Evaluation

of

Harmful

Algal

Bloom

Outreach

Activities

With

an

apparent

increase

of

harmful

algal

blooms

-LRB-

HABs

-RRB-

worldwide

healthcare

providers

public

health

personnel

and

coastal

managers

are

struggling

to

provide

scientifically-based

appropriately-targeted

HAB

outreach

and

education

Since

the

Florida

Poison

Information

Center-Miami

with

its

day/year

free

Aquatic

Toxins

Hotline

-LRB-

-RRB-

available

in

several

languages

has

received

over

HAB-related

calls

As

part

of

HAB

surveillance

all

possible

cases

of

HAB-related

illness

among

callers

are

reported

to

the

Florida

Health

Department

This

pilot

study

evaluated

an

automated

call

processing

menu

system

that

allows

callers

to

access

bilingual

HAB

information

and

to

speak

directly

with

a

trained

Poison

Information

Specialist

The

majority

-LRB-

-RRB-

of

callers

reported

satisfaction

with

the

information

and

many

provided

specific

suggestions

for

improvement

This

pilot

study

the

first

known

evaluation

of

use

and

satisfaction

with

HAB

educational

outreach

materials

demonstrated

that

the

automated

system

provided

useful

HAB-related

information

for

the

majority

of

callers

and

decreased

the

routine

informational

call

workload

for

the

Poison

Information

Specialists

allowing

them

to

focus

on

callers

needing

immediate

assistance

and

their

healthcare

providers

These

results

will

lead

to

improvement

of

this

valuable

HAB

outreach

education

and

surveillance

tool

Formal

evaluation

is

recommended

for

future

HAB

outreach

and

educational

materials

Introduction

Harmful

algal

blooms

-LRB-

HABs

-RRB-

are

caused

by

blooms

of

algae

known

as

phytoplankton

these

include

organisms

such

as

dinoflagellates

diatoms

and

cyanobacteria

-LSB-

-RSB-

HABs

can

occur

in

all

aquatic

environments

In

marine

environments

they

are

also

known

as

red

tides

because

some

of

these

organisms

can

change

the

color

of

the

water

to

red

or

brown

HABs

may

cause

harm

to

the

environment

and

other

organisms

in

two

ways

First

through

the

severe

overgrowth

of

the

HAB

organisms

that

depletes

oxygen

in

the

local

environment

and

second

when

the

HAB

organisms

produce

extremely

potent

natural

toxins

Phytoplankton

are

the

base

of

the

marine

food

web

thus

the

toxins

they

produce

can

bioconcentrate

in

organisms

higher

up

in

the

food

chain

Several

human

illnesses

are

caused

by

the

ingestion

of

seafood

contaminated

with

the

natural

toxins

produced

by

the

HAB

organisms

-LSB-

-RSB-

In

addition

to

exposure

through

seafood

ingestion

environmental

exposures

can

occur

through

skin

contact

with

contaminated

water

or

by

inhalation

when

the

HAB

organisms

are

broken

up

by

waves

and

their

toxins

become

aerosolized

For

example

human

exposure

to

aerosols

containing

brevetoxins

from

the

Florida

red

tide

dinoflagellate

Karenia

brevis

has

been

associated

with

reports

of

respiratory

distress

particularly

but

not

exclusively

in

persons

with

asthma

as

well

as

increased

emergency

room

admissions

for

pneumonia

bronchitis

and

asthma

among

coastal

residents

during

active

Florida

red

tides

-LSB-

-RSB-

With

the

increasing

number

of

persons

interacting

with

the

coastal

areas

-LRB-

both

freshwater

and

marine

-RRB-

and

with

the

apparent

increase

of

HABs

around

the

world

healthcare

providers

public

health

personnel

and

coastal

managers

are

struggling

to

provide

scientifically-based

information

-LSB-

-RSB-

There

is

a

paucity

of

appropriately

targeted

outreach

and

educational

materials

for

healthcare

providers

and

persons

with

possible

exposure

to

the

marine

and

freshwater

toxins

as

well

as

coastal

resource

managers

the

media

and

the

general

public

A

variety

of

educational

outreach

materials

and

services

have

been

created

but

there

has

been

almost

no

formal

evaluation

to

determine

whether

these

materials

are

reaching

their

target

audiences

and

meeting

these

audiences

expectations

Florida

Poison

Information

Centers

The

Florida

Poison

Information

Center/Miami

-LRB-

FPIC/Miami

-RRB-

is

one

of

three

poison

centers

created

in

the

State

of

Florida

in

by

an

act

of

the

Florida

Legislature

-LRB-

FS

-RRB-

and

it

has

rapidly

grown

into

a

cost-effective

model

of

a

poison

center

system

The

FPIC/Miami

is

located

at

the

University

of

Miami

Miller

School

of

Medicine

and

Jackson

Health

Systems

and

directly

serves

the

residents

of

Southeastern

Florida

-LRB-

i.e.

Miami-Dade

Broward

Monroe

Palm

Beach

Lee

and

Collier

counties

-RRB-

and

beyond

with

toll-free

hour/day

services

in

several

languages

For

example

in

the

FPIC/Miami

responded

to

over

calls

of

these

calls

-LRB-

-RRB-

were

for

known

or

suspected

exposures

to

toxic

substances

Approximately

of

the

patients

calling

from

home

were

managed

in

the

home

without

the

need

for

an

emergency

department

visit

The

FPIC/Miami

placed

-LRB-

-RRB-

follow-up

calls

to

continually

assess

this

home

management

The

FPIC/Miami

toxicologists

provided

over

consultations

with

Florida

's

physicians

to

assist

in

the

care

of

hospitalized

patients

In

an

attempt

to

prevent

poisonings

the

FPIC/Miami

educators

coordinated

outreach

and

education

programs

for

the

lay

public

health

professional

and

the

media

They

also

distributed

over

informational

brochures

handouts

and

telephone

stickers

reaching

over

persons

Since

with

funding

from

the

Florida

Department

of

Health

the

National

Center

for

Environmental

Health

-LRB-

NCEH

-RRB-

of

the

Centers

for

Disease

Control

and

Prevention

-LRB-

CDC

-RRB-

and

the

University

of

Miami

National

Institute

of

Environmental

Health

Sciences

-LRB-

NIEHS

-RRB-

Marine

and

Freshwater

Biomedical

Sciences

Center

and

the

National

Science

Foundation

-LRB-

NSF

-RRB-

NIEHS

Oceans

and

Human

Health

Center

the

FPIC-Miami

established

a

toll-free

hotline

-LRB-

-RRB-

dedicated

to

providing

information

concerning

aquatic

health

issues

All

calls

are

answered

by

Poison

Information

Specialists

who

are

highly

trained

physicians

and

nurses

All

calls

are

reported

as

a

form

of

passive

surveillance

of

HAB-related

illness

and

information

requests

to

the

Aquatic

Toxins

Program

-LRB-

-RRB-

and

to

the

Foodborne

Illness

Program

of

the

Florida

Department

of

Health

Recently

the

Hotline

was

expanded

to

include

additional

options

for

callers

Using

an

automated

call

processing

menu

system

the

Aquatic

Toxins

Hotline

now

allows

callers

to

access

information

in

English

or

Spanish

about

the

possible

health

effects

and

locations

of

the

Florida

red

tide

-LRB-

including

the

National

Atmospheric

and

Oceanographic

Administration

-LRB-

NOAA

-RRB-

Harmful

Algae

Bloom

-LRB-

HAB

-RRB-

Bulletin

-RRB-

information

on

ciguatera

fish

poisoning

blue

green

algae

-LRB-

cyanobacteria

-RRB-

and

resources

for

learning

about

general

marine

toxin

issues

as

well

as

still

giving

the

callers

the

opportunity

to

select

to

speak

directly

to

a

Poison

Information

Specialist

if

they

wish

These

materials

were

reviewed

and

developed

with

cooperation

between

the

FPIC-Miami

the

University

of

Miami

the

Florida

Department

of

Health

the

CDC

Mote

Marine

Laboratory

and

the

Florida

Fish

and

Wildlife

Commission

Concurrent

with

this

initiative

a

number

of

academic

public

health

and

community

partners

-LRB-

including

Solutions

to

Avoid

Red

Tide

or

START

-RRB-

collaborated

to

create

a

series

of

informational

signs

advertising

the

Aquatic

Toxins

Hotline

as

a

source

of

information

for

Florida

red

tide

After

focus

groups

with

public

health

personnel

beach

managers

hotel

and

restaurant

owners

and

tourist

boards

these

signs

were

designed

to

be

posted

on

and

near

beaches

and

targeted

at

beach

users

including

tourists

-LRB-

see

Figure

-RRB-

This

Pilot

Study

evaluated

whether

the

new

automated

system

was

used

by

callers

and

whether

the

callers

considered

the

information

to

be

useful

to

them

as

well

as

to

collect

caller

recommendations

for

the

improvement

of

this

service

The

goal

of

this

pilot

study

was

to

evaluate

whether

the

Florida

Aquatic

Toxins

Hotline

was

serving

the

self-identified

needs

of

current

callers

Methods

The

FPIC-Miami

receives

the

telephone

numbers

of

all

callers

to

the

Aquatic

Toxins

Hotline

whether

the

caller

speaks

with

a

Poison

Information

Specialist

or

just

the

automated

system

In

this

evaluation

telephone

numbers

from

the

calls

made

to

the

Aquatic

Toxins

Hotline

from

September

through

January

were

re-contacted

Of

note

throughout

this

study

time

period

there

was

an

active

Florida

red

tide

on

the

West

Coast

of

Florida

This

evaluation

aimed

to

determine

Basic

Caller

demographics

What

information/services

callers

were

seeking

Which

menu

selections

were

the

most

commonly

chosen

Which

menu

options

in

the

caller

's

opinion

were

the

most

helpful

If

callers

were

satisfied

with

the

service

provided

by

the

Aquatic

Toxins

Hotline

If

callers

would

call

again

What

would

in

the

callers

opinion

make

the

Aquatic

Toxins

Hotline

service

more

useful

Trained

interviewers

contacted

the

Aquatic

Toxins

Hotline

callers

by

phone

in

English

and

Spanish

to

explain

the

study

obtained

verbal

consent

for

participation

and

conducted

a

short

scripted

interview

-LRB-

available

on

request

from

the

corresponding

author

-RRB-

Specifically

the

interviewers

asked

a

brief

series

of

questions

about

using

the

Aquatic

Toxins

Hotline

and

about

the

caller

's

perception

of

the

system

's

usefulness

Study

participants

also

had

an

opportunity

to

make

suggestions

to

improve

the

system

No

personal

identifying

information

was

collected

To

maximize

success

in

contacting

callers

the

interviewers

called

each

telephone

number

three

times

-LRB-

once

during

a

week

day

once

during

a

week

night

and

once

during

the

weekend

-RRB-

in

an

attempt

to

re-contact

the

specific

person

who

had

called

the

Aquatic

Toxins

Hotline

This

study

was

approved

by

both

the

University

of

Miami

and

Florida

Department

of

Health

Human

Subject

Committees

The

data

were

collected

into

a

Study

Access

Database

The

data

were

analyzed

using

SAS

version

The

data

were

analyzed

by

frequencies

as

a

descriptive

process

Results

The

Aquatic

Toxins

Hotline

has

experienced

increasing

use

over

the

past

few

years

particularly

during

periods

of

active

Florida

red

tides

Since

its

inception

in

there

have

been

calls

to

the

Hotline

with

occurring

between

and

during

alone

calls

were

made

to

the

Aquatic

Toxins

Hotline

During

the

study

period

from

Sept

through

Jan

there

were

calls

made

to

the

hotline

with

-LRB-

-RRB-

answered

by

a

Poison

Information

Specialist

of

these

calls

were

for

Florida

red

tide

for

ciguatera

for

jellyfish

each

for

PSP/pufferfish

scombroid

and

stingrays

and

blue

green

algae

This

means

that

only

of

the

callers

selected

to

speak

with

a

Poison

Information

Specialist

from

the

automated

HAB

Menu

in

English

or

Spanish

while

the

vast

majority

-LRB-

-RRB-

only

accessed

the

automated

HAB

menu

The

Interviewers

attempted

to

contact

each

of

the

callers

during

the

study

period

as

described

above

Even

though

the

follow-up

calls

were

made

within

two

months

of

the

original

contact

only

a

small

percentage

-LRB-

-RRB-

of

the

callers

was

able

to

be

re-contacted

Inability

to

re-contact

these

individuals

was

due

to

a

variety

of

factors

such

as

no

answer

despite

repeated

calls

answering

machines

and

no

real

contact

number

-LRB-

i.e.

original

call

made

from

hotel

hospital

etc

-RRB-

Nevertheless

once

contacted

all

callers

-LRB-

-RRB-

agreed

to

participate

A

total

of

callers

were

successfully

re-contacted

and

agreed

to

participate

Of

these

only

callers

reported

that

they

recalled

making

a

call

to

the

Florida

Aquatic

Toxins

Hotline

and

thus

these

callers

were

considered

the

participants

in

this

Pilot

Study

Of

note

both

the

actual

number

of

respondents

and

the

percentages

are

reported

below

because

not

all

subjects

answered

all

the

questions

The

majority

of

the

participants

were

women

-LRB-

-LSB-

-RSB-

-RRB-

self-described

as

white

-LRB-

-LSB-

-RSB-

-RRB-

and

non

Hispanic

-LRB-

-LSB-

-RSB-

-RRB-

whose

primary

language

was

English

-LRB-

-LSB-

-RSB-

-RRB-

and

with

a

mean

age

and

standard

deviation

of

years

-LRB-

see

Table

-RRB-

The

participants

were

predominantly

from

Florida

-LRB-

-LSB-

-RSB-

-RRB-

with

the

majority

from

the

West

coast

of

Florida

-LRB-

-LSB-

-RSB-

-RRB-

particularly

in

Sarasota

the

area

of

recurrent

Florida

red

tide

There

were

-LRB-

-RRB-

callers

from

other

parts

of

the

US

including

from

the

US

Virgin

Islands

-LRB-

data

not

shown

-RRB-

The

majority

of

the

participants

had

heard

about

the

Aquatic

Toxins

Hotline

from

a

website

-LRB-

-LSB-

-RSB-

-RRB-

newspaper

or

magazine

article

-LRB-

-LSB-

-RSB-

-RRB-

a

friend

or

personal

recommendation

-LRB-

-LSB-

-RSB-

-RRB-

or

a

sign

-LRB-

-LSB-

-RSB-

-RRB-

Many

of

the

participants

-LRB-

-LSB-

-RSB-

-RRB-

called

with

general

information

questions

of

note

of

the

participants

reported

calling

because

they

or

someone

they

cared

for

was

sick

or

believed

they

had

been

exposed

to

a

toxin

Overall

the

majority

of

participants

-LRB-

-LSB-

-RSB-

-RRB-

reported

that

they

received

an

answer

to

the

question

that

prompted

their

call

to

the

Florida

Aquatic

Toxins

Hotline

-LRB-

see

Table

-RRB-

Furthermore

-LRB-

-RRB-

reported

that

the

Florida

Aquatic

Toxins

Hotline

was

easy

to

use

with

only

-LRB-

-RRB-

reporting

otherwise

Finally

-LRB-

-RRB-

of

the

participants

reported

that

they

would

be

likely

to

use

the

Florida

Aquatic

Toxins

Hotline

again

and

-LRB-

-RRB-

stated

that

they

would

recommend

the

Hotline

to

others

With

regards

to

the

specific

information

supplied

by

the

automated

system

-LRB-

-RRB-

of

participants

reported

speaking

with

a

Poison

Information

Specialist

among

these

participants

-LRB-

-RRB-

were

satisfied

and

only

two

-LRB-

-RRB-

were

dissatisfied

-LRB-

see

Table

-RRB-

Among

those

participants

who

used

the

automated

HAB

menu

the

largest

number

-LRB-

-LSB-

-RSB-

-RRB-

reported

listening

to

the

NOAA

HAB

Bulletin

which

gives

current

and

forecasted

information

on

the

location

of

the

Florida

red

tides

among

these

participants

-LRB-

-RRB-

were

satisfied

and

seven

-LRB-

-RRB-

were

dissatisfied

The

second

largest

group

-LRB-

-LSB-

-RSB-

-RRB-

reported

listening

to

general

information

about

Florida

red

tides

among

these

participants

-LRB-

-RRB-

expressed

satisfaction

and

-LRB-

-RRB-

were

dissatisfied

Participants

also

reported

listening

to

information

about

other

marine

toxin

issues

-LRB-

-LSB-

-RSB-

-RRB-

with

-LRB-

-RRB-

expressing

satisfaction

Ciguatera

fish

poisoning

-LRB-

-LSB-

-RSB-

-RRB-

with

-LRB-

-RRB-

expressing

satisfaction

and

blue

green

algae

-LRB-

cyanobacteria

-RRB-

-LRB-

-LSB-

-RSB-

with

-LRB-

-RRB-

expressing

satisfaction

At

the

end

of

the

questionnaire

the

participants

were

asked

if

there

was

anything

in

particular

they

thought

should

be

done

to

improve

the

usefulness

of

the

Florida

Freshwater

and

Marine

Health

Hotline

as

an

open

ended

question

The

majority

of

the

comments

can

be

summarized

as

a

-RRB-

wanting

more

up-to-date

information

-LRB-

particularly

geographic

location

of

the

Florida

red

tides

-RRB-

on

even

a

daily

basis

and

b

-RRB-

wanting

to

speak

with

a

person

directly

or

listening

to

an

automated

system

Specifically

the

participants

recommended

that

the

Hotline

provide

the

geographic

location

of

the

Florida

red

tides

on

a

map

provide

more

information

to

tourists

in

hotels

and

make

sure

that

the

Poison

Information

Specialists

were

up-to-date

with

HAB

and

Florida

red

tide

information

Discussion

This

study

was

the

first

known

systematic

evaluation

of

the

use

of

and

satisfaction

with

educational

outreach

materials

for

HABs

specifically

evaluating

callers

to

an

automated

Florida

Aquatic

Toxins

Hotline

of

the

South

Florida

Poison

Information

Center

The

majority

-LRB-

-RRB-

of

participants

reported

that

they

were

satisfied

with

the

information

provided

by

the

Aquatic

Toxins

Hotline

that

they

would

use

the

Hotline

again

and

that

they

would

recommend

the

Hotline

to

others

Furthermore

even

when

evaluating

the

specific

services

offered

by

the

Aquatic

Toxins

Hotline

automated

system

-LRB-

including

speaking

directly

with

a

Poison

Information

Specialist

-RRB-

the

high

level

of

satisfaction

persisted

Of

interest

only

of

participants

reporting

hearing

about

the

Aquatic

Toxins

Hotline

from

signs

with

the

rest

learning

about

the

hotline

from

a

website

-LRB-

-RRB-

newspaper

or

magazine

article

-LRB-

-RRB-

or

friend

-LRB-

-RRB-

Our

evaluation

demonstrated

that

the

primary

advantage

of

the

new

automated

system

is

that

it

provided

useful

HAB

information

for

the

large

majority

of

the

callers

without

requiring

contact

with

a

Poison

Information

Specialist

By

decreasing

the

workload

of

purely

informational

calls

for

the

Poison

Information

Specialists

the

Hotline

allows

the

Specialists

to

focus

on

those

persons

who

need

immediate

assistance

Limitations

As

seen

in

previous

studies

using

the

Poison

Information

data

-LSB-

-RSB-

only

a

small

percentage

-LRB-

-RRB-

of

the

callers

were

able

to

be

re-contacted

This

lack

of

participation

in

telephone

surveys

is

part

of

an

unfortunate

and

growing

nationwide

trend

recognized

by

the

evaluation

research

community

-LSB-

-RSB-

Furthermore

the

majority

of

the

study

participants

-LRB-

-RRB-

gave

zip

codes

of

addresses

within

Florida

since

the

follow-up

calls

were

made

two

months

from

the

original

call

it

would

appear

that

the

majority

of

the

participants

were

either

part-time

seasonal

-LRB-

i.e.

snow-birds

-RRB-

or

permanent

residents

Based

on

the

review

of

the

zip

codes

and

area

codes

of

all

Hotline

callers

many

were

likely

tourists

and

thus

were

lost

to

follow-up

when

they

could

not

be

reached

two

months

after

their

original

call

to

the

Hotline

-LSB-

-RSB-

In

addition

these

participants

were

not

entirely

representative

of

the

overall

Hotline

caller

population

since

a

greater

proportion

-LRB-

-RRB-

reported

speaking

with

a

Poison

Information

Specialist

than

was

reported

-LRB-

-RRB-

for

the

entire

population

of

callers

to

the

Florida

Aquatic

Toxins

Hotline

during

the

study

period

However

based

on

the

limited

demographic

information

the

participants

interviewed

were

similar

to

those

contacted

in

other

studies

which

have

used

the

overall

South

Florida

Poison

Information

Center

Caller

Database

i.e.

predominantly

white

non

Hispanic

older

females

-LSB-

-RSB-

We

did

not

identify

many

Hispanic

callers

in

this

study

Of

note

most

of

the

relevant

marine

HAB

activity

is

concentrated

on

the

Gulf

Coast

while

the

majority

of

Florida

's

Hispanic

population

is

concentrated

on

the

east

coast

of

Florida

Finally

there

were

missing

data

-LRB-

e.g.

out

of

persons

did

not

report

their

gender

-RRB-

Discussion

and

Recommendations

This

Pilot

Evaluation

Study

demonstrated

that

the

additional

outreach

provided

by

the

Hotline

was

successful

in

getting

information

to

people

who

wanted

it

It

also

suggested

that

there

needs

to

be

increased

outreach

and

education

efforts

using

a

range

of

media

to

inform

additional

target

populations

about

the

Aquatic

Toxins

Hotline

such

as

residents

and

tourists

and

their

healthcare

providers

For

example

it

has

been

shown

in

the

case

of

ciguatera

fish

poisoning

that

even

in

an

endemic

area

such

as

Miami

-LRB-

Florida

-RRB-

the

majority

of

healthcare

providers

do

not

recognize

nor

-LRB-

more

important

from

the

point

of

view

of

HAB

surveillance

-RRB-

do

they

report

cases

of

ciguatera

to

the

Health

Department

even

though

it

is

a

reportable

illness

throughout

the

US

-LSB-

-RSB-

Therefore

additional

outreach

and

education

not

only

to

the

possible

victims

of

HAB-related

illness

but

also

for

their

healthcare

providers

is

needed

Recommendations

for

improving

the

Aquatic

Toxins

Hotline

automated

HAB

system

include

the

following

-RRB-

Existing

automated

materials

need

to

be

revised

to

incorporate

an

ongoing

evaluation

component

with

a

focus

on

tourist

not

just

resident

callers

and

in

coordination

with

ongoing

efforts

in

providing

geographic

location

-LSB-

-RSB-

-RRB-

Existing

materials

will

need

constant

updating

and

-RRB-

Ongoing

training

should

be

provided

for

the

Poison

Information

Specialists

-LRB-

as

well

as

other

healthcare

providers

-RRB-

concerning

the

possible

exposures

and

health

effects

from

the

aquatic

toxins

as

new

HAB

research

data

and

resources

are

made

available

-LSB-

-RSB-

In

addition

as

different

HAB

educational

outreach

materials

and

services

are

created

these

materials

and

services

need

to

be

evaluated

before

and

after

implementation

in

the

target

populations

to

improve

their

quality

and

utility

Based

on

the

participation

rate

in

this

study

rapid

follow

up

is

necessary

to

ensure

an

effective

evaluation

This

will

require

the

incorporation

of

additional

resources

and

collaboration

with

investigators

with

expertise

in

outreach

education

and

evaluation

in

future

HAB

activities

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

poison information centers
outreach and education
florida red tide
ciguatera fish poisoning
blue green algae
cyanobacteria
brevetoxins
karenia brevis

harmful algal bloom (hab)
ciguatoxins
human health effects
neurotoxic shellfish poisoning (nsp)
paralytic shellfish poisoning (psp)
solutions to avoid red tide (start)


C1q deficiency promotes the production of transgenic-derived IgM and IgG3 autoantibodies in anti-DNA knock-in transgenic mice C1q-deficient mice have been shown to develop a lupus-like disease and to display an impaired clearance of apoptotic cells that are enriched in lupus autoantigens . However , the role of C1q in the regulation of autoreactive B cells remains debatable . To explore this we crossed MRL/Mp C1q-deficient mice with knock-in transgenic -LRB- Tg -RRB- mice expressing an anti-ssDNA antibody -LRB- VH3H9R and VH3H9R/VLκ8R -RRB- . Analysis of the VH3H9R mice showed that in the absence of C1q higher titres of Tg-derived IgM and IgG3 anti-ssDNA antibodies were detectable . In contrast , in the VH3H9R/VLκ8R C1q-deficient animals no increase in Tg antibody levels was observed . In both models the lack of C1q induced a marked reduction of marginal zone B cells and this was paralleled by a significant increase in the percentage of plasmocytes . Thus , one could postulate that in the absence of C1q the failure to clear efficiently dying cells provides an additional stimulus to the autoreactive Tg B cells resulting in their emigration from the marginal zone B cell compartment with subsequent increase in plasmocytes . However , the lack of C1q led to an increased production of Tg IgM and IgG3 antibodies only in VH3H9R mice indicating that additional genetic susceptibility factors are required to break self-tolerance . 1 Introduction Hereditary deficiencies of early components of the classical pathway of the complement system are known to predispose to systematic lupus erythematosus -LRB- SLE -RRB- . Among these , C1q deficiency exhibits the strongest association with prevalence greater than 90 % suggesting that a physiological activity of the early part of the classical pathway normally protects against the development of SLE -LRB- Pickering et al. , 2000 -RRB- . Mice with targeted deletion of the C1q gene -LRB- C1qa − / − -RRB- developed a spontaneous lupus-like disease characterised by the development of anti-nuclear autoimmunity and glomerulonephritis associated with the presence of multiple apoptotic bodies -LRB- Botto et al. , 1998 -RRB- . Introgression of C1q deficiency onto different genetic backgrounds revealed that in mice C1q operates as a disease modifier . C57BL/6 . C1qa − / − mice displayed no increase of IgG autoantibodies -LRB- autoAbs -RRB- or glomerulonephritis -LRB- Mitchell et al. , 2002 -RRB- , whilst C1q deficiency backcrossed onto the lupus-prone MRL/Mp background accelerated both the onset and the severity of the autoimmune disease -LRB- Mitchell et al. , 2002 -RRB- . Consistent with these observations , C1q reconstitution by bone marrow transplant attenuated the autoimmune disease present in MRL/Mp . C1qa − / − mice -LRB- Cortes-Hernandez et al. , 2004 -RRB- . Currently , there are two main hypotheses to explain the role of complement in the development of SLE , neither of which is mutually exclusive . The first model , defined as the tolerance hypothesis , proposes a role for complement in determining the activation thresholds of lymphocytes , whereby complement enhances presentation of autoantigens to self-reactive immature B cells resulting in their elimination -LRB- Prodeus et al. , 1998 -RRB- . The second one , known as the ` waste disposal ' hypothesis , suggests that in addition to its role in the clearance of immune complexes , complement is involved in the physiological disposal of apoptotic cells -LRB- Botto et al. , 1998 -RRB- , that have been shown to express lupus autoantigens on their surface -LRB- Casciola-Rosen et al. , 1994 -RRB- . C1q plays a significant role in the clearance pathway of cellular debris by binding directly or indirectly to apoptotic blebs where it activates complement and mediates phagocytosis by professional and non-professional phagocytes -LRB- Korb and Ahearn , 1997 ; Mevorach et al. , 1998 ; Nauta et al. , 2004 ; Quartier et al. , 2005 ; Taylor et al. , 2000 -RRB- . Therefore , improper removal of dying cells in the setting of C1q deficiency could result in the stimulation of autoreactive cells leading to autoimmunity . Immunoglobulin Tg models have been instrumental in understanding B cell regulation revealing several key mechanisms , including receptor editing , deletion , anergy and ignorance . The hen egg lysozyme -LRB- HEL -RRB- -- anti-HEL -LRB- IgHEL -RRB- Tg model in particular has been widely used to demonstrate elimination of self-reactive clones by membrane-bound expressed antigen , anergy induction by soluble antigen -LRB- sHEL -RRB- or ignorance when the amount of antigen is so low that it does not reach the threshold to induce anergy . More recently , it has been shown that intracellular membrane-bound HEL failed to induce tolerance and was instead autoimmunogenic positively selecting IgHEL B1 cells and inducing large numbers of IgM autoantibody-secreting plasma cells -LRB- reviewed in Ferry et al. , 2006 -RRB- . Taken together , these findings suggest that in this model the fate of self-reactive B cells is determined not only by the abundance , the avidity of the target self-antigen and the affinity of the B cell receptor but also by the location of the `` auto '' - antigen . The role of complement has been tested in the IgHEL Tg -LRB- MD4 -RRB- -- sHEL -LRB- ML5 -RRB- model by crossing the double Tg mice with mice deficient in C1q , C4 , C3 or CD21/CD35 -LRB- Cutler et al. , 2001 ; Prodeus et al. , 1998 -RRB- . IgG anti-HEL Abs remained undetectable in all complement deficient mice , but C4 and CD21/CD35 deficient B cells displayed reduced surface IgM modulation , indicating a lower degree of anergy induction in these mice -LRB- Prodeus et al. , 1998 -RRB- . However , this was not observed in the C1q deficient mice -LRB- Cutler et al. , 2001 -RRB- . This discrepancy could either indicate that C4 operates independently from C1q or reflect differences in the genetic background of the mice used -LRB- Cutler et al. , 2001 -RRB- . Nevertheless sHEL is not the ideal model to study the maintenance of tolerance in SLE as it is neither a natural autoantigen , nor are soluble plasma proteins typically targeted by the SLE autoAbs . More recently , a model targeting an SLE antigen , DNA , was generated by Weigert and co-workers -LRB- Chen et al. , 1995 -RRB- . In this model the rearranged variable heavy chain -LRB- VH -RRB- gene derived from a double stranded DNA-binding hybridoma developed in the autoimmune strain MRL/Mp . lpr/lpr , was inserted at the Igh locus and was referred to as VH3H9R . In contrast to conventional Tg mice , this knock-in model allows the Tg locus to undergo normal editing , isotype switching and somatic mutation . A variety of light chains can combine with the VH3H9 to yield anti-DNA Abs -LRB- Radic et al. , 1991 -RRB- but only few light chains are able to `` silence '' VH3H9R so that it no longer binds to DNA . By virtue of this characteristic the mice expressing only the VH3H9 chain -LRB- VH3H9R mice -RRB- can generate anti-DNA specificities . However , when the VH3H9R mice were crossed with a Tg knock-in light chain VLκ8 -LRB- Prak and Weigert , 1995 -RRB- to generate monospecific Tg mice -LRB- VH3H9R/VLκ8R mice -RRB- -LRB- Chen et al. , 1997a -RRB- , this combination of heavy and light chain V regions -LRB- VH3H9/VLVκ8 -RRB- bound only ssDNA and not dsDNA -LRB- Prak and Weigert , 1995 -RRB- . Previous studies with the VH3H9R mice have shown that the autoreactive Tg B cells accumulated in the splenic marginal zone and were regulated by anergy on non-autoimmune backgrounds such as BALB/c -LRB- Chen et al. , 1995 ; Erikson et al. , 1991 -RRB- and C57BL/6 -LRB- Fukuyama et al. , 2005 ; Sekiguchi et al. , 2002 -RRB- . However , tolerance could be broken in this model if T cell help was provided in the form of a chronic graft versus host disease -LRB- Sekiguchi et al. , 2002 -RRB- . Consistent with these observations , the double VH3H9R/VLκ8R knock-in Tg B cells were regulated by anergy in non-lupus prone mice -LRB- BALB/c -RRB- , whilst in autoimmune prone MRL/Mp . lpr/lpr animals Tg B cells escaped tolerance induction and underwent class-switching and affinity maturation -LRB- Brard et al. , 1999 -RRB- . These experiments suggested that the lpr mutation in the MRL background allowed the Tg autoreactive B cells to receive T cell help during a germinal center reaction . To determine whether C1q is involved in selection of self-reactive B cells , we bred the C1q-deficient mice with the VH3H9R and the VH3H9R/VLκ8R mice and monitored the regulation and activation of anti-DNA Tg B cells over a period of 10 months . The mice in this study were on the autoimmune prone background MRL/Mp expressing the CD95 -LRB- Fas -RRB- gene . The analysis of these mice revealed that the lack of C1q can influence the levels of IgM and IgG3 Tg-derived antibodies only in the VH3H9R model . 2 Materials and methods 2.1 Mice MRL/Mp mice were obtained from Harlan Olac , Bichester , UK . MRL/Mp . C1qa − / − deficient mice were generated as previously described -LRB- Mitchell et al. , 2002 -RRB- . VH3H9R.MRL / Mp -LRB- Chen et al. , 1995 -RRB- , VLκ8R.MRL / Mp -LRB- Prak and Weigert , 1995 -RRB- and VH3H9R/VLκ8R . MRL/Mp mice were kindly provided by Prof. M. Weigert -LRB- Gwen Knapp Center for Lupus and Immunology Research , University of Chicago , Chicago , IL -RRB- . MRL/Mp . C1qa − / − mice were crossed with the VH3H9R/Vκ8R . MRL/Mp mice and the resulting VH3H9R/VLκ8R . MRL/Mp . C1qa + / − were then crossed either with MRL/Mp or MRL/Mp . C1qa − / − in order to generate littermate animals . Mice were bled every 3 months starting from 2 months of age and at 10 months they were sacrificed . The mice were genotyped by PCR using specific primers . PCR primers were as follow : mC1qA/5 ′ -LRB- 5 ′ - GGGGCCTGTGATCCAGACAGG-3 ′ -RRB- , mC1qA/In2 − -LRB- 5 ′ - TAACCATTGCCTCCAGGATGG-3 ′ -RRB- and neo -LRB- 5 ′ - GGGGATCGGCAATAAAAAGAC-3 ′ -RRB- for the C1q genotyping ; MW114 -LRB- 5 ′ - CTGTCAGGAACTGCAGGTAAGG-3 ′ -RRB- and MW162 -LRB- 5 ′ - CATAACATAGGAATATTTACTCCTCGC-3 ′ -RRB- for the VH3H9R genotyping -LRB- Erikson et al. , 1991 -RRB- ; MW133 -LRB- 5 ′ - GGTACCTGTGGGGACATTGTG-3 ′ -RRB- and MW157 -LRB- 5 ′ - AGCACCGAACGTGAGAGG-3 ′ -RRB- for the VLκ8R genotyping -LRB- Carmack et al. , 1991 -RRB- . Animals were kept under specific pathogen-free conditions . All animal care and procedures were conducted according to institutional guidelines and approved by the local ethical committee . 2.2 Flow cytometry Flow cytometry was performed using a four-color staining of cells and analyzed with a FACSCalibur ™ -LRB- Becton Dickinson , Mountain View , CA -RRB- . The following Abs were used : anti-B220 -LRB- RA3-6B2 -RRB- , anti-CD5 -LRB- 53-7.3 -RRB- , anti-CD11b -LRB- M1-70 -RRB- , anti-CD19 -LRB- 1D3 -RRB- , anti-CD23 -LRB- B3B4 -RRB- , anti-CD21 / CD35 -LRB- 7G6 -RRB- , anti-CD90.2 -LRB- 53-2.1 -RRB- , anti-CD138 -LRB- 281-2 -RRB- , anti-IgM -LRB- II/41 -RRB- . All Abs were purchased from BD Biosciences Pharmingen -LRB- San Diego , CA -RRB- with the exception of the anti-VH3H9 idiotype -LRB- 1.209 -RRB- , a kind gift from Prof. M. Weigert -LRB- Gay et al. , 1993 -RRB- . Biotinylated Abs were detected using an allophycocyanin-conjugated streptavidin Ab -LRB- BD Biosciences Pharmingen -RRB- . Staining was performed in the presence of saturating concentration of 24G2 mAb -LRB- anti-FcRII/III -RRB- . Data were analyzed using WinMDI software -LRB- Version 2.8 ; Scripps Institute -RRB- . 2.3 Serological analyses Serum levels of IgM and IgG were quantified by ELISA , as described previously -LRB- Cortes-Hernandez et al. , 2004 -RRB- . Anti-ssDNA Abs , anti-chromatin Abs , anti-histone Abs and anti-dsDNA Abs were measured by ELISA as described previously -LRB- Burlingame and Rubin , 1990 ; Emlen et al. , 1990 -RRB- . Microtiter plates were coated with ssDNA prepared from calf thymus DNA -LRB- Sigma -RRB- , chromatin -LRB- Lorne Laboratories Ltd. , Reading , UK -RRB- or histone -LRB- Calbiochem , Merck Biosciences , Darmstadt , Germany -RRB- . For detecting anti-dsDNA Abs , plates were coated with streptavidin -LRB- Sigma -RRB- . ΦX174 double-stranded plasmid DNA -LRB- Promega , Southampton , UK -RRB- was biotinylated with Photoprobe biotin -LRB- Vector Laboratories , Peterborough , UK -RRB- and added to the streptavidin . Serum samples were diluted appropriately in PBS 2 % BSA , 0.05 % Tween-20 , 0.02 % NaN3 . Bound Abs were detected with alkaline phosphatase conjugated goat anti-mouse IgG -LRB- γ-chain specific -RRB- -LRB- Sigma -- Aldrich , Dorset , UK -RRB- and anti-mouse IgM -LRB- Southern Biotechnology Associates , Inc. , Birmingham , AL -RRB- . All autoAb results are expressed in arbitrary ELISA units -LRB- AEU -RRB- in reference to a standard curve derived from serum pools containing high titers of autoAbs . The idiotype + Abs were captured with the anti-VH3H9 idiotype -LRB- 1.209 -RRB- -LRB- Gay et al. , 1993 -RRB- and detected with alkaline phosphatase conjugated goat anti-mouse IgM or IgG subclasses specific Abs -LRB- Southern Biotechnology Associates -RRB- . The standard curve was derived from an IgM VH3H9R/Vκ8R mAb provided by Prof. Weigert or sera containing high IgG subclasses titers of the Tg . 2.4 Renal assessment Proteinuria was assessed using Haema-combistix -LRB- Bayer Diagnostics , Newbury , UK -RRB- . Kidneys were fixed in Bouin 's solution and paraffin embedded , and sections were stained with periodic acid -- Schiff reagent . Glomerular histology was graded in a blind fashion scored on a scale of 0 -- 4 , as described before -LRB- Carlucci et al. , 2007 -RRB- . 2.5 Statistics The data are presented as median , with range of values in brackets , unless otherwise stated . The non-parametric Mann -- Whitney U-test was applied throughout with differences being considered significant for p-values < 0.05 . Statistics were calculated using GraphPad Prism Version 3.0 -LRB- GraphPad Software , San Diego , CA -RRB- . 3 Results 3.1 Expression of Tg autoantibodies C1q deficiency has been shown to accelerate the onset and progression of SLE in MRL/Mp mice -LRB- Mitchell et al. , 2002 -RRB- , but the mechanisms underlying these effects are still uncertain . The VH3H9R/VLκ8R Tg alleles were transferred onto C1q-deficient mice by crossing VH3H9R/VLκ8R . MRL/Mp with MRL/Mp . C1qa − / − and experimental cohorts of littermate mice were generated . Whilst in previous studies the expression of the Tg alleles was monitored by measuring the Tg-specific immunoglobulin allotype -LRB- Steeves and Marion , 2004 -RRB- , this analysis could not be applied in these mice as the Tg alleles and the endogenous immunoglobulin allotype were indistinguishable . Nevertheless , we were able to detect the combination of the VH3H9 with the VLVκ8 using an idiotype specific monoclonal Ab named 1.209 -LRB- Gay et al. , 1993 -RRB- . As the 1.209 mAb can also recognize the VH3H9 paired with other endogenous light chains such as VLκ4 -LRB- Gay et al. , 1993 -RRB- , this anti-idiotype Ab allowed us not only to assess the VH3H9R/VLκ8R Tg B cells but also some of the VH3H9R Tg B cells . C1q deficiency did not affect the proportion of peripheral blood B cells expressing the Tg in neither of the two models -LRB- VH3H9R/VLκ8R . MRL/Mp . C1qa − / − : 89.1 ± 1.1 % versus VH3H9R/VLκ8R . MRL/Mp : 90.7 ± 1.5 % , p = 0.4118 ; VH3H9R.MRL / Mp.C1qa − / − : 25.3 ± 1.6 % versus VH3H9R.MRL / Mp : 22.3 ± 2.3 % p = 0.1669 -RRB- . The low proportion of idiotype + B cells in the VH3H9R.MRL / Mp mice indicated that most of the B cells in these mice had either paired the VH3H9R Tg allele with a light chain that was not recognised by the anti-idiotype Ab , or edited the VH3H9R allele , or used the endogenous immunoglobulin allele . In the VH3H9R/VLκ8R . MRL/Mp . C1qa − / − mice the B cell receptor was down-modulated to the same extent as in the VH3H9R/VLκ8R . MRL/Mp mice -LRB- data not shown -RRB- . Serological analyses were performed in order to establish whether the Tg autoAbs were expressed . The different cohorts of mice were bled at different time points , and the data presented here are those obtained at 10 month of age when the mice were sacrificed . Substantial levels of idiotype + IgM , IgG3 , IgG2a and IgG2b Abs were detected in the VH3H9R/VLκ8R . MRL/Mp and in VH3H9R.MRL / Mp mice indicating that the anti-DNA knock-in Tg alleles on this background were not regulated by anergy unlike when on the C57BL/6 background -LRB- Fig. 1 -RRB- . In the VH3H9R/VLκ8R . MRL/Mp . C1qa − / − mice the levels of idiotype + Abs were similar to those detected in the strain-matched C1q-sufficient animals . In contrast , the VH3H9R.MRL / Mp.C1qa − / − mice had significantly increased levels of idiotype + IgM and IgG3 Abs compared to the VH3H9R.MRL / Mp mice -LRB- Fig. 1A and B -RRB- , but similar levels of idiotype + IgG2a and IgG2b Abs -LRB- Fig. 1C and D -RRB- . We then measured the levels of IgM and IgG autoAbs . A significant increase in the titre of IgM anti-ssDNA Abs was observed in the VH3H9R/VLκ8R . MRL/Mp . C1qa − / − mice compared to the VH3H9R/VLκ8R . MRL/Mp mice -LRB- Fig. 2A -RRB- . Of note , the VH3H9R/VLκ8R . MRL/Mp . C1qa − / − mice displayed only markedly increased levels of IgM autoAbs directed against ssDNA -LRB- the specificity encoded by the Tg alleles -RRB- but not against other lupus autoantigens such as dsDNA , histone or chromatin -LRB- Fig. 2B -- D -RRB- . In the VH3H9R.MRL / Mp mice the pairing of the VH3H9 with endogenous L chains could yield to more specificities . Consistent with this , VH3H9R.MRL / Mp.C1q − / − mice had significantly increased levels of IgM anti-ssDNA , anti-dsDNA , anti-histone and anti-chromatin Abs -LRB- Fig. 2A -- D -RRB- . We then analysed the levels of IgG autoAbs and found that these were similar between the C1q-sufficient and - deficient cohorts -LRB- Supporting information Table S1 -RRB- . 3.2 Renal assessment At 10 month of age all the animals were sacrificed . VH3H9R/VLκ8R . MRL/Mp and VH3H9R.MRL / Mp animals showed histological evidence of a mild glomerulonephritis -LRB- median score 1.0 , range : 0.0 -- 3.0 and 1.5 , range : 1.0 -- 3.0 , respectively -RRB- reminiscent of the disease observed in MRL/Mp non-Tg mice -LRB- Mitchell et al. , 2002 -RRB- . C1q-deficiency in MRL/Mp mice has previously been shown to worsen the kidney pathology -LRB- Mitchell et al. , 2002 -RRB- . However , in the Tg mice the absence of C1q did not exacerbate the renal disease compared to the strain-matched C1q-sufficient mice -LRB- VH3H9R/VLκ8R . MRL/Mp . C1qa − / − mice : median score 1.0 , range : 0.0 -- 3.0 , p = 0.4287 ; VH3H9R.MRL / Mp.C1qa − / − mice : median range 2.0 , range : 1.0 -- 2.0 , p = 0.4564 -RRB- . 3.3 Flow cytometric analysis of splenic and peritoneal B cells In order to determine if the serological data were accompanied by phenotypic changes in T and B lymphocytes , we performed a comprehensive analysis of the various splenic and peritoneal subpopulations . Cells of at least 7 mice from each cohort were analysed by FACS at the time of the sacrifice using the combinations of markers shown in Table 1 . In agreement with the findings in other genetic backgrounds -LRB- Li et al. , 2002 -RRB- , the VH3H9R.MRL / Mp mice had a larger marginal zone -LRB- MZ -RRB- B cell population compared to VH3H9R/VLκ8R . MRL/Mp -LRB- Fig. 3A -RRB- and an increased proportion of idiotype + B cells in the MZ compartment compared to follicular -LRB- FO -RRB- compartment -LRB- 5.9 ± 1.0 versus 4.2 ± 0.6 , paired t-test p = 0.0157 -RRB- -LRB- Table 1 -RRB- . More interestingly , in the VH3H9R.MRL / Mp.C1qa − / − mice , which produced higher levels of IgM autoAbs , the MZ B cell population was significantly decreased compared to the VH3H9R.MRL / Mp animals -LRB- Fig. 3A -RRB- and less idiotype + B cells were found in the MZ compared to the FO compartment -LRB- 3.4 ± 0.7 versus 4.8 ± 1.1 , paired t-test p = 0.0042 -RRB- -LRB- Table 1 -RRB- . In the VH3H9R/VLκ8R . MRL/Mp mice there was no preferential localization of idiotype + B cells in the MZ zone -LRB- paired t-test p = 0.056 -RRB- -LRB- Table 1 -RRB- . C1q deficiency was associated with a significant reduction in the percentage of MZ B cells -LRB- Fig. 3A -RRB- and a decrease of idiotype + B cells in the MZ compared to the FO area -LRB- 4.2 ± 0.9 versus 17.5 ± 3.2 , paired t-test p = 0.0013 -RRB- . These findings indicated that MZ B cells might have been activated in the absence of C1q . Activated MZ B cells have been shown to migrate into the T cell zone and differentiate into plasma cells . Indeed , the percentage of plasma cells was increased in the two C1q-deficient cohorts compared to the respective MRL/Mp controls -LRB- Fig. 3B -RRB- . The peritoneal cavity in mice has been shown to be a site where some self-reactive B cells can escape tolerance -LRB- Fagarasan et al. , 2000 -RRB- and B-1 cells , one of the major sources of circulating IgM , accumulate . More cells were recovered from the peritoneum of VH3H9R.MRL / Mp.C1qa - / - mice compared to VH3H9R.MRL.Mp mice -LRB- p = 0.0051 -RRB- but not in the VH3H9R/VLκ8R . MRL/Mp . C1qa − / − versus VH3H9R/VLκ8R . MRL/Mp animals -LRB- p = 0.1923 -RRB- -LRB- Table 1 -RRB- . When the peritoneal B cell subpopulations were analyzed in more detail , all subsets were increased in the VH3H9R.MRL / Mp.C1qa - / mice compared to the VH3H9R.MRL.Mp mice -LRB- Table 1 -RRB- . On the contrary , in the VH3H9R/VLκ8R C1q-deficient animals only B-1a cells were slightly increased compared to their wild type strain-matched controls -LRB- Table 1 -RRB- , but this did not reach statistical significance -LRB- p = 0.0518 -RRB- . We then analysed the VH3H9/VLκ8 idiotype expression on the different peritoneal B cells . Surprisingly in the peritoneum of the VH3H9R/VLκ8R . MRL.Mp and VH3H9R.MRL.Mp mice the majority of the B cells were not expressing the VH3H9/VLκ8 idiotype -LRB- Table 1 -RRB- . Of note the C1q-deficient mice had similar percentage of idiotype + B cells in the different peritoneal B cell subpopulations compared to C1q-sufficient mice . The reduced proportion of idiotype + B cells in the peritoneum compared to the peripheral blood and the spleen -LRB- data not shown -RRB- suggests that in this organ the B cells either preferentially used the endogenous alleles or had been activated and undergone editing . 4 Discussion C1q deficiency in humans and in mice has been associated with the development of a lupus-like illness . However , the role of C1q in the regulation of autoreactive B cells remains debatable . Here , we explored the regulation of autoreactive B cells by C1q using anti-DNA knock-in Tg models -LRB- VH3H9R/VLκ8R and VH3H9R -RRB- on the lupus prone MRL/Mp genetic background . The analysis of these mice revealed that the MRL/Mp background was in itself sufficient to allow the expression of anti-DNA Tg autoAbs and that the lack of C1q modified this effect only in the single Tg model -LRB- VH3H9R -RRB- . However , in the absence of C1q the MZ B cell compartment was significantly reduced in both models and this was accompanied by an increase in plasmocytes . Recently the VH3H9R/VLκ8R anti-DNA knock-in Tg model has been widely used for investigating the mechanisms of regulation of autoAb production in murine models of SLE . There is an increasing evidence that the VH3H9R/VLκ8R and VH3H9R anti-DNA Tg B cells , which have a relatively weak affinity to ssDNA , are regulated by anergy on normal genetic backgrounds such as BALB/c and C57BL/6 , but could be induced to lose tolerance when transferred onto lupus models such as -LRB- NZB × NZW -RRB- F1 or MRL/Mp . lpr/lpr -LRB- Brard et al. , 1999 ; Chen et al. , 1995 ; Erikson et al. , 1991 ; Fukuyama et al. , 2005 ; Sekiguchi et al. , 2002 -RRB- . In light of these observations it was important for our study to establish whether the autoimmune prone MRL/Mp background was in itself capable of breaking tolerance . MRL/Mp mice are known to develop a mild autoimmune disease which can be accelerated with different disease-modifying genes such as Yaa -LRB- Merino et al. , 1989 -RRB- , lpr and gld -LRB- Cohen and Eisenberg , 1991 -RRB- . The analysis of the idiotype + -LRB- VH3H9R/VLκ8R -RRB- Abs revealed that the Tg MRL/Mp mice indeed had in circulation these idiotype + Abs -LRB- IgM , IgG2a , IgG2b , IgG3 -RRB- indicating that a break of tolerance had spontaneously occurred in these mice . One explanation for this is an intrinsic defect in MRL/Mp B cells and there is some evidence in support of this . MRL/Mp mice have been reported to exhibit a defect in maintaining the developmental arrest of VH3H9/VLλ anti-dsDNA conventional Tg B cells -LRB- Mandik-Nayak et al. , 1999 , 2000 -RRB- and to have a spontaneous B cell hyperactivity in the absence of Ag in the IgHEL experimental model -LRB- Nijnik et al. , 2006 -RRB- . However , the VH3H9/VLλ anti-dsDNA autoreactive B cells , despite not being any longer developmentally arrested as in a BALB/c mice , exhibited follicular exclusion and failed to differentiate into plasma cells -LRB- Mandik-Nayak et al. , 1999 -RRB- . Furthermore Tg MRL/Mp mice expressing IgHEL have been shown to be able to down-regulate their B cell receptor and to be unable to secrete detectable levels of anti-HEL Abs in the presence of sufficient amount of sHEL -LRB- Nijnik et al. , 2006 -RRB- . Similarly anti-laminin Tg MRL/Mp mice were found to be tolerant -LRB- Rudolph et al. , 2002 -RRB- . Another potential explanation for the break of the B cell tolerance in the VH3H9R/VLκ8R anti-DNA Tg mice , is that in this model the MRL/Mp background was able to provide sufficient T cell help and the presence of idiotype + IgG subclasses favour this hypothesis . We next examined whether C1q could modulate the phenotype of the anti-ssDNA Tg B cells . In the VH3H9R/VLκ8R . MRL/Mp mice the absence of C1q increased significantly the circulating levels of IgM against ssDNA but not against other autoantigens including dsDNA . As the pairing of VH3H9 with VLκ8 prevents the binding of VH3H9 to dsDNA , the elevated amount of IgM anti-ssDNA observed could have been the result of an increased Tg expression . However , the idiotype analysis failed to demonstrate a difference in the levels of IgM idiotype + Abs between VH3H9R/VLκ8R . MRL/Mp . C1qa − / − and VH3H9R/VLκ8R . MRL/Mp mice , questioning whether the source of the increased levels of IgM anti-ssDNA was indeed the Tg B cells . On the other hand , the VH3H9R.MRL / Mp.C1qa − / − mice displayed significantly higher levels of IgM and IgG3 idiotype + Abs . As the VH3H9R heavy chain can pair with different endogenous light chains generating a wider range of autoAbs , other specificities were tested . Indeed the absence of C1q increased significantly IgM levels against all the lupus autoantigens analysed . To gain insight into the mechanisms regulating the autoAb production in the C1q-deficient mice we then carried a detailed analysis of the different B cell populations . Several studies have proposed a possible role for MZ B cells in the development of lupus in mouse models . Although studies in immunoglobulin Tg mice have shown that autoreactive B cells can accumulate in the marginal zone under various experimental situations -LRB- Li et al. , 2002 ; Mandik-Nayak et al. , 1997 , 1999 , 2006 ; Qian et al. , 2004 ; Wen et al. , 2005 -RRB- , it remains to be established whether MZ B cells secrete pathogenic autoAbs in any model of lupus . B cells producing potentially pathogenic autoAbs are thought to home to the MZ -LRB- Chen et al. , 1997b ; Li et al. , 2002 -RRB- and sequestration to this site is believed to prevent them from entering into the germinal centres and developing the properties of pathogenic B cells . However , recent studies in lupus-prone mice have reported both enlargements -LRB- Grimaldi et al. , 2001 ; Mackay et al. , 1999 ; Wither et al. , 2000 -RRB- and impaired development of the MZ B cell compartment -LRB- Amano et al. , 2003 ; Samardzic et al. , 2002 -RRB- . Consistent with previous observations in the VH3H9R/VLκ8R model -LRB- Wen et al. , 2005 -RRB- , the MZ was found to be enlarged in the VH3H9R.MRL / Mp mice but no accumulation of MZ B cells was observed in VH3H9R/VLκ8R . MRL/Mp mice . Importantly C1q deficiency decreased dramatically the proportion of MZ B cells in these mice and this was accompanied by an increase in the percentage of plasma cells . More interestingly , in the C1q-deficient Tg mice we observed a significant disappearance of idiotype + B cells from the MZ suggesting that these cells had been activated . Marginal zone B cells bearing low affinity self-reactive BCR can react to repetitive Ag and produce natural autoAbs of the IgM isotype upon contact with blood-borne pathogens or self Ag -LRB- Oliver et al. , 1999 -RRB- . Moreover , MZ B cells can undergo T cell independent switching to IgG , IgA and IgE in response to pathogen associated molecular patterns -LRB- PAMP -RRB- . As C1q has been shown to be involved in the clearance of apoptotic cells , which are enriched in the typical lupus autoantigens , one could postulate that the increased IgM Tg secretion and the reduction of the MZ B cells in the VH3H9R.MRL / Mp.C1qa − / − mice might be related to a failure to clear antigens associated with dying cells . In the absence of C1q the ineffectively cleared autoAgs could stimulate the autoreactive MZ Tg B cells to differentiate into plasmocyte resulting into the decrease of MZ B cells and increase of circulating IgM autoAbs . Similarly , in the spleens of VH3H9R/VLκ8R . MRL/Mp . C1qa − / − mice significantly less idiotype + B cells were present in the MZ compartment and more plasmocytes were found . However , these cellular changes were not paralleled by an increase of idiotype + IgM Abs in circulation indicating in this model they were not sufficient to induce autoantibody production . Another potential source of IgM autoAbs are the B1 cells . B1 cells have long been associated with the secretion of natural Abs against self and foreign pathogens , which can occur without obvious inflammatory response . The importance of B1 cells in the pathogenesis of lupus continues to be debated . A major argument for a role of B1a cells in mouse lupus relates to the expansion of this subset in NZB and -LRB- NZB × NZW -RRB- F1 mice . Reduction of B1 cells for instance via intraperitoneal injection of H2O delayed disease onset and reduced disease severity in -LRB- NZB × NZW -RRB- F1 mice -LRB- Murakami et al. , 1995 -RRB- . However , the expansion of B1a cells was shown not to be critical for the production of IgM or IgG autoAbs in this murine model of SLE -LRB- Atencio et al. , 2004 -RRB- . The Abs secreted by B1 cells tend to be polyreactive with low-affinity cross-reactivity to a variety of self Ags and these characteristics are very different from the pathogenic IgG Abs produced by lupus mice . In our experimental model , we found an increase in the total number of peritoneal and B1 cells in the VH3H9R.MRL / Mp.C1qa − / − mice compared to their wild type counterparts -LRB- Table 1 -RRB- . A similar trend was observed for the B1a cells in the VH3H9R/VLκ8R . MRL/Mp . C1qa − / − mice but this did not reach statistical significance . However , in both models the percentages of idiotype + B1 cells between C1q-deficient and - sufficient mice were similar . These findings would indicate the lack of C1q may favour the expansion of peritoneal B1 cells and that these cells might have contributed to the increase of serum IgM autoAb levels observed in the VH3H9R.MRL / Mp C1q-deficient mice . Supporting this hypothesis , a recent study showed that C1q deficiency increases the positive selection of B-1 cells and IgM autoAb production by a membrane-bound intracellular auto-Ag -LRB- Ferry et al. , 2007 -RRB- . In conclusion , using mice expressing site-directed transgenes for anti-DNA autoAbs we have shown that : -LRB- i -RRB- the MRL/Mp background was in itself capable of inducing the expression of anti-DNA Tg autoAbs , and -LRB- ii -RRB- VH3H9R.MRL / Mp.C1qa − / − could influence the production of Tg-derived IgM and IgG3 autoAbs possibly as a result of an impaired disposal of cellular debris . However , we found no evidence of a direct role of C1q in the regulation of self-reactive conventional B cells . Further studies on non-lupus prone genetic backgrounds such as C57BL/6 will be necessary to determine if C1q deficiency can play a more substantial role in shaping the repertoire of the autoreactive B cells .

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

autoimmunity
complement
b cells
transgenic

ab, antibody
aeu, arbitrary elisa units
fo, follicular
hel, hen egg lysozyme
mz, marginal zone
tg, transgenic
sle, systematic lupus erythematosus
rodent


Embolism of the popliteal artery after anterior cruciate ligament reconstruction : a case report and literature review Arterial complications after anterior cruciate ligament reconstruction -LRB- ACLR -RRB- are rare . We present a case report of a 44-year-old male patient with a subtotal occlusion of the popliteal artery , with sensory loss in the foot , 17 days after ACLR . Embolectomy and anticoagulant therapy led to full recovery of the peripheral arterial circulation . The sensory loss of the foot also fully recovered . To our knowledge , this is the first case report of an embolus of the popliteal artery after ACLR without relation to graft fixation . A literature review on vascular complications after ACLR is presented . Introduction Vascular complications after anterior cruciate ligament reconstruction -LRB- ACLR -RRB- are rare . Few peer reviewed case reports have been reported with various techniques of reconstruction -LSB- 1 , 3 -- 9 -RSB- . Allum did not report vascular lesions as a complication after ACLR in a review article on this subject -LSB- 2 -RSB- . The origin of vascular lesions after ACLR may be venous or arterial -LSB- 4 -RSB- . We present a case of arterial embolism of the popliteal artery after ACLR . A literature review on vascular complications after ACLR is presented . Case report A 44-year-old male has a previous medical history of open medial and lateral ligament repair of the right knee 15 years previously -LRB- motor accident -RRB- . Functional instability of the right knee due to ACL deficiency was the reason for referral to our service . There were no signs of posterior nor posterolateral instability . An ACLR was performed with a quadruple hamstring graft . The graft was fixed with a Bone Mulch Screw on the femoral side and a WasherLoc device in the tibia -LRB- Arthrotek , Inc. . Warsaw , USA -RRB- . The latter is a spiked washer with bicortical screw fixation . Total tourniquet time was 90 min . No complications were noted during or after surgery . Thromboprophylaxis for deep venous thrombosis was given by means of Low Molecular Weight Heparin -LRB- 2500 IE daily -RRB- during hospital stay . The hospital recovery was uneventful . On the 17th day of post surgery , he experienced pain and swelling in the popliteal fossa of the right knee . The complaints partially resolved with physiotherapy . Two days later , the fossa pain returned with alterations of skin color , sensory loss and an increasing cold right foot . He was referred to a vascular surgeon . Adequate dorsal pedal and posterior tibial pulses were noted . Duplex ultrasound examination showed no sign of venous thrombosis . Angiography revealed a subtotal occlusion of the popliteal artery at the level of the superior genicular artery -LRB- Fig. 1 -RRB- . An embolectomy was performed using a Fogarty catheter inserted in the femoral artery . The pedal pulses were diminished after embolectomy and a second angiography was performed . The occlusion at the level of the popliteal artery was no longer detected . No further emboli were noted , however , the peripheral flow qualified as too slow and suspect of small distal occlusions . Anticoagulant therapy with intravenous heparin as well as epidural analgesia was administered until complete recovery of peripheral circulation . The patient developed a superficial infection of the groin wound , treated by antibiotics . He was mobilized and discharged after 8 days . Sensory loss of the foot slowly recovered after 4 months . Vascular analysis in rest and strenuous activity was performed at 4 months . He had no more complaints , symmetrical ankle-brachial index in both legs and intact pulses at the foot and ankle . Vascular analysis did not reveal any other possible cause for arterial emboli . The patient has full range of motion of the right knee with a Lachman and anterior drawer test of 0 -- 2 mm -LRB- International Knee Documentation Committee -RRB- and absent pivot shift test . Fig. 1Angiography of the right knee showing subtotal occlusion of the popliteal artery at the level of the superior genicular artery -LRB- reproduced with permission from -LSB- 4 -RSB- -RRB- Discussion Vascular complications after ACLR are rare . The origin of vascular lesions may be venous or arterial -LSB- 4 -RSB- . A case of fatal pulmonary embolism after ACLR with venous origin has been published recently -LSB- 5 -RSB- . Hypothesis of the cause was a hereditary coagulopathy . Arterial lesions of the popliteal artery after ACLR have been presented in few peer reviewed case reports even with an all-inside technique of arthroscopic ACL reconstruction and fixation as well as any type of graft -LSB- 4 -RSB- . Roth et al. -LSB- 8 -RSB- described an occlusion of the proximal popliteal artery . A composite graft consisting of a polypropylene ligament augmentation and the middle third of the quadriceps-patellar tendon was fixed to the lateral femur with a staple . The artery was trapped between the graft and the femur . A saphenous bypass was performed 6 weeks post surgery . Spalding et al. -LSB- 9 -RSB- reported a case of unilateral claudication 8 years after ACL reconstruction with use of a Gore-Tex ligament . A cyst had formed around the femoral insertion of the ruptured synthetic ligament and was excised without vascular repair . Evans et al. -LSB- 3 -RSB- reported a pseudoaneurysm of the medial inferior genicular artery following ACL reconstruction using a central third patellar tendon graft fixed with interference screws . At 5 weeks , ligation of the artery and removal of the thrombus led to full recovery . The cause of the lesion was elevation of the periosteum on the medial side of the tibia for tibial tunnel preparation . Aldridge et al. -LSB- 1 -RSB- described an avulsion of the middle genicular artery after a bone-patellar tendon-bone autograft fixed with interference screws . Surgical exploration at 4 weeks revealed a tear in the popliteal artery . There was no rupture of the posterior capsule . Probable cause for the avulsion of the middle genicular artery was the debridement of the femoral ACL remnant tissue . We have previously published two cases of popliteal artery lesions caused by a drill for bicortical tibia fixation after quadruple hamstring ACLR -LSB- 6 , 7 -RSB- . In the first case , the drill had caused an intimal lesion at the level of the infragenicular popliteal artery which led to the pseudoaneurysm . Vascular repair was performed 12 days after ACL reconstruction but sensory loss of the saphenous and medial plantar nerves was still present at 4 months follow-up -LSB- 7 -RSB- . The second case was a simultaneous traumatic pseudoaneurysm and thrombosis of the popliteal artery after ACLR . At surgical exploration , the thrombosis was in line with the drill hole for bicortical tibial fixation . There was no apparent relation of the femoral fixation device to the pseudoaneurysm of the supragenicular popliteal artery . This pseudoaneurysm was thought to be pre-existent . The pseudoaneurysm was ligated and a venous jump graft was performed to bypass the thrombosis located more distally in the popliteal artery -LSB- 6 -RSB- . In this review of the literature on arterial complications after ACLR , all cases are associated with direct damage to the popliteal artery at time of ACLR -LSB- 1 , 3 , 6 -- 9 -RSB- . There was no apparent direct damage to the popliteal artery in the 44-year-old male patient presented in this case report . The popliteal artery occlusion was not in line with either the femoral nor tibial fixation device . Vascular analysis did not reveal any pre-existent vascular causes for arterial embolus formation proximal to the popliteal artery . Our hypothesis of the cause was the traumatic knee dislocation 15 years previously . Precursors could have been pre-existent intimal vascular damage or adhesions of the artery at the level of the superior genicular artery in combination with the use of the tourniquet and ACLR . Conclusion Awareness of possible arterial complications after ACL reconstruction is essential for early diagnosis . Clinical symptoms of pain in the popliteal fossa and sensory deficits in lower leg and foot should prompt the physician to analyze possible injuries of the popliteal artery . The differential diagnosis should include compartment syndrome and deep venous thrombosis . Doppler examination as well as intact pedal arterial pulses are unreliable in diagnosing arterial lesions after ACL reconstruction . Contrast - , CT - or MRI-angiography are the diagnostic tools of choice . Immediate surgical exploration is indicated to limit limb ischemia and neurological damage -LSB- 4 -RSB- .

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