Outcome

of

paediatric

intensive

care

survivors

The

development

of

paediatric

intensive

care

has

contributed

to

the

improved

survival

of

critically

ill

children

Physical

and

psychological

sequelae

and

consequences

for

quality

of

life

-LRB-

QoL

-RRB-

in

survivors

might

be

significant

as

has

been

determined

in

adult

intensive

care

unit

-LRB-

ICU

-RRB-

survivors

Awareness

of

sequelae

due

to

the

original

illness

and

its

treatment

may

result

in

changes

in

treatment

and

support

during

and

after

the

acute

phase

To

determine

the

current

knowledge

on

physical

and

psychological

sequelae

and

the

quality

of

life

in

survivors

of

paediatric

intensive

care

we

undertook

a

computerised

comprehensive

search

of

online

databases

for

studies

reporting

sequelae

in

survivors

of

paediatric

intensive

care

Studies

reporting

sequelae

in

paediatric

survivors

of

cardiothoracic

surgery

and

trauma

were

excluded

as

were

studies

reporting

only

mortality

All

other

studies

reporting

aspects

of

physical

and

psychological

sequelae

were

analysed

Twenty-seven

studies

consisting

of

survivors

met

the

selection

criteria

Distinct

physical

and

psychological

sequelae

in

patients

have

been

determined

and

seemed

to

interfere

with

quality

of

life

Psychological

sequelae

in

parents

seem

to

be

common

Small

numbers

methodological

limitations

and

quantitative

and

qualitative

heterogeneity

hamper

the

interpretation

of

data

We

conclude

that

paediatric

intensive

care

survivors

and

their

parents

have

physical

and

psychological

sequelae

affecting

quality

of

life

Further

well-designed

prospective

studies

evaluating

sequelae

of

the

original

illness

and

its

treatment

are

warranted

Introduction

The

development

of

paediatric

intensive

care

has

contributed

to

improved

survival

rates

in

children

with

critical

illnesses

-LSB-

-RSB-

Consequently

new

disease

patterns

have

emerged

due

to

long-term

complications

and

effects

of

the

original

illness

and

its

treatment

In

addition

to

survival

and

morbidity

physical

and

psychological

sequelae

as

well

as

the

quality

of

life

-LRB-

QoL

-RRB-

in

survivors

and

in

their

families

are

important

outcome

measures

Historically

outcome

research

in

paediatrics

is

either

based

on

an

age-specific

approach

such

as

follow-up

studies

of

premature

infants

-LSB-

-RSB-

or

on

a

more

disease-oriented

approach

such

as

follow-up

studies

in

survivors

of

cardiothoracic

surgery

or

trauma

-LSB-

-RSB-

These

studies

have

shown

substantial

physical

psychological

and

neuro-cognitive

sequelae

interfering

with

daily

life

and

normal

development

In

addition

effects

on

parents

and

siblings

have

been

shown

-LSB-

-RSB-

Evaluative

research

of

adult

intensive

care

survivors

showed

the

effect

of

intensive

care

treatment

per

se

Irrespective

of

the

underlying

illnesses

sequelae

on

all

domains

with

effects

on

QoL

were

found

-LSB-

-RSB-

In

multi-disciplinary

paediatric

intensive

care

unit

-LRB-

PICU

-RRB-

populations

reports

on

outcome

are

scarce

-LSB-

-RSB-

Based

on

these

observations

we

believe

that

follow-up

research

of

paediatric

intensive

care

survivors

and

their

families

is

needed

to

evaluate

-LRB-

-RRB-

physical

sequelae

and

their

impact

during

growth

and

development

-LRB-

-RRB-

psychological

sequelae

in

patients

and

their

families

and

their

impact

on

the

QoL

of

patients

and

family

members

and

-LRB-

-RRB-

the

need

for

treatment

and

support

after

discharge

The

aim

of

this

article

is

to

provide

an

overview

of

the

available

literature

concerning

the

different

domains

of

QoL

-LRB-

i.e.

physical

psychological

and

social

functioning

-RRB-

in

children

surviving

paediatric

intensive

care

including

the

effect

on

parents

and

to

suggest

directions

for

future

follow-up

research

Methods

To

identify

studies

eligible

for

this

review

we

searched

Medline

-LRB-

-RRB-

EMBASE

-LRB-

-RRB-

CINAHL

-LRB-

-RRB-

pre-CINAHL

and

the

Cochrane

Library

-LRB-

-RRB-

in

March

In

the

search

strategy

all

terms

mapped

to

the

appropriate

MeSH/EMTREE

subject

headings

and

exploded

were

used

among

them

were

paediatric

intensive

care

unit

-LRB-

PICU

-RRB-

septic

shock

respiratory

insufficiency

meningococcal

disease

central

venous

catheterisation

intubation

physical

and

psychological

sequelae

post-traumatic

stress

disorder

-LRB-

PTSD

-RRB-

QoL

health

status

and

long-term

outcome

Definitions

Functional

health

is

defined

as

an

individual

's

ability

to

perform

normal

daily

activities

to

fulfil

usual

roles

and

to

maintain

health

and

well-being

QoL

is

defined

as

an

individual

's

perception

of

their

position

in

life

in

the

context

of

the

culture

and

value

systems

and

in

relation

to

their

goals

expectations

standards

and

concerns

-LSB-

-RSB-

Health-related

QoL

-LRB-

HRQoL

-RRB-

is

defined

as

QoL

in

which

a

dimension

of

personal

judgement

over

one

's

health

and

disease

is

added

-LSB-

-RSB-

Study

selection

Studies

were

selected

for

review

if

they

met

two

inclusion

criteria

-LRB-

-RRB-

study

of

a

representative

population

of

PICU

survivors

-LRB-

defined

as

a

population

consisting

of

medical

and/or

surgical

PICU

patients

years

old

-RRB-

and

-LRB-

-RRB-

evaluation

of

physical

sequelae

measurement

of

QoL

or

functional

health

days

after

PICU

discharge

Because

of

the

limited

number

of

studies

the

measurement

tools

did

not

need

to

be

standardised

Studies

with

a

retrospective

and

prospective

design

were

included

Excluded

were

-LRB-

-RRB-

studies

in

homogeneous

PICU

populations

-LRB-

e.g.

survivors

of

cardiothoracic

surgery

and

trauma

-RRB-

reporting

diagnosis-related

outcome

in

particular

but

not

intensive

care

treatment

as

such

and

-LRB-

-RRB-

studies

evaluating

mortality

only

Results

Eligible

studies

and

quality

of

the

studies

Twenty-seven

studies

were

found

in

which

one

or

more

aspects

of

long-term

sequelae

in

PICU

survivors

and/or

their

families

were

described

The

patient

characteristics

populations

measurement

tools

and

outcomes

are

described

in

Tables

and

The

quality

criteria

are

described

in

Table

of

the

studies

met

all

of

the

quality

criteria

In

studies

describing

the

same

outcome

aspect

differences

in

study

population

follow-up

time

and

measurement

tools

make

the

comparison

and

synthesis

of

results

difficult

Table

characteristics

measurement

tools

physical

and

functional

health

outcomeReferencePopulationa

nbAgec

-LRB-

yrs

-RRB-

Follow-up

timed

-LRB-

yrs

-RRB-

Severity

of

illnesseLOSf

-LRB-

days

-RRB-

Measurement

toolOutcome

-LRB-

n

-RRB-

gInterpretation

of

spastic

quadriplegiaMajority

of

children

surviving

meningococcal

disease

neurologically

normal.n

-LRB-

-RRB-

Median

hearing

major

resuscitation

-LRB-

CPR

-RRB-

normal

mild

moderate

severe

of

survivors

of

CPR

neurological

normal.n

-LRB-

-RRB-

same

as

prior

to

CPRLocation

underlying

cause

and

duration

of

CPR

determinants

of

outcome

normal

or

mild

disability

moderate

severe

of

survivors

of

CPR

neurological

normal.n

-LRB-

-RRB-

POPCiLocation

underlying

cause

and

duration

of

CPR

not

determinants

of

outcome

Acquired

brain

injury

GCS

good

recovery

moderate

severe

disabilityMajority

of

children

with

acquired

brain

injury

dead

or

disabled.n

-LRB-

-RRB-

BSID-IIkBSID-II

normal

cerebral

palsy

cognitive

delayNeuro-developmental

normal

mild

moderate

severe

disability

persistent

vegetative

of

survivors

of

CPR

neurological

normal.n

cause

and

duration

of

CPR

determinants

of

outcome

NANAChest

during

exerciseARDS

survivors

at

risk

for

hypoxaemia

during

exercise.n

-LRB-

-RRB-

Pulmonary

reduced

diffusion

disease

with

PRISM

NAPulmonary

wheezing

for

which

salbutamolARDS

survivors

possibly

at

risk

for

mild

obstructive

lung

disease.n

obstructive

NANAPulmonary

restrictive

or

obstructive

diseaseARDS

survivors

at

risk

for

restrictive

and

obstructive

lung

disease.n

-LRB-

-RRB-

Electrocardiography

EchocardiographyCardiac

function

normal

in

NANAPulmonary

limitations

in

activityARDS

survivors

at

risk

for

restrictive

and

obstructive

lung

disease.n

restrictive

septic

NANAPulmonary

normal

lung

functionMeningococcal

septic

shock

survivors

at

risk

for

hypoxaemia

during

exercise.n

during

PRISM

NAChest

-LRB-

-RRB-

restrictive

or

obstructive

diseaseARDS

survivors

at

risk

for

restrictive

and

obstructive

lung

disease.n

-LRB-

-RRB-

Pulse

-LRB-

-RRB-

decreased

diffusion

capacityPulmonary

sepsis

with

renal

replacement

NAPICUGlomerular

filtration

rate

-LRB-

GFR

-RRB-

decreased

GFR

proteinuria

hypertensionChildren

surviving

acute

renal

failure

due

to

septic

shock

at

risk

for

long-term

renal

dysfunction.n

-LRB-

-RRB-

Median

parenchymal

defectProtein

excretion

in

proteinuriaDMSA

drowning

admitted

to

PRISM

NAFunctional

health

severe

neurological

impairmentMajority

of

near-drowning

survivors

lead

a

normal

life.n

-LRB-

-RRB-

GCS

primary

physician

or

examination

by

study

normal

or

mildly

health

moderate

or

severe

handicapMajority

of

PICU

survivors

seem

to

lead

a

normal

life.n

Written

questionnaire

or

telephone

contact

with

the

specialist

physician

or

mild

will

lead

independent

meningitis

with

respiratory

PRISMNAFunctional

health

normalHalf

of

children

surviving

severe

bacterial

meningitis

seem

to

lead

a

normal

life.n

-LRB-

-RRB-

Mean

Telephone

independentRange

partially

dependentaStudied

population

bn

studied

patients

-LRB-

eligible

patients

-RRB-

cAge

of

patients

at

admission

to

PICU

-LRB-

mean

SD

or

range

-RRB-

dFollow-up

time

-LRB-

mean

SD

or

range

-RRB-

eSeverity

of

illness

-LRB-

PRISM

Glasgow

Coma

Score

-LRB-

GCS

-RRB-

Glasgow

Meningococcal

Septicaemia

Prognostic

Score

-LRB-

GMSPS

-RRB-

and/or

parent

rating

-RRB-

fLength

of

stay

-LRB-

LOS

-RRB-

in

PICU

and/or

hospital

-LRB-

mean

SD

or

range

-RRB-

gn

examined

patients

-LRB-

eligible

patients

-RRB-

hPCPC

Paediatric

Cerebral

Performance

Category

iPOPC

Paediatric

Overall

Performance

Category

jGOS

Glasgow

Outcome

Scale

kBSID-II

Bayley

Scales

of

Infant

Development-IITable

characteristics

measurement

tools

and

psychological

and

quality

of

life

-LRB-

QoL

-RRB-

outcomeReferencePopulationa

nbAgec

-LRB-

yrs

-RRB-

Follow-up

timed

-LRB-

yrs

-RRB-

Severity

of

illnesseLOSf

-LRB-

days

-RRB-

Measurement

toolOutcome

-LRB-

n

-RRB-

gInterpretation

of

children

and

PICU

outcome

Children

Behaviour

high

-LRB-

-RRB-

SDQ

high

-LRB-

-RRB-

IES

PTSD

-LRB-

-RRB-

PICU

survivors

and

their

mothers

at

risk

for

psychological

distress

and

PTSD.n

-LRB-

-RRB-

Child

Behaviour

Check

List

SDQh

IESiMothers

GHQ

high

-LRB-

-RRB-

IES

high

-LRB-

-RRB-

Mother

GHQj

Parent

rating

outcome

Children

PTSD

-LRB-

-RRB-

PICU

-LRB-

-RRB-

ward

IES

high

-LRB-

-RRB-

PICU

-LRB-

-RRB-

wardPICU

survivors

and

their

parents

at

risk

for

psychological

distress

and

PTSD.n

-LRB-

-RRB-

Child

PTSDk

SDQh

IESi

depression

anxiety

CSIlParents

PTSD

-LRB-

-RRB-

PICU

-LRB-

-RRB-

wardGeneral

wardParent

GHQj

IESi

depressionn

-LRB-

-RRB-

Children

and

PRISM

Hospital

Psychological

outcome

IES

and

CMFS

dependent

on

invasive

procedures

CMFS

and

CHLOC

on

ageStress

symptoms

in

children

possibly

dependent

on

number

of

invasive

procedures.n

-LRB-

-RRB-

Children

IESi

CMFSm

CHLOCnGeneral

wardn

-LRB-

-RRB-

PRISMNAPsychological

outcome

IES

higher

in

high

risk

not

decreasing

over

time

IES

related

with

invasive

proceduresStress

symptoms

possibly

dependent

on

invasive

procedures

Stress

symptoms

not

decreasing

over

time.n

risk

Low

risk

Children

IESi

CMFSm

CHLOCnHigh

risk

High

risk

disease

PICU

and

wardMedian

GMSPS

PICU

LOS

outcome

Child

PTSD

-LRB-

-RRB-

PICU

survivors

and

their

parents

at

risk

for

psychological

distress

and

PTSD.Children

and

parentsParent

rating

median

LOS

SDQh

IESiMothers

PTSD

-LRB-

-RRB-

n

-LRB-

-RRB-

Parent

GHQj

IESiFathers

PTSD

-LRB-

-RRB-

PRISM

PICU

outcome

ASD

of

PICU

survivors

with

ASD

more

at

risk

for

PTSD.n

-LRB-

-RRB-

Parent

rating

Acute

Stress

Disease

symptomsPTSD

PRISM

PICU

Psychological

outcome

mothers

Mothers

PICU

more

stress

Stress

decreases

over

time

in

all

groupsMothers

of

PICU

survivors

at

risk

for

psychological

distress

families

at

risk

for

dysfunctioning.n

rating

Parental

Stress

Scale

families

dysfunctioningGeneral

wardFAM

IIIpn

Disease

outcome

parents

High

psychological

distress

in

mothers

and

fathers

not

decreasing

over

timeMothers

and

fathers

of

PICU

survivors

at

risk

for

psychological

distress

mothers

PRISMNAQoL

equal

to

before

of

PICU

survivors

seem

to

have

the

same

QoL

as

before

admission

normal

QoL.n

-LRB-

-RRB-

n

improvedn

PRISMPICU

QoL

of

PICU

survivors

seem

to

have

good

QoL.n

-LRB-

-RRB-

n

deterioratedn

normal

after

RAHCs

measure

of

normal

of

PICU

survivors

seem

to

have

normal

QoL.n

-LRB-

-RRB-

Mean

fair

poor

QoL

HSUIt

GOSuHSUI

-LRB-

-RRB-

normal

-LRB-

very

-RRB-

poor

of

PICU

survivors

seem

to

have

good

QoL.n

-LRB-

-RRB-

n

GOS

-LRB-

-RRB-

normal

mild

disability

moderate/severe

seem

to

have

normal

functional

health.n

aStudied

population

bn

studied

patients

-LRB-

eligible

patients

-RRB-

cAge

of

patients

at

admission

to

PICU

-LRB-

mean

SD

or

range

-RRB-

dFollow-up

time

-LRB-

mean

SD

or

range

-RRB-

eSeverity

of

illness

-LRB-

PRISM

Glasgow

Coma

Score

GMSPS

and/or

parent

rating

-RRB-

fLength

of

stay

-LRB-

LOS

-RRB-

in

PICU

and/or

hospital

-LRB-

mean

SD

or

range

-RRB-

gn

examined

patients

-LRB-

eligible

patients

-RRB-

hSDQ

Strength

and

Difficulties

Questionnaire

iIES

Impact

of

Event

Scale

jGHQ

General

Health

Questionnaire

kPTSD

Post-traumatic

stress

disorder

lCSI

Child

Somatization

Inventory

mCMFS

Child

Medical

Fears

Scale

nCHLOC

Child

Health

Locus

Control

Symptom

Revised

pFAM

III

Family

Assessment

Measure

III

qFILE

Family

Inventory

of

Life

Events

and

Change

rMAHSC

Multi-attribute

health

status

classification

sRAHC

Royal

Alexandra

Hospital

for

Children

tHSUI

Health

State

Utility

index

uGOS

Glasgow

Outcome

ScoreTable

assessment

of

reviewed

studiesReferenceSelection

bias

excludedaSelective

loss

to

follow-up

excludedbExposure

clearly

definedcOutcome

clearly

defineddControl

group

bias

excluded

-LRB-

i.e.

exclusion

of

of

the

studied

population

excluded

-RRB-

bSelective

loss

to

follow-up

excluded

-LRB-

i.e.

description

of

patients

lost

to

follow-up

and

comparison

with

those

remaining

in

the

study

-RRB-

cExposure

clearly

defined

-LRB-

i.e.

clear

definition

of

the

studied

population

-RRB-

dOutcome

clearly

defined

-LRB-

i.e.

clear

definition

of

outcome

measures

-RRB-

eComparison

with

control

group

-LRB-

i.e.

children

admitted

to

general

ward

-RRB-

Physical

and

neuro-cognitive

sequelae

-LRB-

Table

-RRB-

In

studies

that

included

in

total

patients

aspects

of

physical

and

neuro-cognitive

sequelae

were

evaluated

Neurological

evaluation

was

conducted

in

five

studies

including

survivors

The

majority

of

the

children

were

neurologically

normal

In

the

remaining

children

disabilities

such

as

hearing

loss

coordination

cognition

and

developmental

problems

turned

out

to

be

severe

-LSB-

-RSB-

Pulmonary

evaluation

was

conducted

in

six

studies

including

patients

-LSB-

-RSB-

Restrictive

and

obstructive

disease

and

hypoxaemia

during

exercise

was

found

Cardiac

evaluation

was

conducted

in

two

studies

including

survivors

-LSB-

-RSB-

No

abnormalities

were

found

except

for

left

ventricular

hypertrophy

in

one

child

Renal

evaluation

was

conducted

in

one

study

including

survivors

-LSB-

-RSB-

In

two

children

glomerular

filtration

was

impaired

one

had

hypertension

and

one

had

proteinuria

Psychological

sequelae

-LRB-

Table

-RRB-

Various

questionnaires

were

used

Cut-off

points

for

the

diagnosis

of

PTSD

differed

between

studies

but

all

of

them

showed

high

scores

for

PTSD

in

children

and

parents

Psychological

evaluation

of

children

was

conducted

in

five

studies

including

children

-LSB-

-RSB-

Symptoms

of

PTSD

were

found

in

of

evaluated

children

In

one

study

a

relation

was

found

between

invasive

procedures

and

high

scores

-LSB-

-RSB-

Psychological

evaluation

of

parents

was

conducted

in

six

studies

including

parents

of

children

-LSB-

-RSB-

Symptoms

of

PTSD

were

found

in

of

evaluated

parents

In

some

studies

a

relation

was

found

between

high

scores

and

illness

severity

as

perceived

by

parents

-LSB-

-RSB-

In

one

study

these

high

scores

decreased

over

time

-LSB-

-RSB-

Functional

health

and

QoL

-LRB-

Tables

and

-RRB-

Evaluation

of

functional

health

was

conducted

in

three

studies

including

children

-LSB-

-RSB-

The

majority

of

the

children

seemed

to

have

normal

functional

health

the

remainder

was

found

to

be

seriously

impaired

Evaluation

of

QoL

was

conducted

in

four

studies

including

children

-LSB-

-RSB-

QoL

was

evaluated

using

three

different

questionnaires

In

the

majority

of

children

the

QoL

was

normal

or

equal

to

the

QoL

before

PICU

admission

In

all

studies

some

of

the

children

had

poor

QoL

Discussion

Only

studies

consisting

of

PICU

survivors

met

our

inclusion

criteria

The

small

numbers

heterogeneity

of

the

studied

populations

and

the

used

measurement

tools

the

frequent

use

of

non-validated

measurement

tools

and

the

various

aspects

of

outcomes

studied

make

aggregation

of

the

data

and

therefore

strong

conclusive

statements

difficult

Physical

sequelae

The

reviewed

studies

report

distinct

physical

sequelae

including

neurological

abnormalities

in

PICU

survivors

Standardised

neurological

examination

of

PICU

survivors

was

validated

in

but

very

few

studies

have

been

carried

out

since

-LSB-

-RSB-

As

neurological

problems

have

a

great

impact

on

daily

life

standardised

evaluation

and

adequate

support

and

rehabilitation

seem

to

be

relevant

similar

to

in

NICU

survivors

-LSB-

-RSB-

Follow-up

studies

evaluating

lung

function

in

children

are

hampered

by

the

small

incidence

of

severe

respiratory

insufficiency

in

children

-LSB-

-RSB-

In

adult

respiratory

distress

syndrome

-LRB-

ARDS

-RRB-

the

recovery

of

lung

function

is

shown

during

the

first

year

and

physical

limitations

seem

to

be

partly

dependent

on

lung

function

-LSB-

-RSB-

In

infants

and

children

post-natal

lung

growth

may

contribute

to

the

improvement

of

lung

function

after

critical

illness

In

addition

to

lung

function

the

long-term

effect

of

small

airway

disease

should

be

evaluated

for

instance

in

children

with

respiratory

syncitial

virus

infection

Data

on

the

structured

evaluation

of

cardiac

and

renal

function

in

paediatric

and

adult

ICU

survivors

is

not

available

In

young

children

septic

shock

and

the

need

for

vasoactive

support

of

the

circulation

may

interact

with

the

developing

myocardium

and

may

have

persistent

effects

on

cardiac

growth

and

function

-LSB-

-RSB-

Complications

of

intensive

care

procedures

per

se

-LRB-

e.g.

vascular

complications

due

to

intra-vascular

catheters

and

side-effects

of

ototoxic

drugs

and

sedatives

-RRB-

are

not

evaluated

-LSB-

-RSB-

One

can

assume

the

exact

incidence

of

physical

sequelae

to

be

higher

than

has

been

reported

so

far

Psychological

sequelae

and

functional

health

and

QoL

In

the

reviewed

studies

psychological

sequelae

have

been

established

in

of

survivors

and

their

parents

The

comparison

of

findings

is

hampered

due

to

different

measurement

tools

and

cut-off

points

for

the

diagnosis

of

PTSD

and

various

follow-up

intervals

Risk

factors

accounting

for

hampered

psychological

outcome

could

be

diverse

-LRB-

severity

of

illness

being

removed

from

one

's

child

having

been

witness

to

the

accident

mental

health

family

functioning

social

support

coping

strategies

and

lack

of

information

from

the

medical

team

-RRB-

-LSB-

-RSB-

Psychological

support

to

improve

coping

strategies

and

prevent

over-protection

might

improve

psychological

outcome

in

children

and

parents

-LSB-

-RSB-

Further

research

is

essential

to

establish

the

appropriate

time

and

extent

of

the

psychological

support

needed

Cognitive

sequelae

have

rarely

been

studied

in

the

reviewed

studies

Adequate

neuro-cognitive

evaluation

is

both

expensive

and

time-consuming

Studies

in

neonatal

ICU

survivors

show

substantial

cognitive

dysfunction

with

great

impact

on

daily

life

-LSB-

-RSB-

Consequently

early

intervention

education

and

rehabilitation

are

expected

to

improve

daily

life

-LSB-

-RSB-

A

majority

of

PICU

survivors

seem

to

have

unchanged

functional

health

and

good

QoL

In

the

reviewed

studies

functional

health

is

evaluated

by

telephone

interviews

-LSB-

-RSB-

In

most

of

these

studies

the

physician

rather

than

the

child

or

its

parents

evaluates

functional

health

Ideal

-LRB-

HR

-RRB-

QoL

questionnaires

should

measure

all

aspects

of

QoL

and

preferably

be

filled

in

by

the

children

themselves

Proxy

investigation

of

functional

health

and

-LRB-

HR

-RRB-

QoL

-LRB-

in

children

years

of

age

-RRB-

is

second

best

-LSB-

-RSB-

Besides

the

pre-morbid

state

is

probably

an

important

factor

which

is

difficult

to

assess

-LSB-

-RSB-

Suggestions

for

future

follow-up

research

The

reviewed

studies

have

a

number

of

methodological

limitations

Heterogeneity

is

the

most

important

one

Consensus

on

all

aspects

of

follow-up

research

is

essential

for

well-founded

conclusions

For

example

structured

and

standardised

evaluation

of

-LRB-

-RRB-

organ

system

function

with

a

validated

tool

such

as

the

Paediatric

Logistic

Organ

Dysfunction

-LRB-

PELOD

-RRB-

score

-LSB-

-RSB-

-LRB-

-RRB-

neuro-cognitive

function

-LRB-

-RRB-

complications

of

PICU

treatment

and

-LRB-

-RRB-

-LRB-

HR

-RRB-

QoL

are

warranted

Multi-centre

studies

as

proposed

by

the

Collaborative

Pediatric

Critical

Care

Research

Network

-LRB-

CPCCRN

-RRB-

with

a

uniform

approach

will

provide

answers

either

in

general

PICU

cohorts

or

in

disease-oriented

study

groups

-LSB-

-RSB-

In

conclusion

this

review

indicates

that

PICU

survivors

and

their

parents

may

have

substantial

physical

and

psychological

sequelae

interacting

with

QoL

Because

of

longer

life

expectancy

longer

follow-up

time

is

warranted

emphasising

the

consequences

for

health

care

in

children

We

believe

that

paediatric

intensivists

and

psychologists

should

be

involved

as

core

members

of

follow-up

teams

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

paediatric intensive care unit
quality of life
health status

outcome assessment (health care)
post-traumatic stress disorder


Coronary fly-through or virtual angioscopy using dual-source MDCT data Coronary fly-through or virtual angioscopy -LRB- VA -RRB- has been studied ever since its invention in 2000 . However , application was limited because it requires an optimal computed tomography -LRB- CT -RRB- scan and time-consuming post-processing . Recent advances in post-processing software facilitate easy construction of VA , but until now image quality was insufficient in most patients . The introduction of dual-source multidetector CT -LRB- MDCT -RRB- could enable VA in all patients . Twenty patients were scanned using a dual-source MDCT -LRB- Definition , Siemens , Forchheim , Germany -RRB- using a standard coronary artery protocol . Post-processing was performed on an Aquarius Workstation -LRB- TeraRecon , San Mateo , Calif. -RRB- . Length travelled per major branch was recorded in millimetres , together with the time required in minutes . VA could be performed in every patient for each of the major coronary arteries . The mean -LRB- range -RRB- length of the automated fly-through was 80 -LRB- 32 -- 107 -RRB- mm for the left anterior descending -LRB- LAD -RRB- , 75 -LRB- 21 -- 116 -RRB- mm for the left circumflex artery -LRB- LCx -RRB- , and 109 -LRB- 21 -- 190 -RRB- mm for the right coronary artery -LRB- RCA -RRB- . Calcifications and stenoses were visualised , as well as most side branches . The mean time required was 3 min for LAD , 2.5 min for LCx , and 2 min for the RCA . Dual-source MDCT allows for high quality visualisation of the coronary arteries in every patient because scanning with this machine is independent of the heart rate . This is clearly shown by the successful VA in all patients . Potential clinical value of VA should be determined in the near future . Introduction Coronary fly-through or virtual angioscopy -LRB- VA -RRB- using various generations of computed tomography -LRB- CT -RRB- devices has been reported in a number of publications since its first mention in 2000 -LSB- 1 -- 3 -RSB- . Main reason for the minimal research on this topic was the limited application because of the requirements for an optimal CT scan and time consuming post-processing . Recent advances in post-processing software facilitate easy construction of VA , but until now image quality of coronary CT was insufficient in most patients . The introduction of dual-source multidetector CT -LRB- MDCT -RRB- has shown a more stable quality of data in all patients because of higher temporal resolutions , which could prove to enable VA in all patients and allow investigation of the clinical value of this visualisation technique -LSB- 4 -- 6 -RSB- . Materials and methods Twenty patients -LRB- 13 male , mean age 60 ± 13 years old -RRB- were selected at random from patients scanned for coronary artery disease using a standard coronary artery protocol on a dual source MDCT -LRB- Definition , Siemens , Forchheim , Germany -RRB- . Post-processing was performed on an Aquarius Workstation -LRB- TeraRecon , San Mateo , Calif. -RRB- . The software allows an automatic vessel fly-through , which provides inside front and rear views , together with a view of the path and location on the surface of the heart -LRB- Fig. 1 -RRB- . Besides this , three multiplanar reformation planes -LRB- sagittal , coronal and axial -RRB- are shown for the location of the camera . Length travelled per major branch was recorded in millimetres , together with the time required in minutes to perform the evaluation per major branch . Ostia of side branches were visualised , but no fly-through of those side branches was performed . Fig. 1Large trajectory visualisation of the right coronary artery -LRB- RCA -RRB- Results VA could be performed semi-automatically with minor user interaction in every patient for each of the major coronary arteries using dual-source CT -LRB- DSCT -RRB- . The mean -LRB- range -RRB- length of the automated fly-through was 80 -LRB- 32 -- 107 -RRB- mm for the left anterior descending -LRB- LAD -RRB- , 75 -LRB- 21 -- 116 -RRB- mm for the left circumflex artery -LRB- LCx -RRB- , and 109 -LRB- 21 -- 190 -RRB- mm for the RCA . Calcifications were clearly visualised as white structures floating inside the lumen . Stenoses were visualised when present by indentations of the lumen wall -LRB- Fig. 2 -RRB- . Visibility of the actual size of the stent lumen was still limited , but fly-through was possible in the stents we found -LRB- Fig. 3 -RRB- . Fig. 2Visibility of a stenotic lesionFig . 3Stent visibility The major side branches of the main coronary arteries could be detected during the fly-through -LRB- Fig. 4 -RRB- . No fly-through was performed through those side branches . Fig. 4Visibility of major side branches The mean time required was 3 min for LAD , 2.5 min for LCx , and 2 min for the RCA . Discussion The introduction of DSCT enables 100 % visualisation of all major arteries and thus demonstrates a major gain in image quality compared with electron beam CT -LRB- EBCT -RRB- and MDCT , with only 76 % and 84 % assessable , respectively -LSB- 3 -RSB- . Furthermore , the time required for processing the data decreased to about 2.5 min compared with the 6-13 min required in 2002 -LRB- Table 1 -RRB- . Notice that the times shown in the table for EBCT and MDCT do not include the actual computing time , which could be hours with the hard - and software at that time . Currently , all computation and visualisation is in real time and thus included in the 2.5 min required for a coronary fly-through of a major branch using DSCT data . Table 1Results of an earlier study using EBCT and four-slice MDCT -LRB- 4-MDCT -RRB- -LSB- 3 -RSB- in comparison with the DSCT results of the current study EBCT -LRB- 2002 -RRB- 4-MDCT -LRB- 2002 -RRB- DSCT -LRB- 2006 -RRB- LAD Percentage assessable14/15 -LRB- 93 % -RRB- 14/15 -LRB- 93 % -RRB- 20/20 -LRB- 100 % -RRB- Mean preparation time -LRB- min -RRB- 12133 Mean number of key frames1316n.a.LC Percentage assessable11/15 -LRB- 73 % -RRB- 10/15 -LRB- 67 % -RRB- 20/20 -LRB- 100 % -RRB- Mean preparation time -LRB- min -RRB- 7102.5 Mean number of key frames913 * n.a.RCA Percentage assessable9/15 -LRB- 60 % -RRB- 14/15 -LRB- 95 % -RRB- 20/20 -LRB- 100 % -RRB- Mean preparation time -LRB- min -RRB- 6102 Mean number of key frames814n.a . Total percentage assessable34/45 -LRB- 76 % -RRB- 38/45 -LRB- 84 % -RRB- 60/60 -LRB- 100 % -RRB- The number of key frames is no longer applicable -LRB- n.a. -RRB- for the current software tools that can perform the coronary fly-through in real time Different artefacts still hamper the fly-through . First , non-optimal reconstruction will present itself as a sudden change in the form of the vessel wall . The effects are shown in Fig. 5 , and it will be clear that these artefacts can be easily recognised because of the non-natural shape of the wall . A more difficult artefact is cause by motion . Here , the artefact can be easily appreciated on the three-dimensional view , but in the fly-through it presents as a apparent stenosis -LRB- Fig. 6 -RRB- . Fig. 5Problems with artefacts in non-optimal reconstructed datasetsFig . 6Problems with motion artefacts causing apparent stenoses Finally , surgical clips in bypasses still pose a problem as already shown an earlier publication using EBCT -LSB- 1 -RSB- -LRB- Fig. 7 -RRB- . Fig. 7Visualisation is hampered by presence of surgical clips in a bypass graft All three articles that -LRB- to our best knowledge -RRB- are available on this topic conclude with similar remarks -LSB- 1 -- 3 -RSB- . They all state that the coronary fly-through or virtual coronary angioscopy -LRB- VCA -RRB- is a promising technique that , however , requires higher quality of the datasets and better image processing software . A shortcoming of our study was the lack of a `` gold standard '' to confirm the findings of the coronary fly-through . Conclusion Dual-source MDCT provides a high quality visualisation of the coronary artery tree , independently of the heart rate . This is clearly shown by the successful VCA in all 20 patients in this study . With recent advancements of image processing software and CT acquisition techniques , the conditions have been met for providing the technical basis for the clinical validation of VCA and the potential clinical value of VCA should be determined in the near future .

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

coronary fly-through
post-processing
dual-source ct


Rab

proteins

and

Rab-associated

proteins

major

actors

in

the

mechanism

of

protein-trafficking

disorders

Ras-associated

binding

-LRB-

Rab

-RRB-

proteins

and

Rab-associated

proteins

are

key

regulators

of

vesicle

transport

which

is

essential

for

the

delivery

of

proteins

to

specific

intracellular

locations

More

than

human

Rab

proteins

have

been

identified

and

their

function

has

been

shown

to

depend

on

their

interaction

with

different

Rab-associated

proteins

regulating

Rab

activation

post-translational

modification

and

intracellular

localization

The

number

of

known

inherited

disorders

of

vesicle

trafficking

due

to

Rab

cycle

defects

has

increased

substantially

during

the

past

decade

This

review

describes

the

important

role

played

by

Rab

proteins

in

a

number

of

rare

monogenic

diseases

as

well

as

common

multifactorial

human

ones

Although

the

clinical

phenotype

in

these

monogenic

inherited

diseases

is

highly

variable

and

dependent

on

the

type

of

tissue

in

which

the

defective

Rab

or

its

associated

protein

is

expressed

frequent

features

are

hypopigmentation

-LRB-

Griscelli

syndrome

-RRB-

eye

defects

-LRB-

Choroideremia

Warburg

Micro

syndrome

and

Martsolf

syndrome

-RRB-

disturbed

immune

function

-LRB-

Griscelli

syndrome

and

Charcot

Marie

Tooth

disease

-RRB-

and

neurological

dysfunction

-LRB-

X-linked

non-specific

mental

retardation

Charcot

Marie

Tooth

disease

Warburg

Micro

syndrome

and

Martsolf

syndrome

-RRB-

There

is

also

evidence

that

alterations

in

Rab

function

play

an

important

role

in

the

progression

of

multifactorial

human

diseases

such

as

infectious

diseases

and

type

diabetes

Rab

proteins

must

not

only

be

bound

to

GTP

but

they

need

also

to

be

prenylated

i.e.

bound

to

the

cell

membranes

by

isoprenes

which

are

intermediaries

in

the

synthesis

of

cholesterol

-LRB-

e.g.

geranyl

geranyl

or

farnesyl

compounds

-RRB-

This

means

that

isoprenylation

can

be

influenced

by

drugs

such

as

statins

which

inhibit

isoprenylation

or

biphosphonates

which

inhibit

that

farnesyl

pyrophosphate

synthase

necessary

for

Rab

GTPase

activity

Conclusion

Although

protein-trafficking

disorders

are

clinically

heterogeneous

and

represented

in

almost

every

subspeciality

of

pediatrics

the

identification

of

common

pathogenic

mechanisms

may

provide

a

better

diagnosis

and

management

of

patients

with

still

unknown

Rab

cycle

defects

and

stimulate

the

development

of

therapeutic

agents

Introduction

Germline

mutations

in

the

genes

involved

in

the

Ras

mitogen-activated

protein

kinase

-LRB-

MAPK

-RRB-

pathway

explain

a

number

of

neuro

cardio

facio

cutaneous

syndromes

as

reported

in

a

paper

recently

published

in

this

journal

-LSB-

-RSB-

Ras-associated

binding

-LRB-

Rab

-RRB-

proteins

are

small

GTPases

of

the

Ras

superfamily

that

continuously

cycle

between

the

cytosol

and

different

membranes

In

Pfeffer

described

different

Rab

GTPases

that

have

been

identified

as

prenylated

proteins

localized

in

distinct

membrane-bound

compartments

-LSB-

-RSB-

Prenylated

proteins

are

proteins

bound

to

isoprenes

which

are

intermediaries

in

the

cholesterol

synthesis

At

the

time

of

the

writing

this

article

more

than

human

Rabs

had

been

identified

-LSB-

-RSB-

This

high

number

of

different

Rab

proteins

highlights

their

importance

in

the

regulation

of

vesicle

trafficking

processes

including

vesicle

formation

motility

tethering

and

fusion

to

the

acceptor

membrane

and

signaling

to

other

organelles

The

ability

of

Rabs

to

perform

these

different

tasks

in

a

co-ordinated

and

regulated

manner

originates

in

their

highly

dynamic

conformation

and

mobility

and

from

their

interaction

with

different

Rab-associated

proteins

as

illustrated

in

the

Rab

GTPase

cycle

-LRB-

Fig.

-RRB-

Rabs

can

cycle

between

the

active

GTP-bound

and

the

non-active

GDP-bound

forms

assisted

by

different

Rab-associated

proteins

such

as

the

GEFs

-LRB-

guanine

nucleotide-exchange

factors

-RRB-

and

GAPs

-LRB-

GTPase-activating

proteins

-RRB-

respectively

-LSB-

-RSB-

This

conformational

cycle

is

the

main

driving

force

for

the

on/off

switch

mechanism

of

Rabs

and

their

ability

to

both

regulate

binding

to

downstream

effectors

and

perform

the

desired

function

Rabs

are

initially

synthesized

as

soluble

proteins

in

the

cytosol

where

they

are

first

recognized

by

a

soluble

chaperone-like

protein

named

REP

-LRB-

Rab

escort

protein

-RRB-

-LSB-

-RSB-

The

REP

brings

the

Rab

to

the

RabGGT

-LRB-

Rab

geranylgeranyl

transferase

-RRB-

for

the

addition

of

generally

two

geranylgeranyl

groups

These

prenyl

groups

are

added

by

covalent

thioether

bonds

to

cysteine

residue

-LRB-

s

-RRB-

located

at

the

C-terminus

of

the

Rab

protein

-LSB-

-RSB-

This

post-translational

modification

of

Rabs

is

needed

to

allow

for

the

attachment

of

the

Rab

proteins

into

the

lipid

bilayer

of

the

organelle

consequently

Rabs

are

considered

to

be

peripheral

membrane

proteins

-LRB-

Fig.

-RRB-

After

performing

their

function

Rab

proteins

are

extracted

from

the

membranes

by

RabGDI

-LRB-

GDP

dissociation

inhibitor

-RRB-

and

they

remain

in

the

cytosol

until

they

are

needed

again

-LSB-

-RSB-

Statins

are

known

to

inhibit

protein

isoprenylation

As

such

they

are

able

to

reduce

the

production

of

amyloid-beta

and

inhibit

preamyloid

catabolism

in

the

lysosomes

which

suggests

their

possible

application

in

the

treatment

of

Alzheimer

-LSB-

-RSB-

However

the

therapeutic

role

of

statins

in

Rab-related

disorders

has

not

yet

been

established

The

potent

nitrogen-containing

biphosphonates

such

as

pamidronate

alendronate

ibandronate

and

zoledronate

inhibit

a

key

enzyme

farnesyl

pyrophosphate

synthase

which

is

necessary

for

Rab

activity

hence

their

inhibitory

action

on

osteoclast

activity

-LSB-

-RSB-

Fig.

Ras-associated

binding

-LRB-

Rab

-RRB-

protein

GTPase

cycles

Rab

proteins

are

intrinsically

soluble

and

require

a

post-translational

modification

for

membrane

association

They

first

associate

with

a

Rab

escort

protein

-LRB-

REP

-RRB-

and

form

a

stable

complex

that

is

the

substrate

for

the

subsequent

dual

prenylation

of

C-terminal

cysteine

motifs

via

Rab

geranylgeranyl

transferase

-LRB-

RabGGT

-RRB-

RabGGT

consists

of

two

different

functional

subunits

-LRB-

RabGGTα

and

RabGGTß

-RRB-

After

lipid

tranfer

REP

delivers

the

prenylated

Rab

to

the

donor

membrane

-LRB-

the

REP

cycle

is

shown

with

blue

arrows

-RRB-

In

the

absence

of

REP

or

RabGGT

Rab

proteins

remain

in

the

cytosol

in

an

inactive

state

The

transfer

of

Rab

proteins

between

acceptor

and

donor

membranes

is

facilitated

by

the

GDP

dissociation

inhibitor

-LRB-

GDI

-RRB-

-LRB-

the

GDI

cycle

is

shown

with

red

arrows

-RRB-

Both

REP

and

GDI

bind

the

GDP-bound

inactive

form

of

Rab

After

REP

or

GDI

dissociate

from

Rabs

at

the

donor

membrane

Rabs

can

cycle

between

the

inactive

-LRB-

GDP-bound

-RRB-

and

active

-LRB-

GTP-bound

-RRB-

states

Rab

proteins

are

activated

by

guanine

nucleotide

exchange

factors

-LRB-

RabGEFs

-RRB-

and

deactivated

by

GTPase

activating

proteins

-LRB-

RabGAPs

-RRB-

which

accelerate

the

slow

intrinsic

rates

of

nucleotide

exchange

and

GTP

hydrolysis

In

the

active

state

Rabs

interact

with

structurally

and

functionally

diverse

effectors

including

cargo

sorting

complexes

on

donor

membranes

motor

proteins

involved

in

vesicular

transport

and

tethering

complexes

that

regulate

vesicle

fusion

with

acceptor

membranes

Evolutionarily

conserved

Rabs

are

usually

expressed

in

all

cell

and

tissue

types

and

regulate

more

fundamental

vesicle

transport

pathways

whereas

the

less

conserved

Rab

family

members

function

in

more

specific

pathways

which

are

often

organ-dependent

Defects

in

intracellular

vesicle

trafficking

underlie

a

large

variety

of

human

diseases

including

pathologies

associated

with

defects

in

Rabs

or

Rab-associated

proteins

-LSB-

-RSB-

In

this

review

we

deal

with

the

role

of

different

Rab

and

rab-associated

proteins

in

inherited

-LRB-

Table

-RRB-

as

well

as

certain

multifactorial

human

diseases

in

different

pediatric

disciplines

In

addition

the

review

will

also

focus

briefly

on

recent

advances

in

understanding

human

disease

through

the

study

of

Rab

proteins

from

in

vitro

and

mouse

knockout

studies

Table

monogenic

diseases

caused

by

a

Ras-associated

binding

-LRB-

Rab

-RRB-

protein

or

Rab-associated

protein

defectDiseaseOMIMGeneRabRab-associatedDescriptionGriscelli

syndrome

type

recessive

albinism

neurological

impairmentGriscelli

syndrome

type

recessive

albinism

hemophagocytic

syndrome

sometimes

secondary

neurological

impairmentGriscelli

syndrome

type

recessive

progressive

loss

of

vision

beginning

at

an

early

age

and

the

choroid

and

retina

undergo

complete

atrophyNon-specific

mental

affected

males

show

moderate

to

severe

mental

retardation

carrier

females

may

also

be

mildly

affectedCharcot-Marie-Tooth

disease

type

dominant

peripheral

sensory

neuropathy

with

late

onset

muscle

weakness

foot

ulcers

and

infectionsWarburg

Micro

recessive

microcephaly

microcornea

congenital

cataract

mental

retardation

optic

atrophy

and

hypogenitalismMartsolf

recessive

congenital

cataracts

hypogonadism

and

mild

mental

retardation

Rab

proteins

in

skin

and

hear

pigmentation

Griscelli

syndrome

-LRB-

GS

-RRB-

is

an

autosomal

recessive

disorder

that

causes

partial

albinism

-LSB-

-RSB-

There

are

three

variants

of

this

disease

one

is

a

purely

hypopigmentation

disorder

-LRB-

GS

type

III

-RRB-

and

two

in

addition

to

the

pigmentation

defect

are

also

associated

with

immunological

defects

-LRB-

GS

type

II

-RRB-

or

with

primary

neurological

dysfunctions

-LRB-

GS

type

I

-RRB-

Griscelli

syndrome

type

II

with

immunological

defects

is

caused

by

missense

mutations

in

the

gene

encoding

-LSB-

-RSB-

This

Rab

regulates

the

movement

of

melanosomes

to

the

cell

periphery

of

melanocytes

and

also

regulates

the

secretion

of

lytic

granules

in

cytotoxic

T

lymphocytes

-LRB-

CTL

-RRB-

-LSB-

-RSB-

The

lack

of

thus

causes

pigment

anomalies

and

dysfunctional

T

lymphocytes

which

is

in

agreement

with

the

defects

observed

in

the

patients

that

present

with

albinism

and

hemophagocytic

syndrome

The

GS

type

I

with

neurological

symptoms

is

caused

by

mutations

in

the

gene

encoding

the

motor

protein

myosin

Va

-LSB-

-RSB-

a

putative

effector

that

drives

the

peripheral

distribution

of

melanosomes

along

actin

filaments

-LSB-

-RSB-

As

myosin

Va

does

not

participate

in

the

exocytosis

of

lytic

granules

of

CTL

the

inactivation

of

this

protein

does

not

lead

to

immunological

symptoms

Finally

GS

type

III

in

patients

with

typically

pigment

abnormalities

without

additional

features

is

caused

by

a

mutation

in

the

MLPH

gene

encoding

melanophilin

A

direct

interaction

exists

between

the

SHD

domain

of

melapholin

and

and

between

the

C-terminal

domain

of

melanophilin

and

myosin

Va

-LSB-

-RSB-

Therefore

although

the

complex

is

crucial

for

the

trafficking

of

melanosomes

neurodevelopmental

abnormalities

are

specific

for

the

deficiency

and

uses

a

different

effector

for

its

function

in

the

CTL

Rab

proteins

in

immunology

It

has

already

been

mentioned

that

GS

type

II

patients

with

mutations

develop

the

hemophagocytic

syndrome

which

is

characterized

by

episodes

of

life-threatening

uncontrolled

T

lymphocyte

and

macrophage

activation

-LSB-

-RSB-

During

these

episodes

activated

T

cells

and

macrophages

infiltrate

various

organs

-LRB-

including

the

brain

-RRB-

leading

to

tissue

damage

organ

failure

and

death

in

the

absence

of

immunosuppressive

therapy

In

practice

only

bone-marrow

or

cord

blood

stem

cell

transplantation

can

be

curative

for

this

condition

-LSB-

-RSB-

The

important

function

of

Rab

proteins

in

intracellular

traffic

endocytosis

and

exocytosis

explains

their

role

in

counteracting

bacterial

and

viral

infections

Phagocytosis

by

macrophages

leads

to

the

destruction

of

bacteria

Endocytosis

leads

to

the

formation

of

phagosomes

but

phagosomes

fuse

with

lysosomes

-LRB-

phagolysosomes

-RRB-

resulting

in

the

release

of

toxic

products

that

kill

the

bacterium

as

well

as

the

release

of

the

degradation

products

by

exocytosis

-LSB-

-RSB-

However

some

bacteria

survive

this

process

and

avoid

bactericide

by

escaping

from

the

phagosomes

into

the

cytoplasm

-LSB-

-RSB-

Listeria

monocytogenes

is

a

paramount

example

It

has

been

shown

that

the

stimulation

of

macrophages

with

interferon

-LRB-

IFN

-RRB-

γ

a

cytokine

that

is

secreted

by

natural

killer

and

T

helper

cells

results

in

the

expression

of

at

least

proteins

including

-LSB-

-RSB-

In

L.

monocytogenes-infected

macrophages

overexpression

of

stimulates

the

intracellular

degradation

of

the

pathogen

-LSB-

-RSB-

It

has

been

suggested

that

participates

in

the

active

recruitment

of

to

phagolysosomes

which

is

important

for

the

destruction

of

the

bacterium

-LSB-

-RSB-

Other

microbes

develop

antiphagocytosis

-LRB-

e.g.

Yersinia

enterocolitica

-RRB-

or

inhibit

the

formation

of

phagosomes

-LRB-

e.g.

Salmonella

typhimurium

-RRB-

In

Salmonella

infections

there

is

evidence

that

is

stabilized

on

the

phagosome

by

the

Salmonella

type

III

secretory

protein

SopE

-LSB-

-RSB-

SopE

has

GEF

activity

for

stimulating

activated

GTP-bound

and

preventing

its

membrane

extraction

by

RabGDI

A

general

conclusion

that

can

be

drawn

is

that

parasites

recruit

endocytic

Rabs

to

prevent

or

delay

the

formation

of

the

degradative

phagolysosome

in

the

host

organism

Viruses

enter

host

cells

by

receptor-mediated

endocytosis

but

they

usually

escape

from

endosomal

vesicles

to

enter

either

the

cytosol

or

the

nucleus

where

they

replicate

The

binding

of

hepatitis

C

virus

-LRB-

HCV

-RRB-

non-structural

protein

-LRB-

NS

-RRB-

with

GTPase-activating

protein

suggests

that

viruses

are

able

to

subvert

host

cell

machinery

in

this

way

because

this

binding

is

necessary

for

HCV

replication

depletion

decreases

HCV-RNA

levels

which

could

lead

to

therapeutic

applications

-LSB-

-RSB-

Rab

proteins

in

vision

Choroideremia

is

an

X-linked

disease

that

involves

the

degeneration

of

the

retinal

pigment

epithelium

and

the

adjacent

choroid

and

retinal

photoreceptor

cell

layers

leading

to

blindness

The

mutated

gene

in

the

choroideremia

is

one

of

the

two

REP

isoforms

-LSB-

-RSB-

Although

the

other

isoform

seems

to

be

sufficient

for

the

geranylgeranylation

of

all

Rab

GTPases

in

all

tissues

the

retinal

pigment

epithelium

seems

to

be

the

exception

A

role

for

in

human

disease

has

not

yet

been

discovered

is

essential

for

the

efficient

geranylgeranylation

of

in

the

retinal

pigment

epithelium

Thus

a

lack

of

leads

to

a

lack

of

functional

specifically

in

the

retinal

pigment

epithelium

-LSB-

-RSB-

The

degeneration

of

this

epithelium

and

its

adjacent

layers

may

be

due

to

deficient

melanosome

transport

and

consequently

a

lack

of

protection

against

harmful

light

exposure

Rab

proteins

in

neurology

A

subgroup

of

patients

with

X-linked

non-specific

mental

retardation

have

mutations

in

the

gene

for

one

of

the

GDI

isoforms

GDI-α

-LSB-

-RSB-

This

isoform

is

particularly

abundant

in

the

brain

and

dysfunctional

membrane

recycling

of

one

or

more

Rab

GTPases

in

brain

synapses

leading

to

aberrant

neurotransmission

is

likely

to

underlie

the

symptoms

in

this

disease

Hereditary

sensory

and

autonomic

neuropathies

-LRB-

HSAN

-RRB-

known

for

many

years

as

familial

dysautonomia

in

Jewish

patients

are

characterized

by

the

loss

of

sensation

for

pain

and

temperature

alacrima

excessive

sweating

and

the

absence

of

fungiform

tongue

papillae

The

onset

is

congenital

and

the

transmission

is

autosomal

recessive

The

clinical

symptoms

of

HSAN

have

been

classified

into

five

types

-LSB-

-RSB-

HSAN

type

II

encompasses

patients

with

Charcot

Marie

Tooth

disease

type

II

This

is

an

axonal

neuropathy

with

a

median

motor

conduction

velocity

of

m/s

This

peripheral

neuropathy

starts

in

the

second

or

third

decade

of

life

and

presents

as

mixed

motor

and

sensory

involvement

associated

with

ulcers

Four

mutations

in

the

gene

have

been

identified

in

four

families

with

Charcot

Marie

Tooth

disease

type

-LRB-

-RRB-

with

symptoms

leading

to

ulcerations

and

amputations

-LSB-

-RSB-

However

a

hallmark

feature

of

patients

with

HSAN

type

is

lancinating

pain

which

is

not

commonly

observed

in

Moreover

HSAN

type

is

associated

with

a

mutation

in

serine

palmitoyl

transferase

-LRB-

SPT

-RRB-

a

rate-limiting

step

in

the

sphingo-lipid

synthesis

This

overlapping

of

the

two

syndromes

is

possibly

explained

by

being

involved

in

both

the

endocytosis

and

transport

of

sphingolipids

-LSB-

-RSB-

The

importance

of

Rab

proteins

in

brain

development

and

function

is

even

more

emphasized

by

the

recent

discoveries

of

the

involvement

of

proteins

in

human

disease

-LSB-

-RSB-

is

the

most

abundantly

expressed

protein

in

the

brain

where

it

is

present

in

all

synapses

and

involved

in

calcium-dependent

neurotransmitter

release

-LSB-

-RSB-

The

activity

of

proteins

is

tightly

regulated

by

which

specifically

converts

active

to

the

inactive-GDP

form

Germline-inactivating

mutations

in

the

catalytic

subunit

of

-LRB-

-RRB-

cause

Warburg

Micro

syndrome

an

autosomal

recessive

disorder

characterized

by

ocular

defects

-LRB-

microphthalmos

microcornea

congenital

cataracts

and

optic

atrophy

-RRB-

neurodevelopmental

defects

-LRB-

microcephaly

cortical

gyral

abnormalities

such

as

pachygyria

and

polymicrogyria

hypoplasia

of

the

corpus

callosum

severe

mental

retardation

and

spastic

cerebral

palsy

-RRB-

and

hypothalamic

hypogenitalism

-LSB-

-RSB-

Mutations

in

the

non-catalytic

subunit

of

-LRB-

-RRB-

have

been

described

in

patients

with

Martsolf

syndrome

an

autosomal

recessive

disorder

with

a

similar

but

milder

phenotype

than

the

Warburg

Micro

syndrome

including

congenital

cataracts

hypogonadism

and

mild

mental

retardation

-LSB-

-RSB-

The

precise

mechanisms

whereby

the

and

mutations

cause

the

human

phenotype

is

still

unclear

Rab

proteins

in

endocrinology

Abnormal

trafficking

of

the

insulin-sensitive

glucose

transporter

has

been

described

in

patients

with

diabetes

type

Glucose

transporter

accumulates

in

the

dense

membrane

compartments

suggesting

that

defects

in

membrane

trafficking

may

be

involved

in

insulin

resistance

-LSB-

-RSB-

has

been

found

to

be

implicated

in

biogenesis

sorting

and

exocytosolic

movement

but

more

studies

are

needed

to

define

the

exact

role

of

this

and

other

Rabs

and

Rab-associated

proteins

in

trafficking

-LSB-

-RSB-

mutations

in

humans

or

mice

do

not

yet

exist

is

necessary

for

insulin

secretion

-LSB-

-RSB-

Diabetes

mellitus

is

characterized

by

both

low

insulin

and

high

glucagon

levels

These

conditions

have

been

shown

recently

to

be

due

to

the

expression

of

an

inducible

cAMP

early

repressor

-LRB-

ICER

-RRB-

-LSB-

-RSB-

resulting

in

a

repression

of

the

transcription

of

the

insulin

gene

and

associated

with

a

diminished

level

of

granuphilin

a

protein

However

patients

with

GS

type

II-homozygous

deficiency

in

do

not

have

diabetes

mellitus

The

control

of

thyroid

hormone

production

depends

on

endocytic

catalysts

and

the

tandem

regulators

and

which

have

been

found

in

excess

in

thyroid

adenomas

-LSB-

-RSB-

Rab

proteins

in

nephrology

Nephrogenic

diabetes

insipidus

-LRB-

NDI

-RRB-

is

characterized

by

the

inability

to

concentrate

the

urine

in

response

to

the

antidiuretic

hormone

vasopressin

-LRB-

AVP

-RRB-

Under

normal

conditions

binding

of

AVP

to

the

vasopressin

receptor

-LRB-

-RRB-

leads

to

the

insertion

of

aquaporin

-LRB-

-RRB-

water

channels

in

the

apical

membrane

of

the

renal

collecting

duct

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

The

defect

can

be

inherited

and

is

either

X-linked

due

to

mutations

in

the

genes

encoding

the

or

autosomal

recessive

or

dominant

in

mutations

in

the

gene

The

trafficking

of

in

the

cell

and

its

internalization

are

cAMP

and

especially

-LSB-

-RSB-

However

mutations

in

humans

or

mice

are

not

yet

described

Fig.

of

-LRB-

-RRB-

mediated

water

transport

by

vasopressin

-LRB-

AVP

-RRB-

Shown

is

a

nephron

with

a

magnified

principal

cell

In

this

cell

the

vasopressin

receptor

-LRB-

-RRB-

stimulatory

GTP

binding

protein

-LRB-

Gs

-RRB-

adenylate

cyclase

-LRB-

AC

-RRB-

ATP

cAMP

and

phosphorylated

proteins

-LRB-

O-P

-RRB-

are

indicated

This

figure

has

been

used

with

permission

of

the

American

Journal

of

Physiology

and

Renal

Physiology

Conclusion

The

Rab

GTPases

are

a

large

family

of

proteins

with

a

variety

of

regulatory

functions

in

membrane

trafficking

The

central

role

of

these

proteins

has

become

clear

during

the

past

decade

as

part

of

the

progress

that

has

been

made

in

understanding

in

detail

the

mechanistic

principles

of

transport

vesicle

formation

movement

and

fusion

Sequencing

of

the

human

genome

has

enabled

researchers

to

realize

the

diversity

of

the

Rab

gene

family

although

the

functions

of

the

majority

of

the

gene

products

are

still

unknown

The

availability

of

complete

genomic

sequences

as

well

as

the

important

advances

in

molecular

and

cell

biological

methods

that

have

already

been

made

hold

the

promise

of

significant

progress

being

booked

in

our

understanding

of

Rab

function

in

the

near

future

The

identification

of

further

genes

involved

not

only

in

monogenic

but

also

in

common

multifactorial

human

vesicle-trafficking

disorders

will

result

in

a

better

understanding

of

this

complex

transport

pathway

but

more

importantly

will

also

lead

to

opportunities

to

develop

novel

treatments

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