Transforming growth factor beta 1 suppresses acute and chronic arthritis in experimental animals

Transforming growth factor beta 1 suppresses

acute and chronic arthritis in experimental

animals.

M E Brandes, … , Y Ogawa, S M Wahl

J Clin Invest.

1991;

87(3)

:1108-1113.

https://doi.org/10.1172/JCI115073

.

Systemic administration of the cytokine, TGF beta 1, profoundly antagonized the

development of polyarthritis in susceptible rats. TGF beta 1 administration (1 or 5

micrograms/animal), initiated one day before an arthritogenic dose of streptococcal cell wall

(SCW) fragments, virtually eliminated the joint swelling and distortion typically observed

during both the acute phase (articular index, AI = 2.5 vs. 11; P less than 0.025) and the

chronic phase (AI = 0 vs. 12.5) of the disease. Moreover, TGF beta 1 suppressed the

evolution of arthritis even when administration was begun after the acute phase of the

disease. Histopathological examination of the joint revealed the systemic TGF beta 1

treatment greatly reduced inflammatory cell infiltration, pannus formation, and joint erosion.

Consistent with the inhibition of inflammatory cell recruitment into the synovium, TGF beta 1

reversed the leukocytosis associated with the chronic phase of the arthritis. Control animals

subjected to the same TGF beta 1 dosing regimen displayed no discernable

immunosuppressive or toxic effects even after 4 wk of treatment. These observations not

only provide insight into the immunoregulatory effects of TGF beta, but also implicate this

cytokine as a potentially important therapeutic agent.

Research Article

Rapid Publication

Transforming Growth Factor /1 Suppresses Acute

and

Chronic Arthritis

in

Experimental Animals

MaryE.Brandes,* JaniceB.Allen,*Yasushi Ogawa,t and SharonM.Wahl*

*Cellular Immunology Section, Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland20892;andtCeltrixLaboratories, Collagen Corporation, Palo Alto, California 94303

Abstract

Systemic administration of thecytokine,

TGF,f1,

profoundly

antagonizedthe development ofpolyarthritisinsusceptible

rats.

_TGF,81

administration (1 or5

,g/animal),

initiated one day before an arthritogenic dose ofstreptococcal cell wall

(SCW) fragments, virtually eliminated thejoint swelling and

distortion typicallyobservedduringboth the acutephase

(artic-ularindex,AI = 2.5 vs. 11; P<0.025)and thechronicphase

(Al=0 vs. 12.5) of the disease. Moreover,

TGF,61

suppressed theevolution ofarthritiseven whenadministrationwasbegun

afterthe acutephase ofthedisease. Histopathological

examina-tion of thejointrevealed thesystemic

TGF,81

treatmentgreatly reduced inflammatorycellinfiltration, pannus formation, and

jointerosion. Consistent with the inhibition ofinflammatory

cellrecruitment intothesynovium,

TGF,81

reversedthe

leuko-cytosis associatedwiththe chronic phase of the arthritis. Con-trolanimalssubjectedtothe same

TGF,&1

dosingregimen dis-played no discernable immunosuppressive or toxiceffectseven

after4 wkoftreatment. Theseobservations notonly provide insight intothe immunoregulatory effects of

TGF#,

but also

implicate thiscytokine as apotentially important therapeutic agent.(J. Clin. Invest. 1991. 87:1108-1113.) Keywords:

cyto-kine * inflammation - immunosuppression *

leukocytosis.

leukocyte

Introduction

The potentimmunosuppressive effects ofthe

cytokine,

TGF#,

suggestthatit may be valuable in thetreatment ofdiseasestates

characterized by aberrant function of the immune system.

TGF,B hasbeen shown invitroto inhibit

proliferation

of

thy-mocytes(1),TandBlymphocytes

(2-5),

andthe less

differen-tiatedhematopoietic progenitorcells(6-9).In

addition,

it sup-presses B cell production ofIgGandIgM(2)andantagonizes theimmunoregulatory effects of interleukins- 1, -2,and-3 (2-4,

10), colony stimulating factors(8, 10), andinterferons alpha

(11)and gamma(12). Although TGF3has been shown invitro to havea

myriad

of

immunosuppressive

effects,

its

ability

to

Address

correspondence

andreprint requeststoDr. M. E. Brandes,

Building 30,Room329,NIDR, National Institutes ofHealth,

Be-thesda,MD20892.

Receivedfor publication27September1990andinrevisedform27

November 1990.

block immune responses in vivo, in particular, chronic inflam-matorylesions, has not been evaluated. Since many of the im-munecell functions influencedby TGF, are involved in the sequence of eventsleadingtoconnective tissuedestruction in arthritic lesions, the ability of TGF,Btoinhibitthesepathways maybe effective in suppressing the pathogenesis ofthis chronic inflammatory disease.

This study examines the effectiveness of systemic adminis-tration of TGF,3l in altering the progression of an inducible arthritis in experimental animals.Asingle injection of strepto-coccal cell wall

(SCW)'

fragments into susceptible rats induces anacuteinflammation ofthejoints, followed by the develop-mentof chronic proliferative and erosive disease (13, 14). The chronic arthritic condition has been identified as aTcell and monocyte-mediated immune response (15) and thus could po-tentially be modulated byanimmunosuppressive agent suchas

TGF,B.The resultsof this study show thatdailyadministration of TGFj3 1 sharply curtails the evolution of both the acute and chronic phases of the disease.Furthermore,the same

_TGF#

1 dosingregimen did not have noticeable toxic or suppressive effects on normal animals.

Methods

Reagents.TGF,B1 was purified to homogeneity from bovine bone (16, 17) andby SDS-PAGEmigrated as a single band.

_TGF#

l was dissolved in 12mM HCI, 20% ethanol (2.5 ,ugTGFlI/Ml,stock) andstored at -70°C inaliquots.Ratserumalbumin (RSA;Sigma Chemical Co., St. Louis, MO)wasdissolved in 8Murea, 10%acetic acid, pH 3.5 (10 mg/ml), and filtered(0.45 um). Thealbumin was eluted from a C18 reverse-phase HPLC column (2.2x25 cm; Vydac, Hesperia, CA)with

alinearacetonitrilegradient(22.5to54%[vol/vol]) in0.1%(vol/vol)

trifluoroacetic acid, aprocedure thatprevious studies had shown to

cause a99%reduction in the endotoxin levels as determined by

Limu-lus amebocytelysateassay(18).The RSA waslyophilizedandstored

at40C.

Arthritis induction and

TGFP3

administration. Specific pathogen-free Lewisfemalerats(- 100 g) (HarlanSprague Dawley, Inc., Indian-apolis, IN)wereinjected withpeptidoglycan-polysaccharide fragments (30Agrhamnose/g body mass) derived from groupASCWtoinduce

anerosivepolyarthritisaspreviously described(13, 14).Thearthritic responsewasquantifiedbydeterminingthearticular index(Al).Each ofthe four distaljointswasscoredblindedon ascaleof0-4onthebasis of swelling, redness,anddegree ofdeformityofnormal contours. The

individualscores were summed toarriveatthewholeanimal score,a

possiblemaximumof 16. Jointscores(Al)wereaveragedfor each

The Journal of ClinicalInvestigation,Inc.

Volume87,March 1991, 1108-1113

1.Abbreviations usedinthis paper:Al,articularindex;RSA,rat serum

albumin; SCW, streptococcalcellwall.

groupof animals and reportedasaverage±SEM, unlessotherwise indi-cated.StatisticalsignificancewasascertainedusingtheStudent's ttest.

Inindividualexperiments,groups consisted oftwo tofive animals.

TGFP

I wasintraperitoneally injected daily forintervalsspecified foreachexperiment, upto 32 d. The

TGF#

I stockwasdiluted ina

vehicle of RSA inPBS (I mg/ml) to 0.1-5.0

;g

TGF*1/2 mlvehicle immediately before intraperitoneal administration. Control animals received an equal volume (2 ml) of either the vehicleorPBS. The vehicle wasfoundtocontain . 25 pg/ml endotoxin (limit of detec-tion) (18).

Lightmicroscopy.Atdefined intervalsduringtheevolutionof the arthritic response, jointtissues from control and arthritic rats were

excised and fixed inasolution of 10% bufferedformalin,embedded in

paraffin, sectioned(8 Mm), andstained withhematoxylin and eosin. Tissue to be used forantibodystainingtoidentifySCWdepositswas

fixed andsectioned inasimilar manner, then treatedasbefore (19). Cell isolation, culture, andanalysis. Atselected intervals,blood smears,hematocrits,and total white cellcounts(ZBI CoulterCounter;

CoulterElectronics,Inc.,Hialeah,FL)wereobtained for each animal. Atthetime of tissue harvest, PBMCswereisolated fromheparinized

bloodbydensity gradientcentrifugation through Histopaque 1083

(SigmaChemicalCo.). Spleencellswereobtainedasdescribed(5),

sus-pendedin DME(Mediatech, Inc.,Washington, DC)containing50,g/

mlgentamicinsulfate,2mMglutamine,0.5 uMfB-mercaptoethanol,

and 1%ratserum, andplatedin 96-wellflat-bottomedplates(Costar Corp., Cambridge, MA) (4x

10'/200

Ml perwell).Proliferation was assessed in the presence orabsence of stimuli: ConA (Calbiochem-BehringCorp.,SanDiego, CA)andPHA(BurroughsWellcomeCo., Greenville,NC),aspreviouslydescribed(5,15).After 68 h ofculture,

the cellswerepulsed for4 hwith 0.5 MCi/well of[3H]thymidine

([3H]TdR,

spact 6.7 Ci/mmol) (Schwarz/Mann, Orangeburg, NY).

Thecultureswereharvested usinganautomated harvester(Skatron,

Inc.,Sterling, VA) and theamountofincorporatedradioactivitywas determined inaliquidscintillationcounter(1205Beta-plate;

Pharma-cia,Inc.,Gaithersburg, MD).

16

x

Lii

0

z

0

C.)

,_-:

12

8

4

0

Results

Suppression

ofacute

and chronic arthritis by

TGFf1I.

To assess

theeffects of

_TGF(#

asatherapeuticagentforarthritis,TGF( I wasadministered daily to Lewis rats at doses of0.1, 1.0, and 5.0

jg/I00

grat,

beginning

1 dbeforethe

injection

of SCW which initiatedthearthritis. SCW-treatedanimals,which did not re-ceive

_TGF#

1, displayed the acute and chronic joint swelling anddeformity which is typical of SCW-induced arthritis (Fig. 1). However, when 5Mgof

TGF#

1/ratwas administered daily during the evolution of the arthritic response, the animals

dis-playeda very bluntedacute inflammatory phase (Al=2.5±1.5

vs. 11±0.9;P < 0.025;day 5) and virtually no chronic inflam-matory phase (AI=0vs.

12.5+0.5;

day21). The striking

dimi-nution ofthe acuteand chronic components of the evolving

SCW-induced polyarthritis was also noted in those animals thatreceivedonly 1 ,ug

_TGF#

1 daily.Theseanimalsdisplayed

minimaljointinflammation duringtheacute phase of the

ar-thritis (Al = 1.8±1.2; P <0.005)and

during

the chronic phase aswell(Al=

1.81.0;

P<0.005).Even adose as low as0.1 g

TGF#

1/animal daily resulted in a decrease in the severity of

thejoint inflammation during the acute and chronic phases and adelayin the onsetofthechronicinflammation (Fig. 1).

Controlanimalsnotreceiving SCW,butdosed intraperitone-ally

daily

with

TGF#1

(5ug/animal), vehicle (1 mgRSA/ml PBS),orPBShad nosynovialpathology.

Suppression

ofestablished

arthritis

by

TGFf3I. Because of

the profound effect of TGF Il on the evolution of arthritic lesionswhenadministrationwas begunbeforethe onsetof

de-tectable inflammation, we nextevaluated whetherTGF, 1

could independently suppresschronicinflammatoryevents.

TGF#

1 administration (5

gg/animal

perday) was begun on

Figure1. Modulationof

SCW-induced arthritis by

,.0

various doses ofTGF3l.

Animalswereinjected

withSCWonday 0. Some animals receivedno addi-tionaltreatment(circles);

othersweretreatedwith

TGFjB6 (i.p.)dailyat0.1

(invertedtriangles), 1.0

(squares),or5.0

1g/ani-mal(triangles) beginning ,~ theday before SCW

in-jection.Eachpoint

repre-sentsthemeanjointscore

for each group of animals (n= 3-5).Data for all groups of control animals (PBS,vehicle,and

U TGF,l-injected) are not

shown; all control ani-mals hadmeanjoint

scoresofzerothroughout

theexperiment.The

ex-32

periment withsome

modificationswas

re-peated three times with similar results.

0

4

8

12

1 6

20

24

28

16

0

z

C.)

12

8

4

A

t .A

-7,' ,/.

S. >1

.'44.)(ki. A'

0 l ll1,, I,

0 4 8 12 1 6 20 24 28

DAYS AFTER SCW INJECTION

Figure2.Theeffect ofTGF,B1 treatmenton thechronic phase of the arthritis. Lewisrats wereinjectedonday 0 with SCW and their Al determineddailythereafter. On day 10, half of the animals(filled circles)werebegunon adailydosingregimen of5,ugTGFfl/animal

(i.p.). Joint scores for control animalswere asindicated in Fig. 1.

day 10 for a group of SCW-injected animals and a group of control animals and continued for 18 d(Fig. 2). Before this

point,allanimalshad verysimilarAl scores (Al= 5.4±0.7; day 10). However, once daily administration of

TGF#

Iwasbegun, the scores ofthetreated groupdivergedfrom that of the un-treated group.

TGF#l

effectivelysuppressed thechronic phase of thearthritis. On day 21,the Alof the untreated group was

10.9±0.9 whilethatoftheTGFf3ltreated group was3.7±0.7; P

<0.0001. Additional studies examined the timing with which TGFf1 must beadministered to avert the acute and chronic

inflammation.

One study examined theeffect of

TGFf3l

on arthriticlesions after connective tissue destructionwasalready

apparent. Whendaily injectionsof

_TGF#

1 werebegun on day

21, well into the chronic destructive phase, no significant

changeoccurred in the Aloftheanimals(Al = 13.3±0.3 for

SCWanimalsvs. Al = 11.0±2.0 forSCW+ TGF,B animals; day 30). Furthermore, asingle injection of

TGF,3l

1 dbefore SCW administration did not diminish the acute or chronic phasesofthearthritis (Al= 10.4±0.8 forSCWanimalsvs. Al

= 10.2±0.3 for SCW+TGFf animals; day 21).

Histopathological

analysis.Inorder to define the potential mechanismswherebyTGFf1 reversed the

inflammatory

pro-cessesleadingtojointdestruction, cellulareventswere

moni-tored byhistopathologic evaluation. Thejoint tissues from SCW-treated animals exhibitedthelesionsthattypifychronic erosivearthritis: extensive synovial hyperplasia withan

accu-mulation ofmononuclear cells (macrophages,lymphocytes,

and fibroblast-like cells),pannusinvasion into thebone with

subsequent bone erosionandcartilage destruction,andfibrous tissuereplacement of thejointspace(Fig.3 B). However, treat-mentofSCW-injectedanimals with 5

MAg

of

_TGF#

Ieliminated

theinflammationandjoint erosionand thesynovialtissue

ex-hibitedrelatively normalmorphology(Fig.3C).Thesynovial

tissuefromanimals treatedwith 1.0 and 0.1

Mg

ofTGF, 1 was

slightly

hypertrophied withanaccumulation ofmononuclear cells(notshown); however,thecellularinfiltratewasvery

lim-ited relativetothatofthe untreated SCWanimals.In

addition,

4

'w&

_M.

f

V}%1

W

t99.

.,' ' '7 '.'

~~~~~~~~~~~~~~~~~i,

Cj

s

i

-t

t;'

Figure 3. Histologic comparison ofjointandsynovialtissueafter SCWandTGF Il treatments.Jointtissue fromcontrolrats(A)and

ratsinjectedwithSCW (BandC)washarvested 33 dafter SCW in-jection. Some of the animalsweredoseddailywith 5 qg TGF,1 (A

andC).Magnification, 16.

there was no apparent bone orcartilage erosion. Tissue from control animals injected daily with vehicle orTGFfl (5 ,ug)

(Fig. 3 A)exhibitednormal morphology. TGFfll administra-tion didnotappeartoaffectdisseminationordepositionof the

injectedSCW asshownbyimmunoperoxidase staining of tis-suewith anantibodytoSCW,nordid it appeartochange the retention timeofthe SCW in the tissues (not shown).

Inhibition of

letukocytosis

by

TGFO3.

The marked reduc-tion ininflammatorycell infiltratepromptedsubsequent analy-sisoftheeffects of TGF3 1 oncirculating hematopoieticcells. On day 4, the number ofcirculatingWBCswaselevated for all

SCW-treated animals, regardless of any TGFJ 1 treatment:

21.9±1.5 x 103/mm3 (n =20) forallSCW-injected animalsvs.

7.0±0.3x 103/mm3(n = 14) (P<0.0001)forcontrols(Fig. 4 A). However, by day 32, daily systemic administration of TGF1 had significantly suppressedthe elevated WBCcount

associated with theinflammationinadose-dependentmanner

(Fig. 4 B). Hematocrits measured on day 4 and 32 were not

suppressed (datanotshown).

Absenceofsideeffectsin

_{TGF,01-treated}

animals. Inspiteof theimpact ofsystemically administeredTGFf 1 on

arthritis,

it had no apparent side effects nordid it cause

generalized

im-25

E

E

0

x

0

_R

0 0

0

-J

0

w

m

3i.

LLI

m

z

20

15

10

5

0

50

40

30

20

10

0

PBS VEH.

TGF,

0.0

0.1 1.0 5.0 CONTROLS SCW +

TGF,

(pIg/rat)

Figure4. Alterations in number ofcirculating WBCs afterTGFOI

treatment.TotalWBC count for control andSCW-injectedanimals weredetermined on day 4 (A) and day 32 (B). Some of the animals were treateddaily with

TGF(

1 daily as described in Fig. 1. The data

is expressedas meannumberofWBCsx 10-3/mm3blood+SEM (n

=_{3-10)for each group.}

mune suppression. Spleen cells isolated from TGFf-treated controlanimals incorporated equivalentamountsof[3H]TdR

(1 18,700±1,600 cpm) when stimulated with ConA as the vehi-cle-treated control animals (125,800±16,000 cpm). All SCW-treated animals, treated andnottreatedwith TGF3, exhibited suppressed spleen cell proliferation,asreported(5). PBMC

iso-lated from the various groups of animals followed the same trends.TGF,B also didnotaffect the normal weight gain of the control rats (TGF,B-treated 164.0±2.3 g vs. vehicle-treated 165±3.1 g; day 32), nor did it alter the slower rate of weight gain typically observed in SCW-treated animals: 142.4±4.0 g (n = 5) for SCW rats compared with 149.2±6.1 g (n = 5) for SCW rats treated with TGFfl1.

TGF#1

injections to control animals did not cause noticeable grossormicroscopic patholo-gies, however daily intraperitoneal injections of TGFf 1 to the SCW-injected animals exaggerated the production of connec-tive tissue in the abdominal wallat the injection sites and surrounding the organs of the abdominal cavity.

Discussion

Daily intraperitoneal administration of TGFl1 to SCW-in-jected animals resulted ina markedsuppression of theacute

and chronic phases of SCW-induced arthritis. An intraperito-neal route of cytokine deliverywas chosen over intravenous

injectionbecause theserumcomponent,

a2-macroglobulin,

is known to effectively bind TGFi3 (20). In addition, first-pass

hepatic extraction has been demonstratedtoefficiently elimi-natethe moleculefrom the blood(21).Byintraperitoneal

ad-ministration, the retention time of the bioactivitywasclearly sufficient todramatically influence thecourseofevents respon-sible forjointdestruction.

Thedecreasedinflammatorycellrecruitmentinto the syno-vium of the TGF, I1-treated animals may be dueto an inhibi-tion of theSCW-induced leukocytosis. SCW-treated animals

typically manifest anincreased numberofcirculating leuko-cytes whichserve as areservoirofcellsfor recruitment into the

jointsand othersites ofchronicinflammation(22). Treatment with TGF,B 1 was found to suppress the increase in the number ofcirculating leukocytes,suggestingthat theinhibition of

leu-kocytosismaybe important inpreventing thearthritic condi-tion.Thiseffectwasnotnotedduring the acute phase, but was observedconsistentlyin thechronicphase and was dependent

on the amount of

_TGFOI

administered. The inhibition of SCW-induced leukocytosis may be due to a decrease in the proliferation of hematopoietic precursor cells in the bone marrow. Administration of TGF,B1 to mice via the femoral artery hasrecently been demonstrated to cause the partial

inhi-bitionof bonemarrowproliferation(9).Several invitro studies supportthis observation (6-8). Thus, the limited recruitment of

inflammatory

cells intothejoint maybedue, in part, to a lower numberof circulating WBCs.

Inaddition to reduced numbers of circulating WBCs, che-motaxis ofinflammatorycells into the joint may also be altered by the treatment with

TGF31.

TGF,B has been identified as a potent monocyte chemotactic factor (23), and its importance

as asynovial chemotactic factor was shown in a recent study in which a local injection of

TGFfl

into normal rat joints caused

an immediateinflux of inflammatory cells (19). Hence, a high concentration of circulatingTGFflmay effectively eliminate a

synovium-centered TGF,B gradient, inhibiting

TGFf-me-diated inflammatory cell influx. This mechanism is consistent with the decreased number of inflammatory cells found in the

joint. Inaddition to changes in the TGF, gradient, systemic administrationof TGFf3 may alter inflammatory cell expres-sion of TGF,B receptors. Exposure of circulating human

mono-cytes to TGF,B effectively downregulates TGF,3 receptor

ex-pression (Brandes and Wahl,inpreparation),in contrast to the lack ofligand-induced receptor downregulation observed in

othercellpopulations (24). Thus,adiminishedpool

ofcirculat-ing WBC and a decreased chemotactic response to TGF,B

might effectively restrict synovialinflammatory events

depen-dentoncellrecruitment. TGF,3 has also been shown to inhibit

neutrophil adhesion to endothelialcells, the event preceding

cell migration into the tissue (25). Systemicadministration of

TGF#

1 maydecrease blood celladhesionto the endothelium, thusalsocontributingtothelimitedinflammatory cell recruit-mentintothejoint.Thoseinflammatorycells that are able to

infiltratethetissuemayalso exhibit suppressed function, since

TGFf#

hasrecently beenshowntodecrease IL- 1 receptor

ex-pression (26) and the production of superoxideradical both in

vitro (27) and in vivo (28; and Allen, Brandes, Ogawa, and

Wahl, manuscript in preparation).

Additionaleffects of TGF,3 identified using in vitrosystems mayalso contributetothe ability of TGF,3toameliorate SCW arthritis. Forexample,

TGF,?

inhibitsIL-1-induced chondro-cyte proteaseactivity andcartilage proteoglycan degradation (29). Furthermore, TGF,B inhibits the formationof

osteoclast-like cells inlong termhumanmarrowcultures(30)andinhibits

bone resorption (31). Thus, systemic

TGF#31

administration may act

directly

tocontain the bone and cartilage erosion, as well asindirectlybysuppressing inflammatorycellinfiltration. Studiesare inprogress to document the contribution of these

and/or other pathways relevanttoTGF,3 suppression oferosive arthritis.

Surprisingly, therewasnoevidence of abnormalitiesinthe numberorin vitroproliferative behavior of cells isolatedfrom

thespleen and peripheral bloodof

_TGF#

1-treated control ani-mals.Moreover, TGF, didnotfurther inhibitthealready

sup-pressed proliferation of spleen cells and PBMC from SCW-in-jected animals. Thus, either generalized immunosuppression does not occur, orit is reversible. In

addition,

treatment of control animals with

TGF#

Ididnotaffecttheirnormalweight

gain

or causenoticeable

pathologies.

This is in contrastto

previous studies in mice injected subcutaneously with>20

_,g

of

TGF(31

perday. The mice developed

anemia,

thrombocyto-penia, increased white cellcount(32),and20-30% loss in body

weight

during the 14-d

dosing

regimen (J.A.Carlino, personal communication).

TGF#l

wasshown inthisstudy toeffectively inhibit the

development ofaninduced arthritic condition inrats,likely via its immunoregulatory effects(33) andits inhibitionof

connec-tivetissue degradation (34). Although TGF3has been

adminis-teredin vivoforacuteinflammatoryevents(28,35),thisis the

firststudy toaddress thetherapeutic potential of

TGF,3

forthe treatment of chronicinflammatory diseases. Our data suggest that

TGF,?

iseffectiveincontrollingthediseaseprocess, while

producingminimal sideeffectsdespiteitspresence in

non-physiological quantities. If

TGF,3

can successfully treat im-munesystem disorders, and do sowith limited toxic effects, then it may warrant consideration as avaluable therapeutic

agent.

Ackcnowledaments

Theauthors thankDr. UweMai and Dr.NancyMcCartney-Francis for

helpful discussions.

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