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Showa v niU J Med icS O
r i g i n a l
M o n o c l o n a l
dybontiA ot Very eatL ‑negit nA4 (VLA‑4) stcetorP nikS spalF tsinagA I
s c h e m i a
‑sufrepeR i o n
:yruj nI An Exper 加latne1 y duSt Rni s ta
Y a s u s h i
KOGA
へ
ihsoyusaY ,ASOT Nozomi TOSA a, niriN neirdA naeJ reiviV Mandrano No b u h i r o
SATO dna aynihS YOSHIMOTO
D epnrαtr 削 tof Pl 仏雪cit α】d R'1 uretsnoc
Show ，αvinU eris 砂loohcS of Medi ,cie n 1
‑‑5,8 ,iado nataH ‑awag,aunkihS yo Tok ‑2,461668 .nap aJ
* To whom gnidnopsenoc dluohs bedesserdda .
Name d na rdda 白sof c gnidnopseno :ro htua Y
a s u s h i
KOGA, MD D
e p a r t m e n t
ofcitsalP dna evitcmtsnoceR yregruS Showa ytisrevinU loohcS Mof enicide
1
‑‑58 ,iad, o nataH ‑awaganihuSk , o okyT ‑2416668 , napaJ . e
‑iam l
: oaltsrif @.liamg moc T
e l
: +‑18‑3‑48783458 , :xaF +‑18‑3‑4873 3819
A r gninnu : eltit Mab ot VLA‑4 stcetorp niks palf tsniaga /1 R yrujni
A b s t r a c t
: nI tnecer ,sra ey rav i uso n oieshda molecu l se have been di.derevo cs V
e r y
etal negitna ‑4 (VLA‑4) sa one of t eh nirgetni ylimaf si an onisheda m
o l e c u l e
gniyalp n a tnatropmi elor mni gnitargi nd a gnitartlifni escytpholym ot t
h e
etis of .noitamma lfni nI eht tneserp ,ydu ts we i detagitsevn er ethwh eht a
d m i n i s t r a t i o n
of antiVLA‑4 tna ibody secuder eht n otimamlanfi ni 25 male S
p r a g u e
‑Dawley tar model (‑522g)052 of a imehcis noisufreper yrujni niks palf . A saicifrepu
l
cirtsagipe suonevoiretra elcidep palf thiw a s ezi of 45 ×30mm was i
n t r o d u c e d
on eht thgir laniugni aera and eht amp cl was removed retfa 9 srouh f
o r
.aimehc si neetfiF steunmi roirp ot eht mp cla er lesae , eht star were dedivid i
n t o
n a ti‑anLA‑V4 deretsinimda (0.20mg /)gk tnemtaert p roug (n=)Ol , a s
a l i n e
‑aert t e d
lortnoc up gro (n= )01 a sham ro uporg taht sah been clamped rof 5 m
i n u t e s
erndu eht same noitidnoc (n=)S. The star were dweollof up rof en sev d
a y s
. The aera of s devivru niks palf ni eht mtaert en t p ougr (87 .0%) was s
i g n i f i c a n t l y
proved im compared ot taht of eht lortnoc up rog (81 .3%). As f ro eht h
i s t o l o g i c a l
sgnidnif y rtomamlanfi sgnidnif , hcsu sa ereves ryoatmmlanfi llec i
n f i l t r a t i o n
and edema were dveersob ni eht lortnoc ,puorg tub esoht of eht t
r e a t m e n t
up rog were dilm and showed a t eussi erutcurts ralimis ot taht of eht sham .puorg Based on eseht stluser , ti sah n eeb detsgegus taht anti‑VLA‑4
m o n o c l o n a l
ydobinta can be sedu sa an noit‑aimtmnaalfni .tne ga However , f
u r t h e r
eiduts s era yrassecen eot etadicul sti deliated mechanism .
Key words: noi‑sauifmreehpecsri jni ury , y1ev etal ‑enignta(4‑LAV)4 , oniesdha m
o l e c u l e
, itna VLA ‑4 adobitn , y niks palf
2
I n t r o d u c t i o n
I n
tnecer sraey , suoirav n oisehad eslcuelmo have been .derev ocsid A s seire of t
h e s e
sleueclom dellac llec noieshda s elcueolm era devlovni ni ralullec i
n t e r a c t i o n
and yalp tnatropmi selor ni niotmamlafni and eht noitaluger of i
m m u n o r e a c t i o n
1). They yalp an tnatropmi elor on eht ssergorp of i
n f l a m m a t i o n
by detavitca setycocuel gnirehda on v ralucsa lailehtodne .s llec The t
i s s u e
noitartlifni ssecorp ni n iotmamlanfi noitcaer can be deifissalc otni eht f
o l l o w i n g
eerht :sp ets gnillor pets , gnorts noieshda pets , d an noitargimsnart .pe ts I
n
e cha ,pe ts id 百tnere onisehda eslcueolm era .devlo vni
The phenomenon dellac a imehcis noisufreper yrujni si known ot ylap i
m p o r t a n t
selor ni eht mechanism of s nik palf lavivrus and .sis orcen yllaicepsE , i
t
sah been detseggus taht eht es nghac tsniaga ralucsav lailehtodne sllec ni i
s c h e m i a
noisufreper yrujni era devlovni ni gnitneverp eht niks palf lavivrus ot c
a u s e
sisorcen 2). ‑6 To e etadicul eht mechanisms of s nik palf lavivrus and n
e c r o s i
s , a r ta model of iamehcsi noisufreper yrujni niks palf sah been d
e v e l o p e d
. reoferehT , we b eveile ti si lufesu ot ezilitu siht tar model of i
s c h e m i a
noisufreper yrujni niks palf ot dnatserdnu eht evitasuac stnega r
e s p o n s i b l e
rof 6noitammalfni )'7. nirgetnI sa one of eht niosheda eslucleom si known ot be ylhgih devlovni rni noitaluge raof ilullecretn e rroalullecartx xitram a
d h e s i o n
r iarolullecartn langis noitcudsnart aiv nosiehad ). 8
Very etal ‑negit n4a (VLA‑4 aniereh 立)re ,which sngolbe ot eht nirgetni ,ylim af i
s
deredisnoc ot be i devlovn hitw eht noitargim of tesocymphly ot eht etis of
i n f l a m m a t i o n 9
). ,suhT we c deirra tuo eht tnmeierpex ot etagitsevni r hehetw eht a
d m i n i s t r a t i o n
of ai‑nt‑VLA4 ydobitna tsniaga VLA‑4 secuder eht n iotmaamlnfi r
e a c t i o n
nro to ,dna fi ti,seod how much ti s eod gnisu eht iamehcsi noisufreper i
n j u r y
niks palf model , and we r trope eht stluser gnola htwi erutaretil .weiv er
M a t e r i a l s
and Methods I
s c h
e
，
αηi noisuf−reper yrujni niks lfαp modelT w e n t y
‑ evif male Sprague‑Dawley star (‑8 01 weeks of ega , 225‑ 250 grams , S
a i t a m a
ltaneimrexpE Animals Supply o.C , .dtL , ,amatiaS )napaJ were edsu ni t
h e
.tnemire pxe The slamina were housed ni ire‑wesh m segac and nevig 企ee a
c c e s s
ot tellep teid (Nosan ,noitaro proC Kanagawa, )napaJ d an gniknird terwa (pat
)ret.aw
Ketamine edirolhcordyh ( 52 mg /)gk was deretsinimda yllaenotirepartni sa a
n e s t h e s i a
. retfA niap noitanimile eht abdomen was evhas d and 45 × 53 m m p
e d i c l e d
niks spalf hitw laicifrepus cirtsagipe sniev and seiretra sa selcidep were d
e s i g n e d
tni eh thgir laniugni noiger and .deta vele Then eht niks palf selcidep were clamped .(gi F )1 ). 6 The laimna nteimrpeex was dtecdunco llacihte y d base on t eh Showa ytisrevinU l maian t enmriexpe .seni lediug
l s c
h emi c tni erναf
The cimehcsi lavretni was 9 sruoh , ro 5 ms eutin rof eht sham .puorg
4
A n t i b o d y
α isnimd noitart
The ‑i tynraev etal ‑negitn 4a (VLA‑4) y doibtna (.0 02 mg /)gk (onal monocl a
n t i
‑tar
VLA‑4: CD49d) (‑TA,2 Cape Cod ,noitar oprocnI h,utlmoFa MA, USA) a
g a i n s t
ryev etal ‑negit n4a (VLA‑4) was deretsinimda by suonevartni noitcejni t
o
eht ldauac nive 51 estnuim eroefb gnisaeler eht .pmacl deziliretS enilas ta t
h e
same sedo was deretsinimda tni eh same manner a csa .lor tno
E x p e r i m e n t a l
roupsg
An itna VLA‑4 ydboitan tnemtaert group (n=)Ol and a p lacigoloisyh enilas c
o n t r o l
(=n)lO were ed rvseob over 7 d.sya A s nik palf was detavele ni eht same manner ni a sham group (=n)5, tub eht clamp was desaeler a食r 5 me .estuni The salmnia were deiznahtue ta eht end of eht ‑7day noitavresbo by an overdose
of a,aise htsen and lacigolotsih ioninatxame of t eh niks spalf was d cteducon (g Fi 2
).
Method of evula α noit S
k i n
spalf were detaulave by scopic macro noitarvesbo and lacigolotsih a
n a l y s i s .
1
) Sin k palf lavivrus aera oitar
The aera oitar of t eh niks palf lavivrus snoiger and citorcen snoiger was c
a l c u l a t e d
by t ecar based on eht teaplmet method of Ms irro te .l,a and eht savivru
l
aera oitar was deniatbo I)̲ o
2
) lacigolotsiH n ionatamiex
Samples were edrpaepr by h‑nliyxtoan emiseo .gni niats eussiT plessam were t
h e n
edervbso rof ionatmmflain sgnidnif and rieht tnetxe , and rof eht noitidnoc w
i t h i n
od blo .sle ssev
S
t αtsi cit αl an αl sisy
The atad were edsesrxpe sa mean 土SD. Mann‑Whitney lacitsitats sisylana was deilppa ot etaulave lavivrus ecnereffid between lortnoc and tnemteart g
r o u p s
. secnereffiD of p< .0 50 were dreedinsoc be ot yllacitsitats .tna cifingis
R e s u l t s S k i n
palf r νus νiααl rear αt oi
Noncomparative sisylana of t eh tnemtaert group edretsinimda anti‑VLA‑4 a
n t i b o d y
and eht lortnoc oup gr dreetsiinmda lacigoloisyhp enilas was .dectudnco
Comparison of m opicroscac sgnidnif showed taht niks palf lavivrus ni eht t
r e a t m e n t
oup gr was ,odog d an taht niks palf morphology and roloc were esolc t
o
eht sham .puorg nI eht lortnoc ,puorg however , niks spalf showed e rvees i
n f l a m m a t i o n
accompanied by laitrap sisorcen (.giF )3. I
n
comparison of eht mean niks palf lavivrus aera oitar ni each group on day 7 a
f t e r
noisufreper , improved niks palf lavivrus noitar was ed rvbseo with s
t a t i s t i c a l l y
tnacifingis ecnereffid (p<.0)500 ta .870 土39.8% ni eht ‑9 hour i
s c h e m i a
yodbinta noitartsinimda group compared ot .813 士19.6% ni eht lortnoc
6
group (.giF )4.
H i s t o l o g i c a l
xe α niotanim I
n
eht tnemtaert ,puorg ghoultha ion lammatinf sgnidnif were observed and d
i s o r g a n i z e d
erutcurts was yllaitrap served ob on t eh niks ,ecaf rus eht slevel of i
n f l a m m a t i o n
llec noitartlifni and edema were ylevitaler mild , ralucsav lumep was ,devreser p h itw eussit erutcurts ralimis ot esoht of eht sham .puorg nI c
o n t r a s t
, ni eht lortnoc ,puorg ation inflamm llec,no itartlifni edema and sisorcen
were .devrsebo nI noitidda , ralcusav lumen was dellif with setycorhtyre , i
n d i c a t i n g
eht ontimaorf of blood ,sto lc which was yllacitamard tnereffid from t
h e
noitidnoc of v ralucsa lumen of t eh ntemtaetr group (.giF )5.
D i s c u s s i o n
Very etal ‑negitna 4 (VLA‑4) si one of eht ateb 1 nirgetni seilimaf and osla known sa a hlpa 4 ba te ,1 nir getni CD49d /CD29, ro lymphocyte Peyer s tch pa HEV n siodhea cule mole (2.PL¥lAι)2. tI stsisnoc of a r mediroeteh of a l 50kD ( on SDS‑PAGE under d ducere )noitidn oc a phla 4 cn iha tinubus and a 130kD of b
e t a
1 inahc 11tinubus ). Both aphla 4 n ihac and ateb 1 cn iah era e pyt I membrane snietorp ning span llec membrane once and g havin rieht N ‑ nus1mite
on t eh roiretxe edis of eht llec and eht C‑us ninten on eht micaspltocy edis of eht c
e. ll The aphla 4 c niha (999 amino )sdica si detsisnoc of an ralullecartxe domain ( 944 amino ). sdic a th wi seven tandem staeper n,domai a t snar membrane
