Modulation

of

Aire

regulates

the

expression

of

tissue-restricted

antigens

Intrathymic

expression

of

tissue-restricted

antigens

-LRB-

TRAs

-RRB-

has

been

viewed

as

the

key

element

in

the

induction

of

central

tolerance

and

recently

a

central

role

for

the

autoimmune

regulator

-LRB-

Aire

-RRB-

has

been

suggested

in

this

process

The

aim

of

this

study

was

to

establish

whether

down

or

up-regulation

of

Aire

leads

to

alterations

in

TRA

expression

and

whether

this

is

limited

to

thymic

epithelial

cells

This

study

also

characterized

whether

TRAs

follow

Aire

expression

during

normal

development

and

whether

thymic

microenvironment

plays

a

role

in

the

expression

of

Aire

and

TRAs

We

did

several

in

vivo

and

in

vitro

experiments

to

manipulate

Aire

expression

and

measured

expression

of

four

TRAs

-LRB-

Trefoil

Major

urinary

and

Salivary

-RRB-

by

real-time

RT-PCR

Aire

had

an

allele

dose-dependent

effect

on

TRA

expression

in

the

thymuses

of

mice

from

two

strains

and

Balb/c

but

had

no

effect

on

TRA

expression

in

the

lymph

nodes

In

the

thymus

Aire

and

TRAs

were

both

localized

in

the

medulla

and

were

co-expressed

during

normal

development

and

involution

In

the

primary

stromal

cells

as

well

as

thymic

epithelial

cell

line

the

adenoviral

over-expression

of

Aire

resulted

in

an

increase

in

TRA

expression

By

manipulating

in

vitro

organ-cultures

we

showed

that

thymic

microenvironment

plays

a

dominant

role

in

Aire

expression

whereas

TRAs

follow

the

same

pattern

The

data

underline

a

direct

role

for

Aire

in

TRA

expression

and

suggest

that

modulation

of

Aire

has

a

potential

to

control

central

tolerance

and

autoimmunity

Introduction

Thymus

has

an

essential

role

in

establishing

immune

tolerance

Previous

studies

have

demonstrated

that

tissue-restricted

antigens

-LRB-

TRAs

-RRB-

are

expressed

in

thymus

and

that

this

expression

is

needed

for

the

deletion

of

self-reactive

T

cells

-LRB-

Kyewski

and

Klein

-RRB-

A

central

feature

in

this

process

is

the

promiscuous

expression

of

TRAs

by

epithelial

cells

in

thymic

medullary

region

where

the

TRAs

are

presented

and

encountered

by

the

thymocytes

leading

to

the

induction

of

tolerance

either

by

clonal

deletion

or

functional

inactivation

-LRB-

Derbinski

et

al.

-RRB-

In

this

context

medullary

thymic

epithelial

cells

-LRB-

mTEC

-RRB-

highly

express

MHC

molecules

with

costimulatory

signals

and

act

as

professional

antigen

presenting

cells

-LRB-

APCs

-RRB-

in

thymus

A

detailed

study

of

the

gene

expression

pattern

in

mTEC

revealed

that

many

of

the

TRAs

and

in

particular

almost

all

putative

autoantigen

targets

of

experimental

animal

models

and

human

diseases

are

expressed

by

mTEC

-LRB-

Derbinski

et

al.

Gotter

and

Kyewski

-RRB-

Altogether

the

pool

of

promiscuously

expressed

genes

in

thymus

appears

to

be

highly

diverse

including

tissue

and

sex-specific

genes

and

genes

specifically

involved

in

development

-LRB-

Kyewski

and

Klein

-RRB-

An

important

molecule

in

regulation

of

TRA

expression

in

mTEC

is

autoimmune

regulator

-LRB-

Aire

-RRB-

-LRB-

Nagamine

et

al.

-RRB-

The

Aire

protein

has

several

features

such

as

SAND

and

PHD

finger

domains

that

are

characteristic

to

proteins

involved

in

transcriptional

control

and

has

been

reported

to

bind

directly

to

DNA

-LRB-

Kumar

et

al.

-RRB-

and

to

a

common

transcriptional

regulator

and

histone

acetyltransferase

CREB

binding

protein

-LRB-

CBP

-RRB-

-LRB-

Pitkanen

et

al.

-RRB-

In

the

thymus

and

cell

lines

the

Aire

protein

is

subcellularly

located

to

the

nuclear

bodies

-LRB-

Bjorses

et

al.

Heino

et

al.

-RRB-

which

have

been

associated

with

several

functions

including

modulation

of

chromatin

structure

transcriptional

control

DNA

repair

and

antiviral

response

-LRB-

Everett

and

Chelbi-Alix

-RRB-

Initial

studies

have

shown

the

Aire

protein

to

be

predominantly

expressed

in

mTECs

and

suggest

it

has

a

role

in

regulation

of

immune

tolerance

-LRB-

Blechschmidt

et

al.

Heino

et

al.

-RRB-

In

humans

mutations

in

AIRE

cause

autoimmune-polyendocrinopathy-candidiasis

ectodermal

dystrophy

-LRB-

APECED

-RRB-

a

syndrome

characterized

by

the

presence

of

autoantibodies

to

multiple

self

antigens

and

lymphocytic

infiltration

of

endocrine

glands

leading

to

autoimmune

endocrine

disorders

-LRB-

Perheentupa

Peterson

and

Peltonen

-RRB-

In

agreement

with

the

human

disease

the

Aire

deficient

mice

have

autoantibodies

and

tissue

infiltration

although

the

full

development

of

autoimmune

disease

appears

to

depend

on

the

genetic

background

of

the

mouse

-LRB-

Anderson

et

al.

Kuroda

et

al.

Ramsey

et

al.

-RRB-

The

Aire

deficiency

affects

negative

selection

since

there

is

a

complete

failure

to

delete

the

organ-specific

thymocytes

in

this

mouse

model

-LRB-

Liston

et

al.

-RRB-

More

importantly

the

microarray

analysis

of

mTEC

population

shows

a

decreased

or

abolished

expression

of

multiple

tissue

specific

genes

in

the

Aire

deficient

mouse

suggesting

thus

that

Aire

plays

a

role

in

modulating

TRAs

in

the

mTEC

-LRB-

Anderson

et

al.

Derbinski

et

al.

Jiang

et

al.

-RRB-

This

study

aims

to

further

clarify

whether

Aire

can

directly

regulate

the

TRA

expression

by

analyzing

the

expression

of

four

antigens

in

several

experimental

settings

where

Aire

's

expression

has

been

modulated

The

study

aims

to

establish

whether

there

is

a

dose-dependent

correlation

between

the

number

of

Aire

allele

copies

and

TRA

expression

level

in

thymic

epithelial

cells

and

whether

TRAs

are

co-expressed

with

Aire

during

thymic

development

and

involution

We

also

studied

whether

the

over-expression

of

Aire

as

a

sole

factor

is

sufficient

to

induce

TRA

expression

and

whether

thymic

microenvironment

plays

a

role

in

the

expression

of

Aire

and

TRAs

Material

and

methods

Mice

and

cell

cultures

Aire

deficient

mice

-LRB-

and

Balb/c

background

-RRB-

were

generated

at

The

Walter

and

Eliza

Hall

Institute

-LRB-

Melbourne

Australia

-RRB-

The

inserted

targeting

construct

containing

LacZ

gene

replaced

mouse

Aire

exon

For

genotyping

the

genomic

DNA

was

extracted

using

JetQuick

Tissue

DNA

Spin

Kit

-LRB-

Genomed

-RRB-

and

wild-type

-LRB-

WT

-RRB-

and

knockout

-LRB-

KO

-RRB-

alleles

were

amplified

using

primers

or

As

shown

in

Fig.

primers

pair

and

detect

WT

and

KO

alleles

respectively

Thymuses

from

to

WT

Aire

HET

-LRB-

heterozygote

-RRB-

and

Aire

KO

mice

were

used

Embryonic

-LRB-

and

-RRB-

newborn

neonatal

and

adult

-LRB-

weeks

months

and

months

-RRB-

mouse

tissues

were

used

in

developmental

dynamics

analysis

Mice

were

maintained

at

the

mouse

facility

of

the

Institute

of

Molecular

and

Cell

Biology

Tartu

University

TEC

cell

line

-LRB-

Mizuochi

et

al.

-RRB-

was

kindly

provided

by

G.

Holländer

-LRB-

University

of

Basel

Switzerland

-RRB-

Human

embryonic

kidney

-LRB-

-RRB-

cells

were

cultured

in

Dulbecco

's

modified

Eagle

's

medium

-LRB-

DMEM

-RRB-

supplemented

with

fetal

calf

serum

-LRB-

FCS

-RRB-

U/ml

penicillin

μg/ml

streptomycin

and

μg/ml

amphotericin

B

-LRB-

Gibco

BRL

-RRB-

EGFP

and

Aire

adenovirus

construction

and

infection

The

pAdTrack-CMV

-LRB-

Stratagene

-RRB-

vector

expressing

enhanced

green

fluorescence

protein

-LRB-

EGFP

-RRB-

gene

was

used

as

pAd-GFP

plasmid

The

mouse

Aire

gene

was

amplified

from

pcAire

vector

-LRB-

Heino

et

al.

-RRB-

using

the

primers

tttgtcgac

and

and

cloned

into

SalI

and

NotI

sites

of

pAdTrack-CMV

resulting

in

AdAire-GFP

cells

-LRB-

Invitrogen

-RRB-

which

constitutively

express

AdEasy

deleted

genes

in-trans

were

used

for

expression

analysis

of

adenoviral

vectors

and

for

virus

growth

To

make

recombinant

adenoviruses

pAd-GFP

and

pAdAire-GFP

plasmids

were

recombined

with

The

amplification

and

harvest

process

was

repeated

to

generate

higher

titer

viral

stocks

Subsequently

the

lyzates

from

several

amplification

steps

were

purified

by

CsCl

gradient

centrifugation

-LRB-

He

et

al.

-RRB-

Virus

bands

was

collected

and

mixed

with

preservation

buffer

-LRB-

mM

Tris

pH

mM

NaCl

BSA

glycerol

-RRB-

We

quantified

the

Ad-GFP

and

AdAire-GFP

virus

particles

by

absorbance

measurement

at

nm

to

be

equivalent

to

particles

with

virus

titers

pfu/ml

The

adenoviruses

were

next

verified

for

their

expression

by

infection

of

cells

for

h

and

analyzed

by

immunoblotting

with

rabbit

polyclonal

anti-mouseAire

and

mouse

monoclonal

-LRB-

Ambion

-RRB-

The

signal

was

detected

by

Supersignal

West

Pico

Chemoluminescent

Substrate

-LRB-

Pierce

Biotechnology

-RRB-

according

to

the

manufacturer

's

instructions

For

adenoviral

infection

of

thymic

primary

culture

and

TEC

the

cells

were

infected

with

Ad-GFP

and

AdAire-GFP

at

approximately

confluence

Cells

were

incubated

in

μl

serum-free

OptiMEM

-LRB-

U/ml

penicillin

μg/ml

streptomycin

and

μg/ml

amphotericin

B

-RRB-

and

infected

with

equal

amounts

of

virus

for

h

The

infection

rate

was

further

assessed

by

quantitative

real-time

PCR

analyzing

intake

of

viral

genomic

DNA

Thymic

stromal

cell

isolation

Small

cuts

were

made

into

the

capsules

of

thymi

dissected

from

eight

to

twelve

mice

and

thymocytes

were

released

by

repetitive

pipeting

The

remaining

thymic

fragments

were

incubated

in

mg/ml

dispase/collagenase

-LRB-

Roche

-RRB-

and

μg/ml

DNase

I

-LRB-

AppliChem

-RRB-

in

PBS

at

°C

for

min

with

gentle

agitation

using

a

Pasteur

pipette

every

min

Released

cells

were

collected

to

separate

fractions

and

fresh

enzyme

solution

was

added

four

times

The

cells

were

resuspended

in

mM

EDTA

in

ml

of

Isolated

cells

were

used

either

for

stromal

cell

isolation

or

for

growing

thymic

primary

culture

For

thymic

primary

culture

the

isolated

cells

were

cultured

for

days

in

DMEM

supplemented

with

FCS

For

stromal

cell

isolation

cells

were

counted

in

each

fraction

To

obtain

cells

required

for

isolation

the

final

fraction

was

used

first

and

then

collected

backwards

through

the

collected

fractions

The

fractions

were

pooled

and

passed

through

a

μm

mesh

to

remove

clumps

Cell

sorting

For

depletion

MicroBeads

-LRB-

Miltenyi

Biotec

-RRB-

were

used

according

to

manufacturers

instructions

For

cortical

epithelial

cell

-LRB-

cTEC

-RRB-

isolation

the

cells

were

stained

with

Ab

-LRB-

-RRB-

followed

by

goat

anti-rat

IgG

Microbeads

-LRB-

Miltenyi

Biotech

-RRB-

and

AutoMACS

separation

-LRB-

isolation

mode

Possel-S

-RRB-

The

positive

fraction

-LRB-

-RRB-

contained

magnetically

bound

cTEC-s

For

mTEC

isolation

cells

were

stained

with

-LRB-

anti-EpCAM

generated

from

a

hybridoma

cell

line

-RRB-

followed

by

goat

anti-rat

IgG

Microbeads

-LRB-

Miltenyi

Biotec

-RRB-

and

separation

as

previously

described

The

purity

of

mTEC

and

cTEC

was

as

assessed

by

staining

with

FITC

-LRB-

Miltenyi

Biotech

-RRB-

and

anti-I-Ab

PE

-LRB-

BD

Biosciences

-RRB-

using

FACSCalibur

flow

cytometer

-LRB-

BD

Biosciences

-RRB-

Immunofluorescence

and

microscopy

Cryostat

sections

-LRB-

-RRB-

of

fresh-frozen

mouse

thymus

were

thaw-mounted

onto

SuperFrost

Plus

microscope

slides

-LRB-

Menzel

Gläzer

-RRB-

and

fixed

in

cold

acetone

-LRB-

°C

-RRB-

for

min

Sections

were

permeabilized

in

Triton

normal

goat

serum

-LRB-

DAKO

-RRB-

for

min

Slides

were

blocked

with

normal

goat

sera

for

min

at

room

temperature

and

incubated

with

rat

monoclonal

-LRB-

-RRB-

or

rabbit

polyclonal

anti-mAIRE

-LRB-

-RRB-

and

then

incubated

with

Alexa

Fluor

goat

anti-rat

IgG

-LRB-

H

L

-RRB-

or

Alexa

Fluor

goat

anti-rabbit

-LRB-

Fab

-RRB-

-LRB-

both

from

Molecular

Probes

Eugene

-RRB-

followed

by

washing

six

times

in

PBS

The

slides

were

incubated

with

μg/ml

DAPI

-LRB-

Roche

-RRB-

and

mounted

with

fluorescent

mounting

medium

-LRB-

DAKO

-RRB-

The

images

were

acquired

by

fluorescence

microscopy

-LRB-

Eclipse

Nikon

Melville

NY

-RRB-

Thymic

reaggregate

organ

culture

Reaggregated

thymic

organ-cultures

were

done

as

described

previously

-LRB-

Jenkinson

et

al.

-RRB-

Briefly

thymic

stromal

cells

from

mice

were

prepared

by

disaggregating

fetal

thymic

lobes

which

had

been

previously

cultured

for

days

in

mM

deoxyguanosine

-LRB-

Sigma

St.

Louis

MO

-RRB-

using

trypsin

-LRB-

Life

Technologies

Grand

Island

NY

-RRB-

Reaggregates

were

formed

by

mixing

together

stromal

cells

and

thymocytes

at

a

cell

ratio

and

cultured

for

days

at

°C

Real-time

PCR

RNA

was

isolated

using

TRIzol

-LRB-

Invitrogen

Life

Technologies

-RRB-

and

reverse-transcribed

to

cDNA

using

the

SuperScript

III

Reverse

Transcriptase

-LRB-

Invitrogen

Life

Technologies

-RRB-

Real-time

PCR

was

performed

with

the

ABI

Prism

Fast

Real-Time

PCR

System

instrument

-LRB-

Applied

Biosystems

-RRB-

using

qPCR

SYBR

Green

Core

Kit

-LRB-

Eurogentec

-RRB-

according

to

the

manufacturer

's

instructions

except

that

mM

concentration

was

used

The

amplification

program

included

an

initial

denaturation

step

at

°C

for

min

followed

by

denaturation

at

°C

for

s

and

annealing

and

extension

at

°C

for

min

for

cycles

SYBR

Green

fluorescence

was

measured

after

each

extension

step

and

the

specificity

of

amplification

was

evaluated

by

melting

curve

analysis

Primers

used

to

amplify

specific

gene

products

from

murine

cDNA

were

sense

antisense

Aire

Aire

sense

antisense

sense

antisense

sense

antisense

sense

antisense

The

relative

gene

expression

levels

were

calculated

using

the

comparative

Ct

-LRB-

ΔΔCt

-RRB-

method

-LRB-

according

to

Applied

Biosystems

-RRB-

where

the

relative

expression

is

calculated

as

and

where

Ct

represents

the

threshold

cycle

Every

sample

was

run

in

three

parallel

reactions

Results

Decrease

in

Aire

expression

down-regulates

the

TRA

expression

in

a

dose-dependent

manner

We

initially

set

out

to

confirm

by

real-time

PCR

the

previously

published

array

results

-LRB-

Derbinski

et

al.

-RRB-

demonstrating

decreased

TRA

mRNA

levels

in

the

Aire

deficient

mouse

In

order

to

study

whether

the

self

antigen

expression

is

dependent

on

Aire

we

chose

four

TRAs

and

which

were

downregulated

in

the

Aire

deficient

mouse

according

to

publicly

available

microarray

data

-LRB-

Derbinski

et

al.

-RRB-

Throughout

the

study

we

normalized

our

data

to

the

expression

level

of

keratin

-LRB-

-RRB-

mRNA

which

in

thymus

is

specifically

expressed

in

epithelial

cell

fraction

and

is

not

influenced

by

Aire

gene

expression

-LRB-

Anderson

et

al.

Derbinski

et

al.

-RRB-

The

real-time

PCR

analysis

showed

almost

complete

absence

of

TRA

mRNA

signal

in

Aire

KO

thymus

samples

which

was

uniformly

seen

with

all

four

antigens

studied

-LRB-

Fig.

-RRB-

Furthermore

Aire

allele

dose-dependency

was

observed

as

heterozygous

mouse

thymus

consistently

showed

lower

expression

levels

compared

to

the

WT

thymus

levels

The

expression

level

of

all

four

TRAs

in

heterozygous

mice

thymus

was

approximately

of

the

expression

level

in

WT

mice

In

order

to

determine

whether

Aire

's

effect

on

TRA

expression

depends

on

the

genetic

background

we

also

measured

expression

levels

of

the

four

TRAs

in

Aire

KO

and

Aire

HET

mice

backcrossed

to

Balb/c

WT

mice

-LRB-

Fig.

-RRB-

Again

we

observed

a

clear

allele

dose-dependency

for

all

TRAs

studied

and

almost

no

expression

of

the

TRAs

in

the

Aire

KO

mouse

We

next

determined

whether

Aire

has

a

similar

effect

on

TRA

expression

in

the

lymph

nodes

and

quantified

the

expression

of

TRAs

in

the

lymph

nodes

from

mice

Although

we

could

clearly

detect

Aire

mRNA

in

the

lymph

nodes

at

level

that

was

even

higher

than

the

one

of

the

whole

thymus

-LRB-

Fig.

-RRB-

most

of

the

analyzed

TRAs

were

undetectable

or

close

to

the

detection

limit

The

higher

expression

of

Aire

in

lymph

nodes

was

relative

to

the

epithelial

cell

marker

limiting

the

detection

of

Aire

mRNA

signal

to

the

epithelial

cell

fraction

However

the

mRNA

signal

for

the

was

clearly

present

in

lymph

node

samples

but

interestingly

did

not

depend

on

the

presence

of

Aire

-LRB-

Fig.

-RRB-

In

order

to

establish

whether

Aire

co-localizes

in

the

thymus

with

TRAs

we

purified

the

thymic

mTEC

based

on

the

cell-surface

marker

EpCAM

-LRB-

Fig.

-RRB-

and

analyzed

the

expression

of

the

TRA

genes

As

seen

in

Fig.

the

expression

of

the

and

antigens

was

limited

to

the

mTEC

population

i.e.

the

cell

population

of

Aire

expression

The

cTEC

population

showed

a

very

low

expression

for

all

four

TRAs

both

in

the

WT

as

well

as

Aire

KO

mouse

Collectively

these

data

show

that

Aire

dose-dependently

regulates

TRA

expression

in

thymus

but

not

in

the

lymph

nodes

and

confirms

by

real-time

PCR

the

previously

published

microarray

data

suggesting

that

both

Aire

and

TRAs

are

predominantly

expressed

in

thymus

medullary

epithelium

TRAs

follow

the

expression

of

Aire

during

normal

development

and

involution

Thus

far

the

expression

of

Aire

and

TRAs

has

been

studied

in

fetal

or

neonatal

mice

using

WT

versus

Aire

KO

mice

If

the

expression

of

self

antigens

is

directly

dependent

on

Aire

this

should

be

evident

throughout

the

development

of

thymic

tissue

However

the

thymic

cell

content

and

volume

changes

significantly

during

development

To

limit

our

analysis

to

the

epithelial

cell

subsets

only

we

normalized

our

data

again

to

the

gene

Thymuses

from

different

embryonic

neonatal

young

or

adult

developmental

stages

were

analyzed

for

the

Aire

and

TRA

expression

-LRB-

Fig.

-RRB-

Very

low

Aire

expression

was

detectable

already

at

day

but

showed

a

significant

increase

at

Thus

the

start

of

Aire

expression

coincides

with

the

influx

of

the

first

wave

of

the

hematopoietic

cells

to

the

thymus

We

observed

the

highest

expression

level

at

postnatal

and

a

gradual

decrease

thereafter

until

the

very

last

time-point

studied

Aire

expression

was

however

clearly

present

even

in

mice

The

expression

of

TRAs

closely

followed

the

pattern

of

Aire

reaching

their

peak

at

followed

by

an

obvious

decrease

suggesting

a

role

for

Aire

in

their

regulation

Over-expression

of

Aire

results

in

an

increase

in

TRA

expression

Although

the

lack

of

Aire

has

been

shown

to

have

a

negative

effect

on

TRA

expression

the

ability

of

Aire

as

a

single

factor

to

up-regulate

TRA

expression

has

not

been

demonstrated

We

used

an

adenoviral

expression

system

-LRB-

AdAire-GFP

versus

Ad-GFP

-RRB-

to

determine

whether

the

specific

over-expression

of

Aire

has

any

effect

on

TRAs

Infection

with

the

AdAire-GFP

resulted

in

an

increased

production

of

Aire

protein

-LRB-

Fig.

-RRB-

The

AdAire-GFP

infection

also

resulted

in

an

increase

of

expression

of

all

four

TRAs

studied

which

was

detected

in

primary

thymic

stromal

cells

-LRB-

Fig.

-RRB-

as

well

as

in

thymic

medullary

epithelial

cell

line

TEC

-LRB-

Fig.

-RRB-

The

results

demonstrate

that

Aire

can

indeed

act

as

a

single

inducer

of

TRAs

in

thymic

epithelial

cells

even

in

the

absence

of

signals

from

other

cell-types

normally

present

in

thymus

Thymic

microenvironment

is

needed

for

the

expression

of

Aire

and

TRAs

Although

the

precise

mechanism

is

not

fully

understood

it

has

been

demonstrated

that

the

complex

structure

of

thymus

is

essential

for

mTECs

to

function

properly

-LRB-

Anderson

et

al.

-RRB-

To

study

whether

the

microenvironment

plays

a

role

in

Aire

and

TRA

expression

we

used

ex

vivo

culture

of

thymocyte-depleted

dissaggregated

and

reaggregated

thymic

stromal

cells

As

shown

in

Fig.

the

disaggregation

of

thymus

to

culture

resulted

in

a

dramatic

decrease

in

expression

of

Aire

as

well

as

most

of

the

TRAs

This

effect

was

at

least

partly

reversed

by

reaggregation

suggesting

a

critical

role

for

the

thymic

microenvironment

in

Aire

as

well

as

TRA

expression

Discussion

In

this

paper

we

report

Aire

dependent

expression

of

four

TRAs

in

mouse

thymus

TRA

expression

was

previously

reported

to

be

substantially

decreased

in

Aire

deficient

mTEC

subpopulations

by

microarray

analysis

-LRB-

Anderson

et

al.

Derbinski

et

al.

-RRB-

which

prompted

us

to

follow

the

expression

of

four

TRAs

as

marker

antigens

All

four

genes

have

highly

selective

tissue

specific

expression

For

example

is

restricted

to

mucin

producing

epithelial

cells

with

high

expression

in

stomach

and

intestine

-LRB-

Hoffmann

and

Jagla

Karam

et

al.

-RRB-

is

expressed

in

salivary

and

lacrimal

glands

-LRB-

Dickinson

et

al.

-RRB-

and

is

expressed

in

liver

but

also

in

salivary

lacrimal

and

mammary

glands

-LRB-

Shaw

et

al.

-RRB-

Of

the

two

insulin

genes

present

in

the

mouse

genome

and

encoded

from

separate

loci

we

selected

due

to

its

high

expression

in

the

thymus

as

well

as

pancreatic

beta

cells

and

previously

reported

Aire

dependent

expression

pattern

-LRB-

Chentoufi

and

Polychronakos

Derbinski

et

al.

-RRB-

Furthermore

it

has

been

reported

that

deficient

mouse

with

low

insulin

expression

in

thymus

has

T

cell

reactivity

to

proinsulin

-LRB-

Chentoufi

and

Polychronakos

-RRB-

The

expression

signal

of

all

four

TRAs

tested

was

readily

detectable

in

whole

thymus

and

sorted

mTEC

samples

by

real-time

PCR

analysis

Interestingly

the

TRA

expression

in

heterozygous

mouse

thymus

both

on

and

Balb/c

background

was

repeatedly

only

and

not

of

the

expression

level

seen

in

WT

mouse

which

would

be

expected

when

one

of

the

Aire

genomic

alleles

remains

intact

This

expression

at

lower

levels

than

expected

in

the

heterozygous

mouse

suggests

Aire

haploinsufficiency

in

regulation

of

target

TRA

genes

In

haploinsufficiency

of

transcriptional

regulators

only

one

intact

gene

copy

is

not

sufficient

for

the

functional

activity

of

the

regulated

target

gene

product

The

phenomenon

has

been

explained

by

a

stochastic

expression

model

where

diploid

cells

have

a

higher

probability

than

haploid

cells

in

maintaining

the

abundance

of

an

expressed

gene

product

above

a

low

threshold

level

-LRB-

Cook

et

al.

Kaern

et

al.

-RRB-

Further

support

to

Aire

haploinsufficiency

comes

from

the

report

by

Liston

et

al

-LRB-

Liston

et

al.

-RRB-

demonstrating

that

the

loss

of

one

copy

of

the

Aire

allele

in

TCR-insHEL

double

transgenic

mice

caused

severe

functional

defects

in

negative

selection

of

autoreactive

T

cells

and

resulted

in

pancreatic

cell

insulitis

with

diabetes

incidence

comparable

to

the

Aire

KO

mouse

It

should

be

noted

however

that

the

activation

of

target

genes

by

a

transcriptional

factor

in

vivo

can

be

dependent

on

the

specific

gene

and

the

physiological

context

such

as

in

the

case

of

cardiac

transcription

factor

-LRB-

Jay

et

al.

-RRB-

A

recent

study

reported

that

lymph

node

stroma

can

also

express

Aire

as

well

as

a

range

of

TRAs

and

contributes

accordingly

to

tolerance

induction

-LRB-

Lee

et

al.

-RRB-

Here

we

report

that

although

Aire

as

well

as

expression

is

clearly

present

in

the

lymph

nodes

of

WT

mice

the

lack

of

Aire

does

not

lead

to

a

decrease

in

expression

in

the

Aire

KO

mice

The

data

suggests

that

unlike

in

the

thymus

other

Aire-independent

factors

are

likely

to

control

expression

in

the

lymph

nodes

Regarding

the

rest

of

the

TRAs

studied

we

found

very

low

if

any

expression

by

real-time

PCR

which

did

not

allow

us

to

quantify

the

changes

In

order

to

determine

whether

the

thymic

expression

of

Aire

and

TRAs

follow

the

same

pattern

during

development

we

monitored

the

expression

throughout

mouse

development

from

to

months

We

detected

an

increase

in

Aire

expression

at

day

which

is

in

concordance

with

earlier

reported

results

-LRB-

Sousa

Cardoso

et

al.

-RRB-

The

expression

was

at

its

highest

at

and

decreased

thereafter

but

was

still

present

even

in

mouse

tissue

The

expression

of

TRAs

followed

a

similar

pattern

to

Aire

expression

indicating

a

correlation

between

the

amount

of

Aire

and

TRAs

The

data

demonstrate

that

the

dynamics

of

Aire

and

TRA

expression

closely

follow

the

dynamics

of

thymic

function

in

general

being

most

active

during

the

postnatal

period

and

followed

by

a

gradual

decline

in

activity

-LRB-

Gray

et

al.

-RRB-

Aire

dependent

TRA

expression

is

further

illustrated

by

adenoviral

experiments

enforcing

Aire

expression

in

thymic

epithelial

cells

We

show

that

over-expression

of

Aire

as

a

single

factor

is

sufficient

to

induce

the

expression

of

all

four

TRAs

studied

providing

evidence

that

modulation

of

Aire

can

directly

lead

to

alterations

in

TRA

levels

and

may

thus

also

affect

the

maintenance

of

central

tolerance

The

finding

that

Aire

in

addition

to

primary

thymic

stromal

cells

can

also

modulate

TRA

levels

in

the

thymic

medullary

epithelial

cell

line

suggests

that

there

is

no

need

for

other

cell

types

for

the

Aire-induced

up-regulation

of

TRAs

to

occur

The

disruption

of

normal

thymic

architecture

is

known

to

affect

the

expression

pattern

and

functionality

of

thymus

and

it

has

been

suggested

that

interactions

between

epithelial

cells

and

thymocytes

control

the

development

of

the

thymic

microenvironment

and

T

cell

development

-LRB-

Van

Ewijk

et

al.

-RRB-

Although

the

maturation

of

thymic

epithelial

stroma

during

the

fetal

period

apparently

occurs

independently

of

thymocyte-derived

signals

-LRB-

Jenkinson

et

al.

-RRB-

and

is

mainly

regulated

by

thymic

mesenchyme

-LRB-

Jenkinson

et

al.

-RRB-

thymocytes

deliver

signaling

molecules

which

are

needed

to

maintain

the

normal

adult

thymic

microenvironment

For

example

lymphotoxin

that

signals

through

the

lymphotoxin

receptor

and

directs

the

alternative

NfkappaB

pathway

is

needed

for

development

of

the

thymic

medullary

compartment

Consequently

lymphotoxin

receptor

deficient

mouse

thymus

had

subnormal

levels

of

Aire

and

TRAs

-LRB-

Boehm

et

al.

Chin

et

al.

-RRB-

Thymic

medullary

atrophy

and

lower

expression

of

Aire

and

TRAs

have

been

reported

in

mouse

models

deficient

in

several

genes

involved

in

the

NFkappaB

pathway

such

as

NIK

RelB

or

suggesting

an

important

role

of

this

pathway

in

development

of

thymic

medulla

-LRB-

Akiyama

et

al.

Burkly

et

al.

Kinoshita

et

al.

Zhang

et

al.

-RRB-

A

recent

study

suggests

that

Aire

deficiency

may

also

cause

changes

in

the

organization

and

composition

of

the

medullary

epithelial

compartment

-LRB-

Gillard

et

al.

-RRB-

Thus

it

is

presently

unknown

whether

the

reduced

levels

of

TRA

expression

seen

in

Aire

KO

mice

are

predominantly

the

result

of

changes

in

transcriptional

activity

or

changes

in

thymic

epithelial

cell

development

In

this

study

we

show

a

sustained

Aire

and

TRA

expression

in

treated

FTOC

which

rapidly

disappeared

after

the

disruption

of

the

three-dimensional

thymic

meshwork

into

two-dimensional

culture

Aire

as

well

as

TRA

expression

was

regained

in

RTOC

however

the

presence

of

thymocytes

did

not

further

augment

this

effect

These

results

show

that

Aire

and

TRA

expression

is

dependent

on

the

three-dimensional

structure

of

epithelial

microenvironment

This

expression

however

seems

to

be

independent

of

the

presence

of

thymocytes

being

in

line

with

previous

data

demonstrating

the

Aire

expression

signal

in

RAG

deficient

and

transgenic

mice

in

which

T-cell

development

is

blocked

-LRB-

Jenkinson

et

al.

Zuklys

et

al.

-RRB-

In

conclusion

we

show

that

Aire

has

a

dose-dependent

effect

on

TRA

expression

in

thymus

but

not

in

the

lymph

nodes

Both

Aire

as

well

as

TRAs

localize

in

the

thymic

medulla

and

are

co-expressed

during

normal

development

and

involution

We

also

show

that

Aire

can

directly

induce

TRA

expression

in

medullary

epithelial

cells

although

the

thymic

microenvironment

plays

a

crucial

role

for

the

maximal

expression

to

occur

Our

data

suggest

a

clear

correlation

between

the

expression

of

Aire

and

TRAs

and

indicate

that

approaches

to

stimulate

Aire

expression

in

thymic

epithelium

could

be

considered

to

modulate

tolerance

induction

to

peripheral

antigens

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

autoimmune regulator
thymus
autoimmunity


Respondent-Driven Sampling in a Study of Drug Users in New York City : Notes from the Field Beth Israel Medical Center -LRB- BIMC -RRB- , in collaboration with the Centers for Disease Control -LRB- CDC -RRB- and the New York State Department of Health -LRB- NYSDOH -RRB- , used respondent-driven sampling -LRB- RDS -RRB- in a study of HIV seroprevalence among drug users in New York City in 2004 . We report here on operational issues with RDS including recruitment , coupon distribution , storefront operations , police and community relations , and the overall lessons we learned . Project staff recruited eight seeds from a syringe exchange in Lower Manhattan to serve as the initial study participants . Upon completion of the interview that lasted approximately 1 h and a blood draw , each seed was given three coupons to recruit three drug users into the study . Each of the subsequent eligible participants was also given three coupons to recruit three of their drug-using acquaintances . Eligible participants had to have : injected , smoked or snorted an illicit drug in the last 6 months -LRB- other than marijuana -RRB- , aged 18 or older , adequate English language knowledge to permit informed consent and complete questionnaire . From April to July 2004 , 618 drug users were interviewed , including 263 -LRB- 43 % -RRB- current injectors , 119 -LRB- 19 % -RRB- former injectors , and 236 -LRB- 38 % -RRB- never injectors . Four hundred sixty nine -LRB- 76 % -RRB- participants were men , 147 -LRB- 24 % -RRB- were women , and two -LRB- < 1 % -RRB- were transgender . By race/ethnicity , 285 -LRB- 46 % -RRB- were black , 218 -LRB- 35 % -RRB- Hispanic , 88 -LRB- 14 % -RRB- white , 23 -LRB- 4 % -RRB- mixed/not specified , and four -LRB- < 1 % -RRB- native American . Interviews were initially done on a drop-in basis but this system changed to appointments 1 month into the study due to the large volume of subjects coming in for interviews . Data collection was originally proposed to last for 1 year with a target recruitment of 500 drug users . Utilizing RDS , we were able to recruit and interview 118 more drug users than originally proposed in one quarter of the time . RDS was efficient with respect to time and economics -LRB- we did not have to hire an outreach worker -RRB- and effective in recruiting a diverse sample of drug users . Background A variety of sampling methods have been used to recruit hard-to-reach populations , such as drug users , into research studies . These methods include snowball sampling , targeted sampling , and time-space sampling .1 -- 3 Despite widespread use of these methods , they are not without important limitations . Snowball sampling begins with a set of initial study participants , called seeds , who refer other eligible respondents . Once these persons are interviewed , they are also asked to bring in or provide references for other potential respondents . This process continues until the sample size is achieved . Snowball sampling can provide easy access to hidden populations , but it is most often biased because it is usually done out of convenience rather than randomly .1 -- 3 Targeted sampling involves thoroughly collecting preliminary data to determine various characteristics such as locations where a sample congregates and demographic characteristics such as race , gender , and age .1,2,4 Similar to snowball sampling , using targeted sampling to study drug users may result in a selection bias because less-visible , isolated drug users may not be found at the sites where other drug users are sampled and , thus , may be overlooked . Time-space sampling involves preliminary research by ethnographers to determine when and where hidden populations congregate .3,5 Once a list of times and sites is determined , researchers then randomly choose times to visit those sites to collect data . Time-space sampling has limited use with drug users because making an exhaustive list of where drug users congregate could be a very protracted and expensive process , as drug-use sites change frequently in response to environmental factors such as police presence . Respondent-driven sampling -LRB- RDS -RRB- ,1 a relatively new method being used to draw probability samples of hidden populations , incorporates some methods of snowball sampling , such as chain referral sampling , but includes additional provisions to minimize bias . It produces population estimates that are asymptotically unbiased , which means that bias is only on the order of 1 / -LSB- sample size -RSB- , so bias is minimal in samples of substantial size .6 This allows for probability-based inferences based on the social network of the sample . This paper reports on the use of RDS to recruit participants for a study of risk behavior and HIV seroprevalence among drug users in New York City . The study was conducted by Beth Israel Medical Center in collaboration with the Centers for Disease Control and Prevention and the New York State Department of Health . We discuss the operational issues faced with RDS such as recruitment , coupon distribution , storefront operations , police and community relations , and the overall lessons learned . Methods In April 2004 , Beth Israel Medical Center staff recruited eight seeds from a syringe exchange in Lower Manhattan . Each seed was screened for eligibility before being given a coupon to come back for an interview . The seeds were asked about their drug use , including mode of use and drug preparation techniques . Those who claimed to be an injection drug user -LRB- IDU -RRB- were also asked to show track marks . Although not a requirement of RDS , the seeds were recruited to resemble the race and gender profile of drug users in Lower Manhattan .7 They were asked to come to a research storefront in Lower Manhattan the following day to complete a computer-assisted interviewer-administered personal interview -LRB- CAPI -RRB- and to have their blood drawn for an HIV test . Each seed , and subsequent study subject , received $ 20 compensation for their time . When subjects arrived at the storefront , they were questioned by the study screener to ensure eligibility . Once they were deemed eligible by the screener , each subject was assigned a unique code to serve as their study identification . This code included the following information : the first two letters of the last name , first letter of mother 's first name , the last two digits of the birth year , one letter for the person 's race , and one letter for gender . This code was used to identify blood work and questionnaires and could be regenerated if the subject forgot it . No names or other identifying information were asked . After being assigned a study code , subjects met with an interviewer to be consented and interviewed . The interview had three parts , an hour-long questionnaire , administered by the interviewer , HIV pre-test counseling and a blood draw for an HIV test . The interview consisted of a structured questionnaire which took approximately 1 h and asked about drug-use frequency , drug and sexual risk behavior , syringe acquisition , and knowledge of HIV and hepatitis B and C . After the interview , HIV counseling , and blood collection , each subject was given three coupons to recruit three other drug users into the study . The subjects were briefly trained on how to recruit others , with specific emphasis on the recruitment of friends and acquaintances who use drugs . Eligible respondents had to have done the following : injected , smoked , or snorted an illicit drug in the past 6 months -LRB- those who smoked only marijuana were not eligible for the study -RRB- ; turned age 18 years or older by the time of the interview ; been able to speak English adequately to consent to the study and complete the questionnaire ; and lived , bought , and/or used drugs on the Lower East Side of Manhattan . Subjects could have , however , lived in the larger New York metropolitan area , as long as they bought or used their drugs on the Lower East Side of Manhattan . Coupons contained the time that the storefront opened -LRB- 9:00 a.m. -RRB- and a unique number -LRB- to make each one distinct -RRB- . To make them difficult to duplicate , coupons were printed on thick cardstock paper with color images . To track the coupons and payment for each respondent , we used custom-developed software for RDS called IRIS Plus . Information such as respondent 's unique code , physical traits , coupon number , and the numbers of the coupons each respondent distributed were all recorded in IRIS Plus . This information enabled us to link coupons together , determine when respondents should be paid and who gave coupons to whom . This software also helped to prevent the redemption of duplicated coupons because the database would not accept duplicate coupon numbers . When respondents came in to make an appointment , the coupon was checked in the IRIS Plus database to verify that it had not been used previously . Additionally , each person 's code was checked in IRIS Plus after being screened to determine whether a person with that code had previously been enrolled in the study . If another study subject had that code , the screener looked at the physical traits listed for that person for verification . Additionally , if the screener felt that a person looked familiar , she would search for the person 's physical traits in IRIS Plus to see if another person with similar characteristics was previously enrolled .1 On July 2 , 2004 , we ceased coupon distribution to give potential subjects 2 weeks to redeem remaining coupons . On July 16 , 2004 , we concluded data collection , and the study officially concluded on July 30 , 2004 . The last 2 weeks of July were set aside to allow the remainder of subjects to come in for their HIV test results . Results From April to July 2004 , 618 respondents were interviewed , including 263 -LRB- 43 % -RRB- current injectors -LRB- had injected drugs within the past 6 months -RRB- , 119 -LRB- 19 % -RRB- former injectors -LRB- had injected drugs more than 6 months prior to the interview -- who used drugs in the past 6 months by other modes of administration -RRB- , and 236 -LRB- 38 % -RRB- never injectors -LRB- those that had never injected any drugs but used non-injection drugs in the past 6 months -RRB- . The mean age of respondents was 44 years , with a mean age of first drug use at 19 years and mean age of first injection drug use at 22 years -LRB- among current and former drug injectors -RRB- . By gender , 469 -LRB- 76 % -RRB- were men , 147 -LRB- 24 % -RRB- were women , and two -LRB- < 1 % -RRB- were transgender . By race/ethnicity , 285 -LRB- 46 % -RRB- were black ; 218 -LRB- 35 % -RRB- , Hispanic ; 88 -LRB- 14 % -RRB- , white ; 23 -LRB- 4 % -RRB- , mixed race or unspecified race ; and four -LRB- < 1 % -RRB- , native American . Seed Recruitment To control the flow of recruitment , we recruited only eight seeds : two white men , one white woman , two Hispanic men , one Hispanic woman , one black man , and one black woman , all of whom identified themselves as active IDUs . A relatively small number of seeds was recruited so that we could assess how productive they were and then recruit more if necessary , without being too overwhelmed with respondents . Respondent Recruitment Our initial goal was to interview 500 drug users within 1 year . After 4 months , 618 drug users had been interviewed . Respondent recruitment -LRB- those people recruited after the seeds -RRB- happened much faster than we had expected . In the first couple of weeks , recruitment was slow but quickly picked up as more coupons got out into the community . Interviews were initially done on a drop-in basis , but this system changed to appointments after 1 month into the study due to the large volume of respondents coming in for interviews . As the flow of potential subjects continued to be rapid and at times overwhelming , we were also forced to post-date all coupons by 2 days . Therefore , when a respondent was given a coupon , that person could not be interviewed or make an appointment until the date written on the coupon . This system was helpful in pacing the number of people coming into the storefront for interview appointments but perhaps choosing a smaller number of seeds may have alleviated the need to implement these changes altogether . Police and Community Relations As previously noted , as the study progressed and more coupons were distributed in the community , the number of people coming into the storefront for interviews increased dramatically . For the first 2 weeks , the interviewers would arrive in the morning to find 10 to 20 people waiting for them . People reported arriving as early as 7:00 a.m. so that they could be interviewed that day . Once crowds began to form outside the storefront , neighboring businesses complained to our staff or called the police to file complaints . The police came to speak with staff on three separate occasions regarding complaints of noise and loitering . At that point we decided to move to an all-appointment system . Instead of telling people that their recruits could show up and be interviewed , recruits were told to first come into the storefront to be screened , and that if they passed the screener , they could make an appointment . Lessons Learned Were we to do another RDS study in New York City , we would do the following : We would have a phone number so that screening and appointments for potential participants could be done by telephone . This would lessen the amount of traffic in the storefront and reduce some confusion . Once participants arrived for their interviews , we would then screen them about their drug use . If we were looking to recruit a large number of drug users in a short amount of time , we would again recruit eight seeds . The eight seeds recruited for this study seemed to be an appropriate number because we were trying to maximize our time in the field for data collection . Although the first 2 weeks of the study were relatively slow-paced , it gave the field staff time to adjust to their new environment and new roles and make any necessary changes before they became too overwhelmed . Drug users in New York City are generally a very overstudied population , and thus we expected a large portion of the seeds to show up for their interviews . In cities where the population is not as familiar with research , researchers may want to recruit more seeds , considering that many may not return for the initial interview . Another alternative , however , if time permits , is to recruit a smaller number of seeds , give them a few weeks to return for their interview and then recruit more seeds , if necessary . It was our intention to recruit more seeds if our initial eight were not productive , but this was not necessary . We would start with an appointment-only system . We began with a drop-in system for interviews because our time to collect data was very limited and this was the most efficient use of the interviewers ' time . For instance , if a person dropped in and wanted to be interviewed , we could do it on the spot if the person with a scheduled interview had not shown up . Even though this system worked for us initially , it soon became unwieldy , and we were forced to change a month into the study . People adjusted very quickly , and many people reported being relieved because they did n't have to wait in front of the storefront for 2 h in the morning before staff arrived . We would be very rigid about our appointments . In this study we were very strict about appointment times and conveyed this to each participant . If persons with appointments were more than 5 min late and someone else arrived to schedule an appointment but was free to complete it then , we took the person who dropped in rather than waiting for the person with the scheduled appointment to show up . We rarely had interviewers sitting around with nothing to do . There was almost always someone who dropped in when someone else was late for an appointment . We would meet with local business owners to inform them of who we are and what we were doing prior to the start of the study . We would invite them to tell us if they had any concerns . We would not disclose the type of research being done or with whom , but it would be helpful to let the surrounding business owners know that they may see an increase in the number of people in or around the storefront . We would remain at the storefront 6 weeks after ending coupon distribution . Coupon distribution ended a full 4 weeks before we vacated the storefront , and data collection ended 2 weeks before . The timing of both of these allowed for a fair amount of those with coupons to redeem them , for those with outstanding incentives to collect their money , and for those with outstanding test results to come in for them . Two additional weeks would have been helpful to allow for remaining coupons to be redeemed . Because the storefront we used stayed open for other research studies , we were able to leave contact information for our interviewers in case any respondents showed up for their HIV test results . Within the month after we left the storefront , we received two additional calls for HIV test results . Our interviewers met the respondents at a park and a coffee shop to give them their results . The other storefront staff reported a handful of people coming in with coupons after the study closed , but they did not report any incidents with those people that were unable to redeem them . Finally , we did not pay out any uncollected incentives after July 30 , 2004 . Some of the lessons that we learned about conducting a study using RDS may be specific to New York City . Because there was an abundance of willing and able participants , we had to take several measures to make data collection more manageable . These measures may not be necessary or practical for researchers in cities where the drug-using population is smaller , less willing to participate in research , or both . Conclusions Using RDS , we were able to recruit and interview 118 more drug users than originally proposed in one third of the time . In our experience , RDS was efficient with respect to time and economics -LRB- we did not have to hire an outreach worker -RRB- and effective in garnering a diverse sample of drug users . We were able to interview many more drug users than expected in a short period of time with very minimal recruitment effort . In summary , RDS can be an efficient and effective form of recruitment , particularly for research involving drug users or other hidden populations .

O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O

respondent-driven sampling (rds)
time-space sampling