Coxsackie–adeno i us ecep o
exp ession is enhanced in panc eas
om pa ien s wi h ype 1 diabe es
M Hodik,
1
M Anagandula,
1
J Fuxe,
2
L K og old,
3
K Dahl-Jø gensen,
3
H Hyö y,
4,5
L Sa mien o,
6
G F isk,
1
POD-V Conso ium
To ci e: Hodik M,
Anagandula M, Fuxe J, e al.
Coxsackie–adeno i us
ecep o exp ession is
enhanced in panc eas om
pa ien s wi h ype 1 diabe es.
BMJ Open Diabe es Resea ch
and Ca e 2016;4:e000219.
doi:10.1136/bmjd c-2016-
000219
▸Addi ional ma e ial is
a ailable. To iew please isi
he jou nal (h p://dx.doi.o g/
10.1136/bmjd c-2016-
000219).
Recei ed 29 Feb ua y 2016
Re ised 8 June 2016
Accep ed 9 July 2016
Fo numbe ed a ilia ions see
end o a icle.
Co espondence o
D Gun F isk; gun.e gida.
[email p o ec ed]
ABSTRACT
Objec i es: One o he heo ies connec ing
en e o i us (EV) in ec ion o human isle s wi h ype 1
diabe es (T1D) is he de elopmen o a e ile ield in
he isle s. This implies induc ion o app op ia e
p o eins o he i al eplica ion such as he coxsackie–
adeno i us ecep o (CAR). The aim o his s udy was
o in es iga e o wha ex en CAR is exp essed in
human isle s o Lange hans, and wha condi ions ha
would change he exp ession.
Design: Immunohis ochemis y o CAR was pe o med
on pa a in-embedded panc ea ic issue om pa ien s
wi h T1D (n=9 ecen onse T1D, n=4 long-s anding
T1D), isle au oan ibody-posi i e indi iduals (n=14) and
non-diabe ic con ols (n=24) indi iduals. The exp ession
o CAR was also examined by e e se ansc ip ion PCR
on mic odissec ed isle s (n=5), exoc ine issue (n=5) and
on explan ed isle s in ec ed wi h EV o exposed o
chemokines p oduced by EV-in ec ed isle cells.
Resul s: An inc eased equency o pa ien s wi h T1D
and au oan ibody-posi i e indi iduals exp essed CAR in
he panc eas (p<0.039). CAR s aining was de ec ed mo e
equen ly in panc ea ic isle s om pa ien s wi h T1D
and au oan ibody-posi i e subjec s (15/27) compa ed
wi h (6/24) non-diabe ic con ols (p<0.033). Also in
explan ed isle s cul u ed in UV- ea ed cul u e medium
om coxsackie i us B (CBV)-1-in ec ed isle s, he
exp ession o he CAR gene was inc eased compa ed
wi h con ols. Lase mic odissec ion o panc ea ic issue
e ealed ha CAR exp ession was 10- old highe in
endoc ine compa ed wi h exoc ine cells o he panc eas.
CAR was also exp essed in explan ed isle s and he
exp ession le el dec eased wi h ime in cul u e. CBV-1
in ec ion o explan ed isle s clea ly dec eased he
exp ession o CAR (p<0.05). In con as , in ec ion wi h
echo i us 6 did no a ec he exp ession o CAR.
Conclusions: CAR is exp essed in panc ea ic isle s o
pa ien s wi h T1D and he exp ession le el o CAR is
inc eased in explan ed isle s exposed o
p oin lamma o y cy okines/chemokines p oduced by
in ec ed isle s. T1D is associa ed wi h inc eased le els
o ce ain chemokines/cy okines in he isle s and his
migh be he mechanism behind he inc eased
exp ession o CAR in TID isle s.
INTRODUCTION
Type 1 diabe es (T1D) is a li elong disease
cha ac e ized by he loss o o se e ely
educed numbe o insulin-p oducing βcells
in he isle o Lange hans, p esence o isle
au oan ibodies and, especially in younge
indi iduals, insuli is consis ing o infil a ion
o he isle s p edominan ly by CD8+ T cells
and mac ophages. The e iology o he disease
is unclea bu i has been shown ha gene ic
ac o s, especially genes in he human leuco-
cy e an igen (HLA) complex, a e o majo
impo ance o he pa hogenesis. In addi ion,
se e al s udies ha e shown ha en i onmen-
al ac o s likely con ibu e o disease de el-
opmen .
12
En e o i uses (EVs), pa icula ly
he coxsackie i us B (CBV), a e among he
main en i onmen al candida es and nume -
ous s udies ha e shown associa ion be ween
hese gu i uses and T1D by di e en ech-
niques.
3–10
In addi ion, expe imen s using
isola ed human isle s ha e shown ha CBVs
a e able o in ec and eplica e in insulin-
p oducing βcells.
411–13
CBV belong o he human en e o i us
species B (HEV-B) species. Thei genome is
Signi icance o his s udy
Wha is al eady known abou his subjec ?
▪I is known ha en e o i us, especially
Coxsackie i us B, o en can be de ec ed in
human isle s o Lange hans in ype 1 diabe es
cases.
Wha a e he new indings?
▪I is no known i and o wha ex en isle cells
exp ess app op ia e i us ecep o s and i he
exp ession di e s be ween ype 1 diabe es
cases, p e-diabe ic cases and non-diabe es
indi iduals.
How migh hese esul s change he ocus o
esea ch o clinical p ac ice?
▪Ou inding ha he Coxsackie-Aden i us-
Recep o (CAR) is mo e equen ly exp essed in
panc eas om p e-diabe ic and in ype 1 dia-
be es cases compa ed o con ol indi iduals
sugges he p esence o a e ile ield in panc eas
in he wo o me g oups.
BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 1
Open Access Resea ch
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om
∼7500 nucleo ides long and p o ec ed by a capsid com-
posed o 180 subuni s o he capsid p o eins VP1–VP4.
The i ions use a ious ecep o molecules o en e
he cell. The main ecep o o CBVs is he igh junc-
ion p o ein Coxsackie–adeno i us ecep o (CAR),
which belongs o he la ge amily o adhesion molecules;
howe e , o he ecep o s ha e also been shown o be
impo an o i us in e naliza ion.
14 15
The ull-leng h
CAR p o ein comp ises wo ex acellula
immunoglobulin-like domains (D1 and D2), one ans-
memb ane helix and an in acellula C- e minal
domain.
16 17
A leas fi e iso o ms o CAR a e known,
wo ansmemb ane iso o ms, which di e only a hei
C- e minal, and h ee lacking he ansmemb ane
domain ha we e shown o be sec e ed in ans ec ed
HeLa cells.
18–20
CBV binds he dis al end o he D1
domain in he canyon o he i us capsid,
21
whe eas he
in acellula C domain has been shown o be dispensable
o in ec ion.
22
The issue dis ibu ion o CAR in human
issues has been mainly s udied a he ansc ip ional
le el; CAR is exp essed in a a ie y o human o gans,
including he panc eas.
19 23
The p o ein is highly
exp essed in he b ain and hea du ing mu ine emb y-
onic de elopmen , bu exp ession declines apidly a e
bi h.
24 25
Exp ession o he CAR p o ein in human
adul panc eas has been demons a ed in duc al epi he-
lial cells,
26
isle cells,
27
and exoc ine acina cells (h p://
www.p o eina las.o g) by immunohis ochemis y (IHC).
Which o he iso o ms o he CAR p o ein is p esen in
panc eas, and he biological unc ions o hese a e no
known. A monoclonal an ibody di ec ed agains he
ex acellula CAR domain
28
blocked eplica ion o CBV-4
and CBV-5 in explan ed isle s, an indi ec p oo o exp es-
sion and unc ion in human isle s.
4
Fu he mo e, he
exp ession o he CAR p o ein has been no ed o be
inc eased du ing healing and inflamma ion
29 30
and
down egula ed in a ious human cance s.
31
The main aim o his s udy was o in es iga e he
exp ession o he CAR gene and p o ein in panc ea ic
issue, ob ained om di e en g oups o pa ien s wi h
T1D, and o compa e i wi h non-diabe ic con ol indi i-
duals. We also wan ed o s udy how EV in ec ion o
i us-induced inflamma o y media o s egula e he
exp ession o CAR in human isle s.
MATERIALS AND METHODS
Tissue specimen
Panc ea ic pa a fin-embedded issue om he panc ea ic
head o ail we e ob ained om non-diabe ic dono s
wi h isle cell au oan ibodies (n=14), dono s wi h ecen
onse T1D (n=9, bo h om o gan dono s and om he
DiViD s udy), dono s wi h long-s anding T1D (n=5), and
om con ol, au oan ibody-nega i e, non-diabe ic o gan
dono s (n=24). Tissues om o gan dono s we e p o-
ided by he Ne wo k o Panc ea ic O gan Dono s wi h
Diabe es (nPOD) and he Uppsala biobank. Panc eas
biopsies we e ob ained om he DiViD biobank, Oslo,
No way (see online supplemen a y able S1).
Human isle and cell cul u es
Panc ea ic isle s we e isola ed om he panc eas o
o gan dono s acco ding o he p o ocol by Go o e al.
32
Isle s we e hen handpicked unde a ligh mic oscope o
u he inc ease pu i y o >90%. Isle s we e cul u ed ee
floa ing in six-well pla es o suspension cul u es
(Sa s ed , Nümb ech , Ge many) in 3 mL RPMI-1640
5.5 mM glucose (SVA, Uppsala Sweden) supplemen ed
wi h 10% e al bo ine se um (FBS) (Gibco, In i ogen,
S ockholm, Sweden). G een Monkey Kidney (GMK)
cells we e cul u ed in 96-well pla es (Co ning, New Yo k,
USA) in Eagle’s minimum essen ial medium (EMEM)
(SVA, Uppsala Sweden) supplemen ed wi h 10% FBS
(Gibco, In i ogen, S ockholm, Sweden). All cells we e
cul u ed a 37°C in 5% CO
2
.
P olonged cul u e o isle s
Human isle s, isola ed and u he pu ified by handpick-
ing, we e cul u ed as desc ibed abo e wi h change o
cul u e medium e e y hi d day. To al RNA was ex ac ed
on days 1, 4, 8 and 13 (numbe o dono s=2) pos isola-
ion o analyze he genes coding o CAR and insulin.
E hics
The wo k pe o med on human issue was in acco dance
wi h he p inciples exp essed in he Decla a ion o
Helsinki and he Eu opean Council’s Con en ion on
Human Righ s and Biomedicine. Isola ion o human
isle s o Lange hans and he esea ch pe o med on
hese was app o ed by he Regional E hics Commi ee in
Uppsala, Sweden. Collec ion o panc ea ic issue in he
DiViD s udy was app o ed by he No wegian Go e nmen
Regional E hics Commi ee in Oslo, No way.
Vi us
Th ee s ains o CBV-1 we e used in he gene exp ession
s udy: CBV-1-7-10796 and CBV-1-11-10802 we e isola ed
in A gen ina du ing 1983 and 1998, espec i ely,
whe eas CBV-1-3-10790 was isola ed in he USA in 1973.
All s ains we e ob ained om he Cen e o Disease
Con ol and P e en ion (CDC), A lan a, Geo gia, USA.
In addi ion, a s ain o echo i us 6 (Echo-6-2C) was
included in his s udy. This s ain was isola ed in Cuba
2011 om a pa ien wi h meningi is as a pa o he
Public Heal h Su eillance s a egies in Cuba on ci cula -
ing EV in meningi is cases. The CBV-1 s ains we e
chosen since CBV-1 has been shown o use CAR as i s
main ecep o . The Echo-6-2C s ain was chosen since
his se o ype does no use CAR as a ecep o and has
been shown o bind o CD55/decay accele a ing ac o .
33
Immunohis ochemis y
Pa a fin-embedded o malin-fixed panc ea ic issue as
well as pa a fin-embedded o malin-fixed explan ed
isle s o Lange hans we e sec ioned (5 µm) and d ied on
Supe os glass slides (Menzel-Gläze , Fische scien ific,
B aunschweig, Ge many), ollowed by de-pa a finiza ion
and ehyd a ion in 99.70% e hanol. An igen e ie al
2BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219
Isle s udies
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om
was pe o med in TE-bu e , pH 9 (DAKO, Glos up,
Denma k) in a s eam boile and pe meabilized in TBS
con aining 0.05% TWEEN 20. Endogenous pe oxidase
was blocked by he use o a eady- o use pe oxidase
blocke (DAKO, Glos up, Denma k). A e insing,
he sec ions we e incuba ed wi h p ima y an ibodies,
mouse an i-CAR (RmcB clone, Millipo e, Bille ica,
Massachuse s, USA) dilu ed 1:1000. Incuba ion wi h
hese an ibodies was pe o med a oom empe a u e o
1 hou and he isualiza ion was achie ed wi h he an i-
mouse En ision-ki (Dako, Glos up, Denma k) using
3,3’-diaminobenzidine (DAB) as subs a e ch omogen.
Fo double s aining wi h CAR and ch omog anin A
(mouse, eady o use, Dako, Glos up), he sec ions we e
isualized using a Polink DS MM D ki polyme -HRP&AP
double s aining ki (Golden B idge, Mukil eo,
Washing on, USA).
Vi us in ec ion
Handpicked human isle s o Lange hans we e inocula-
ed wi h any o he i us s ains o ob ain a final concen-
a ion o 10
3
issue cul u e in ec ious dose (TCID)
50
.
Aliquo s o he cul u e medium we e collec ed on he
day o in ec ion and e e y day un il day 3 pos in ec ion
(p.i.) o i us eplica ion measu emen by TCID
50
i a-
ions, and s o ed a −20°C. On day 3 p.i., all isle s and
all cells de ached om he isle s du ing cul u e we e
spun down and washed be o e o al RNA was ex ac ed.
Isle s inocula ed wi h he CBV-1-11 s ain we e also ha -
es ed o wes e n blo (WB) analysis on day 3 p.i. Fo
IHC analysis, isle s om he same dono we e inocula ed
wi h CBV-1-11 a a final concen a ion o 10
2.5–3
TCID
50
o mock-in ec ed. On day 4 p.i., hey we e fixed and
embedded.
Analysis o i us eplica ion
Vi us eplica ion was de e mined by TCID
50
i a ions o
samples o cul u e medium on an in house cell line o i-
gina ing om GMK cells, ob ained om Go henbu g
Uni e si y, Sweden, as desc ibed p e iously.
34
The
samples we e collec ed om he in ec ed isle cul u es
on he indica ed days p.i.
T ea men wi h i us-induced p oin lamma o y chemokines
The e ec o inflamma o y media o s on he exp ession
le el o he CAR gene was analyzed by ea ing
explan ed isle s wi h UV- ea ed cul u e medium om
CBV-1-in ec ed human isle s ( wo dono s) and by
adding CXCL10 (Pep o ech, S ockholm, Sweden) a
final concen a ions o 0.1, 10 and 50 ng/mL ( ou
dono s) o 1 and 4 days. To al RNA was ex ac ed and
he exp ession o he gene encoding CAR was analyzed
by quan i a i e e e se ansc ip ion PCR.
RNA ex ac ion and cDNA syn hesis
Th ee days p.i., isola ed human panc ea ic isle s we e
washed in phospha e-bu e ed saline (PBS) and lysed by
using RLT bu e (Qiagen, Sollen una, Sweden) on
QIAsh edde spin columns (Qiagen, Sollen una,
Sweden). To al RNA was ex ac ed om he in ec ed
and con ol isle s by using he RNAeasy mini ki
(Qiagen, Sollen una, Sweden) wi h G DNA elimina o
column o emo e he genomic DNA. RNA concen a-
ion and quali y we e de e mined wi h Nanod op
(The mo Scien ific, B aunschweig, Ge many). Up o
50 ng o o al RNA pe sample was e e se ansc ibed o
cDNA in a eac ion mix u e o 0.5 µL andom p ime
(300 ng/μL, In i ogen, S ockholm, Sweden), 1 µL
RNaseOUT (40 U/µL), 4 µL 5× fi s s and bu e , and
1 µL 0.1 M di hio h ei ol (DTT) acco ding o he manu-
ac u e ’s ins uc ions. Reac ion was ca ied ou a 25°C
o 10 min, 42°C o 55 min, and 15 min a 75°C.
Real- ime PCR
The mRNA exp ession le el o genes encoding CAR,
insulin, and amylase was analyzed wi h eal- ime PCR.
The eac ion mix u e consis ing o 10 µL Sybe G een
mas e mix (Applied Biosys ems, S ockholm, Sweden),
2 µL p ime , and 1 µL cDNA was p epa ed acco ding
o he manu ac u e ’s ins uc ions. The eac ion was
ca ied ou in a 96-well op ical pla e on a S ep one
plus Real- ime PCR sys em (Applied Biosys ems,
S ockholm, Sweden). The cycling condi ions we e 40
cycles o 15 s a 94°C, 30 s a 55°C and 30 s a 68°C.
P edesigned p ime s (Qun i ec , Qiagen, Sollen una
Sweden) we e used o de ec ion o CAR and 18s. Also,
o amylase and insulin, comme cially a ailable p ime s
we e used o analyses o he LMC samples. Real- ime
PCR da a we e analyzed by he compa a i e del a c
me hod. The exp ession le el o he gene encoding CAR
was no malized o he exp ession o he 18s housekeep-
ing gene by sub ac ing he 18s c alue om CAR gene c
alues. The ela i e gene exp ession le els we e calcula ed
by using he 2^-
dc
o mula and p esen ed as he clus e ed
columns in MS Excel. Mel cu e analysis was used o
e i y he specifici y o final PCR p oduc s.
Wes e n blo
Exp ession o he CAR p o ein was analyzed in
explan ed human isle s in ec ed wi h CBV-1-11 and
mock in ec ed by WB using a polyclonal an ibody
(α-CAR20) as p e iously desc ibed,
35
using an an ibody
agains calnexin (BioNo dika Sweden AB, S ockholm) as
loading con ol.
S a is ical analyses
Linea eg ession and ORs wi h 95% CIs we e used o
es he likelihood o CAR posi i i y in g ouped T1D
dono s and non-diabe ic con ols. Owing o low sample
sizes, he likelihood o CAR posi i i y in he sepa a e
T1D g oups was only measu ed be ween au oan ibody-
posi i e dono s and non-diabe ic con ols. Di e ences in
gene exp ession le els be ween con ol and
i us-in ec ed samples, be ween di e en dono s and
be ween he CBV-1 s ains we e analyzed wi h he
K uskal-Wallis es (co esponding o he pa ame ic
BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 3
Isle s udies
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om
one-way analysis o a iance), when compa ing da a
om mo e han wo independen g oups, and he
Mann-Whi ney es (co esponding o he pa ame ic
independen samples - es ), when compa ing da a om
wo independen g oups. Co ela ion analyses be ween
he exp ession o amylase and exoc ine issue, insulin
and endoc ine issue, and CAR and endoc ine/exoc ine
issue we e pe o med wi h Spea man’s . The p<0.05
was conside ed s a is ically significan . All ba s a e p e-
sen ed as mean+SEM.
RESULTS
Immunohis ochemis y
CAR posi i i y was significan ly highe in he combined
T1D and isle au oan ibody-posi i e g oup (15/27 com-
pa ed wi h con ols (6/24) in linea eg ession
(p<0.033), wi h an OR o 3.877 and be ween isle
au oan ibody-posi i e subjec s and non-diabe ic con ols
(p<0.039)). The exac figu e o his highe equency o
CAR-posi i e s aining in he endoc ine isle s o ecen
onse T1D was 5/9, isle au oan ibody-posi i e 8/14, and
long-s anding subjec s wi h T1D 2/4 compa ed wi h con-
ols 6/24. To be conside ed posi i e, he sec ion s ained
posi i e o CAR in a ew up o se e al isle s. In
CAR-nega i e sec ions om ei he g oup o dono s, no
s aining o CAR was seen. The posi i e CAR s aining
e ealed a memb anous pa e n in he endoc ine issue
(figu e 1A), sugges ing he p esence o he ansmem-
b aneous pa o CAR.
A somewha less in ense cy oplasmic/memb anous CAR
s aining was also obse ed in cells in he exoc ine pa o
he panc eas in all dono s, wi h CAR-posi i e endoc ine
cells. In addi ion, in fi e dono s wi h T1D, posi i e s aining
o CAR in duc al epi helial cells was seen. In he majo i y
(30/51) o he panc ea ic sec ions (mainly con ols), no
s aining o he ex acellula domain o CAR was seen in
he endoc ine o exoc ine issue.
Vi us eplica ion
All EV s ains/se o ypes used o in ec ion eplica ed in
he isle s. The mean i us i e inc ease in he cul u e
medium du ing 3 days p.i. was 10
1.8+0.28
(CBV-1-11, n=6),
10
2.5+0.4
(CBV-1-3, n=7), 10
2.5+0.40
(CBV-1-7, n=6) and
10
4.5+0.19
(echo i us 6-2C, n=5) TCID
50
/200 µL, espec -
i ely (figu e 2).
Exp ession o CAR mRNA
In unin ec ed con ol isle s (n=3), cul u ed o an
ex ended pe iod pos isola ion, he exp ession le el
o CAR dec eased linea ly wi h he ime o cul u e
(figu e 3). Isle s in ec ed in i o wi h h ee s ains o
CBV-1 e ealed a significan (p<0.001) educ ion in
CAR coding mRNA compa ed wi h unin ec ed con ol
isle s om he same dono s (n=6). The e we e no di e -
ences be ween he CBV-1 s ains in ha espec .
Howe e , in isle s in ec ed wi h echo i us-6-2C, which does
no use CAR as a ecep o , no educ ion o he exp ession
o CAR mRNA was seen compa ed wi h con ol (figu e
4), indica ing ha he e ec was no due o i us-induced
β-cell dea h. Compa ing he exp ession le el o CAR
be ween he di e en CBV-1 s ains and he Echo-6-2C
e ealed ha he di e ence was simila o ha o he di -
e ence be ween he con ol and he CBV-1-in ec ed isle s.
Figu e 2 Mean i us i e inc ease in human isle s, om
con ol dono s, in ec ed in i o wi h CBV-1-3 (n=7), CBV-1-7
(n=6), CBV-1-11 (n=6) and Echo-6-2C (n=5). CBV,
coxsackie i us B.
Figu e 1 (A) Posi i e CAR s aining in one dono wi h
GAD65 and IA-2 au oan ibodies. (B) CAR s aining in isola ed
human isle s, om con ol o gan dono , in ec ed wi h a CBV-1
s ain. CAR, coxsackie–adeno i us ecep o ; CBV,
coxsackie i us B.
4BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219
Isle s udies
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om
CBV-1-11 and CBV-1-3 di e ed somewha mo e compa ed
wi h Echo-6-2C (p<0.009) han CBV-1-7 (p<0.004). In
explan ed isle s cul u ed in UV- ea ed cul u e medium
om in ec ed isle s om o gan dono s, a clea inc ease o
CAR mRNA was seen a e 4 days o cul u e compa ed
wi h isle s cul u ed in UV- ea ed isle cul u e medium
om con ols (figu e 5).
Wes e n blo
The educ ion o he CAR gene seen in isle s in ec ed
wi h s ains o CBV-1 was also confi med on he p o ein
le el. The e was a dec eased exp ession o he CAR
p o ein ela i e o he housekeeping p o ein in isle s
in ec ed wi h CBV-1-11 compa ed wi h ha in mock-
in ec ed isle s om he same dono (figu e 6).
DISCUSSION
In his s udy, we ound ha equency o CAR exp ession
was inc eased in he panc ea ic endoc ine issue om
au oan ibody-posi i e non-diabe ic dono s and subjec s
wi h T1D compa ed wi h con ols. This indica es ha
CAR may p omo e i us in ec ion and up ake in endo-
c ine cells o panc ea ic isle s, p e iously shown o be
pe missi e o i us in ec ion bo h in i o
4
and in i o.
12
The eason o he highe pe cen age CAR posi i i y in
pa ien s wi h T1D and p ediabe es is no known, bu
s udies ha e shown ha CAR exp ession can be induced
by inflamma ion and issue damage.
29
CAR has been
shown o be an inducible p o ein and inc eased exp es-
sion o CAR has been epo ed in myocy es in dila ed
ca diomyopa hy
30
and in hea s om subjec s wi h myo-
ca dial in a c ion.
24
Inc eased CAR exp ession has also
been associa ed wi h an ongoing inflamma ion
29
and
du ing o ma ion o cell- o-cell con ac due o issue
des uc ion.
24 25
In ac , we ound ha UV- ea ed
cul u e supe na an s o CBV-in ec ed isle s induced
simila CAR exp ession on cul u e isle s, sugges ing ha
Figu e 4 Mean CAR mRNA exp ession le els in isola ed
human isle s om con ol dono s in ec ed in i o wi h di e en
EV s ains. CAR exp ession le els we e dec eased in all isle s
in ec ed wi h s ains o CBV-1 compa ed wi h he
mock-in ec ed con ol (p<0.05). In isle s in ec ed wi h he
non-CAR using E6-2C s ain, no a ec on he CAR exp ession
le el was seen. Ba s ep esen mean±SEM. CAR, coxsackie–
adeno i us ecep o ; CBV, coxsackie i us B; E6-2C,
Echo-6-2C; EV, en e o i us.
Figu e 3 mRNA exp ession le els o he gene encoding
CAR in isola ed human isle s om con ol dono s, cul u ed o
p olonged ime (n=2). Isle s we e cul u ed o 1, 4, 8 and
12 days and CAR mRNA exp ession le els we e quan i ied
wi h qPCR. CAR, coxsackie–adeno i us ecep o ; qPCR,
quan i a i e PCR.
Figu e 5 mRNA exp ession le els o he gene encoding
CAR in explan ed human isle s om con ol dono s, cul u ed
in UV- ea ed cul u e medium de i ed om CBV-in ec ed
human isle s and om mock in ec ed isle s. CAR mRNA
exp ession le els we e quan i ied wi h qPCR on day 4 o
cul u e. CAR, coxsackie–adeno i us ecep o ; CBV,
coxsackie i us B; qPCR, quan i a i e PCR; UV, ul a iole .
Figu e 6 Wes e n blo analysis o explan ed human isle s
om con ol dono s in ec ed in i o wi h CBV-1-11 e ealed
dec eased exp ession o he CAR p o ein compa ed wi h
mock-in ec ed isle s om he same dono (n=1). CAR,
coxsackie–adeno i us ecep o ; CBV, coxsackie i us B.
BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 5
Isle s udies
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om

CBV in ec ion in isle s o Lange hans could be one pos-
sible cause o CAR exp ession in isle s. This supe na an
did no con ain in ec ious i us pa icles, sugges ing ha
he e ec was due o some inflamma o y media o s
induced by he i us in in ec ed isle s. P e ious s udies
ha e shown ha CBV in ec ion o human isle s induces
se e al cy okines and chemokines.
36 37
We es ed he
e ec o one o he mos abundan ly p oduced chemo-
kines, CXCL10, on isle s, bu i ailed o induce CAR.
Thus, u he s udies a e needed o iden i y
i us-induced subs ances ha media e induc ion o CAR
on human isle s. In addi ion o i al ac o s, o he
ac o s, such as he au oimmune p ocess i sel , may be
in ol ed.
Rega dless o he cause o he induc ion o he splice
a ian o CAR ha is used by CBV o in ec ion, induc-
ion o his p o ein may make isle mo e suscep ible o
CBV in ec ion and also p omo e he sp ead o ongoing
in ec ion o o he isle s.
17
The exp ession o CAR in isle s om dono s wi h isle
au oan ibodies o T1D sugges s ha isle s in hese indi i-
duals a e po en ially mo e suscep ible o CBV in ec ion.
In humans, in con as o in mice, i seems ha isle o
Lange hans a e he a ge issue o CBV. In con as o
ou findings in humans, in si u hyb idiza ion in panc e-
a ic issue om mice has ailed o show any exp ession
o CAR mRNA in he isle s
38
ha migh explain why
mouse isle s a e no mally spa ed du ing CBV
in ec ion.
38 39
Mos s udies ha e ailed o de ec EV in human exo-
c ine issue, ha is, in neona es wi h sys emic EV in ec-
ions.
4
In i o s udies, in which p ima y human
exoc ine cells we e inocula ed wi h wo s ains o CBV-5,
ailed o show any eplica ion and no i us pa icles
could be de ec ed by elec on mic oscopy in he cells.
12
S aining o he ex acellula domain o CAR on pan-
c ea ic issue sec ions e ealed ha mos non-diabe ic
dono s did no exp ess he ex acellula domain o CAR.
In con as , CAR (p o ein and gene) was exp essed in
explan ed isle s om all dono s es ed. P olonged
cul u e o isola ed isle s e ealed ha he exp ession o
he CAR gene dec eased wi h ime o cul u e, sugges ing
ha he diges i e p ocess o he panc eas du ing isle
isola ion migh cause an induc ion o CAR.
In i o in ec ion o isle s wi h EVs e ealed ha in
isle s in ec ed wi h CAR using i uses, he exp ession o
CAR was dec eased, in con as in ec ion wi h a
non-CAR using EV, he exp ession le el o CAR was no
a ec ed. One possible explana ion o he educed
exp ession could be a species-specific down egula ion o
i us ecep o s, which migh lead o inhibi ion o in ec-
ion by a second i us, he so-called supe in ec ion
exclusion.
40
This phenomenon has also been desc ibed
o se e al o he i uses
41
and migh a o a slow-
g owing, pe sis en s ain. Whe he he down egula ion
is due o s imula ion o ce ain cy okines, which in
i o ha e been shown o dec ease he exp ession le el
o he CAR gene
42
and also he CAR p o ein by
p omo ion o u no e o CAR p o ein h ough ubiqui-
in–p o easome and nuclea ac o kappa-ligh -chain-
enhance o ac i a ed B cells (NFkB)-dependen pa h-
ways
43
o some o he ac o s, emains o be elucida ed.
A mo e likely explana ion migh be ha CAR is down-
egula ed by hsa-miR-466d; his has ecen ly been
shown in a oden b-cell model challenged wi h
CBV-4.
44
In con as o he dec eased exp ession o CAR du ing
an acu e CBV in ec ion in isle endoc ine cells, he
exp ession o CAR in indi iduals wi h acu e EV in ec ion
in myocy es was inc eased.
30
Also in EV-posi i e cases
wi h dila ed ca diomyopa hy, a disease wi h a ch onic EV
in ec ion, he exp ession o he CAR p o ein was
inc eased compa ed wi h con ols.
45
Sys emic EV in ec-
ion has du ing some epidemics esul ed in se ocon e -
sion o isle - ela ed au oan ibodies, indica ing ha i al
eplica ion has aken place in he isle cells, wi h elease
o in acellula p o eins as a esul .
46
I such an in ec ion,
in a subg oup o in ec ed indi iduals, p og essed o a
ch onic phase wi h a low-g ade i al eplica ion and
ongoing issue damage, CAR migh be induced by EV
in ec ion. This has been shown ega ding myoca di is and
i s p og ession in some indi iduals o dila ed myopa hy.
47
In summa y, his s udy shows ha he CAR p o ein is
exp essed in isle s o Lange hans in a highe pe cen age
o indi iduals wi h T1D o a isk o T1D compa ed wi h
non-diabe ic indi iduals. Inc eased exp ession o CAR is
also seen a e ea men wi h p oinflamma o y cy o-
kines/chemokines, and induc ion o his p o ein in pan-
c ea ic isle s in i o migh induce a e iled field and his
may also p omo e he sp ead o CBV o o he isle and
play a ole in i us-induced T1D.
Au ho a ilia ions
1
Depa men o Immunology, Gene ics and Pa hology, Uppsala Uni e si y, The
Rudbeck Labo a o y, Uppsala, Uppland, Sweden
2
Depa men o Mic obiology, Tumo and Cell biology, Ka olinska Ins i u e ,
S ockholm, Sweden
3
Di ision o Paedia ic and Adolescen Medicine, Oslo Uni e si y Hospi al,
Oslo and Facul y o Medicine, Uni e si y o Oslo, Oslo, No way
4
Depa men o Vi ology, Uni e si y o Tampe e, Tampe e, Finland
5
FimlabLabo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland
6
Au oimmuni y Uni , Depa men o Clinical Sciences, Skåne Uni e si y
Hospi al, Lund Uni e si y, Malmo, Sweden
Acknowledgemen s The au ho s hank Inga Hansson o he echnical
assis ance wi h he IHC.
Collabo a o s POD-V Conso ium, h p://www.jd npod.o g/publica ions/
npod- i al-wo k-g oup/.
Funding This wo k was suppo ed by unding om he Eu opean Union’s
Se en h F amewo k P og amme PEVNET (FP7/2007–2013) unde g an
ag eemen numbe 261441 and om a Diabe es Resea ch Wellness
Founda ion Non-clinical Resea ch Fellowship, Ba ndiabe es onden, Swedish
diabe es associa ion and Ne wo k o Panc ea ic O gan Dono s wi h Diabe es
(nPOD), a collabo a i e ype 1 diabe es esea ch p ojec sponso ed by he
Ju enile Diabe es Resea ch Founda ion In e na ional (JDRF). The DiViD s udy
was unded by he No weigian Sou h Eas Hel h Region, The No o No disk
Founda ion and PEVNET. O gan P ocu emen O ganiza ions (OPO) pa ne ing
wi h nPOD o p o ide esea ch esou ces a e lis ed a h p://www.jd npod.o g/
ou -pa ne s.php.
6BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219
Isle s udies
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om
Compe ing in e es s None decla ed.
E hics app o al The e hical commi ee a Oslo uni e si y hospi al and he
e hical commi ee a Uppsala Uni e si y.
P o enance and pee e iew No commissioned; ex e nally pee e iewed.
Da a sha ing s a emen No addi ional da a a e a ailable.
Open Access This is an Open Access a icle dis ibu ed in acco dance wi h
he C ea i e Commons A ibu ion Non Comme cial (CC BY-NC 4.0) license,
which pe mi s o he s o dis ibu e, emix, adap , build upon his wo k non-
comme cially, and license hei de i a i e wo ks on di e en e ms, p o ided
he o iginal wo k is p ope ly ci ed and he use is non-comme cial. See: h p://
c ea i ecommons.o g/licenses/by-nc/4.0/
REFERENCES
1. Kond asho a A, Reunanen A, Romano A, e al. A six- old g adien
in he incidence o ype 1 diabe es a he eas e n bo de o Finland.
Ann Med 2005;37:67–72.
2. Redondo MJ, Je ey J, Fain PR, e al. Conco dance o isle
au oimmuni y among monozygo ic wins. N Engl J Med
2008;359:2849–50.
3. Elshebani A, Olsson A, Wes man J, e al. E ec s on isola ed human
panc ea ic isle cells a e in ec ion wi h s ains o en e o i us
isola ed a clinical p esen a ion o ype 1 diabe es. Vi us Res
2007;124:193–203.
4. Ylipaas o P, Klingel K, Lindbe g AM, e al. En e o i us in ec ion in
human panc ea ic isle cells, isle opism in i o and ecep o
in ol emen in cul u ed isle be a cells. Diabe ologia
2004;47:225–39.
5. Richa dson SJ, Lee e P, Bone AJ, e al. Exp ession o he
en e o i al capsid p o ein VP1 in he isle cells o pa ien s wi h ype 1
diabe es is associa ed wi h induc ion o p o ein kinase R and
down egula ion o Mcl-1. Diabe ologia 2013;56:185–93.
6. Oika inen S, Ma iskainen M, Tau iainen S, e al. En e o i us RNA in
blood is linked o he de elopmen o ype 1 diabe es. Diabe es
2011;60:276–9.
7. Yoon JW, Aus in M, Onode a T, e al. Isola ion o a i us om he
panc eas o a child wi h diabe ic ke oacidosis. N Engl J Med
1979;300:1173–9.
8. Yin H, Be g AK, Tu emo T, e al. En e o i us RNA is ound in
pe iphe al blood mononuclea cells in a majo i y o ype 1 diabe ic
child en a onse . Diabe es 2002;51:1964–71.
9. F isk G, Fohlman J, Kobbah M, e al. High equency o
coxsackie-B- i us-speci ic IgM in child en de eloping ype I diabe es
du ing a pe iod o high diabe es mo bidi y. J Med Vi ol 1985;17:
219–27.
10. K og old L, Edwin B, Buanes T, e al. De ec ion o a low-g ade
en e o i al in ec ion in he isle s o Lange hans o li ing
pa ien s newly diagnosed wi h ype 1 diabe es. Diabe es
2015;64:1682–7.
11. F isk G, Dide holm H. Tissue cul u e o isola ed human panc ea ic
isle s in ec ed wi h di e en s ains o coxsackie i us B4:
assessmen o i us eplica ion and e ec s on isle mo phology and
insulin elease. In J Exp Diabe es Res 2000;1:165–75.
12. Hodik M, Lukinius A, Ko sg en O, e al. T opism analysis o wo
coxsackie B S ains e eals i us g ow h in human p ima y
panc ea ic isle s bu no in exoc ine cell clus e s in i o. Open Vi ol J
2013;7:49–56.
13. Anagandula M, Richa dson S, Skog O, e al. In ec ion o human
isle s o Lange hans wi h wo s ains o coxsackie B i us se o ype
1; assessmen o i us eplica ion, deg ee o cell dea h and induc ion
o genes in ol ed in he inna e immuni y pa hway. J Med Vi ol
2014;86:1402–11.
14. F isk G, El s om T, Dide holm H. The eplica ion o ce ain
coxsackie B i us s ains in CHO cells. J Vi ol Me hods 2001;98:
161–5.
15. Sha en DR, Ba es RC, Ag ez MV, e al. Coxsackie i uses B1, B3,
and B5 use decay accele a ing ac o as a ecep o o cell
a achmen . J Vi ol 1995;69:3873–7.
16. Be gelson JM, Cunningham JA, D ogue G, e al. Isola ion o a
common ecep o o coxsackie B i uses and adeno i uses 2 and
5. Science 1997;275:1320–3.
17. Be gelson JM, K i hi as A, Celi L, e al. The mu ine CAR homolog is
a ecep o o coxsackie B i uses and adeno i uses. J Vi ol
1998;72:415–19.
18. Exco on KJ, Ganseme ND, Mobily ME, e al. Iso o m-speci ic
egula ion and localiza ion o he coxsackie and adeno i us ecep o
in human ai way epi helia. PLoS ONE 2010;5:e9909.
19. Thoelen I, Magnusson C, Tage ud S, e al. Iden i ica ion o
al e na i e splice p oduc s encoded by he human
coxsackie-adeno i us ecep o gene. Biochem Biophys Res
Commun 2001;287:216–22.
20. Do ne A, Xiong D, Couch K, e al. Al e na i ely spliced soluble
coxsackie-adeno i us ecep o s inhibi coxsackie i us in ec ion.
J Biol Chem 2004;279:18497–503.
21. He Y, Chipman PR, Howi J, e al. In e ac ion o coxsackie i us B3
wi h he ull leng h coxsackie i us-adeno i us ecep o . Na S uc
Biol 2001;8:874–8.
22. Wang X, Be gelson JM. Coxsackie i us and adeno i us ecep o
cy oplasmic and ansmemb ane domains a e no essen ial
o coxsackie i us and adeno i us in ec ion. J Vi ol
1999;73:2559–62.
23. Tomko RP, Xu R, Philipson L. HCAR and MCAR: he human and
mouse cellula ecep o s o subg oup C adeno i uses and g oup B
coxsackie i uses. P oc Na l Acad Sci USA 1997;94:3352–6.
24. Fechne H, Nou sias M, Tschoepe C, e al. Induc ion o
coxsackie i us-adeno i us- ecep o exp ession du ing myoca dial
issue o ma ion and emodeling: iden i ica ion o a cell- o-cell con ac -
dependen egula o y mechanism. Ci cula ion 2003;107:876–82.
25. Honda T, Sai oh H, Masuko M, e al. The coxsackie i us-adeno i us
ecep o p o ein as a cell adhesion molecule in he de eloping
mouse b ain. B ain Res Mol B ain Res 2000;77:19–28.
26. Raschpe ge E, Thybe g J, Pe e sson S, e al. The coxsackie- and
adeno i us ecep o (CAR) is an in i o ma ke o epi helial igh
junc ions, wi h a po en ial ole in egula ing pe meabili y and issue
homeos asis. Exp Cell Res 2006;312:1566–80.
27. D esche KM, Kono K, Bopegamage S, e al. Coxsackie i us B3
in ec ion and ype 1 diabe es de elopmen in NOD mice: insuli is
de e mines suscep ibili y o panc ea ic isle s o i us in ec ion.
Vi ology 2004;329:381–94.
28. Hsu KH, Lonbe g-Holm K, Als ein B, e al. A monoclonal an ibody
speci ic o he cellula ecep o o he g oup B coxsackie i uses.
J Vi ol 1988;62:1647–52.
29. I o M, Kodama M, Masuko M, e al. Exp ession o coxsackie i us
and adeno i us ecep o in hea s o a s wi h expe imen al
au oimmune myoca di is. Ci c Res 2000;86:275–80.
30. Nou sias M, Fechne H, de Jonge H, e al. Human
coxsackie-adeno i us ecep o is colocalized wi h in eg ins alpha( )
be a(3) and alpha( )be a(5) on he ca diomyocy e sa colemma and
up egula ed in dila ed ca diomyopa hy: implica ions o ca dio opic
i al in ec ions. Ci cula ion 2001;104:275–80.
31. Kus e K, Koschel A, Rohwe N, e al. Down egula ion o he
coxsackie and adeno i us ecep o in cance cells by hypoxia
depends on HIF-1alpha. Cance Gene The 2010;17:
141–6.
32. Go o M, Eich TM, Felldin M, e al. Re inemen o he au oma ed
me hod o human isle isola ion and p esen a ion o a closed sys em
o in i o isle cul u e. T ansplan a ion 2004;78:1367–75.
33. Be gelson JM, Chan M, Solomon KR, e al. Decay-accele a ing
ac o (CD55), a glycosylphospha idylinosi ol-ancho ed complemen
egula o y p o ein, is a ecep o o se e al echo i uses. P oc Na l
Acad Sci USA 1994;91:6245–8.
34. Moell A, Skog O, Ahlin E, e al. An i i al e ec o nico inamide on
en e o i us-in ec ed human isle s in i o: e ec on i us eplica ion
and chemokine sec e ion. J Med Vi ol 2009;81:1082–7.
35. Nys öm N, Be g T, Lundin E, e al. Human en e o i us species B in
ileocecal Ch on’s disease. Clin T ansl Gas oen e ol 2013 Jun 27;
4:e38.
36. Olsson A, Johansson U, Ko sg en O, e al. In lamma o y gene
exp ession in coxsackie i us B-4-in ec ed human isle s o
Lange hans. Biochem Biophys Res Commun 2005;330:571–6.
37. Anagandula M, Richa dson SJ, Obe s e MS, e al. In ec ion o
human isle s o Lange hans wi h wo s ains o coxsackie B i us
se o ype 1: assessmen o i us eplica ion, deg ee o cell dea h and
induc ion o genes in ol ed in he inna e immuni y pa hway. J Med
Vi ol 2014;86:1402–11.
38. Mena I, Fische C, Gebha d JR, e al. Coxsackie i us in ec ion o
he panc eas: e alua ion o ecep o exp ession, pa hogenesis, and
immunopa hology. Vi ology 2000;271:276–88.
39. Bopegamage S, Ko aco a J, Va go a A, e al. Coxsackie B i us
in ec ion o mice: inocula ion by he o al ou e p o ec s he panc eas
om damage, bu no om in ec ion. J Gen Vi ol 2005;86(P
12):3271–80.
40. Tsche ne DM, E ans MJ, on Hahn T, e al. Supe in ec ion
exclusion in cells in ec ed wi h hepa i is C i us. J Vi ol
2007;81:3693–703.
41. Simon KO, Ca damone JJ J , Whi ake -Dowling PA, e al. Cellula
mechanisms in he supe in ec ion exclusion o esicula s oma i is
i us. Vi ology 1990;177:375–9.
BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219 7
Isle s udies
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om
42. Vincen T, Pe e sson RF, C ys al RG, e al. Cy okine-media ed
down egula ion o coxsackie i us-adeno i us ecep o in endo helial
cells. J Vi ol 2004;78:8047–58.
43. Gao Y, Lui WY. Syne gis ic e ec o in e e on-gamma and umo
nec osis ac o -alpha on coxsackie i us and adeno i us ecep o
exp ession: an explana ion o cell sloughing du ing es icula
in lamma ion in mice. Biol Rep od 2014;90:59.
44. Lam WY, Cheung AC, Tung CK, e al. miR-466 is pu a i e nega i e
egula o o coxsackie i us and adeno i us ecep o . FEBS Le
2015;589:246–54.
45. Liu Q, Su XJ, Yu Y, e al. Co ela ions among pe sis en i al
in ec ion, hea unc ion and Chinese medicine synd omes in
dila ed ca diomyopa hy pa ien s. Chin J In eg Med
2014;20:928–33.
46. Sa mien o L, Cubas-Duenas I, Cab e a-Rode E. E idence o
associa ion be ween ype 1 diabe es and exposu e o en e o i us
in Cuban child en and adolescen s. MEDICC Re 2013;15:29–32.
47. Chapman NM, Kim KS, D esche KM, e al. 5 e minal dele ions in
he genome o a coxsackie i us B2 s ain occu ed na u ally in
human hea . Vi ology 2008;375:480–91.
8BMJ Open Diabe es Resea ch and Ca e 2016;4:e000219. doi:10.1136/bmjd c-2016-000219
Isle s udies
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om
1 diabe es ypeenhanced in panc eas om pa ien s wi h
adeno i us ecep o exp ession is−Coxsackie
L Sa mien o, G F isk and POD-V Conso ium
M Hodik, M Anagandula, J Fuxe, L K og old, K Dahl-Jø gensen, H Hyö y,
doi: 10.1136/bmjd c-2016-000219
2016 4: BMJ Open Diab Res Ca e
h p://d c.bmj.com/con en /4/1/e000219
Upda ed in o ma ion and se ices can be ound a :
These include:
Re e ences #BIBLh p://d c.bmj.com/con en /4/1/e000219
This a icle ci es 46 a icles, 17 o which you can access o ee a :
Open Access
h p://c ea i ecommons.o g/licenses/by-nc/4.0/non-comme cial. See:
p o ided he o iginal wo k is p ope ly ci ed and he use is
non-comme cially, and license hei de i a i e wo ks on di e en e ms,
pe mi s o he s o dis ibu e, emix, adap , build upon his wo k
Commons A ibu ion Non Comme cial (CC BY-NC 4.0) license, which
This is an Open Access a icle dis ibu ed in acco dance wi h he C ea i e
se ice
Email ale ing box a he op igh co ne o he online a icle.
Recei e ee email ale s when new a icles ci e his a icle. Sign up in he
Collec ions
Topic A icles on simila opics can be ound in he ollowing collec ions
(3)Isle s udies
No es
h p://g oup.bmj.com/g oup/ igh s-licensing/pe missions
To eques pe missions go o:
h p://jou nals.bmj.com/cgi/ ep in o m
To o de ep in s go o:
h p://g oup.bmj.com/subsc ibe/
To subsc ibe o BMJ go o:
g oup.bmj.com on Decembe 1, 2016 - Published by h p://d c.bmj.com/Downloaded om