Stromal

mast

cells

in

invasive

breast

cancer

are

a

marker

of

favourable

prognosis

a

study

of

cases

Purpose

We

have

previously

demonstrated

in

a

pilot

study

of

invasive

breast

cancers

that

mast

cell

-LRB-

MC

-RRB-

infiltrates

within

primary

breast

cancers

are

associated

with

a

good

prognosis

Our

aim

was

to

verify

this

finding

in

a

larger

cohort

of

invasive

breast

cancer

patients

and

examine

the

relationship

between

the

presence

of

MCs

and

other

clinical

and

pathological

features

Introduction

Mast

cells

-LRB-

MCs

-RRB-

are

part

of

the

innate

immune

system

and

are

recruited

to

and

activated

in

the

microenvironment

of

a

developing

tumor

MCs

originate

from

multipotential

hemopoietic

stem

cells

in

the

bone

marrow

and

express

the

proto-oncogene

c-kit

a

transmembrane

type

III

tyrosine

kinase

receptor

protein

-LSB-

-RSB-

After

leaving

the

vascular

bed

they

differentiate

and

acquire

functional

maturity

MC

infiltrates

have

been

described

in

a

variety

of

human

cancers

including

non-small-cell

lung

cancer

-LSB-

-RSB-

breast

cancer

-LSB-

-RSB-

colorectal

cancer

-LSB-

-RSB-

basal

cell

carcinoma

-LSB-

-RSB-

and

pulmonary

adenocarcinoma

-LSB-

-RSB-

MCs

are

attracted

to

the

tumor

by

tumor-derived

chemo

attractants

where

they

either

degranulate

to

release

potential

tumor

cytotoxic

compounds

or

become

innocent

bystanders

depending

on

local

tumor

conditions

-LSB-

-RSB-

There

is

controversy

about

the

pro-

and

anti-tumorigenic

effects

of

MCs

in

different

cancers

The

accumulation

of

MCs

has

been

associated

with

enhanced

growth

and

invasion

of

several

human

cancers

-LSB-

-RSB-

On

the

other

hand

MC

infiltration

has

been

associated

with

good

prognosis

in

breast

-LSB-

-RSB-

ovarian

-LSB-

-RSB-

lung

-LSB-

-RSB-

and

colorectal

-LSB-

-RSB-

carcinomas

Murine

models

have

shown

that

tumor

incidence

and

growth

are

inversely

correlated

with

the

MC

density

in

MC-deficient

mice

compared

to

normal

-LSB-

-RSB-

Our

previously

published

pilot

report

of

case

series

of

breast

carcinomas

showed

that

the

presence

of

stromal

MCs

correlated

with

a

good

prognosis

-LRB-

P

-RRB-

in

invasive

breast

cancer

-LSB-

-RSB-

We

present

here

a

large

tissue

microarray

-LRB-

TMA

-RRB-

study

of

cases

of

invasive

breast

carcinomas

with

clinical

outcome

data

This

study

validates

the

independent

prognostic

significance

of

stromal

MCs

in

breast

cancer

Materials

and

methods

Patient

selection

A

total

of

archival

samples

from

patients

with

invasive

breast

carcinoma

referred

to

the

British

Columbia

Cancer

Agency

between

January

and

September

were

used

for

TMA

construction

The

Clinical

Research

Ethics

Board

of

the

University

of

British

Columbia

approved

the

study

Patients

with

in-situ

disease

metastatic

disease

at

presentation

and

male

breast

cancer

were

excluded

from

analysis

thus

bringing

the

final

tally

to

cases

This

represents

of

all

patients

diagnosed

with

breast

cancer

in

the

province

of

British

Columbia

during

this

time

period

This

large

well

characterized

cohort

is

derived

from

a

consecutive

series

of

patients

who

were

referred

to

the

BC

Cancer

Agency

for

consultation

and

had

tumor

samples

sent

to

a

central

laboratory

at

the

Vancouver

General

Hospital

for

estrogen

receptor

-LRB-

ER

-RRB-

status

Consequently

for

all

of

these

patients

we

have

available

detailed

demographic

and

outcome

data

as

well

as

formalin

fixed

paraffin

embedded

-LRB-

FFPE

-RRB-

primary

tumor

samples

for

immunohistochemical

analysis

Available

clinical

information

includes

age

histology

tumor

grade

tumor

size

lymph

node

status

type

of

local

and

adjuvant

systemic

therapy

and

dates

of

first

recurrence

and

death

Clinical

and

pathological

variables

were

determined

following

well-established

criteria

A

portion

of

this

cohort

of

patients

was

recently

used

in

a

population

study

validating

the

on-line

breast

cancer

prognostic

calculator

ADJUVANT

Online

-LSB-

-RSB-

TMA

construction

The

Vancouver

Hospital

ER

laboratory

retained

single

archival

tumor

blocks

from

each

case

in

this

patient

cohort

The

material

had

been

frozen

prior

to

neutral

buffered

formalin

fixation

All

the

paraffin

sections

were

first

stained

with

H&E

and

reviewed

by

a

pathologist

Representative

tumor

areas

were

circled

and

matched

with

the

donor

blocks

cores

were

punched

out

from

the

donor

blocks

and

embedded

mm

apart

in

recipient

blocks

using

Tissue

Microarrayer

-LRB-

Beecher

instruments

Silver

Springs

MD

-RRB-

These

tissue

arrays

have

been

used

to

test

the

new

ER

antibody

that

improves

the

sensitivity

for

detecting

ER

by

IHC

-LSB-

-RSB-

Immunohistochemistry

-LRB-

IHC

-RRB-

Sections

from

TMA

were

cut

at

μm

and

immunostained

with

antibody

to

KIT

-LRB-

-RRB-

a

transmembrane

tyrosine

kinase

acting

as

a

type

III

receptor

for

MC

growth

factor

Slides

were

also

concurrently

stained

for

ER

and

using

standard

immunoperoxidase

techniques

The

antibodies

and

antigen

retrieval

methods

are

summarized

in

Table

Table

of

antibodies

used

for

immunohistochemistryAntibodyIsotype

CompanyAntigen

retrievalConcentrationc-kitRabbit

monoclonalLab

visionSteam

min

M

TRIS

buffer

-LRB-

pH

-RRB-

ER

monoclonalLab

visionCitrate

buffer

-LRB-

pH

-RRB-

Immunohistochemical

image

processing

and

scoring

The

stained

slides

were

digitally

scanned

with

a

BLISS

automated

digital

imaging

microscope

-LRB-

Bacus

Laboratories

Lombard

IL

-RRB-

which

consists

of

a

microscope

with

a

scanning

stage

video

camera

and

software

designed

for

scanning

TMAs

A

relational

database

was

constructed

using

identification

information

and

immunohistochemistry

scores

for

each

tissue

core

in

the

microarrays

An

internet

website

was

then

constructed

using

this

database

and

a

WebSlideViewer

Java

applet

provided

by

the

manufacturer

to

view

the

microarray

images

and

allow

for

an

image

zooming

functionality

This

website

is

publicly

accessible

through

The

slides

were

scored

manually

by

two

independent

pathologists

blinded

to

the

clinical

outcome

as

previously

described

-LSB-

-RSB-

Total

number

of

stained

MCs

in

each

core

was

recorded

ER

status

was

assessed

using

the

rabbit

monoclonal

antibody

-LRB-

-RRB-

antibody

-LSB-

-RSB-

The

fractions

of

ER

positive

tumor

nuclei

were

scored

as

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

rabbit

monoclonal

from

LabVision

-LRB-

NeoMarkers

-RRB-

was

used

to

stain

the

TMA

slides

and

were

scored

using

Hercept

test

-LRB-

Dako

Corporation

Carpinteria

CA

-RRB-

scoring

system

Final

score

was

derived

using

both

IHC

and

Fluorescent

in

situ

hybridization

-LRB-

FISH

-RRB-

assays

Cases

with

IHC

Herceptest

score

were

scored

as

positive

Those

cases

with

IHC

Herceptest

score

were

re-evaluated

using

FISH

assay

and

only

those

cases

with

FISH

amplification

ratio

were

scored

as

positive

C-Kit

and

ER

scores

were

binarized

for

statistical

analysis

as

follows

no

MCs

any

MCs

ER

nuclei

stained

nuclei

stained

We

excluded

cases

for

which

it

was

not

possible

to

assign

a

score

to

the

immunostaining

-LRB-

insufficient

invasive

tumor

in

the

core

or

missing

core

-RRB-

Statistical

analysis

The

raw

scores

were

entered

into

an

Excel

database

and

each

TMA

core

was

assigned

a

unique

core

ID

number

The

spreadsheet

was

then

processed

utilizing

TMA-Deconvoluter

software

that

had

been

adapted

for

TMA

analysis

-LSB-

-RSB-

A

database

was

created

by

incorporation

of

all

deconvoluted

marker

data

into

the

clinicopathologic

patient

database

We

applied

a

split-sample

validation

technique

for

our

statistical

analysis

Our

patient

cohort

was

stratified

into

eight

subgroups

-LRB-

Table

-RRB-

based

on

adjuvant

treatment

received

and

then

randomized

into

equal

sized

training

and

validation

sets

The

two

groups

were

balanced

with

respect

to

treatment

received

but

there

are

also

no

significant

differences

in

clinical

or

pathological

variables

including

age

tumor

size

and

grade

nodal

status

and

ER

status

For

this

study

and

future

studies

using

this

TMA

the

primary

investigator

is

given

access

to

all

clinical

outcome

and

TMA

data

from

the

training

set

only

The

training

set

is

used

to

generate

and

refine

hypotheses

regarding

the

biomarker

under

study

Significant

findings

are

then

formally

presented

at

a

bimonthly

joint

scientific

group

meeting

of

the

Genetic

Pathology

Evaluation

Centre

-LRB-

GPEC

a

collaborative

group

of

scientists

and

pathologists

-RRB-

and

Breast

Cancer

Outcomes

Unit

-LRB-

BCOU

a

group

of

oncologists

and

epidemiologists

based

at

the

BC

Cancer

Agency

-RRB-

Those

findings

considered

to

be

of

clinical

and

scientific

interest

are

then

re-tested

on

the

validation

set

A

separate

researcher

who

did

not

participate

in

the

training

set

analysis

performs

the

re-testing

on

the

validation

set

Our

statistical

approach

is

intended

to

minimize

false

positive

results

particularly

with

subgroup

analysis

Table

of

clinical-pathological

characteristics

of

the

breast

cancer

patientsTest

setValidation

setn

within

group

within

known

valuesn

within

group

within

known

Age

at

diagnosis

-LRB-

years

-RRB-

Median

-LRB-

range

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Nodal

Number

of

positive

nodesMedian

-LRB-

range

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

ER

Status

at

Tumor

size

-LRB-

cm

-RRB-

Median

-LRB-

range

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Tumor

.66.01034.64.9287639.442.084438.040.231,08348.752.01,15351.954.9

Statistical

analysis

was

performed

using

SPSS

In

univariate

analysis

breast-cancer

specific

survival

-LRB-

BCSS

-RRB-

was

estimated

using

Kaplan

Meier

-LRB-

KM

-RRB-

curves

and

significant

differences

determined

by

Log

rank

tests

For

BCSS

survival

time

was

censored

at

the

date

of

death

if

the

cause

was

not

breast

cancer

or

at

the

date

of

last

follow-up

if

the

patient

was

still

alive

at

the

end

of

the

study

period

patients

with

unknown

cause

of

death

were

excluded

from

BCSS

analysis

Cox

proportional

hazards

models

were

used

to

calculate

adjusted

hazard

ratios

accounting

for

covariates

Kendall

's

tau-b

and

the

Mann-Whitney

tests

were

used

to

measure

the

correlation

of

c-kit

status

to

pathological

variables

All

statistical

tests

were

two-sided

The

Bonferroni

correction

for

multiple

comparisons

was

applied

during

validation

set

analysis

and

the

alpha

value

for

each

comparison

is

n

where

n

is

the

total

number

of

comparisons

X-tile

analysis

We

also

used

X-tile

software

-LSB-

-RSB-

to

find

the

optimal

cut-off

point

for

the

total

number

of

MCs

that

will

predict

prognosis

in

breast

cancer

patients

X-tile

program

split

the

cohort

randomly

into

a

matched

training

and

validation

set

as

a

method

for

selecting

optimal

cut-points

It

than

calculated

a

P

value

for

every

possible

division

of

the

cohort

expression

data

A

two-dimensional

graph

with

its

corresponding

survival

curves

was

plotted

where

each

colored

pixel

was

proportional

to

is

χ2

Value

The

program

automatically

calculated

the

maximum

χ2

value

which

served

as

a

cut-point

to

separate

the

number

of

MCs

that

predicted

prognosis

Results

Out

of

cases

on

the

TMAs

we

selected

breast

cancers

that

showed

invasive

tumor

in

the

cores

The

clinico-pathologic

characteristics

of

patients

included

in

the

study

are

depicted

in

Table

The

total

number

of

stained

MCs

was

recorded

as

a

continuous

variable

with

counts

ranging

from

to

MCs

per

core

They

were

seen

as

μm

round

to

oval

mononuclear

cells

with

granular

cytoplasm

and

single

oval

nucleus

The

cytoplasmic

granules

were

ganglion

net

or

crystal-shaped

-LRB-

Fig.

-RRB-

Fig.

core

showing

stromal

mast

cells

stained

with

c-kit

-LRB-

-RRB-

Magnification

MCs

are

seen

as

brown

granular

stained

oval

spindle

or

polygonal

cells

Training

set

results

Survival

analysis

A

total

of

patients

were

included

in

the

training

set

analysis

After

excluding

cases

which

had

insufficient

invasive

tumor

missing

core

or

un-interpretable

staining

pattern

cases

were

carried

forward

for

the

analysis

Out

of

these

MCs

were

present

in

-LRB-

-RRB-

cases

The

mean

survival

time

of

patients

with

presence

of

stromal

MCs

was

years

-LRB-

CI

-RRB-

compared

to

years

-LRB-

CI

-RRB-

for

those

who

did

not

have

positively

stained

MCs

in

their

tumor

stroma

KM

survival

analysis

-LRB-

Fig.

-RRB-

showed

that

the

presence

of

stromal

MCs

was

a

favourable

prognostic

marker

in

the

entire

training

set

-LRB-

BCSS

years

Log

rank

-LSB-

Mantel

Cox

-RSB-

P

-RRB-

Fig.

survival

curve

for

all

patients

in

training

set

-LRB-

a

-RRB-

and

validation

set

-LRB-

b

-RRB-

with

presence

of

stromal

mast

cells

Correlation

with

other

biomarkers

There

was

positive

correlation

between

MCs

and

ER

-LRB-

Kendall

's

tau-b

-LSB-

τb

-RSB-

P

-RRB-

-LRB-

τb

P

-RRB-

and

-LRB-

τb

P

-RRB-

and

negative

correlation

between

MCs

and

EGFR

-LRB-

τb

P

-RRB-

and

-LRB-

τb

P

-RRB-

in

the

training

set

analysis

-LRB-

Table

-RRB-

As

these

correlations

were

either

not

significant

or

extremely

weak

they

were

not

carried

forward

to

the

validation

set

for

further

analysis

Table

between

mast

cells

and

other

biomarkersTraining

setMast

cellsERKendall

's

Significance

-LRB-

-RRB-

EGFRKendall

's

tau-b

Significance

-LRB-

-RRB-

's

Significance

-LRB-

-RRB-

's

tau-b

Significance

-LRB-

-RRB-

's

Significance

-LRB-

-RRB-

N

scorable

for

both

markersAll

scores

binarized

as

detailed

in

the

text

Nodal

status

KM

survival

analysis

showed

no

statistically

significant

difference

in

the

survival

between

tumors

with

and

without

MCs

in

node-negative

-LRB-

BCSS

years

Log

rank

-LSB-

Mantel

Cox

-RSB-

P

-RRB-

and

a

significant

difference

in

the

node-positive

group

-LRB-

BCSS

years

Log

rank

-LSB-

Mantel

Cox

-RSB-

P

-RRB-

Hence

this

result

was

also

not

carried

forward

to

the

validation

set

Multivariate

analysis

Cox

proportional

hazard

model

was

used

to

carry

out

the

multivariate

analysis

and

included

age

tumor

grade

tumor

size

nodal

status

ER

and

as

independent

predictors

of

BCSS

All

the

above

variables

achieved

statistical

significance

as

shown

in

Table

-LRB-

a

-RRB-

Presence

of

MCs

achieved

statistical

significance

-LRB-

P

-RRB-

with

a

HR

CI

Table

proportional

hazard

regression

analysis

showing

hazard

ratios

and

P-values

in

patients

with

invasive

breast

carcinomaSignificanceHRBCSS

CI

for

HRLowerUpper

-LRB-

a

-RRB-

Training

setMast

Size

of

the

lesion

cm

vs.

cm

vs.

Grade

Grade

vs.

Grade

Nodal

Age

vs.

vs.

vs.

-LRB-

b

-RRB-

Validation

setMast

Size

of

the

lesion

cm

vs.

cm

vs.

Grade

Grade

vs.

Grade

Nodal

Age

vs.

vs.

vs.

Grade

well

differentiatedGrade

moderately

differentiatedGrade

poorly

differentiatedHR

adjusted

hazard

ratio

CI

confidence

interval

and

BCSS

Breast

Cancer

Specific

Survival

Validation

set

results

Survival

analysis

This

group

included

the

remaining

patients

from

the

whole

cohort

The

mean

age

at

diagnosis

was

years

and

the

median

follow-up

was

years

The

median

tumor

size

was

cm

of

patients

had

Grade

tumors

were

node

positive

and

were

ER

positive

After

excluding

cases

that

had

insufficient

invasive

tumor

missing

core

or

un-interpretable

staining

pattern

cases

were

carried

forward

for

the

analysis

Out

of

these

MCs

were

present

in

-LRB-

-RRB-

cases

KM

survival

analysis

-LRB-

Fig.

-RRB-

showed

that

the

presence

of

stromal

MCs

was

a

favourable

prognostic

marker

in

the

validation

set

-LRB-

P

-RRB-

Multivariate

analysis

Cox

proportional

hazard

model

was

used

to

carry

out

the

multivariate

analysis

and

included

age

tumor

grade

tumor

size

nodal

status

ER

and

as

independent

predictors

of

BCSS

All

the

above

variables

achieved

statistical

significance

as

shown

in

Table

-LRB-

b

-RRB-

Presence

of

MCs

did

not

achieve

statistical

significance

-LRB-

P

-RRB-

but

retained

a

similar

HR

CI

as

in

the

training

set

analysis

X-tile

analysis

results

We

assessed

the

association

between

patient

outcome

and

number

of

positively

stained

MCs

in

the

stroma

of

tumors

using

X-tile

software

This

software

allowed

us

to

define

an

optimal

cut-off

point

that

defined

the

number

of

MCs

needed

to

predict

good

prognosis

in

the

cancer

patients

The

program

divided

the

population

into

low

and

high-risk

groups

based

on

the

number

of

MCs

detected

It

converted

continuous

data

into

ordinal

classes

for

statistical

analysis

The

X-tile

plot

showed

that

breast

cancer

patients

with

any

number

of

stromal

MCs

had

better

prognosis

compared

to

those

who

have

no

MC

infiltration

in

their

stroma

MCs

ranging

between

and

in

the

tumor

stroma

were

present

in

-LRB-

-RRB-

patients

and

were

grouped

together

in

the

low

population

group

of

the

training

set

The

high

population

group

comprised

of

-LRB-

-RRB-

patients

in

the

training

set

It

reiterated

the

findings

obtained

by

KM

survival

analysis

that

the

presence

of

any

number

of

stromal

MCs

predicts

good

prognosis

in

invasive

breast

cancer

patients

Discussion

The

importance

of

the

reciprocal

relationship

between

tumor

and

stroma

is

being

increasingly

recognized

-LSB-

-RSB-

and

is

the

role

of

stromal

inflammatory

cells

like

MCs

macrophages

fibroblasts

and

T

cell

subtypes

in

cancer

initiation

and

progression

cDNA

microarray

analysis

has

shown

that

genes

expressed

by

stromal

cells

correlate

with

differences

in

the

biology

of

the

tumors

and

are

prognostic

predictors

in

breast

cancer

-LSB-

-RSB-

There

is

an

ongoing

debate

about

possible

detrimental

or

beneficial

effects

of

MC

accumulation

in

the

stroma

of

solid

tumors

In-vitro

studies

have

shown

that

MC

inhibits

tumor

growth

-LSB-

-RSB-

This

is

in

agreement

with

other

studies

showing

increase

in

MC

count

in

early

stage

of

non-small

cell

lung

cancer

and

thereby

supporting

their

anti-tumor

role

-LSB-

-RSB-

Increased

islet/stromal

MC-ratio

and

presence

of

tumor

islet

MCs

were

shown

to

be

independent

good

prognostic

indicators

in

non-small-cell

lung

cancer

-LSB-

-RSB-

Recent

studies

in

ovarian

cancer

-LSB-

-RSB-

and

colorectal

cancer

-LSB-

-RSB-

have

also

confirmed

their

correlation

with

prognosis

MCs

secrete

factors

like

heparin

-LRB-

-RRB-

and

vascular

endothelial

growth

factor

that

promote

neovascularization

histamine

which

suppresses

immune

response

platelet-derived

growth

factor

nerve

growth

factor

and

stem-cell

factors

which

are

mitogenic

and

proteases

that

promote

metastasis

-LSB-

-RSB-

On

the

other

hand

MCs

can

inhibit

tumor

growth

by

releasing

endogenous

peroxidase

that

is

cytotoxic

to

mammalian

tumor

cells

-LSB-

-RSB-

by

natural

cytotoxicity

-LSB-

-RSB-

and

by

recruiting

neutrophils

eosinophils

lymphocytes

and

macrophages

-LSB-

-RSB-

MCs

can

secrete

protective

substances

without

degranulation

in

the

presence

of

tumor-derived

blockers

like

oxidized

polyamines

and

thus

be

detrimental

to

the

tumor

-LSB-

-RSB-

They

can

destroy

tumor

cell

surface

structures

directly

and

indirectly

in

a

fashion

similar

to

the

effect

of

arginase

-LSB-

-RSB-

MCs

can

also

inhibit

tumor

growth

by

secreting

beneficial

cytokines

like

and

tumor

necrosis

factor-α

that

induce

apoptosis

of

endothelial

cells

-LSB-

-RSB-

and

chondroitin

sulphate

which

inhibits

metastases

-LSB-

-RSB-

Cathepsin

G

secreted

by

MC

activates

platelets

lymphocytes

and

macrophages

and

is

known

to

be

cytotoxic

to

some

mammalian

cells

-LSB-

-RSB-

Its

inhibition

by

a

tumor

cell

product

was

implicated

in

the

progression

of

advanced

squamous

cell

tumors

-LSB-

-RSB-

suggesting

its

cytotoxic

capability

against

target

cells

MCs

secrete

chymase

which

stimulates

apoptosis

in

different

target

cells

-LSB-

-RSB-

and

inhibits

angiogenesis

by

exerting

cytotoxic

effect

on

vascular

smooth

muscles

-LSB-

-RSB-

MC

tryptase

stimulates

inflammation

and

recruits

fibroblasts

leading

to

tumor

fibrosis

and

thereby

limiting

tumor

growth

and

metastasis

-LSB-

-RSB-

Tryptase

however

is

also

implicated

in

angiogenesis

and

its

effect

correlated

with

a

better

response

to

chemotherapy

in

ovarian

cancer

This

suggests

that

MC

tryptase-mediated

fibrosis

and

angiogenesis

may

be

responsible

for

the

good

prognostic

effect

seen

in

advanced

ovarian

cancer

-LSB-

-RSB-

In

this

TMA

study

we

showed

that

the

presence

of

even

one

or

more

MCs

in

the

tumor

microenvironment

was

sufficient

to

exert

a

positive

prognostic

effect

Our

finding

supports

the

idea

of

assessing

inflammatory

cell

infiltrates

as

prognostic

markers

in

cancer

In

our

previously

published

pilot

report

we

had

observed

no

correlation

between

the

presence

of

stromal

MCs

with

either

B-cells

-LRB-

-RRB-

or

T-cells

-LRB-

-RRB-

-LSB-

-RSB-

and

hence

we

did

not

stain

for

these

inflammatory

cells

in

this

study

We

also

did

not

find

a

statistically

significant

prognostic

effects

of

MCs

in

the

node-negative

group

in

the

training

set

analysis

This

differs

from

our

earlier

published

report

-LSB-

-RSB-

on

a

case

series

which

concluded

that

the

presence

of

MCs

was

a

favourable

prognostic

factor

in

the

node-negative

patients

-LRB-

P

-RRB-

but

not

the

node-positive

group

-LRB-

P

-RRB-

This

finding

underscores

the

importance

of

validating

clinically

relevant

findings

on

a

larger

series

of

patients

and

using

the

test-validation

approach

to

arrive

at

a

meaningful

conclusion

A

number

of

studies

have

elaborated

on

the

role

of

immune

cells

in

cancer

etiopathogenesis

T

regulatory

-LRB-

Treg

-RRB-

cells

are

known

to

be

associated

with

poor

outcome

in

ovarian

cancer

-LSB-

-RSB-

and

the

initial

results

of

a

clinical

trial

aimed

at

specifically

eliminating

Treg

cells

has

shown

promising

results

-LSB-

-RSB-

Lymphoma-associated

macrophages

have

been

used

to

predict

the

outcome

of

follicular

lymphoma

-LSB-

-RSB-

and

reactive

macrophages

are

used

to

predict

breast

prostate

ovarian

and

cervical

cancer

outcome

-LSB-

-RSB-

Tumor-infiltrating

neutrophils

are

used

to

predict

outcome

in

patients

with

adenocarcinoma

of

the

bronchioloalveolar

carcinoma

subtype

-LSB-

-RSB-

and

gastric

carcinoma

-LSB-

-RSB-

In

a

recent

report

it

was

concluded

that

the

type

density

and

location

of

immune

cells

within

the

tumor

samples

are

a

better

predictor

of

patient

survival

than

the

histopathological

methods

currently

used

to

stage

colorectal

cancer

-LSB-

-RSB-

In

conclusion

we

confirm

our

earlier

pilot

study

findings

and

confirm

that

stromal

MCs

correlate

with

a

good

prognosis

in

a

large

cohort

of

invasive

breast

cancer

patients

with

long-term

follow-up

It

highlights

the

critical

role

that

the

host

stromal

reaction

in

particular

the

inflammatory

cell

infiltrate

plays

in

modulating

cancer

progression

MCs

can

be

used

as

markers

for

risk

stratification

in

invasive

breast

cancers

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


Purification and bioactivity of exendin-4 , a peptide analogue of GLP-1 , expressed in Pichia pastoris Exendin-4 , a peptide analogue of glucagon-like peptide-1 -LRB- GLP-1 -RRB- , has been developed for treatment of type 2 diabetes . Herein , the secretive exendin-4 fusion protein , expressed by methanol induction in Pichia pastoris system , was purified to homogeneity by chromatography followed by enterokinase cleavage of the fusion protein and subsequent purification of the recombinant exendin-4 . Purity of the recombinant exendin-4 was 95.6 % . Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo . Introduction Exendin-4 is a 39 amino acid peptide -LRB- 53 % structural homology to GLP-1 -RRB- and was first isolated from the salivary secretions of Gila monster lizard -LRB- Heloderma suspectum -RRB- -LRB- Eng et al. 1992 -RRB- . It shares many of the glucoregulatory actions with GLP-1 -LRB- Buse et al. 2004 ; Kendall et al. 2005 -RRB- and has aroused great attention for its potential for the treatment of diabetes . Clinical and non-clinical studies have shown that exendin-4 has several beneficial anti-diabetic actions that include glucose-dependent enhancement of insulin secretion , glucose-dependent suppression of inappropriately high glucagon secretion , slowing of gastric emptying , reduction of food intake and body weight , and an increase in β-cell mass -LRB- Buse et al. 2004 ; Edwards et al. 2001 ; Kendall et al. 2005 ; Kolterman et al. 2003 ; Xu and Kaneto 2006 -RRB- . For many years exendin-4 has been synthesized chemically at a high price and is thus unsuitable for mass production . It therefore needs to be produced in quantity by genetic recombinant technology . Exendin-4 has been successfully expressed in E. coli system and it proved having glucose-lowering action in vivo -LRB- Yi et al. 2006 ; Yin et al. 2005 -RRB- . However , no successful expression in other expression systems has been reported until now . For large-scale production , our laboratory recently constructed a recombinant Pichia pastoris that can produce exendin-4 constitutively and secrete it into the broth -LRB- Zhuang et al. 2007 -RRB- . In this study , the purification and bioactivity of recombinant exendin-4 expressed in P. pastoris are reported . Materials and methods Microorganism and expression of exendin-4 fusion protein Pichia pastoris GS115 was transformed with plasmid pPIC9 containing the gene encoding exendin-4 , which was obtained from GenBank database -LRB- Accession No . AAB22006 -RRB- . The details of the vector construction and transformation were described elsewhere -LRB- Zhuang et al. 2007 -RRB- . Recipes and fermentation procedures were all followed by the supplier 's protocol and were described briefly as follows . Fermentation of the pPIC9/exendin -4 transformant of was carried out using a 30 l fermentor . After 48 h of growth at 28 °C , the transformant P. pastoris was induced by methanol for 72 h . The culture was centrifuged at 5,000 g for 15 min to collect the supernatant which was used as the source of recombinant protein . Purification of fusion protein For the purification of exendin-4 , the total protein in the supernatant was first concentrated by ultrafiltration using a 10 kDa molecular mass cutoff membrane . The fusion protein was subsequently purified by ion-exchange chromatography , hydrophobic interaction chromatography -LRB- HIC -RRB- and size-exclusion chromatography . The void volume and eluted fractions from the three chromatographic columns were monitored at 280 nm . Fractions were collected and subjected to SDS-PAGE analysis . The purification steps were shown in Table 1 . Table 1Summary of exendin-4 purificationPurification stepTotal protein -LRB- mg -RRB- Protein of interesta -LRB- mg -RRB- Recovery -LRB- % -RRB- Fusion exendin-4Broth supernatant1 ,640580100 DEAE A-251,20039067 Phenyl FF76028549G-7541021437Recombinant exendin-4G-25214264.5 The concentrated broth supernatant was dialyzed with buffer A -LRB- 20 mM Tris/HCl , pH 6.0 , 1 mM EDTA -RRB- and was applied to a DEAE Sephadex A-25 -LRB- DEAE A-25 -RRB- -LRB- 100 ml bed volume -RRB- equilibrated with buffer A beforehand , at 1 ml/min . The column was washed with 300 ml buffer A and then eluted with a linear gradient of 0 to 1 M NaCl in buffer A . The eluted fractions containing exendin-4 were pooled and dialyzed . The desalted protein was mixed with ammonium sulfate at 1 M and applied onto a phenyl-Sepharose Fast Flow -LRB- Phenyl FF -RRB- column -LRB- 50 ml bed volume -RRB- pre-equilibrated with buffer B -LSB- 20 mM Tris/HCl , pH 6.0 , 1 M -LRB- NH4 -RRB- 2SO4 , 1 mM EGTA -RSB- at 1 ml/min , the column was washed with 150 ml buffer B and eluted with a linear -LRB- NH4 -RRB- 2SO4 gradient from 1 to 0 M in buffer B . The peak with exendin-4 was concentrated and dialyzed with buffer C -LRB- 50 mM PBS , pH 6.0 , 10 % -LRB- v/v -RRB- glycerol -RRB- . The last step of purification was through a Sephadex G-75 column -LRB- 1 × 50 cm -RRB- pre-equilibrated with buffer C at 0.5 ml / min . For recombinant protein purification , the pool of fractions containing purified fusion protein from the three columns was dialyzed in 20 mM Tris/HCl -LRB- pH 6.0 -RRB- aThe amount of protein of interest was determined by quantifying the amount in each gel lane by densitometry -LRB- Totallab V1 .11 software -RRB- Cleavage of the fusion protein by enterokinase After desalting , the fusion protein was incubated with enterokinase -LRB- 1 U/1 mg fusion protein -RRB- at 37 °C for 8 h to obtain the recombinant exendin-4 . The reaction mixture was analyzed by Tricine SDS-PAGE . The recombinant exendin-4 was purified by Sepharose G-25 column and its purity was assayed by HPLC . SDS-PAGE , Tricine SDS-PAGE , immunoblotting and protein determination Proteins were analyzed by SDS-PAGE under reducing conditions using 12 % -LRB- v/v -RRB- gels . Tricine SDS-PAGE was carried out with the supplier 's protocol -LRB- Amersham Bioscience -RRB- . The separated proteins were stained with Coomassie Brilliant Blue R-250 or electroblotted to nitrocellulose membrane for western blotting . After blocking with 3 % -LRB- v/v -RRB- non-fat milk in 0.05 % Tween/PBS -LRB- PBST -RRB- , the membrane was washed 3 times with PBST for 10 min and incubated further with a 1:2,000 dilution of polyclonal rat anti exendin-4 antibody for 1 h , followed by washing as was described above . The membrane was incubated with a 1:1,000 dilution of HRP-conjugated goat anti-rat IgG as the second antibody , washed with the same procedure as above and revealed with DAB substrate -LRB- Sigma -RRB- . Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard . Biological activity assay The effects of recombinant protein exendin-4 on plasma glucose concentration and insulin levels were examined using 8 week-old male Wistar rats . Animals were housed six per cage -LRB- three repetition -RRB- at 24 °C environmental conditions with free access to food and water . They were allowed one week to adapt to their environment before the experiment . Food was withdrawn for 24 h before intraperitoneal injection of glucose -LRB- 20 mM/kg body weight -RRB- alone or in combination with exendin-4 -LRB- 10 nmol/kg body wt -RRB- . Test solutions were administered in a final volume of 1 ml/kg body weight -LRB- O'Harte et al. 2000 -RRB- . Blood samples were collected at 15 , 30 and 60 min after the injection from the eye socket with capillary into chilled heparin Eppendorf tubes . Blood samples were centrifuged and plasma samples were stored at − 20 °C before determination . Plasma glucose and insulin level were assayed respectively by a glucose assay kit and insulin enzyme linked immunosorbent assay kit -LRB- Dingguo , Beijing -RRB- . Results were expressed as mean ± SE and were analyzed by one-way analysis of variance -LRB- ANOVA -RRB- by SPSS 11.0 software . Differences were considered significant at P < 0.05 . Results Expression and detection The results of expression and detection exendin-4 fusion protein are given in Fig. 1 . Fig. 1Expression and detection of exendin-4 fusion protein . -LRB- a -RRB- SDS-PAGE analysis of the expression level of fusion exendin-4 at different time in hours . After 56 h , the amount of the fusion protein expression reached the maximum -LRB- 580 mg/l -RRB- . Fifteen micro liter of supernatant were applied to a 12 % gel and then stained with R-250 . Lane 1 , control transformant GS155/pPIC9 after 72 h culture ; Lanes 2 -- 10 , recombinant GS115/pPIC9/exendin -4 after 8 , 16 , 24 , 32 , 40 , 48 , 56 , 64 and 72 h of culture ; The position of the target proteins induced by methanol is indicated by arrow . -LRB- b -RRB- Western blotting analysis of expressed protein in P. pastoris . Lanes 1 -- 8 expressed protein of recombinant GS115/pPIC9/exendin -4 after 0 , 8 , 16 , 24 , 32 , 40 , 48 and 56 h , lane 9 expressed protein of control transformant GS115/pPIC9 Purifying the fusion protein and obtaining the recombinant exendin-4 by enterokinase cleave Purification of the fusion protein and production of the recombinant exendin-4 by enterokinase cleavage are given in Table 1 and further details were shown in Fig. 2 . Fig. 2 -LRB- a -RRB- Purification of the exendin-4 fusion protein by three chromatography steps . Lane 1 , broth supernatant ; lane 2 , DEAE A-25 ; lane 3 , Phenyl FF ; lane 4 , G-75 ; M is protein marker -LRB- KDa -RRB- . -LRB- b -RRB- Purification of the recombinant protein exendin-4 by a G-25 column . Lane 1 , reaction mixture of fusion protein cleaved by enterokinlase . Recombinant exendin-4 -LRB- 4.3 kDa -RRB- is marked by arrow ; lane 2 , the non-target protein eluted from the G-25 column ; lane 3 , purified recombinant peptide exendin-4 from G-25 column . -LRB- c -RRB- . The purity analyzed by HPLC was 95.6 % which was performed on a 250 mm × 4.6 mm C5 column . The column was eluted with the linear gradient of acetonitrile -LRB- 90 -- 0 % -RRB- in 0.1 % trifluoroacetic acid for 35 min at 0.5 ml/min . The target peak was at 18.3 min Bioactivity of exendin-4 in vivo Figure 3 showed the results of bioactivity of exendin-4 in vivo . Compared with the control group , plasma glucose concentration in GLP-1 group was significantly reduced -LRB- P < 0.05 -RRB- at 30 min , and the same phenomenon was observed after 60 min in exendin-4 group -LRB- Fig. 3a -RRB- . There was no difference between the glucose-lowering capabilities of treatment group and control group before 15 min . The capabilities of stimulating insulin secretion showed significant response to GLP-1 and exendin-4 supplementation . The maximal increase in insulin in rat treated with GLP-1 was 2 times higher than the control group before 30 min . The plasma insulin concentration of exendin-4 treated group was also significantly raised -LRB- P < 0.05 -RRB- at 30 min compared with the control group and the capabilities of stimulating insulin secretion by exendin-4 could keep to 60 min -LRB- Fig. 3b -RRB- . Fig. 3Biological activity assay of exendin-4 in rats . The capabilities of glucose-lowering -LRB- a -RRB- and insulin-releasing -LRB- b -RRB- after intraperitoneal glucose alone -LRB- 20 mM/kg -RRB- -LRB- control group -RRB- , or glucose in combination with either exendin-4 -LRB- trial group -RRB- or GLP-1 -LRB- positive group -RRB- -LRB- 10 nmol/kg -RRB- . The time of injection is indicated by the arrow -LRB- 0 min -RRB- . Values are mean ± SE -LRB- n = 6 -RRB- . Asterisk denotes statistically significant differences -LRB- P < 0.05 -RRB- between control and trial -LRB- or positive -RRB- groups by one-way ANOVA Discussion For many years , the production of exendin-4 was by chemical synthesis which necessitated a high cost . Recently , bioengineering methods of producing exendin-4 have appeared . Yi et al -LRB- 2006 -RRB- and Yin et al -LRB- 2005 -RRB- cloned the exendin-4 gene and obtained stable expression in E. coli . However , the E. coli-expressed exendin-4 existed initially in inclusion bodies and bioactivity was realized only after renaturation , which made the purification process complicated and led to a low yield . Furthermore , clinical application of bacterially produced products may be affected by the possible presence of endotoxins that sometimes contaminate protein preparations expressed by E. coli . We have several reasons to select P. pastoris as an alternative recombinant expression host for exendin-4 synthesis . P. pastoris has the advantages of large production , genetically stable expression strains , the potential to secrete recombinant proteins into culture medium , and simple inexpensive culture conditions -LRB- Patrick et al. 2005 -RRB- . In this study , the highest expression level of fusion protein exendin-4 was estimated at about 580 mg/l culture , which was higher than E. coli system . In addition , Pichia pastoris-synthesized recombinant exendin-4 was effectively purified from the culture medium through four chromatograph steps in this study . The whole purification process was simple and easy . These properties make P. pastoris a superior system to E. coli for preparation of exendin-4 . To the best of our knowledge , this is the first study to directly investigate the effects of recombinant exendin-4 expressed in P. pastoris on glucose-lowering and insulin-releasing in vivo . The results suggested that the ability of native peptide GLP-1 to reduce plasma glucose to a certain level was at 30 min , while the activity of exendin-4 appeared at 60 min -LRB- Fig. 3a -RRB- . It indicated that the exendin-4 has a longer duration of action and a greater potency than GLP-1 -LRB- Fig. 3a -RRB- . Insulin is one of the most important hormonal immune responses and is linked to glucose-lowering responses . Although reports on influence of administration of exenatides on insulin activity are not very consistent , this function can be enhanced by injection administration of exendin-4 or its analogue -LRB- Gedulin et al. 2007 ; Kolterman et al. 2003 ; Nielsen et al. 2004 ; Xu and Kaneto 2006 -RRB- . The present study observed similar phenomenon . Furthermore , we observed that the capabilities of stimulating insulin secretion by GLP-1 was maintained up to 30 min , while exendin-4 was up to 60 min -LRB- Fig. 3b -RRB- . Thus the half-life of exendin-4 in plasma is longer than GLP-1 . There are two main reasons for this . One reason is that the exendin-4 lacks many of the neutral endopeptidase substrate sites present in GLP-1 and has a nine-AA sequence at the C-terminus absent from GLP-1 -LRB- Hupe et al. 1995 -RRB- . Another one is that the N-terminal sequence -LRB- His : Gly : Glu -RRB- of exendin-4 is not recognized by dipeptidylpeptidase -LRB- IV -RRB- -LRB- DPP -LRB- IV -RRB- -RRB- , which rapidly cleaves the His : Ala : Glu sequence found on the N-terminus of GLP-1 -LRB- O'Harte et al. 2000 -RRB- . In our opinion , we should not rule out the presumption that the exendin-4 -LRB- 4.3 kDa -RRB- has higher molecular weight than GLP-1 -LRB- 3.1 KDa -RRB- , which can reduce the glomerular filtration in vivo . It is important to note that in the three independent experiments , the recombinant exendin-4 did not have obvious effects on one or two rats of the six . It was probably because of individual differences . The results of the three independent experiments were comparable . According to the research , the recombinant exendin-4 may also have effects at smaller doses but remains to be proved . In summary , we have achieved high-level secretion of biologically active exendin-4 in P. pastoris . The result of the study indicated that although the heterogeneity-expressed exendin-4 in P. pastoris was slightly different from native GLP-1 in biological value , it still maintained good bioactivity . High purity and stability of the recombinant peptide of exendin-4 after purification makes it possible to be a therapeutical drug in the future . In the next step , we will focus on structure analysis and evaluation of the safety of exendin-4 .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Clinical review : Outreach -- a strategy for improving the care of the acutely ill hospitalized patient We examined the literature relating to the safe care of acutely ill hospitalized patients , and found that there are substantial opportunities for improvement . Recent research suggests substantial benefit may be obtained by systems of outreach care that facilitate better integration , co-ordination , collaboration and continuity of multidisciplinary care . Herein we review the various approaches that are being adopted , and suggest the need for continuing evaluation of these systems as they are introduced into different health care systems . Introduction Health care providers in many countries are increasingly aware that quality of care can be improved by measures to reduce errors . Evidence suggests that the greatest opportunities for improvement may be in the management of the acutely ill hospitalized patient , where uncertainty , urgency and lack of integration substantially increase the risk of errors leadinf to adverse outcomes . Different approaches to this problem have evolved , including hospitalists in the USA , medical emergency teams in Australia , and outreach care in the UK . Critical care has a central role to play in all three developments , which share the common aim of improving the safe care of acutely ill patients as they travel through the health care system . We will review the background , methods , roles and benefits of these various systems which we group under the heading of ` outreach ' care . Method We focused on identifying research publications that examined ways of improving the integration of critical care with acute care services as a means of improving the safe care of acutely ill hospitalized patients . Publications were initially identified by an electronic search of Medline and Cinahl , and the cited references provided additional material . The initial date range searched was 1995 -- 2003 to ensure that current research and up to date literature was reviewed . However , this produced only a limited number of references , and the time period was therefore extended to 1990 . Keywords searched singularly and in combination were ` acute pain team ' , ` suboptimal care ' , ` patient at risk ' , ` critical illness ' and ` cardiopulmonary resuscitation ' . Only English language references were included in the analysis . Background Trends in hospital care Hospitals are becoming increasingly complex environments because of developments in medical technology , more potent treatments , and an ageing and dependent population of patients . The proportion of emergency admissions continues to rise in most countries -LSB- 1 -RSB- , whereas the stock of hospital beds has fallen . Combined with political demands for cost containment , this has resulted in shorter hospital stays , and increased bed occupancy and throughput -LSB- 2 -RSB- . At the same time there has been a trend toward greater transparency in decision making , rising public expectations , and improved public access to information about quality of care and outcomes from medical interventions . These changes have coincided with increasing difficulty with the recruitment and retention of trained nurses -LSB- 3 -RSB- , and constraints on doctors ' hours of work . The implementation of the European Working Time Directive -LSB- 4 -RSB- , and similar trends in the USA -LSB- 5 -RSB- , means that proportionately fewer staff are available to manage this increased workload , particularly the out-of-hours and emergency elements . Shorter training times reduce the expertise acquired through apprenticeship . The net effect is that sicker patients are receiving care from fewer and less experienced staff . Error and quality in health care These pressures have been accompanied by a growing awareness of the problem of error in health care and an exponential increase in litigation . The much quoted Institute of Medicine report ` To Err is Human ' -LSB- 6 -RSB- estimated that between 44 000 and 98 000 patients die each year in the USA as a result of preventable clinical errors . Studies in Australia and the UK suggest that around 10 -- 16 % of hospitalized patients experience an adverse event related to clinical care , with a mortality rate in these patients of 5 -- 8 % , and overall financial costs in the range US $ 4.7 -- 29 billion -LSB- 7-9 -RSB- . Clinical error is now widely recognized as a systems problem ; that is , adverse events affecting individual patients are usually caused by a sequence of events in the macro - and micro-environment that involve deficiencies in the structure and organization of health care , and are not simply a consequence of human error by the responsible clinician who is the terminal link in the chain . To improve safety and quality in health care requires a systems approach in which all participants -- politicians , administrators and health care professionals -- assume responsibility for patient outcomes . This ambitious task requires focusing of efforts on those patients most at risk . Identifying at-risk populations Acutely ill patients in general The risk of error and adverse outcomes would appear to be higher in the context of acute and emergency care , for example in emergency departments -LSB- 7 -RSB- or in general medical -LRB- rather than elective surgical -RRB- admissions -LSB- 8 -RSB- . The elderly are more susceptible -LSB- 7 -RSB- ; they are also more likely to be admitted as emergencies and exposed to emergency surgery -LSB- 10 -RSB- . Inexperienced clinicians and unsupervised trainees -LRB- who often deliver first-line care out-of-hours -RRB- have a higher error rate -LSB- 11,12 -RSB- . The risk for an adverse event increases by approximately 6 % per day for patients admitted with emergency conditions -LSB- 13 -RSB- , and is much increased in severely ill patients who undergo life-saving invasive interventions -LSB- 14 -RSB- . Discontinuities in care -LSB- 15 -RSB- created by shift working or poor information transfer contribute to error by failing to recognize trends in a deteriorating patient 's condition . Postoperative patients In the UK the 1993 National Confidential Enquiry into Perioperative Deaths showed that two thirds of perioperative deaths occurred 3 or more days after surgery when the patient had been returned to the ward . The majority of these deaths were from cardiorespiratory complications , and many were considered preventable by earlier identification and treatment -LSB- 16 -RSB- . The 1999 report -LSB- 17 -RSB- analyzed deaths within 30 days of a surgical procedure in elderly patients -LRB- > 90 years -RRB- , and found that suboptimal fluid management was a major cause of serious postoperative morbidity and mortality in this group . It recommended more accurate monitoring and recording of fluid balance and earlier recognition and correction of problems as a means of reducing the incidence of postoperative complications . The report also identified deficiencies in multidisciplinary care , despite the high incidence of comorbid disease in these elderly patients , and recommended more collaborative working between surgeons , anaesthetists and physicians with expertise in the care of the elderly . This type of audit based on a large observational database is essential for identifying current practice and opportunities for improvement . However , recommendations were based on peer review and data from questionnaires provided by assessors who were unblinded to clinical outcomes , examining only those patients who died -- there are no denominator data . Cardiopulmonary arrest Hospitalized patients who undergo cardiopulmonary resuscitation commonly exhibit premonitory signs and symptoms many hours before the cardiac arrest -LSB- 18-21 -RSB- . Schein and coworkers -LSB- 18 -RSB- studied 64 patients following cardiac arrest , and reported that 84 % of vital signs charts showed an acute deterioration in the patient 's condition prior to arrest . Franklin and Mathew -LSB- 20 -RSB- examined the case notes of 150 consecutive patients who had suffered a cardiac arrest on a general ward , and found documented prior clinical deterioration in 99 -LRB- 66 % -RRB- . A common finding was the failure of the nurse to notify a physician of deterioration in the patient 's condition . The interface with critical care Critical illness increases the opportunity for clinical error -LSB- 22,23 -RSB- , and this is related at least in part to the complexity of diseases , the multiplicity of therapies , frequent invasive interventions and , within the intensive care unit -LRB- ICU -RRB- , the intensity of monitoring and observation , which may paradoxically increase the apparent error rate in this environment simply by improved detection . Iatrogenic complications are a common cause for ICU admission -LSB- 24 -RSB- , and suboptimal care before referral to intensive care is associated with a markedly increased mortality -LSB- 25 -RSB- . Premature discharge from intensive care of patients recovering from critical illness is also associated with a markedly increased hospital mortality -LSB- 26 -RSB- , supporting the view that organizational aspects of clinical care profoundly influence patient outcomes -LSB- 27 -RSB- . Common errors include lack of attention to detail , poor communication , failures of organization , lack of knowledge , failure to appreciate clinical urgency , insufficient supervision and failure to seek advice . Treatment limitation decisions , futility , and end of life care Studies in the USA and Europe have demonstrated considerable diversity of practice and opportunities for improving autonomy , patient centred decision making , and quality of end-of-life care -LSB- 28,29 -RSB- . The Study to Understand Prognosis and Preferences for Outcomes and Risks of Treatment in the USA -LSB- 29 -RSB- demonstrated that 31 % of the cohort of patients admitted to intensive care would have preferred not to be resuscitated , but that clinicians were aware of this preference in only 47 % of the subset and in half it had not been documented . Aarons and Beeching -LSB- 30 -RSB- surveyed the use of ` do not resuscitate ' orders in a community hospital in the UK and suggested that end-of-life care of dying patients and their families in hospital could be substantially improved in terms of symptom relief , communication and respect for the patients ' wishes . They also concluded that poor decision making by health care workers could be improved by education . One consequence of a lack of communication between staff and a reluctance to discuss these difficult issues with patients and families is the inappropriate resuscitation and subsequent admission to the ICU of patients for whom further intervention would be futile and add to the burden of suffering . A more appropriate approach to management of acutely ill patients at risk for critical illness or cardiac arrest would be to prevent these complications , or agree treatment goals and limitations , by earlier recognition of simple warning signs . We consider the various approaches that are being adopted below . Systems for earlier recognition and management of patients at risk The problems described above require a systems approach to improving the safe care of the acutely ill hospitalized patient . The USA , the UK , Australia , Canada and Denmark are instituting national systems for improving patient safety -LSB- 31-33 -RSB- . Within this general framework , three countries have specifically identified the need to improve the care of acutely ill patients . In the USA the Leapfrog Group -LSB- 34 -RSB- has recommended that ICUs be managed by intensivists -- doctors specifically accredited in critical care medicine -- while in a parallel development many hospitals are appointing ` specialist generalists ' -LRB- ` hospitalists ' -RRB- to provide inpatient care on the wards -LSB- 35 -RSB- . Australia has promoted the establishment of medical emergency teams -LRB- METs -RRB- led by doctors as an alternative to cardiac arrest teams -LSB- 36,37 -RSB- ; and the UK has implemented the recommendation of the expert group report ` Comprehensive Critical Care ' -LSB- 38 -RSB- to establish multidisciplinary outreach care . All these developments are based on the concept of earlier intervention by people with appropriate knowledge and skills in managing acutely ill patients . Is this concept valid , and which model is the best ? Structures and processes for early intervention In the UK a survey of intensive care facilities found that , in 94 % of the units questioned , staff regularly visited wards in response to requests for advice from medical and nursing members of the admitting team -LSB- 39 -RSB- , demonstrating that this is clearly an important role for intensive care staff . However , there is little information describing the experiences and perspectives of ward based staff who care for acutely ill patients outside the intensive care environment . Gibson -LSB- 40 -RSB- found that ward staff often lacked confidence and felt ill prepared to deal with acutely ill unstable patients , and that they experienced increased stress and anxiety . Such emotions are unlikely to enhance staff retention or reduce sickness rates . It therefore makes sense to put in place systems that improve the support not only of sick patients but also of the staff responsible for their care , and that reduce discontinuities in clinical care . The key to this is empowerment through adequate resourcing and training , and simple methods of clinical monitoring . Three models are currently employed : hospitalists in the USA , the MET in Australia , and outreach care in the UK . Hospitalists Hospitalists are internists who specialize in acute hospital medicine . This new speciality has appeared in the USA during the past 8 years in response to perceived difficulties with primary care clinicians maintaining continuity of care for their patients admitted to hospital . Currently at around 5000 , their numbers are expected to increase rapidly . Hospitalists are usually salaried employees of managed care organizations , which favour their development as a means of reducing costs and duration of hospital stay -LSB- 41,42 -RSB- . The training of hospitalists is rooted primarily in internal medicine , but there is no national core curriculum . They do not appear to have responsibility for surgical patients , and therefore can not be considered generalists in the sense of dealing with all acutely ill patients . The relationship between hospitalists and intensivists has not been defined -LSB- 43 -RSB- , although there clearly are opportunities for interaction . Hospitalists occupy a role that is close to that of the general physician in UK hospitals , except that the latter group often have a subspeciality . This model is suited to the system of care in the USA , where primary care physicians often have continuing responsibility for inpatient care . It might also be of value to other health care systems in which increasing specialization is creating a need for generalist acute care clinicians in hospital practice . Acute pain teams A joint expert committee report from a Royal College of Surgeons and College of Anaesthetists working party -LSB- 44 -RSB- reviewed the evidence related to postoperative pain management and recommended the establishment of an acute pain service in all major hospitals . However , the Audit Commission reported in 1997 -LSB- 45 -RSB- that only 57 % of hospitals in the UK had established a pain service . The situation had improved by 2000 when the Clinical Standards Advisory Group found that 88 % of responding UK hospitals had set up an acute pain service , although in some cases this was only a token service . The 1990 working party did not describe a model for the ideal acute pain team -LSB- 44 -RSB- , and this has led to many inconsistencies in the provision of pain management throughout the UK . Acute pain teams ensure adequate postoperative pain relief by supporting and educating ward staff -LSB- 46 -RSB- . Concerns have been expressed that they may de-skill ward nurses -LSB- 47 -RSB- , although there is no research evidence to support this view . Because more complicated surgery is performed in older patients , who have more comorbidities , we can expect to see an increase in the number of life-threatening postoperative complications -LSB- 16 -RSB- . Surgical patients make up 60 -- 70 % of the workload of ICUs in the UK -LSB- 48,49 -RSB- . It has been suggested that the role of acute pain teams should be extended to identify nonpain problems in patients , and liaise with other specialties to manage them -LSB- 46 -RSB- . Some acute pain services have already extended their role from pain management to include fluid balance , oxygen therapy , management of nausea and vomiting , and anticoagulant prophylaxis -LSB- 50 -RSB- . This role extension requires proper training in the management of acutely ill patients and an understanding of the interface between acute medicine and intensive care . Medical emergency teams The concept of a better integrated , multidisciplinary approach to postoperative care -LSB- 51,52 -RSB- has been extended to other groups of patients in the form of specific groups or teams of clinicians , usually centred on the ICU . Examples of this evolution include postoperative care teams -LSB- 53 -RSB- , patient at risk teams -LSB- 54 -RSB- and METs -LSB- 36,48,52 -RSB- . All suggest that the introduction of such teams to identify and manage complications on the ward might prevent unnecessary admissions to the ICU and reduce morbidity and mortality . The team based approach uses calling criteria based on abnormal clinical or physiological variables , specific conditions , or patients causing concern to ward staff . The research evidence to support the validity of these criteria is limited by the practical and ethical difficulties of conducting randomized controlled trials in the clinical environment . Although postoperative care teams provide additional support , expertise and equipment for postoperative patients -LSB- 54 -RSB- , and similar approaches could improve suboptimal ward care in general -LSB- 25 -RSB- , there are no a priori definitions of inadequate care that have been prospectively calibrated against outcome in this context . However , pragmatic studies of the impact of METs indicate a reduction in cardiac arrest rates and in the use of intensive care resources for cardiac arrest survivors -LSB- 55,56 -RSB- . Critical care outreach ` Outreach ' care is a systems approach for identifying and managing patients at risk of critical illness through collaborative care and education . Rather than providing a service through an external group , it aims to empower ward staff by offering them regular support , usually led by critical care trained nurses visiting the wards , with the facility to call on more expert assistance if required . Currently , critical care is seen as occurring within a defined environment -LRB- the ICU -RRB- and patients must achieve a certain level of severity of illness to merit admission . Outreach services facilitate a more flexible approach that is based on the needs of the patients and the skills and abilities of the ward staff . Both the Audit Commission -LSB- 57 -RSB- and the Department of Health working party report ` Comprehensive Critical Care ' -LSB- 55 -RSB- supported the development of outreach care as a means of improving the care of acutely ill patients in hospital wards . The latter report identified three goals for a critical care outreach team . The first was to identify patients at risk for critical illness and either prevent their admission to ICU by timely interventions at a ward level or ensure early appropriate admission to ICU . The second was to facilitate timely and safe discharge from intensive care by following up patients discharged to the ward . The third was to share ICU skills with ward staff . Given the wide variations in hospital size , specialties , staff expertise and skills , the Department of Health report did not prescribe a standard structure for achieving these three goals . The development of outreach services should not be taken in isolation from other critical care initiatives and should be part of an integrated , multidisciplinary , hospital-wide delivery of critical care services that improves liaison between intensive , high dependency and ward care -LSB- 56 -RSB- . The majority of UK centres have nurse led outreach systems , supported by critical care doctors with sessional recognition for this service . However , the aim of outreach is to diffuse skills across many disciplines , and to enhance collaborative care . Many professional groups can therefore contribute to this approach , including physiotherapists and nutritionists as well as physicians . Studies are needed to evaluate the efficacy of this development , but current experience indicates a strongly favourable response from ward based staff . Standards for the development of outreach care are now available -LSB- 58 -RSB- . Identifying the patient at risk -- scoring systems for decision support Traditionally , the process of identifying critically ill and deteriorating patients has relied on the clinical intuition of staff . The value of experienced clinical judgement is well recognized -LSB- 36,54,59,60 -RSB- , but given the trend toward shorter training times and reduced hours of work in the clinical environment , objective systems are becoming increasingly important because inexperienced practitioners may fail to recognize impending critical illness and the need for assistance -LSB- 61 -RSB- . There are several such systems that utilize combinations of physiological variables as indicators of risk -LSB- 36,54,56,62-66 -RSB- . A summary of component variables is given in Table 1 . Hodgetts and coworkers -LSB- 67 -RSB- analyzed factors that predicted risk for cardiac arrest and could therefore be used as triggers to call for help -LRB- ` activation criteria ' -RRB- . Variables with predictive capacity included chest pain , staff concern , systolic blood pressure , oximetry , pulse and respiratory rate , and temperature . The choice of vital signs is necessarily constrained to those that are easy to record in the ward environment . More finesse may be achieved with the inclusion of laboratory tests such as serum electrolytes , blood sugar and acid-base analysis , or the inclusion of diagnosis , but the problem with this approach is that recording vital signs is often omitted or inaccurate -LSB- 17,54 -RSB- , and the greater the degree of complexity , the more likely it is that errors or omissions will occur . Scoring systems for use in ordinary wards must be simple , and should direct attention to patients who need more intensive observation ; they should be a trigger for investigation , not a precise tool for predicting individual patient outcomes . Thus , although physiological abnormalities may be unreliable predictors of the need for intensive care admission , they remain important stimuli for empowering staff to call for assistance in improving simple aspects of care of acutely ill patients . Does earlier intervention improve outcome ? It may seem unnecessarily argumentative to suggest that something that is so evidently ` good ' requires evaluation . Is it not self-evident that the earlier a life-threatening disease process is identified and treated , the better ? This may be so , but complex systems -- of which the acutely ill patient is one example -- demonstrate sensitivity to initial conditions ; that is , the outcome may be more difficult to predict with earlier application of an intervention . The outcome from cardiopulmonary resuscitation is well defined -- a survival rate of around 10 -- 15 % , with death usually occurring when resuscitation attempts are discontinued . Is it not possible that earlier intervention that prevents cardiac arrest might result in delayed death following prolonged organ system support in the ICU , with the attendant suffering that this may cause ? What is the cost -- benefit of implementing the different models of care ? Who will manage the process of discussing treatment goals or limitations with patients , and what do patients themselves feel about it ? Physiological goals The literature relating to preoperative optimization of systemic oxygen delivery in high-risk surgical patients suggests that early intervention is beneficial -LSB- 68-72 -RSB- . However , a recent large-scale study -LSB- 73 -RSB- conducted in surgical patients did not confirm this in terms of benefit from goal-directed therapy guided by pulmonary artery catheterization , perhaps because patients in the control group were already being optimally managed -LRB- and experienced a low overall mortality -RRB- as a result of improvements in clinical practice derived from earlier research . In critically ill septic patients , early intervention to optimize oxygen delivery with fluid resuscitation , vasoactive drugs and respiratory support appears to reduce mortality -LSB- 74 -RSB- , whereas similar interventions applied later in the course of illness do not -LSB- 75-77 -RSB- . It seems reasonable to conclude that using fluids and supplemental oxygen to optimize circulating volume , cardiac output and systemic oxygen delivery as early as possible in acutely ill hospitalized patients will tend to reduce the incidence and severity of organ dysfunction related to a systemic oxygen debt . These simple measures may need to be supplemented later by more complex interventions , but the ability to manage the initial phase of preventive care should be within the ability of most health care staff . Clinical outcomes Early intervention may reduce morbidity and mortality , but it is also possible that the earlier application of supportive treatment could contribute additional burdens . Buist and colleagues -LSB- 62 -RSB- demonstrated that the introduction of a MET contributed to a reduction in ward cardiac arrest rates , but this was also associated with an increase in emergency admissions to intensive care with no significant change in ICU mortality rates . This suggests that for some patients earlier intervention may have the effect of shifting the burden of mortality from the ward to the ICU , replacing a ` cheap ' death with an expensive one . More recently , however , Bellomo and colleagues -LSB- 63 -RSB- also demonstrated a marked reduction in cardiac arrest rates associated with the introduction of a MET , and a parallel reduction in the use of intensive care resources on cardiac arrest survivors . It seems likely , therefore , that hospitals with high ward based cardiac arrest rates may well benefit from the introduction of systems aimed at identifying and managing sick patients earlier . Further multicentre studies from Australia are awaited . Autonomy and treatment limitation decisions In an ideal world we would all possess clear advance directives giving guidance on how we would wish to be treated given differing circumstances . However , treatment preferences will not be static over time and are likely to be influenced by circumstances and the provision of information . How do patients respond to opportunities to discuss treatment preferences ? Studies conducted in elective or out-patient settings demonstrate that many patients wish to be involved in decisions about treatment goals and intensity . They also demonstrate that the accuracy of information substantially alters preferences for resuscitation -LSB- 78 -RSB- . In the acute care context , however , there is considerable variability in clinical practice ; moreover , many patients may not wish to discuss their preferences -LSB- 79 -RSB- , even though this results in inappropriate treatment decisions -LSB- 80 -RSB- . It is clear that discussions about treatment preferences must be conducted with sensitivity by staff with appropriate experience and training , who have had an opportunity to develop a relationship of trust with the patient . This mandates a collaborative approach between the various medical and nursing teams , the patient and the family . Early intervention may buy time for these complex discussions to take place and an appropriate decision to be made . Changing behaviour through education and training The key to improving safe care of acutely ill hospitalized patients is through team working and education , combined with improvements in resources for integrated delivery of care . In accident and emergency care , team based working reduces clinical error rates and enhances overall quality of care -LSB- 81,82 -RSB- , and the principles of crew resource management drawn from aviation have important messages for clinical practice in this respect , by empowering all members of the team to make contributions to safety -LSB- 83 -RSB- . To achieve this we must incorporate team based attitudes in medical education , starting at undergraduate level and following this through into speciality training so that there is more overlap between disciplines than is currently the case . A competency based core curriculum for acute care that incorporates education in patient safety is essential . This process has started in intensive care medicine -LSB- 84 -RSB- , and it is hoped that other disciplines will follow . Personal responsibility and continuity of care are important features of quality care , and one of the challenges of implementing outreach is that it may encourage the attitude that someone else is responsible for the patient , and thus disem-power and de-skill ward staff -LSB- 85 -RSB- . Team working and continuity of care may be secured through the development of collaboratively produced guidelines or protocols that seek to support and guide the interaction between ward and outreach staff . Outreach staff should also recognize the limitations of their own expertise . Education is a key element in outreach activities , and is a two-way process that requires sharing of expertise , collaborative support , and blurring of traditional boundaries -LSB- 86 -RSB- . Important attitudinal attributes of outreach staff must therefore include the capacity to teach , learn from , and support other clinicians , sometimes under difficult circumstances . Conclusion There are substantial opportunities for improving the safety and quality of care delivered to acutely ill hospitalized patients . The methods which are adopted will vary according to local circumstance , but common elements include the need for better integration of care across disciplines and systems for earlier identification of patients at risk , and we refer to these as ` outreach ' care . We do not yet know which of these various approaches will best improve patient outcomes , and there is a need for prospective studies in this area which take into account the difficulties of using randomization and controls , and which employ long-term follow-up . METs appear to reduce the incidence of cardiac arrests in ordinary wards , and consequential use of scarce intensive care resources . Outreach-based systems which support and educate ward-based staff in delivering clinical care appear to have achieved a high degree of acceptance in the UK . Health care managers need to work closely with clinicians to introduce these methods of team-working into hospital practice , while evaluating their effectiveness . Competing interests None declared . Abbreviations ICU = intensive care unit ; MET = medical emergency team .

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

acute pain team
cardiopulmonary resuscitation
critical care
critical illness
patient at risk
suboptimal care