t i ng . Pat i en t s who have surgi cal exc i s i on for can cer are

s tud i ed as example s of the mod i f i cat i on of s en sory i nput by ablat i on .

III. Major F i n d i ngs

The d i sorders of feed i ng and of upper respi ratory performance s fromimpai rme nt of peri pheral motor performan ce c an be mean i ngfully des cri bedby currently avai lable cli n i cal met hod s . It i s pos s i ble to pred i ctcertai n d i sab i li t i es of progre s s i ve motor d i sorders . In some c i rcums tan ces ,

the impai rme nts are ac ces s i ble to prevent i on or to s pec i f i c therapy .

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IV . S i gn i f i can ce

Adaptat i on s of selected d i agnos t i c met hod s , i n clud i ng sen soryevaluat i on , have en han ced the defi n i t i on of sen sorimotor d i sorders of

the mout h , phary nx and lary n x . T h i s has con tri buted to the recogn i t i onof related elemen t s of separate d i sorders , to an t i c i pat i on of someimpai rmen t s in progres s i ve d i sease s , and to t herapy of d i sorders wh i c hare s hown to have common pat hogen i c mec han i sms .

V . Proposed Course

Cli n i cal s tud i es of subj ec ts hav i ng the above categori es of d i sorderswi ll con t i nue wi t h curren t met hod s .

Our rad i ograph i c s tud i e s of sen sorimotor phary ngeal impai rmen t s are

curren tly un der ac cumulat i on i n to a ge neral volume , Rad i ography p j_ the_

Pharynx , under aut hors h i p of Jame s F . Bosma and Mart i n Don ner , of the

Departmen t of Rad i ology , the Joh n s Hopk i n s Med i cal Cen ter . Publi cat i onwi ll be by Spri nger—Verlag .

Publi cat i on s :

Bosma , J . F . , Phy s i ology of the Mout hChapter i n pre s s . Otolaryngology, Ed i t i on 2 . Eds . Paparella ,

M . M . and Shumric k, D .A . , Ph i ladelph i a , Saun ders Co .

Bosma , J . F . and Don ner , M. , Phy s i ology of the Phary nx , i b i d .

N AM ES,L A BOR ATORY AN D I N ST I TUT E AF F I L I AT I ON S , ANO T I T L E S OF PR I NC I PAL INV EST I G ATOR S AND ALL OTHER

PR OF ES S I ON AL PER S ON N EL EN G AG ED ON THE PR O JECT

Wolf , Robert 0,

Dent i s tWeiffenbach, James M Psyc hologi s tBaum, Bruce J . Den t i s t

OTHER : Cowart , Beverly J . Psy c holog i s tElli s , Charles Studen t trai neeBrown , V i v i an Studen t trai nee

S UMM AR Y OF WO R K (200 wo rd s or le s s und e rl in e keyword s )

The select i on and ref i nemen t of appropri atethe separate measuremen t of the vari ous aspec ts of

primary and con t i nui ng con cern of t h i s proj ec t . Norma vari at i on ntas te percept i on wi t h c hronologi cal age i s be i ng as ses sed by procedureswh i c h quan t i fy the i n ten s i ty and pleasantnes s of the subj ec t

's tas te

experi en ce . Parallel s tud i e s are con cerned wi t h anomalous tas te experi en ce

as a naturally occurri ng phenomenon or as a con sequen ce of experimen talman i pulat i on of d i et . Ot her s tud i e s are d i rec ted s pec i f i cally at tas te

percept i on d i fferen ce between i n d i v i duals wi t h and wi t hout cari es .

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ZOl-002l2-03 CI

V . Proposed Course

Age related c hanges i n the relat i on of suprat hre s hold s t imulusconcen trat i on to the i n ten s i ty and pleasan t ne s s of tas te experi en ce are

be i ng s tud i ed cros s-sect i onally . Ki ndergarten age c h i ldren have alreadybeen tes ted . Add i t i onal groups of elementary and h i gh s c hool age subj ec tsand two adult samples wi ll be te s ted . In a separate , but fully compat i bleeffort , we wi ll collaborate wi t h NIA i n ves t i gators at the Geron tologyRe searc h Cen ter i n Balt imore on longi tud i nal s tud i es of tas te and agi ng .

Stud i es of the relat i on of tas te to cari es s tatus wi ll con t i nue , as

wi ll ot her s tud i es e valuat i ng the reli ab i li ty and eff i c i en cy of cli n i calas ses smen t procedure s . A greater number of pat i ent s wi t h tas teabnormali t i e s will be accepted for e valuat i on .

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Part B The results of the t hree compared pan creat i c func t i ontes t s wi ll d i c tate the con t i nuance of th i s amylase s tudy .

Part C Stud i e s of the mec han i sms of fluori de-i n duced sali vat ionare plan ned . Pert i nen t b i ologi cal mec han i sms to evaluate xeros tomi atreatmen t are under con s i derat i on .

Publ i cat i on s

Jam, I Shoham, M. , Wolf , R. O . and M i s h k i n , S . : Elevated serum amylaseac tiv i ty i n the absence of cli n i cal pan creat i c or sali vary gland d i sease .

Amer . J . Gastroenterol .-488 , 1978 .

Hen k i n , R. I . , Li ppoldt , R. E . , Bi ls tad , J . , Lum, Wolf , R. 0 . and

Edelhock, H. : Frac t i onat i on of human parot i d sali va . J . Bi ol . Chem.

( i n pres s )

Moutsopolus , H.M Kli ppel , J . H. , Parlidis , N , Wolf , R.D . , Sweet , J . B. ,

Chu, F . C . , Stern berg , A. O Tarpley , T .M. : Correlat i ve h i s tologi c and

serologi c f i n d i ngs of s i cca sy n drome i n pat i en ts wi t h sy s temi c lupus and

erythematos i s . Art hri t i s Rheum. ( i n Pre s s )

Wolf , R. O . and Taus s i g , L . M. : Letter to the ed i tor: Serum Sali varyAmylase i n Cy s t i c F i bros i s . Pediat . Res . lO76 , l979

S UMM AR Y OF WOR K (2 00 word s or les s und e rlin e keywo rd s )

T h i s proj ec t i s concerned wi t h mec han i sms of product i on and con trol ofex tri n s i c (i. e . sali va) and i n tri n s i c (e . g . serum sali vary isoamylase )sali vary gland products .

PHS-60 40

A . Human (primari ly rotid) sali va c hemi cal consitiuents andmec han i sms are evalua elated to health and d i sease . Parot i dsali vary flow rate , rotein compos i t i on and enzyme s (part i cularlyly sozyme and amylase are evaluated i n normals and selected d i sorderssuc h as Sj ogren

's

'

syndrome and apht hous s tomat i t i s .

B. The i n tri n s i c secret i on of sali vary isoamylase i n serum of c sticf i bros i s g j

_ tpe_ pancreas and Sj ogren's sy ndrome as well as no

mon key serum and mon key gland homogenates are be i ng s tud i ed . Diagnositcappli cat i on and an un ders tand i ng of the mec han i sms of hyperamylasemi aare be i ng sought .

C . Fluoride i s be i ng s tud i ed as a s i alogogue for preven t i on andallev iation of xerostomic symptoms .

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II. Methods

Part A F i ve con secut i ve two mi nute parot i d sali va sample s are

be i ng collec ted at leas t weekly from AS pat i en ts t hrough at leas t one

complete cycle of ulcers and no ulcers . Ly sozyme i s as se s sed by rad i ald i ffus i on i n agar gel con tai n i ng mi crococ cus lutens subs trate .

Parot i d sali va from Sj ogren's sy n drome pat i en ts i s as ses sed for ly sozyme

le vels i n compari son wi t h a con trol group .

Parot i d sali va samples from pat i ent s wi t h un i lateral parot i d tumorswere quant i tat i vely and qual i tat i vely as se s sed for amy lase and isoamylases

respec t i vely . Parot i d sali va from the non-tumor s i de was the wi t h i nsubj ec t con trol .

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Part B Serum and uri ne pan creat i c and sali vary amy lases weremeasured by separat i ng the i soenzyme s by PAGE and t hen determi n i ng theen zyme act i vity i n seri al s li ces of the gel . Vari ous cli n i cal populat i on sare be i ng as se s sed .

Part C Admi n i s trat i on of a course of fluori de mout h ri n se or

placebo was as se s sed as a s i alogogue i n pat i en ts wi t h Sj ogren's sy n drome ,

Hodgk i n s d i sease treated by rad i at i on , and ot her t herapeut i c head andnec k i rrad i at i on .

111. Maj or F i n d i ngs

Part A A s tat i s t i cally s i gn i f i can t lower lysozyme level wasreported for AS pat i en ts duri ng the ulcerat i ve s tage of the i r d i sease as

compared wi t h t he i r non-ulcerat i ve s tage .

Elevated parot i d sali vary ly sozyme was found i n l7 pat i en ts wi t h

primary Sj ogren's sy ndrome as d i s t i ngui s hed from t hose wi t h Sj ogren

'ssy n drome secon dary to hyperli poprote i nemi a and wi t h normal con trolsubj ects . No d i fference was foun d i n the total parot i d sali vary prote i namong the t hree groups .

Part B

No s tat i s t i cally s i gn i f i can t d i fference could be foun d between the

total parot i d sali vary amylase i n In ternat i onal Un i t s per li ter nor

c hange i n isoamy lase pattern i n the 22 cases of s tudy . In add i t i ont here was no con s i s ten t pattern of i ncreased sali vary or pan creat i cfrac t i on of amylase from t he se pat i ent s .

Part C The fluori de mout h ri n se was s hown to be a s i alogogue . The

total sali vary flow was s i gn i f i can tly i n creased i n the 6 person groupsof pat i en ts hav i ng xeros tomi a i n as soc i at i on wi t h Sj ogren

's sy ndrome ,

Hodgk i n s rad i at i on and ot her t herapeut i c head and nec k i rrad i at i on s .

IV . S i gn i f i can ce

Part Ay

The et i ology of AS i s un known . The decrease of ly sozymei n parot i d sali va duri ng ulcerat i on i n AS pat i en ts i s a f i nd i ng wi t hout

appropri ate i n terpretat i on . The source of t h i s c hange i s remote from

the ulcer s i te . Herpe s lab i ali s of v i ral et i ology i s reported to alsohave decreased ly sozyme duri ng ulcerat i on s . The glan dular source of

ly sozyme i s t hough t to be the ductal cells ; however , macrophages and

monocy tes are also k nown to con s tan tly produce ly sozyme . The cy toplasmof granulocy tes also con tai n s ly sozyme . The explanat i on for the decrease

of ly sozyme wi t h ulcerat i on mi gh t well be i n the dy nami cs of t hese

populat i on s of i n flammat i on part i c i pat i ng cells and i n permeab i li t i es of

the glan d cell membranes .

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In primary Sj ogren's sy n drome the levels of parot i d sali vary ly sozyme

are h i gher t han i n normal subj ects or i n t hose pat i ent s who have Sj ogren's

sy ndrome secon dary to hyperli poprote i nemi a . We pos tulate t hat thesource of ly sozyme i s from the parot i d i n flammatory i n f i ltrate and t hatthe as ses smen t of parot i d sali vary ly sozyme may be a d i agnos t i c adjun c tfor Sj ogren

's sy n drome .

The oral phy s i ologi cal data base of the exten s i ve longi tudi nals tud i es of the NAI normat i ve populat i on at Balt imore i s i n creased by the

i nclus i on of the sali vary amylase parameters .

Parot i d tumors can not be d i fferen t i ated by total amylase values i nthe serum or parot i d sali va , by the isoamylase pat tern s of the serum, bythe serum sali vary pan creat i c rat i o , nor by the parot i d sali vary isoamylase

pattern s compari ng tumor s i de wi t h non-tumor s i de .

Part B The d i agnos t i c value of the serum sali vary pan creat i c rat i oobtai ned by PAGE i n cy s t i c f i bros i s of the pan creas i s be i ng te s ted . A

blood te s t of t h i s nature would be c li n i cally useful because of d i ff i cultyi n obtai n i ng duodenal flui d by i ntubat i on for pan creat i c enzyme te s t i ngi s d i ff i cult i n some i n fan ts and small c h i ldren . A nEw and second tes tut i li z i ng a te s t compoun d of tyros i ne bound to PABA i s gi ven orally .

Normal pan creat i c metaboli sm spli t s the PABA tyosine ban d and PABA i sfoun d i n the uri ne . No PABA i s found i n the uri ne of pat i ents wi t hi nadequate pan creat i c fun c t i on . The PAGE s erum sal i vary pancreat i c rat i oi s the t h i rd te s t i s an N IAMO s tudy to determi ne the appropri atenes s of

the three te s t s .

Part C Fluori de admi n i s tered as a mout h ri n se i n crease s the flowrate i n person s xerostomic from Sj ogren

's sy ndrome , t herapeut i c head and

nec k i rrad i at i on or Hodgk i n's d i sease . The fluori de ri n se eli c i t s a

tran s i en t flow-las t i ng as long as 30 mi nutes after s t imulat i on . T h i sun i que silalogogue wi ll help allev i ate the xerostomic symptoms wh i c h are

a un i versal complai n t among t he se pat i en t s . The result i ng i n crease i nsali vat i on s hould d imi n i s h the cari e s li ab i li ty as soc i ated wi t h -rad i at i ont herapy as well as by d i rec t ac t i on of the fluori de on the teet h .

V . Proposed Course

Part A The temporal dynami c s of ly sozyme i n AS parot i d sali va are

un der s tudy . The dy nami c s of blood ly sozyme wi ll next be s tud i ed as

wi ll the et i ology (gene produc t s or complexe s ) of the i soen zymes of

ly sozyme s prev i ous ly reported .

Ly sozyme i n the parot i d s ali va of primary Sj ogren's sy ndrome pat i en ts

wi ll also be s tud i ed in relat i on to blood parameters and pos s i blei soenzymes .

The results of parot i d amylase analy s i s wi ll be con t i nued , i fmean i ngful correlat i on s are foun d i n the NAI normat i ve agi ng populat i on .

We s hall not con t i nue s tudy of sali va from parot i d tumors .