Intermediate

filament

cytoskeleton

of

the

liver

in

health

and

disease

Intermediate

filaments

-LRB-

IFs

-RRB-

represent

the

largest

cytoskeletal

gene

family

comprising

genes

expressed

in

tissue

specific

manner

In

addition

to

scaffolding

function

they

form

complex

signaling

platforms

and

interact

with

various

kinases

adaptor

and

apoptotic

proteins

IFs

are

established

cytoprotectants

and

IF

variants

are

associated

with

human

diseases

Furthermore

IF-containing

inclusion

bodies

are

characteristic

features

of

several

neurodegenerative

muscular

and

other

disorders

Acidic

-LRB-

type

I

-RRB-

and

basic

keratins

-LRB-

type

II

-RRB-

build

obligatory

type

I

and

type

II

heteropolymers

and

are

expressed

in

epithelial

cells

Adult

hepatocytes

contain

and

as

their

only

cytoplasmic

IF

pair

whereas

cholangiocytes

express

and

in

addition

animals

exhibit

a

marked

susceptibility

to

various

toxic

agents

and

Fas-induced

apoptosis

In

humans

variants

predispose

to

development

of

end-stage

liver

disease

and

acute

liver

failure

-LRB-

ALF

-RRB-

variants

also

associate

with

development

of

liver

fibrosis

in

patients

with

chronic

hepatitis

C.

Mallory-Denk

bodies

-LRB-

MDBs

-RRB-

are

protein

aggregates

consisting

of

ubiquitinated

chaperones

and

-LRB-

-RRB-

as

their

major

constituents

MDBs

are

found

in

various

liver

diseases

including

alcoholic

and

non-alcoholic

steatohepatitis

and

can

be

formed

in

mice

by

feeding

hepatotoxic

substances

griseofulvin

and

3,5-diethoxycarbonyl-1,4-dihydrocollidine

-LRB-

DDC

-RRB-

MDBs

also

arise

in

cell

culture

after

transfection

with

ubiquitin

and

Major

factors

that

determine

MDB

formation

in

vivo

are

the

type

of

stress

-LRB-

with

oxidative

stress

as

a

major

player

-RRB-

the

extent

of

stress-induced

protein

misfolding

and

resulting

chaperone

proteasome

and

autophagy

overload

keratin

excess

transglutaminase

activation

with

transamidation

of

keratin

and

upregulation

Intermediate

filaments

in

health

and

disease

Together

with

the

actin

microfilaments

and

the

microtubules

intermediate

filaments

-LRB-

IFs

-RRB-

are

the

components

of

the

cytoskeleton

of

eukaryotic

cells

which

is

involved

in

the

maintenance

of

cell

shape

locomotion

intracellular

organization

and

transport

-LRB-

Bershadsky

and

Vasiliev

Ku

et

al.

-RRB-

IF

proteins

comprise

a

large

family

which

includes

different

genes

-LRB-

Omary

et

al.

Kim

and

Coulombe

Herrmann

et

al.

Goldman

et

al.

-RRB-

They

are

further

divided

into

six

subtypes

-LRB-

Table

-RRB-

which

are

at

least

in

part

expressed

in

a

cell

type

-LRB-

and

differentiation

-RRB-

dependent

manner

-LRB-

Omary

et

al.

Kim

and

Coulombe

Goldman

et

al.

-RRB-

Accordingly

IFs

serve

as

cell

type

markers

and

antibodies

to

IF

proteins

are

widely

used

today

in

diagnostic

pathology

-LRB-

Wick

Barak

et

al.

-RRB-

Individual

IF

proteins

consist

of

a

conserved

central

coiled-coil

α-helical

rod

domain

-LRB-

interrupted

by

linkers

-RRB-

which

is

flanked

by

N-terminal

-LRB-

head

-RRB-

and

C-terminal

-LRB-

tail

-RRB-

domains

-LRB-

Omary

et

al.

Godsel

et

al.

Kim

and

Coulombe

Herrmann

et

al.

Goldman

et

al.

-RRB-

The

N

and

C-terminal

domains

contribute

to

the

structural

heterogeneity

and

are

major

sites

of

posttranslational

modifications

with

phosphorylation

being

the

best

characterized

one

-LRB-

Omary

et

al.

-RRB-

This

makes

them

important

regulatory

domains

since

dynamic

changes

in

phosphorylation

status

are

responsible

for

alterations

in

IF

dynamics

solubility

and

organization

-LRB-

Omary

et

al.

-RRB-

Table

filament

proteinsTypeName/LocalizationDisease

locationRemarksI

-LRB-

n

-RRB-

-LRB-

epithelia

-RRB-

-LRB-

hair/nail

-RRB-

-LRB-

pI

-RRB-

mucosaType

I/II

obligate

-LRB-

n

-RRB-

-LRB-

epithelia

-RRB-

-LRB-

hair/nail

-RRB-

-LRB-

pI

-RRB-

mucosaType

I/II

obligate

-LRB-

muscle

-RRB-

Muscle

heartDesmin

vimentin

and

GFAP

are

found

in

stellate

cellsVimentin

-LRB-

mesenchymal

-RRB-

Peripherin

-LRB-

neurons

-RRB-

Braina

spinal

cordaGFAP

-LRB-

astrocytes/glia

-RRB-

BrainIVNF-L

-LRB-

neurons

-RRB-

Braina

spinal

cordα-internexin

forms

polymers

with

NFsNF-M

-LRB-

neurons

-RRB-

Braina

spinal

cordaNF-H

-LRB-

neurons

-RRB-

Brain

spinal

cordaSynemin

β

is

also

called

desmuslin.α-internexin

-LRB-

CNS

neurons

-RRB-

Nestin-stem

cell

markerSynemins

-LRB-

muscle

-RRB-

Syncoilin

-LRB-

muscle

-RRB-

NestinVLamin

A/C

-LRB-

ubiquitous

-RRB-

Heart

muscle

fat

premature

aging

complex

defectsThe

only

nuclear

IFs

longer

rod

domainLamin

-LRB-

ubiquitous

-RRB-

OrphanPhakinin

-LRB-

lens

-RRB-

LensBeaded

filaments

in

lens

epitheliaFilensin

-LRB-

lens

-RRB-

Lensa

Not

a

causative

association

variants

represent

a

risk

factor

For

an

overview

about

the

new

keratin

nomenclature

see

Schweizer

et

al.

-LRB-

-RRB-

In

addition

to

the

posttranslational

modification

IF

function

is

modified

and

complemented

through

interaction

with

a

variety

of

IF-associated

proteins

-LRB-

IFAP

Table

Green

et

al.

Omary

et

al.

Kim

and

Coulombe

-RRB-

These

proteins

can

be

subdivided

into

several

subgroups

which

reflect

multiple

IF

functions

-LRB-

Green

et

al.

-RRB-

For

example

IFs

interact

with

a

variety

of

anchoring

proteins

thereby

forming

transcellular

networks

which

contribute

to

proper

tissue

architecture

IFAPs

also

include

several

cytolinker

proteins

which

provide

the

structural

framework

for

coordinated

cytoskeletal

function

-LRB-

Table

-RRB-

By

doing

that

they

are

important

mechanical

stabilizers

and

accordingly

IF

disruption

results

in

increased

mechanical

fragility

-LRB-

Omary

et

al.

Ku

et

al.

Herrmann

et

al.

-RRB-

The

scaffolding

function

of

IFs

is

best

seen

in

IF-deficient

animals

who

exhibit

disrupted

cellular

architecture

protein

mistargeting

as

well

as

alterations

in

organelle

localization

and

function

-LRB-

Toivola

et

al.

-RRB-

In

the

case

of

lamin

deficiencies

the

impairment

of

nuclear

composition

has

profound

impact

on

many

aspects

of

normal

nuclear

functions

such

as

epigenetic

changes

chromatin

organization

or

DNA

transcription

and

repair

-LRB-

Dechat

et

al.

-RRB-

Table

of

IF-associated

proteinsType

of

interactionExamplesFunctionAnchoringDesmoplakin

α-dystobrevinTissue

architectureCytolinkerPlectin

Filaggrin

Cellular

α-B-crystallin

foldingKinasesPKC

regulation

phosphate

sink

cell

cycleAdaptor

protein

effectsMembrane

proteinsTRADD

C-Flip

regulationMotor

proteinsDynein

KinesinMovement

of

IF

components

IFs

are

not

just

simple

cellular

scaffolds

they

rather

build

complex

signaling

platforms

-LRB-

Pallari

and

Eriksson

Kim

and

Coulombe

-RRB-

IFs

interact

with

a

variety

of

enzymatic

and

adaptor

proteins

thereby

affecting

a

multitude

of

cellular

functions

For

example

keratins

associate

with

proteins

in

a

phosphorylation-dependent

manner

and

this

interaction

regulates

cell

growth

and

cell

cycle

progression

-LRB-

Ku

et

al.

Kim

and

Coulombe

-RRB-

IF

phosphorylation

through

associated

kinases

does

not

only

regulate

IF

properties

but

IFs

also

serve

as

phosphate

sponge

thereby

preventing

activation

of

other

potentially

pro-apoptotic

substrates

-LRB-

Omary

et

al.

Ku

et

al.

-RRB-

In

addition

to

that

IFs

also

directly

participate

in

apoptosis

regulation

through

binding

of

several

apoptosis-related

molecules

-LRB-

Marceau

et

al.

-RRB-

In

contrast

to

the

actin

and

tubulin

system

IFs

emerged

later

in

the

evolution

and

are

important

supportive

elements

of

the

cell

rather

than

their

essential

components

Therefore

IF

variants

are

observed

in

various

human

diseases

which

reflect

their

tissue

specific

distribution

whereas

only

few

actin

and

tubulin

variants

have

been

described

so

far

likely

due

to

their

embryolethality

-LRB-

Ku

et

al.

Omary

et

al.

-RRB-

Currently

more

than

diseases

are

caused

by/associated

with

IF

mutations

-LRB-

Omary

et

al.

-RRB-

Among

them

keratin-related

and

lamin-related

disorders

are

the

best

studied

ones

For

example

mutations

in

keratins

cause

a

blistering

skin

disease

termed

Epidermolysis

bullosa

simplex

whereas

mutations

in

lamins

A

and

C

result

among

others

in

different

diseases

including

premature

aging

cardiomyopathy

and

lipodystrophy

-LRB-

Omary

et

al.

Dechat

et

al.

-RRB-

The

disease

relevance

of

IFs

is

also

highlighted

by

a

variety

of

IF-containing

inclusion

bodies

which

represent

the

pathological

hallmarks

of

several

neurodegenerative

muscular

and

other

disorders

-LRB-

Goebel

Ross

and

Poirier

Omary

et

al.

Cairns

et

al.

-RRB-

These

aggregates

share

a

variety

of

features

such

as

presence

of

misfolded

ubiquitinated

structural

proteins

together

with

variable

amounts

of

chaperones

and

-LRB-

Kuusisto

et

al.

Zatloukal

et

al.

Ross

and

Poirier

-RRB-

Among

the

IF-related

inclusions

Mallory-Denk

bodies

-LRB-

MDBs

-RRB-

are

the

most

common

and

also

the

best

studied

ones

due

to

the

availability

of

animal

MDB

models

-LRB-

Denk

et

al.

Yokoo

et

al.

Jensen

and

Gluud

Denk

et

al.

Zatloukal

et

al.

-RRB-

Therefore

one

focus

of

our

review

will

be

to

describe

MDB

as

a

prototype

of

IF-related

inclusion

body

which

should

offer

useful

insights

into

the

formation

of

IF-related

aggregates

in

multiple

human

diseases

Keratins

as

epithelially

expressed

IFs

Keratins

represent

the

largest

subfamily

of

IFs

consisting

of

unique

gene

product

members

-LRB-

Schweizer

et

al.

Kim

and

Coulombe

Godsel

et

al.

-RRB-

which

include

epithelial

and

hair

keratin

members

in

humans

-LRB-

Table

Schweizer

et

al.

-RRB-

Based

on

their

pI

epithelial

keratins

can

be

subdivided

in

types

I

-LRB-

acidic

-RRB-

and

II

-LRB-

basic

-RRB-

corresponding

to

keratins

-LRB-

-RRB-

and

keratins

plus

keratins

-LRB-

-RRB-

respectively

-LRB-

Table

Coulombe

and

Omary

Schweizer

et

al.

-RRB-

Keratins

are

found

as

obligatory

type

I

and

type

II

heteropolymers

-LRB-

i.e.

consisting

of

at

least

one

type

I

and

one

type

II

keratin

-RRB-

and

a

homozygous

disruption

of

a

keratin

results

in

degradation

of

its

keratin

partner

at

the

protein

level

-LRB-

Ku

and

Omary

Omary

et

al.

-RRB-

Similarly

to

IFs

keratins

are

expressed

in

a

tissue-specific

manner

with

different

pairs

being

the

major

cellular

IFs

in

different

cell

populations

-LRB-

Moll

et

al.

Ku

et

al.

Coulombe

and

Omary

-RRB-

For

example

simple

-LRB-

i.e.

single

layered

-RRB-

epithelia

as

found

in

digestive

organs

express

together

with

variable

levels

of

and

depending

on

the

tissue

-LRB-

Moll

et

al.

Ku

et

al.

Coulombe

and

Omary

Ku

et

al.

-RRB-

In

contrast

stratified

epithelia

like

epidermis

express

in

the

basal

and

in

the

suprabasal

keratinocytes

respectively

-LRB-

Moll

et

al.

Lane

and

McLean

Coulombe

and

Omary

-RRB-

Despite

their

similar

molecular

composition

simple

and

stratified

keratins

are

not

interchangeable

as

shown

in

mice

whose

phenotype

was

only

partially

restored

by

addition

of

-LRB-

Hutton

et

al.

-RRB-

Furthermore

keratins

have

their

preferential

binding

partners

in

vivo

and

the

lack

of

such

partner

leads

to

their

rapid

degradation

-LRB-

Magin

-RRB-

This

contrasts

with

the

in

vitro

situation

where

IF

assembly

is

more

promiscuous

-LRB-

Hatzfeld

and

Franke

-RRB-

are

promising

serological

markers

based

on

their

high

abundance

-LRB-

approximately

of

total

liver

protein

-RRB-

and

intracellular

localization

under

basal

conditions

with

release

into

blood

upon

liver

injury

-LRB-

Omary

et

al.

Ku

et

al.

-RRB-

However

one

important

caveat

is

the

fact

that

are

expressed

in

most

simple

epithelial

cells

and

are

therefore

not

liver-specific

-LRB-

Moll

et

al.

Ku

et

al.

-RRB-

The

epitopes

used

in

serologic

diagnosis

can

be

divided

into

two

classes

that

is

non-specific

and

apoptosis-generated

epitopes

The

former

class

constitutes

established

tumor

markers

such

as

tissue

polypeptide

antigen

-LRB-

TPA

represents

total

-RRB-

tissue

polypeptide

specific

antigen

-LRB-

TPS

derived

from

-RRB-

and

CYtokeratin

FRAgment

-LRB-

CYFRA

derived

from

-RRB-

Their

original

clinical

use

was

to

monitor

treatment

response

and

to

detect

recurring

tumors

-LRB-

Barak

et

al.

-RRB-

However

later

studies

showed

that

these

epitope

serum

levels

are

also

elevated

in

non-malignant

diseases

and

might

be

a

general

marker

of

tissue

injury

-LRB-

Gonzalez-Quintela

et

al.

b

Tarantino

et

al.

-RRB-

The

apoptosis-specific

keratin

antibodies

are

based

on

the

finding

that

type

I

keratins

are

cleaved

at

a

conserved

VEMD/VEVD

residue

during

apoptosis

In

addition

to

that

posseses

a

second

specific

caspase-cleavage

site

at

which

is

an

early

event

during

apoptosis

preceding

the

cleavage

at

the

VEMD/VEVD

motif

-LRB-

Oshima

Marceau

et

al.

-RRB-

The

cleavage

of

human

at

can

be

monitored

using

the

antibody

-LRB-

Leers

et

al.

-RRB-

and

ELISA

has

become

a

useful

serologic

test

for

determining

liver

disease

severity

For

example

elevated

serum

titers

can

distinguish

simple

steatosis

from

non-alcoholic

steatohepatitis

-LRB-

Wieckowska

et

al.

-RRB-

and

predict

several

important

prognostic

parameters

in

patients

with

chronic

hepatitis

C

infection

-LRB-

Bantel

et

al.

Volkmann

et

al.

-RRB-

As

another

tool

for

detecting

apoptotic

keratin

fragments

an

antibody

specific

to

the

conserved

cleavage

site

at

was

recently

generated

-LRB-

Tao

et

al.

-RRB-

It

detects

both

mouse

and

human

fragments

and

appears

to

be

more

sensitive

than

the

established

-LRB-

Tao

et

al.

-RRB-

Measuring

the

serum

levels

of

apoptosis-specific

keratin

fragments

should

also

improve

our

understanding

of

chronic

liver

disease

where

apoptotic

cell

death

is

an

important

pathogenic

feature

-LRB-

Malhi

et

al.

-RRB-

In

addition

to

that

monitoring

the

phosphorylation

status

of

the

circulating

keratin

fragments

might

be

useful

Keratins

undergo

dynamic

phosphorylation

during

mitosis

apoptosis

and

a

variety

of

stress

situations

-LRB-

Omary

et

al.

Omary

et

al.

-RRB-

and

their

in

situ

phosphorylation

status

is

a

marker

of

human

liver

disease

progression

-LRB-

Toivola

et

al.

-RRB-

However

it

is

currently

unknown

whether

the

phosphorylation

status

of

the

circulating

keratin

fragments

correlates

with

the

situation

in

situ

Hepatic

phenotype

in

keratin-deficient

transgenic

animals

The

liver

consists

of

different

cell

types

with

characteristic

IF

composition

-LRB-

Table

Omary

et

al.

-RRB-

Adult

hepatocytes

are

unique

among

simple

epithelial

cells

in

that

they

express

exclusively

and

whereas

other

glandular

epithelia

exhibit

a

more

complex

keratin

expression

pattern

-LRB-

Omary

et

al.

Ku

et

al.

-RRB-

The

hepatocytic

keratin

IF

network

is

dense

particularly

around

bile

canaliculi

and

at

the

cell

periphery

and

acts

as

cytoskeletal

backbone

to

the

functionally

more

dynamic

and

contractile

actin

microfilament

system

-LRB-

Zatloukal

et

al.

-RRB-

Biliary

epithelial

cells

differ

from

hepatocytes

by

additional

expression

of

keratin

and

-LRB-

Omary

et

al.

Zatloukal

et

al.

-RRB-

Keratins

in

cholangiocytes

but

not

hepatocytes

exhibit

polarized

and

compartment-specific

expression

pattern

-LRB-

Omary

et

al.

Zatloukal

et

al.

-RRB-

The

biological

significance

of

such

an

expression

is

enigmatic

but

it

may

be

related

to

polarity

and

secretory

processes

Among

non-epithelial

cells

stellate

cells

express

variable

amounts

of

GFAP

desmin

vimentin

and

nestin

dependent

on

their

activation

status

localization

and

other

parameters

-LRB-

Table

Geerts

-RRB-

Table

of

liver

cell

populationsCell

typeIF

cellVimentinStellate

cellGFAP

Vimentin

Desmin

NestinbEndothelial

cellVimentinModified

from

Omary

et

al.

-LRB-

-RRB-

a

During

embryogenesis

hepatocytes

also

express

variable

levels

of

-LRB-

Vassy

et

al.

-RRB-

b

Stellate

cells

represent

a

highly

heterogeneous

population

with

variable

IF

expression

dependent

on

species

activation

status

of

the

cell

location

within

the

hepatic

lobe

and

many

other

parameters

-LRB-

Geerts

-RRB-

Studies

in

keratin

knock-out

mice

revealed

that

the

regular

liver

development

does

not

require

the

presence

of

or

-LRB-

Ku

et

al.

-RRB-

However

knockout

mice

exhibited

mild

chronic

hepatitis

hepatocyte

fragility

and

were

markedly

more

sensitive

to

a

variety

of

stress

conditions

-LRB-

Omary

et

al.

Zatloukal

et

al.

Ku

et

al.

-RRB-

Furthermore

transgenic

mice

were

developed

which

over-express

different

single-amino-acid

variants

-LRB-

Ku

et

al.

-RRB-

Among

them

the

variant

resulted

in

disruption

of

hepatocyte

IF

network

and

exhibited

a

phenotype

reminiscent

of

the

situation

in

mice

-LRB-

Ku

et

al.

-RRB-

mice

also

predisposed

to

development

of

thioacetamide-induced

liver

fibrosis

-LRB-

Strnad

et

al.

-RRB-

Ablations

of

different

phosphorylation

sites

usually

led

to

a

somewhat

milder

phenotype

which

became

apparent

in

stress

situations

but

not

under

basal

conditions

-LRB-

Ku

et

al.

-RRB-

For

example

variant

resulted

in

increased

sensitivity

to

microcystin-LR-induced

liver

injury

whereas

mice

were

predisposed

to

Fas-induced

liver

apoptosis

-LRB-

Ku

et

al.

Ku

and

Omary

-RRB-

The

ablation

of

phosphorylation

site

which

regulates

the

binding

to

proteins

caused

limited

mitotic

arrest

and

accumulation

of

abnormal

mitotic

figures

after

partial

hepatectomy

-LRB-

Ku

et

al.

-RRB-

In

contrast

mice

lacking

did

not

have

an

obvious

liver

phenotype

-LRB-

Tao

et

al.

-RRB-

but

surprisingly

exhibited

skeletal

myopathy

-LRB-

Stone

et

al.

-RRB-

GFAP/vimentin-knockouts

displayed

compromised

astrocytic

function

with

attenuated

reactive

gliosis

but

no

obvious

alteration

in

the

in

vitro

activation

of

hepatic

stellate

cells

despite

the

lack

of

IF

network

-LRB-

Geerts

et

al.

Pekny

and

Pekna

-RRB-

However

in

vivo

studies

are

needed

to

conclusively

address

the

fibrogenic

potency

of

these

transgenic

mice

Keratin

variants

in

liver

disease

The

large

body

of

evidence

from

animal

studies

showing

the

importance

of

for

liver

homeostasis

led

to

a

search

for

keratin

mutations

in

patients

with

liver

diseases

Several

variants

were

found

to

associate

with

the

development

of

cryptogenic

liver

disease

-LRB-

Ku

et

al.

-RRB-

In

subsequent

studies

were

shown

to

represent

susceptibility

genes

for

development

of

end-stage

liver

disease

of

multiple

etiologies

-LRB-

Ku

et

al.

-RRB-

In

particular

biologically

significant

variants

were

found

in

of

liver

explants

-LRB-

-RRB-

but

only

in

out

of

analyzed

blood

bank

donors

which

were

used

as

a

control

group

-LRB-

P

prevalence

CI

-RRB-

Furthermore

variants

associate

with

liver

fibrosis

progression

in

patients

with

chronic

hepatitis

C

-LRB-

Strnad

et

al.

-RRB-

represents

the

most

common

amino

acid

altering

variant

and

it

is

the

only

one

which

was

significantly

associated

with

development

of

end-stage

liver

disease

-LRB-

Ku

et

al.

-RRB-

Larger

studies

are

needed

to

analyze

the

pathological

significance

of

the

other

less

common

variants

-LRB-

Ku

et

al.

-RRB-

In

contrast

to

human

association

studies

experiments

in

mice

were

shown

to

predispose

mainly

to

acute

liver

injury

-LRB-

Ku

et

al.

-RRB-

To

address

this

issue

we

recently

analyzed

a

large

cohort

of

patients

with

acute

liver

failure

-LRB-

ALF

-RRB-

variants

were

significantly

more

frequent

in

total

ALF

patient

cohort

-LRB-

-RRB-

and

in

patients

with

acetaminophen-induced

ALF

-LRB-

-RRB-

when

compared

to

blood

bank

donors

-LRB-

P

for

both

comparisons

-RRB-

Among

the

single

polymorphisms

the

variant

was

found

at

significantly

higher

frequencies

in

the

whole

ALF

cohort

as

well

as

in

the

acetaminophen-induced

ALF

subgroup

-LRB-

frequency

and

respectively

vs.

in

the

control

group

P

for

both

comparisons

-RRB-

In

addition

transgenic

mice

over-expressing

variants

displayed

augmented

acetaminophen-induced

liver

toxicity

In

conclusion

are

also

susceptibility

genes

for

development

of

ALF

and

variants

may

predispose

to

drug-induced

liver

injury

-LRB-

Strnad

et

al.

unpublished

data

-RRB-

Up

to

date

only

one

published

study

analyzed

the

polymorphisms

in

gene

and

found

no

association

between

variants

and

inflammatory

bowel

disease

-LRB-

Tao

et

al.

-RRB-

Interestingly

we

recently

observed

variant

in

three

out

of

patients

with

primary

biliary

cirrhosis

but

was

found

in

control

blood

bank

donors

-LRB-

samples

Zhong

et

al.

unpublished

data

-RRB-

However

larger

studies

are

needed

to

address

the

importance

of

in

biliary

diseases

The

human

variants

described

above

do

not

cause

a

particular

liver

disease

per

se

they

just

pose

a

risk

factor

for

its

development

This

is

different

from

the

situation

in

stratified

epithelia

where

keratin

mutations

result

in

several

monogenic

keratin

diseases

-LRB-

Lane

and

McLean

Omary

et

al.

-RRB-

This

discrepancy

may

be

caused

either

by

the

intrinsic

difference

between

keratins

of

simple

and

stratified

epithelia

-LRB-

Hutton

et

al.

-RRB-

or

by

the

different

localization

of

the

variants

within

the

protein

backbone

To

that

end

disease-causing

keratin

mutations

in

stratified

epithelia

are

clustered

in

the

highly

conserved

helix

initiation

and

termination

motif

whereas

disease-predisposing

variants

are

observed

in

more

variable

domains

-LRB-

Owens

and

Lane

Omary

et

al.

Ku

et

al.

-RRB-

The

disturbance

in

cytoprotective

function

of

keratins

is

the

likely

mechanisms

by

which

and

variants

predispose

to

liver

disorders

For

example

are

anti-apoptotic

proteins

and

this

ability

is

hampered

in

keratin-deficient

animals

-LRB-

Oshima

Ku

and

Omary

Marceau

et

al.

Ku

et

al.

-RRB-

The

ways

of

interaction

between

keratins

and

apoptosis

are

manifold

bind

to

several

apoptotic

proteins

and

type

I

keratins

are

established

caspase

substrates

-LRB-

Oshima

Green

et

al.

Marceau

et

al.

-RRB-

In

addition

serve

as

physiologic

kinase

substrates

in

vitro

and

in

transgenic

mice

and

an

ablation

of

the

phosphorylation

site

or

introduction

of

the

naturally

occurring

variant

leads

to

increased

apoptosis

through

increased

phosphorylation

of

pro-apoptotic

proteins

-LRB-

Ku

et

al.

Ku

and

Omary

-RRB-

The

keratin-mediated

anti-apoptotic

function

may

be

highly

relevant

given

the

importance

of

apoptosis

in

liver

disease

and

the

pro-fibrogenic

properties

of

elevated

rate

of

apoptotic

cell

death

-LRB-

Friedman

Malhi

et

al.

-RRB-

Keratins

exhibit

anti-oxidative

properties

as

they

sequester

oxidatively

damaged

proteins

and

similarly

the

variant

primes

the

liver

towards

oxidative

injury

-LRB-

Tao

et

al.

Zhou

et

al.

-RRB-

This

keratin

property

might

be

helpful

in

attenuating

both

liver

fibrosis

and

acetaminophen-induced

liver

injury

-LRB-

Parola

and

Robino

Jaeschke

et

al.

-RRB-

Keratins

are

also

established

stress-inducible

proteins

which

are

upregulated

both

in

humans

and

mice

under

several

mainly

cholestatic

conditions

-LRB-

Fickert

et

al.

Zatloukal

et

al.

Ku

et

al.

Strnad

et

al.

-RRB-

Keratin

variants

may

interfere

with

keratin

upregulation

or

simply

result

in

decreased

keratin

levels

due

to

protein

instability

-LRB-

as

seen

for

variant

in

the

liver

Ku

et

al.

-RRB-

The

naturally

occurring

variants

interfere

with

basic

IF

properties

such

as

filament

assembly

and

keratin

solubility

-LRB-

Ku

et

al.

Owens

et

al.

Ku

et

al.

-RRB-

Due

to

altered

protein

conformation

some

of

them

impair

potentially

cytoprotective

keratin

phosphorylation

at

adjacent

residues

-LRB-

as

seen

in

and

variant

Ku

et

al.

Ku

and

Omary

-RRB-

variants

may

also

affect

organelle

function

as

seen

in

knockout

mice

which

exhibit

altered

mitochondrial

shape

localization

and

alterations

in

several

mitochondrial

proteins

-LRB-

Toivola

et

al.

-RRB-

Despite

the

various

cytoprotective

effects

of

keratins

their

impact

seems

to

be

limited

to

certain

conditions

For

example

mice

are

predisposed

to

Fas

but

not

TNF-induced

apoptosis

and

the

same

mice

develop

more

pronounced

liver

fibrosis

after

thioacetamide

but

not

after

carbon

tetrachloride

injection

-LRB-

Ku

et

al.

Strnad

et

al.

-RRB-

It

is

also

unknown

which

one

of

the

keratin-mediated

effects

is

important

in

particular

disease

settings

The

recently

established

transgenic

mouse

lines

overexpressing

the

naturally

occurring

and

variants

will

likely

offer

valuable

insights

in

this

respect

-LRB-

Ku

and

Omary

Zhou

et

al.

unpublished

data

-RRB-

Keratin

network

alteration

in

steatohepatitis

of

the

alcoholic

-LRB-

ASH

-RRB-

and

the

non-alcoholic

-LRB-

NASH

-RRB-

type

Steatohepatitis

is

characterized

by

hepatocyte

ballooning

that

is

swelling

and

rounding

with

clearing

of

the

cytoplasm

which

prevails

in

the

perivenular

zone

and

is

often

associated

with

pericellular

fibrosis

predominantly

granulocytic

inflammation

-LRB-

satellitosis

-RRB-

steatosis

-LRB-

usually

of

macrovesicular

type

-RRB-

and

cholestasis

-LRB-

Brunt

Lefkowitch

-RRB-

Ballooned

cells

are

often

associated

with

granular

rope

or

clump-like

cytoplasmic

inclusions

called

Mallory-Denk

bodies

-LRB-

MDBs

-RRB-

-LRB-

originally

also

designated

alcoholic

hyalin

which

is

however

a

misnomer

since

they

are

not

specific

for

alcoholic

etiology

-RRB-

together

with

derangement

or

even

disappearance

of

the

keratin

IF

cytoskeleton

-LRB-

Mallory

Denk

et

al.

Zatloukal

et

al.

Fig.

b

-RRB-

The

disappearance

of

the

keratin

immunostaining

in

ballooned

hepatocytes

is

reasonably

specific

for

ASH

and

NASH

since

it

is

not

seen

in

ballooned

cells

of

viral

hepatitis

or

toxic

damage

and

can

therefore

be

used

as

an

objective

morphologic

parameter

in

grading

of

steatohepatitis

-LRB-

Lackner

et

al.

-RRB-

However

ballooning

-LRB-

with

concomitant

cytoskeletal

alterations

-RRB-

and

MDB

formation

are

not

entirely

interchangeable

since

not

all

ballooned

hepatocytes

contain

MDBs

Fig.

are

readily

detected

by

different

methods

In

clinical

routine

MDBs

are

usually

detected

as

eosinophilic

aggregates

in

standard

hematoxylin

and

eosin

stained

sections

-LRB-

a

-RRB-

After

chromotrope

aniline

blue

staining

MDBs

appear

as

blue

structures

often

with

red

center

-LRB-

b

-RRB-

Immunofluorescence

or

immunohistochemical

staining

represents

a

more

sensitive

method

for

MDB

detection

than

conventional

histological

stainings

but

is

strongly

dependent

on

the

antibody

used

as

well

as

the

staining

protocol

MDBs

can

be

reliably

detected

with

antibodies

against

-LSB-

green

and

redchannel

in

-LRB-

c

-RRB-

and

-LRB-

d

-RRB-

respectively

-RSB-

or

-LSB-

red

channel

in

-LRB-

c

-RRB-

-RSB-

whereas

only

some

MDBs

stain

with

antibodies

to

phosphorylated

keratins

such

as

antibody

-LSB-

green

channel

in

-LRB-

d

-RRB-

-RSB-

In

both

immunofluorescence

pictures

MDBs

are

seen

as

yellow

structure

due

to

co-localization

of

both

visualized

epitopes

MDBs

are

typical

morphological

features

of

ASH

and

NASH

although

NASH

usually

exhibits

slightly

less

prominent

MDBs

than

the

ones

seen

in

ASH

-LRB-

Brunt

Zatloukal

et

al.

-RRB-

MDBs

can

also

be

detected

after

intestinal

bypass

surgery

for

morbid

obesity

in

chronic

cholestasis

particularly

in

late

stages

of

primary

biliary

cirrhosis

Wilson

disease

and

other

types

of

copper

toxicosis

various

metabolic

disturbances

and

hepatocellular

neoplasms

-LRB-

Müller

et

al.

Zatloukal

et

al.

Fig.

-RRB-

In

contrast

MDBs

have

not

been

observed

in

the

context

of

acute

cholestasis

acute

viral

hepatitis

and

a

variety

of

acute

toxic

or

drug-induced

liver

diseases

-LRB-

Jensen

and

Gluud

Zatloukal

et

al.

Ku

et

al.

-RRB-

However

even

in

potentially

MDB-forming

liver

diseases

MDBs

are

found

only

in

a

subset

of

patients

partially

-LRB-

but

not

completely

-RRB-

depending

on

the

sensitivity

of

the

detection

method

used

For

example

when

using

immunohistochemistry

for

keratin

or

ubiquitin

MDBs

were

found

in

about

of

ASH

cases

in

contrast

to

seen

in

hematoxilin-eosin-stained

sections

-LRB-

Ray

-RRB-

Fig.

are

seen

in

various

human

liver

diseases

Immunohistochemical

staining

with

antibody

visualizes

the

presence

of

multiple

irregularly

shaped

aggregates

in

patients

with

alcoholic

steatohepatitis

-LRB-

a

-RRB-

non-alcoholic

steatohepatitis

-LRB-

b

-RRB-

Indian

childhood

cirrhosis

and

-LRB-

c

-RRB-

idiopathic

copper

toxicosis

-LRB-

d

-RRB-

This

suggests

that

MDBs

require

either

a

specific

pathogenetic

constellation

or

genetic

predisposition

for

its

formation

which

is

present

only

in

a

subset

of

patients

Apart

from

MDBs

additional

features

may

be

observed

in

some

chronic

cholestatic

conditions

For

example

a

low

percentage

of

hepatocytes

express

keratin

and

to

a

lesser

extent

keratin

which

indicate

that

these

cells

acquire

features

of

precursor

cells

which

normally

express

keratin

and

during

regeneration

-LRB-

Van

Eyken

et

al.

Zatloukal

et

al.

-RRB-

In

idiopathic

copper

toxicosis

and

hepatocellular

carcinoma

MDBs

may

coincide

with

another

type

of

cytoplasmic

inclusions

termed

intracellular

hyaline

bodies

-LRB-

IHBs

-RRB-

which

share

several

components

with

MDBs

but

do

not

contain

keratins

-LRB-

Stumptner

et

al.

Denk

et

al.

-RRB-

The

ease

of

MDB

detection

makes

them

attractive

morphologic

markers

However

correlation

between

the

clinical

disease

manifestation/progression

on

one

side

and

hepatocyte

ballooning

with

MDB

formation

on

the

other

is

imperfect

For

example

patients

with

severe

clinical

symptoms

of

ASH

sometimes

show

only

moderate

histopathological

alterations

with

few

or

no

MDBs

whereas

patients

with

pronounced

histological

alterations

do

not

necessarily

exhibit

significant

clinical

and

laboratory

abnormalities

-LRB-

Zatloukal

et

al.

-RRB-

Despite

that

controlled

clinical-pathologic

studies

comparing

NASH

patients

with

ambulatory

and

hospitalized

alcoholics

revealed

that

hepatocellular

damage

presence

of

MDBs

inflammation

and

fibrosis

collectively

correlated

with

disease

severity

-LRB-

Cortez-Pinto

et

al.

-RRB-

Also

in

other

studies

hepatocellular

ballooning

and

MDB

formation

was

positively

correlated

with

disease

progression

development

of

fibrosis

and

cirrhosis

and

liver-related

mortality

-LRB-

Orrego

et

al.

Matteoni

et

al.

Gramlich

et

al.

Mendler

et

al.

-RRB-

Morphology

and

composition

of

MDBs

MDBs

are

irregularly

shaped

usually

dense

cytoplasmic

inclusions

of

different

sizes

-LRB-

Mallory

-RRB-

Small

MDBs

arise

in

association

with

IF

bundles

throughout

the

cytoplasm

whereas

larger

MDBs

are

often

seen

in

the

perinuclear

region

-LRB-

Denk

et

al.

Zatloukal

et

al.

Ku

et

al.

-RRB-

Ultrastructurally

they

usually

consist

of

haphazardly

arranged

filamentous

rods

approximately

nm

in

diameter

covered

by

a

fuzzy

coat

-LRB-

designated

as

type

II

by

Yokoo

et

al.

-RRB-

In

addition

MDBs

with

an

electron

dense

granular

to

amorphous

center

-LRB-

designated

type

-RRB-

are

seen

predominantly

in

older

inclusions

and

filaments

in

parallel

arrangement

were

also

described

-LRB-

designated

as

type

I

-RRB-

but

seem

to

be

exceedingly

rare

-LRB-

Yokoo

et

al.

-RRB-

Keratins

and

sequestosome

-LRB-

-RRB-

and

ubiquitin

are

major

and

low

and

high

molecular

weight

heat

shock

proteins

-LRB-

HSP

HSP

-RRB-

but

also

proteins

of

the

protein

degradation

machinery

are

minor

constituents

-LRB-

Denk

et

al.

Riley

et

al.

Zatloukal

et

al.

Ku

et

al.

Figs.

d

Fig.

-RRB-

Keratins

and

have

also

occasionally

been

detected

-LRB-

Cadrin

et

al.

Dinges

et

al.

-RRB-

The

within

MDBs

exhibit

increased

β-sheet

structure

-LRB-

Cadrin

et

al.

Kachi

et

al.

-RRB-

are

hyperphosphorylated

partially

degraded

and

cross-linked

-LRB-

Hazan

et

al.

Zatloukal

et

al.

Cadrin

et

al.

Stumptner

et

al.

Fig.

-RRB-

The

list

of

MDB

components

is

likely

to

grow

since

in

addition

to

intrinsic

components

proteins

non-specifically

incorporated

in

the

aggregate

have

to

be

expected

Pathogenesis

of

MDBs

Studies

on

MDB

formation

and

composition

are

greatly

enhanced

by

the

availability

of

animal

models

MDBs

can

be

induced

under

standardized

conditions

and

their

fate

followed

in

mouse

liver

by

chronic

griseofulvin

or

-LRB-

DDC

-RRB-

administration

-LRB-

Denk

et

al.

Yokoo

et

al.

Denk

et

al.

Zatloukal

et

al.

Fig.

d

-RRB-

Fig.

formed

in

DDC-fed

animals

resemble

inclusion

bodies

observed

in

human

diseases

Liver

sections

were

double

labeled

with

antibodies

to

-LRB-

green

-RRB-

and

-LRB-

red

-RRB-

Samples

from

mouse

fed

DDC

for

weeks

-LRB-

d

-RRB-

and

from

a

patient

with

alcoholic

steatohepatitis

-LRB-

b

-RRB-

exhibit

multiple

irregularly

shaped

inclusion

bodies

which

appear

yellow

due

to

presence

of

both

epitopes

In

contrast

control

human

-LRB-

a

-RRB-

and

mouse

liver

-LRB-

c

-RRB-

display

an

unaffected

keratin

network

with

no

apparent

staining

Note

that

DDC

feeding

leads

to

deposition

of

protoporphyrin

which

can

be

seen

as

occasional

blue

pigment

in

-LRB-

d

-RRB-

In

patients

MDB

formation

is

usually

a

chronic

process

requiring

several

years

of

alcohol

intoxication

or

metabolic

imbalance

that

is

in

association

with

the

metabolic

syndrome

Wilson

disease

other

metabolic

disorders

and

chronic

cholestasis

An

exception

is

idiopathic

copper

toxicosis

of

children

in

which

end

stage

liver

disease

associated

with

MDB

formation

occurs

at

very

early

age

-LRB-

Müller

et

al.

Zatloukal

et

al.

-RRB-

Surprisingly

in

alcoholics

recovered

from

ASH

an

alcohol

excess

may

almost

instantaneously

lead

to

MDB

recurrence

a

situation

which

has

been

compared

to

an

immunologic

response

and

termed

toxic

memory

-LRB-

Jensen

and

Gluud

Denk

et

al.

-RRB-

The

animal

model

of

MDB

formation

does

not

only

reproduce

the

structural

and

morphological

features

seen

in

humans

but

also

the

phenomenon

of

toxic

memory

-LRB-

Fig.

Denk

et

al.

Zatloukal

et

al.

-RRB-

To

induce

MDBs

in

mice

griseofulvin

or

DDC

is

usually

fed

for

months

MDB

formation

in

mice

is

reversible

since

MDBs

disappear

after

recovery

on

standard

diet

for

month

However

after

rechallenge

of

the

recovered

-LRB-

primed

-RRB-

animals

MDBs

reappear

within

a

few

days

-LRB-

Denk

et

al.

Zatloukal

et

al.

-RRB-

Rapid

MDBs

formation

in

primed

mice

is

rather

non-specific

since

it

can

be

triggered

by

a

variety

of

stress

conditions

including

colchicine

-LRB-

but

not

by

lumicolchicine

-RRB-

bile

acids

bile

duct

ligation

several

other

toxins

and

proteasome

inhibitors

which

were

unable

to

induce

MDBs

in

the

naïve

animal

-LRB-

Zatloukal

et

al.

and

references

therein

-RRB-

The

reasons

for

this

phenomenon

are

as

yet

unclear

but

point

to

a

final

common

pathway

activated

by

the

trigger

The

availability

of

cellular

and

animal

models

of

MDB

formation

led

to

valuable

insights

into

the

mechanism

of

MDB

formation

Several

pathogenic

mechanisms

were

implicated

in

this

process

-LRB-

Fig.

Dobson

-RRB-

Enhanced

oxidative

expression

together

with

keratin

protein

degradation

Ad

The

MDB-inducing

conditions

both

in

humans

and

mice

cause

elevated

levels

of

oxidative

stress

-LRB-

Tephly

et

al.

Mehta

et

al.

Dey

and

Cederbaum

Farrell

and

Larter

-RRB-

and

MDBs

themselves

were

shown

to

contain

misfolded

keratins

with

increased

β-sheet

formation

-LRB-

Cadrin

et

al.

Kachi

et

al.

-RRB-

Recent

animal

studies

highlight

the

importance

of

altered

methyl

group

metabolism

and

mitochondrial

stress

in

this

process

-LRB-

Li

et

al.

Zatloukal

et

al.

unpublished

data

-RRB-

During

DDC

detoxification

N-methylprotoporphyrin

is

formed

through

transfer

of

a

methyl

group

to

heme

moieties

-LRB-

Tephly

et

al.

-RRB-

N-methylprotoporphyrin

subsequently

acts

as

a

potent

inhibitor

of

ferrochelatase

thereby

causing

porphyria

-LRB-

Tephly

et

al.

-RRB-

Accordingly

MDB

formation

and

DDC

effects

can

be

effectively

attenuated

by

feeding

S-adenosylmethionine

that

is

a

compound

involved

in

methyl

group

transfer

-LRB-

Li

et

al.

-RRB-

This

is

reminiscent

of

the

situation

in

ASH

which

is

also

associated

with

disrupted

methyl

group

metabolism

-LRB-

Schalinske

and

Nieman

-RRB-

Fig.

formation

results

from

a

complex

interplay

of

several

contributing

factors

Since

the

cytoplasm

represents

a

hydrophilic

milieu

all

exposed

hydrophobic

molecules

-LRB-

depicted

by

red

stretches

within

the

protein

-RRB-

are

predisposed

to

aggregation

Properly

folded

proteins

usually

hide

their

hydrophobic

stretches

inside

but

these

get

exposed

in

nascent

protein

chains

or

after

proteins

become

misfolded

as

a

consequence

of

oxidative

stress

Chaperones

bind

to

these

hydrophobic

residues

and

facilitate

protein

refolding

Alternatively

damaged

proteins

become

polyubiquitinated

and

degraded

either

by

the

proteasomal

or

autophagic

system

MDB-causing

agents

typically

generate

extensive

amount

of

oxidative

stress

with

increased

protein

misfolding

In

addition

chaperone

levels

are

downregulated

and/or

chaperone

function

is

compromised

Dysbalanced

expression

precedes

MDB

formation

likely

increases

keratin

misfolding

and

predisposes

to

posttranslational

modifications

which

may

interfere

with

keratin

refolding

and/or

repair

Accumulated

misfolded

proteins

are

sequestered

as

inclusions

through

the

action

of

also

shuttles

polyubiquitinated

proteins

to

degradative

machineries

However

proteasomal

degradation

might

be

impaired

by

oxidative

stress

may

not

be

able

to

digest

highly

cross-linked

protein

species

or

might

simply

be

overwhelmed

by

the

excessive

supply

On

the

other

hand

autophagy

is

upregulated

during

MDB

formation

in

mice

and

additional

stimulation

of

autophagy

attenuates

MDB

formation

in

certain

conditions

Of

note

supplementation

of

S-adenosylmethionine

or

mitochondrially

targeted

antioxidants

effectively

diminishes

MDB

formation

thereby

pointing

to

a

central

role

of

oxidative

stress

in

MDB

generation

DDC

targets

mitochondria

where

it

reacts

with

cytochrome

-LRB-

Marks

et

al.

-RRB-

An

unbiased

microarray

analysis

identified

cytochrome

-LRB-

Cyp

-RRB-

as

a

major

gene

induced

both

after

DDC

exposure

and

particularly

after

DDC

re-challenge

Moreover

overexpression

spacially

coincides

with

MDB

formation

-LRB-

Zatloukal

et

al.

unpublished

data

-RRB-

is

a

leaky

cytochrome

which

produces

reactive

oxygen

species

-LRB-

Lewis

et

al.

-RRB-

In

that

sense

it

resembles

human

which

was

implicated

as

a

source

of

oxidative

stress

in

the

pathogenesis

of

ASH

and

NASH

-LRB-

Villeneuve

and

Pichette

-RRB-

To

address

the

role

of

mitochondrial

stress

in

MDB

formation

we

re-fed

DDC-primed

mice

with

DDC

alone

or

in

combination

with

the

mitochondria-targeted

antioxidant

mito

Q

-LRB-

Smith

et

al.

-RRB-

Mito

Q

co-administration

attenuated

both

MDB

formation

and

DDC-induced

liver

damage

Therefore

mitochondrial

oxidative

stress

seems

to

be

involved

in

MDB

formation

in

mice

which

is

in

good

concordance

to

the

mitochondrial

dysfunction

seen

both

in

ASH

and

NASH

patients

-LRB-

Pessayre

Mantena

et

al.

Zatloukal

et

al.

unpublished

data

-RRB-

Ad

Keratins

are

major

constituents

of

MDBs

and

both

altered

expression

and

keratin

modification

seems

to

affect

MDB

formation

-LRB-

Zatloukal

et

al.

Ku

et

al.

-RRB-

Griseofulvin/DDC

feeding

leads

to

rapid

induction

of

expression

with

disproportional

levels

-LRB-

Denk

et

al.

-RRB-

The

elevated

ratio

is

crucial

for

MDB

formation

as

shown

in

and

overexpressing

animals

who

are

predisposed

to

MDB

formation

already

upon

short

exposure

to

DDC

and

even

form

MDBs

spontaneously

during

aging

-LRB-

Magin

et

al.

Nakamichi

et

al.

-RRB-

Accordingly

or

overexpressing

mice

are

resistant

to

MDB

formation

and

the

protective

function

of

is

not

affected

by

its

phosphorylation

status

or

mutation

-LRB-

Zatloukal

et

al.

Harada

et

al.

-RRB-

However

the

exclusive

MDB

inducing

property

of

in

vivo

can

not

be

reproduced

in

vitro

where

aggregates

resembling

MDBs

can

also

be

produced

by

transfection

of

-LRB-

Nakamichi

et

al.

Stumptner

et

al.

-RRB-

Among

posttranslational

modifications

MDB

formation

is

associated

with

hyperphosphorylation

and

transamidation

-LRB-

Zatloukal

et

al.

Stumptner

et

al.

-RRB-

In

a

recent

study

ablation

of

phosphorylation

site

in

transgenic

mice

resulted

in

diminished

MDB

formation

after

DDC

exposure

-LRB-

Harada

et

al.

unpublished

results

-RRB-

Several

potential

mechanisms

might

be

responsible

for

this

observation

is

a

well-known

kinase

target

site

and

up-regulation

induces

keratin

network

reorganization

with

subsequent

granule

formation

-LRB-

Ku

et

al.

Wöll

et

al.

-RRB-

kinase-induced

keratin

network

reorganization

might

be

a

necessary

prerequisite

for

MDB

formation

since

kinase

inhibition

prevented

MDB

formation

in

vitro

-LRB-

et

al.

-RRB-

Alternatively

hyperphosphorylation

may

induce

MDB

formation

through

inhibition

of

degradation

which

results

in

increased

ratio

-LRB-

Ku

and

Omary

-RRB-

MDBs

contain

highly

cross-linked

keratins

and

the

ablation

of

tissue

transglutaminase

effectively

inhibits

DDC-induced

MDB

formation

-LRB-

Zatloukal

et

al.

Strnad

et

al.

-RRB-

Since

is

a

much

better

in

vitro

transglutaminase

substrate

than

and

highly

cross-linked

proteins

found

in

MDB-forming

mice

contain

but

not

it

was

suggested

that

excessive

gets

preferentially

transamidated

and

acts

as

a

nucleus

for

MDB

formation

This

is

supported

by

studies

in

transgenic

mice

where

overexpression

accelerates

and

excess

inhibits

not

only

MDB

but

also

cross-link

formation

-LRB-

Strnad

et

al.

-RRB-

Ad

MDBs

were

shown

to

contain

misfolded

keratins

-LRB-

Cadrin

et

al.

Kachi

et

al.

-RRB-

The

protein

misfolding

is

usually

counteracted

by

the

reparative

function

of

chaperones

-LRB-

Ross

and

Poirier

Macario

and

Conway

de

Macario

Bukau

et

al.

-RRB-

however

DDC

feeding

is

associated

with

diminished

chaperone

expression

persistent

chaperone

modifications

and

impairment

of

chaperone

function

-LRB-

Strnad

et

al.

submitted

-RRB-

Similarly

chaperone

function

is

impaired

in

a

rat

model

of

chronic

alcoholic

liver

disease

-LRB-

Carbone

et

al.

-RRB-

Ad

is

a

stress-inducible

protein

with

multiple

functions

which

is

a

constituent

of

multiple

inclusion

bodies

termed

sequestosomes

-LRB-

Shin

Kuusisto

et

al.

Zatloukal

et

al.

Moscat

et

al.

-RRB-

It

binds

proteins

polyubiquitinated

at

lysine

and

shuttles

them

for

proteasomal

or

autophagic

degradation

-LRB-

Vadlamudi

et

al.

Bjorkoy

et

al.

Seibenhener

et

al.

Wooten

et

al.

-RRB-

has

been

shown

to

enhance

aggregate

formation

-LRB-

Donaldson

et

al.

Wang

et

al.

Komatsu

et

al.

Gal

et

al.

Nezis

et

al.

-RRB-

action

is

beneficial

under

normal

conditions

since

it

prevents

accumulation

of

abnormal

proteins

-LRB-

Bjorkoy

et

al.

Ramesh

Babu

et

al.

-RRB-

but

may

become

harmful

when

protein

degradation

is

inhibited

-LRB-

Komatsu

et

al.

-RRB-

There

are

several

lines

of

evidence

implicating

-LRB-

containing

the

ubiquitin

binding

site

-RRB-

in

MDB

formation

In

cell

culture

experiments

protein

aggregates

resembling

MDBs

formed

only

after

co-transfection

but

not

when

and

ubiquitin

were

transfected

alone

or

in

combination

-LRB-

Stumptner

et

al.

-RRB-

Furthermore

was

rapidly

induced

in

DDC-fed

mice

with

aggregates

preceding

the

formation

of

MDBs

-LRB-

Stumptner

et

al.

-RRB-

Furthermore

inhibition

attenuated

whereas

overexpression

enhanced

MDB

formation

in

DDC-primed

hepatocytes

-LRB-

et

al.

-RRB-

Ad

Accumulation

of

protein

aggregates

as

seen

during

MDB

formation

is

counteracted

by

proteasomal

or

autophagic

degradation

-LRB-

Glickman

and

Ciechanover

Williams

et

al.

-RRB-

and

both

degradation

machineries

are

active

in

the

liver

For

example

conditional

knock-out

of

the

autophagy-related

gene

-LRB-

-RRB-

leads

to

development

of

ubiquitin-positive

inclusions

in

hepatocytes

-LRB-

Komatsu

et

al.

-RRB-

Similarly

proteasomes

seem

to

interact

with

MDBs

and

inhibition

of

proteasomal

degradation

leads

to

formation

of

MDB-like

structures

both

in

vitro

and

in

DDC-primed

mice

-LRB-

Harada

et

al.

Bardag-Gorce

et

al.

Riley

et

al.

French

et

al.

-RRB-

Furthermore

autophagic

degradation

is

upregulated

in

DDC-fed

mice

and

its

further

stimulation

with

rapamycin

attenuates

spontaneous

MDB

formation

in

overexpressing

mice

-LRB-

Harada

et

al.

-RRB-

In

contrast

MDB

formation

might

be

facilitated

by

proteasomal

impairment

since

MDBs

are

preferentially

seen

in

proteasome-depleted

hepatocytes

-LRB-

Bardag-Gorce

et

al.

-RRB-

In

addition

an

aberrant

form

of

ubiquitin

termed

-LSB-

UBB

-RSB-

arising

as

a

consequence

of

molecular

misreading

is

observed

in

MDBs

and

may

inhibit

proteasomal

function

-LRB-

McPhaul

et

al.

Lindsten

et

al.

-RRB-

Similarly

alcohol

and

aging

both

potential

inducers

of

MDBs

can

be

associated

with

decreased

proteasome

function

-LRB-

Hayashi

and

Goto

Fataccioli

et

al.

-RRB-

However

aggregate

formation

must

not

always

be

caused

by

a

general

inhibition

in

protein

degradation

The

degradative

capacity

may

simply

be

overwhelmed

by

increased

amounts

of

misfolded

proteins

Alternatively

certain

proteins

may

not

be

easily

degradable

such

as

proteins

with

long

polyglutamine

stretches

-LRB-

Venkatraman

et

al.

-RRB-

The

latter

scenario

may

apply

to

DDC-fed

mice

which

exhibit

an

accumulation

of

ubiquitinated

protein

only

in

the

highly

insoluble

protein

fraction

-LRB-

Strnad

et

al.

-RRB-

This

fraction

is

characterized

by

highly

cross-linked

protein

species

generated

through

extensive

transamidation

which

may

make

them

resistant

to

proteolytic

degradation

-LRB-

Strnad

et

al.

-RRB-

Pathologic

significance

of

MDBs

In

principle

cytoplasmic

protein

aggregates

can

be

beneficial

detrimental

or

inert

depending

on

the

context

In

many

pathologic

situations

aggregation

seems

to

be

a

protective

response

if

the

first

lines

of

defense

that

is

refolding

and

degradation

fail

by

sequestration

of

potentially

harmful

proteins

-LRB-

Arrasate

et

al.

Bodner

et

al.

-RRB-

On

the

other

hand

large

protein

aggregates

can

be

detrimental

either

by

mechanical

interference

with

cellular

transport

processes

-LRB-

e.g.

shown

with

microtubule-dependent

transport

in

neurons

-RRB-

deprivation

of

the

cell

of

vital

components

by

aggregation

or

coaggregation

and

by

overwhelming/inhibiting

the

capacity

of

the

chaperone

system

and/or

the

protein

degradation

machinery

by

indigestible

material

-LRB-

Alonso

et

al.

Stenoien

et

al.

Suhr

et

al.

Bence

et

al.

Lee

et

al.

Grune

et

al.

-RRB-

It

can

be

expected

therefore

that

in

different

cell

systems

and

pathologic

conditions

inclusion

bodies

differ

regarding

their

cellular

effects

and

consequences

MDBs

per

se

do

not

seem

to

compromise

the

viability

of

transfected

tissue

culture

cells

-LRB-

Stumptner

et

al.

-RRB-

or

hepatocytes

in

vivo

who

even

exhibit

an

activated

phenotype

-LRB-

Denk

et

al.

-RRB-

Further

studies

are

needed

to

definitely

characterize

the

molecular

consequences

of

MDB

formation

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

keratin
aggregate
inclusion
variant
p62
ubiquitin
oxidative stress
steatohepatitis


Assessment of normal tricuspid valve anatomy in adults by real-time three-dimensional echocardiography Background The tricuspid valve -LRB- TV -RRB- is a complex structure . Unlike the aortic and mitral valve it is not possible to visualize all TV leaflets simultaneously in one cross-sectional view by standard two-dimensional echocardiography -LRB- 2DE -RRB- either transthoracic or transesophageal due to the position of TV in the far field . Introduction The tricuspid valve -LRB- TV -RRB- is a multi-component complex structure -LSB- 1 -RSB- . In classic anatomic studies the anterior , septal and posterior TV cusps were described -LSB- 2 , 3 -RSB- . Unlike the aortic and mitral valve it is not possible to visualize all TV cusps simultaneously in one cross-sectional view by standard transthoracic two-dimensional echocardiography -LRB- 2DE -RRB- -LSB- 4 -RSB- . During transesophageal 2DE small changes in transducer angle , probe position and rotation may bring to light some additional TV details -LSB- 5 , 6 -RSB- . However , because of the position of the TV in the far field in relation to probe , transesophageal 2DE can still only provide limited information and can also not visualize all TV cusps simultaneously . In three-dimensional -LRB- 3D -RRB- transesophageal image reconstruction and intracardiac echocardiography studies this goal could be achieved but at the cost of some procedural risks and an increase in procedural duration -LSB- 7 , 8 -RSB- . Real-time three-dimensional echocardiography -LRB- RT3DE -RRB- can visualize the atrio-ventricular valves from both the ventricular and atrial side in detail without these limitations -LSB- 9 -RSB- . This study aimed to apply RT3DE for quantitative and qualitative assessment of normal TV anatomy . Subjects and methods In one hundred patients -LRB- mean age 30 ± 9 years , 65 % males -RRB- the TV was examined by transthoracic RT3DE after an informed consent . All patients had sinus rhythm and normal right-sided heart -LRB- normal right ventricular dimensions and function , normal right atrial dimension , trivial or absent tricuspid regurgitation and normal tricuspid valve function -RRB- . Patients with good 2DE image quality only were included . RT3DE was done with a commercially available ultrasound system -LRB- Philips Sonos 7500 , Best , The Netherlands -RRB- attached to a X4 matrix array transducer capable of providing real-time B-mode images . The 3D data set was collected within approximately 5 -- 10 s of breath holding in full volume mode from an apical window and transferred for off-line analysis with TomTec software -LRB- Unterschleissheim , Munich , Germany -RRB- . Data analysis of 3D images was based on a two-dimensional approach relying on images obtained initially from the apical 4-chamber view . The images were adjusted to put the TV in the center of interest . To exclude non-relevant tissue , the TV was sliced between the two narrowest lines by which all parts of the TV leaflets were still in between . The TomTec software allows in this way visualization of the short-axis TV view in a 3D display -LRB- see Fig. 1 -RRB- . RT3DE gain and brightness were adjusted to improve delineation of anatomic structures . The following points were checked for visualization : 1 -RRB- tricuspid annulus diameter and area , 2 -RRB- TV leaflets -LRB- number , mobility , thickness and relation to each other -RRB- , 3 -RRB- TV area , and 4 -RRB- TV commissures -LRB- antero-septal , antero-posterior , and postero-septal -RRB- including the position of their closure lines . All these structures were classified according to a subjective 4-point scale for image quality -LRB- 1 = not visualized , 2 = inadequate , 3 = sufficient and 4 = good -RRB- . Fig. 1TomTec quadri screen display of the tricuspid valve . The upper two images represent two-dimensional views created from the 3D data set -LRB- 4-chamber , left and orthogonal view , right -RRB- . The lower left image represents a two-dimensional short axis view and the lower right image represents the 3D image For quantitative assessment of TV the following RT3DE data were obtained : 1 -RRB- TV annulus diameter defined as the widest diameter that could be measured from an end-diastolic still frame , 2 -RRB- maximal TV annulus area obtained from an end-diastolic still frame and measured by manual planimetry , 3 -RRB- TV area defined as the narrowest part of the TV at the time of maximal opening and measured by manual planimetry , and 4 -RRB- TV commissural width obtained from a late-diastolic still frame using zoom function to avoid underestimation . The images were optimized for each commissure along its plane to measure the maximal width of the angle formed by the two adjacent TV leaflets . To identify the TV leaflets visualized by the standard 2DE images the TomTec quad screen display was used . As seen in Fig. 1 , this screen contains four images ; the upper two images are 2DE images perpendicular to each other , the lower two images are a short-axis 2DE image and a RT3DE image . From the properly chosen two-dimensional image a mid-diastolic frame was selected to visualize the TV leaflets just separated from each other . Each leaflet was defined by a marker , after which this marker position was compared with the RT3DE image to detect which leaflet was shown in the 2DE images . Analysis of images was done by two experienced echocardiographers -LRB- AMA , JSM -RRB- independently . Each one dealt with the full volume image as acquired from echo machine and the selection of cut plane , angulation and gain setting adjustment were dependable on his experience . Statistical analysis All data obtained by RT3DE were presented as mean ± SD . Interobserver and intraobserver agreements for the visualization score were estimated using kappa values for each morphologic feature and classified as poor -LRB- kappa < 0.4 -RRB- , moderate -LRB- kappa 0.4 to 0.7 -RRB- , or good -LRB- kappa > 0.7 -RRB- . Interobserver and intraobserver variabilities for RT3DE measurements was assessed according to the Bland and Altman method in a randomly selected group of 50 patients -LSB- 10 -RSB- Table 1 . Table 1Scores for real-time three-dimensional echocardiography visualization of TV structuresScoreTV annulusTV leafletsTV areaTV commissuresGood -LRB- 4 -RRB- 60 % 80 % 55 % 50 % Sufficient -LRB- 3 -RRB- 30 % 10 % 30 % 20 % Inadequate -LRB- 2 -RRB- 10 % 10 % 15 % 20 % Not visualized -LRB- 1 -RRB- 0 % 0 % 0 % 10 % Mean score3 .5 ± 0.73.7 ± 0.63.4 ± 0.73.1 ± 1.0 Median score3 .03.03.02.5 Abbreviations : TV = tricuspid valve Results Acquisition and analysis of the RT3DE data was performed in approximately 10 min per patient . The TV could be visualized in 90 % of patients enface from both ventricular and atrial aspects in relation to adjacent cardiac structures . In these 90 patients detailed analysis of the TV was performed including tricuspid annulus shape and size , TV leaflets shape , size , and mobility , and commissural width . Tricuspid annulus Tricuspid annulus visualization was good in 54 patients -LRB- 60 % -RRB- , sufficient in 27 patients -LRB- 30 % -RRB- , and inadequate in 9 patients -LRB- 10 % -RRB- . As seen in Fig. 2 , tricuspid annulus shape appeared as oval rather than circular . Tricuspid annulus diameter and area could be measured in 63 patients -LRB- 70 % -RRB- , normal values were 4.0 ± 0.7 cm and 10.0 ± 2.9 cm2 , respectively . Fig. 2Oval-shaped Tricuspid annulus -LRB- the line represent the tricuspid annulus diameter , the dots demark the area -RRB- Tricuspid valve leaflets Visualization of the three TV leaflets -LRB- in motion -RRB- was good in 72 patients -LRB- 80 % -RRB- , sufficient in 9 patients -LRB- 10 % -RRB- , and inadequate in another 9 patients -LRB- 10 % -RRB- . The anterior leaflet was the largest and most mobile of the three leaflets and had a nearly semicircular shape . The septal leaflet was the least mobile and had a semi-oval shape . Its position was parallel to the interventricular septum . The posterior leaflet was the smallest one with variable shape . It was clearly separated from the septal leaflet in all patients but in 10 % of patients it was hard to discriminate the posterior leaflet from the anterior leaflet even during maximal TV opening . From the RT3DE data set all standard two-dimensional TV cross-sections -LRB- apical 4-chamber , parasternal short-axis and parasternal long-axis right ventricular inflow -RRB- were simulated . As seen in Fig. 3 , in the apical 4-chamber view in all patients the septal leaflet was seen adjacent to the septum and the anterior leaflet was seen adjacent to the right ventricular free wall . In the parasternal short-axis view , the posterior leaflet was seen adjacent to the right ventricular free wall in 92 % of patients and in the remaining 8 % no leaflet could be obtained although modification of the cut plane downward could identify this leaflet . In this view the leaflet adjacent to the aorta was the anterior in 52 % and the septal leaflet in 48 % . In the parasternal right ventricular inflow view the leaflets seen were identical to the apical 4-chamber view with in all patients the septal leaflet seen adjacent to the septum and the anterior leaflet seen adjacent to the right ventricular free wall . Fig. 3Identification of the tricuspid valve leaflets seen on two-dimensional imaging . Below the 2D images , percentage of leaflet identification in each standard view depending the RT3DE images Tricuspid valve area Visualization of the triangular shaped TV area was good in 50 patients -LRB- 55 % -RRB- , sufficient in 27 patients -LRB- 30 % -RRB- , and inadequate in 13 patients -LRB- 15 % -RRB- . As seen in Fig. 4 , the anterior and septal leaflets formed the TV area 's angle and the small posterior leaflet formed its base . TV area could be measured in 77 patients -LRB- 86 % -RRB- and mean TV area in these patients was 4.8 ± 1.6 cm2 . Fig. 4Triangular shape TV area and commissural views Tricuspid valve commissures As seen in Fig. 4 , the three TV leaflets were separated from each other by three commissures . The commissures and the direction of closure lines were good visualized in 45 patients -LRB- 50 % -RRB- , sufficient in 18 patients -LRB- 20 % -RRB- , inadequate in 18 patients -LRB- 20 % -RRB- , and not visualized in 9 patients -LRB- 10 % -RRB- . TV commissural width could be obtained in 63 patients -LRB- 70 % -RRB- , mean commissural width in these patients was 5.4 ± 1.5 mm for the antero-septal commissure , 5.2 ± 1.5 mm for the postero-septal commissure , and 5.1 ± 1.1 mm for the antero-posterior commissure , respectively . Visualization and measurement of commissures was relatively easy for the antero-septal commissure and most difficult for the antero-posterior commissure . All measurements are listed in Table 2 as absolute value and indexed to body surface area . Table 2Normal -LRB- absolute and index -RRB- values of tricuspid valve annulus -LRB- diameter and area -RRB- , Tricuspid valve area and the width of the 3 commissuresParameterAbsolute valueIndex valueTricuspid annulus diameter 4.0 ± 0.7 -LRB- cm -RRB- 2.2 ± 0.4 -LRB- cm/m2 -RRB- Tricuspid annulus area 10.0 ± 2.9 -LRB- cm2 -RRB- 5.5 ± 1.6 -LRB- cm2/m2 -RRB- Tricuspid valve area 4.8 ± 1.6 -LRB- cm2 -RRB- 2.7 ± 0.9 -LRB- cm2/m2 -RRB- Antero-septal commissure 5.4 ± 1.5 -LRB- mm -RRB- 2.9 ± 0.8 -LRB- mm/m2 -RRB- Postero-septal commissure5 .2 ± 1.5 -LRB- mm -RRB- 2.9 ± 0.7 -LRB- mm/m2 -RRB- Antero-posterior commissure 5.1 ± 1.1 -LRB- mm -RRB- 2.8 ± 0.6 -LRB- mm/m2 -RRB- Interobserver variability The visualization score between two observers was good for the TV annulus -LRB- kappa value 0.91 -RRB- and TV leaflets -LRB- kappa value 0.71 -RRB- and moderate for the TV commissures -LRB- kappa value 0.59 -RRB- . As seen in Fig. 5 , good interobserver correlations were found for measurement of TV annulus -LRB- r = 0.98 , P < 0.0001 -RRB- and TV area -LRB- r = 0.95 , P < 0.0001 -RRB- and fair correlation was found for TV commissural width -LRB- r = 0.51 , P < 0.001 -RRB- . In the same Figure , the interobserver agreement for TV annulus diameter -LRB- mean difference − 0.28 ± 1.20 mm , agreement : 2.12 , − 2.68 -RRB- , for TV area -LRB- mean difference : 0.17 ± 0.52 cm2 , agreement : 1.21 , − 0.87 -RRB- , and for mean TV commissural width -LRB- mean difference : 0.01 ± 0.62 mm , agreement : 1.25 , − 1.24 -RRB- is displayed . Fig. 5Interobserver correlations -LRB- top -RRB- and Bland -- Altman analysis -LRB- bottom -RRB- of TV annulus , leaflets , and commissures Intraobserver variability The visualization score by the first observer at 2 separate settings was good for the TV annulus -LRB- kappa value 0.92 -RRB- and TV leaflets -LRB- kappa value 0.73 -RRB- and moderate for the TV commissures -LRB- kappa value 0.58 -RRB- . Intraobserver agreement was -LRB- mean difference − 0.26 ± 1.15 mm , agreement : 2.14 , − 2.56 -RRB- for TV annulus diameter , -LRB- mean difference : 0.15 ± 0.50 cm2 , agreement : 1.15 , − 0.85 -RRB- for TV area , and -LRB- mean difference : 0.02 ± 0.60 mm , agreement : 1.22 , − 1.18 -RRB- for the mean TV commissural width . Discussion Two-dimensional echocardiography is a valuable imaging modality for the functional assessment of TV -LSB- 11 -- 13 -RSB- . However , with 2DE it is not possible to visualize all TV cusps simultaneously in one cross-sectional view nor can detailed anatomical information of the TV annulus , leaflets , and commissures be provided . Previous studies and case reports described visualization of TV by RT3DE -LSB- 9 , 14 -RSB- in abnormal states , while this study applied RT3DE for the morphological assessment of the normal TV anatomy . RT3DE allowed analysis of TV annulus , leaflets and commissures in the majority of patients . Beside this morphologic description , quantitative assessment could be obtained . However , it should be noticed that only patients with good 2DE image quality underwent RT3DE . In our experience these patients represent over 50 % of the total number of patients referred to our echocardiographic laboratory . Nevertheless , RT3DE allowed TV analysis to a level quite comparable to that recently reported by others for the mitral valve leaflets -LSB- 15 -RSB- . One of the salient findings in our study was the identification of the TV leaflets as seen in the routine 2DE views . It is still a matter of controversy in echocardiographic textbooks . In one well known echocardiographic textbook -LSB- 16 -RSB- , the leaflet seen in the apical 4-chamber view adjacent to the right ventricular free wall was described as being the anterior or posterior leaflet depending on the exact rotation and angulation of the image plane . However , in our study this leaflet was consistently found to be the anterior leaflet -LRB- see Fig. 6 for explanation -RRB- , as described in another textbook -LSB- 17 -RSB- . Also , in both these echocardiographic textbooks -LSB- 16 , 17 -RSB- in the parasternal short-axis view the leaflet adjacent to the right ventricular free wall was described as being the anterior . However , as shown in Fig. 3 and explained in Fig. 6 , in all patients in whom a leaflet could be identified in our study it was the posterior one . Fig. 6Surgical view of the heart valves demonstrating the range of the two-dimensional echocardiographic 4-chamber and short-axis planes In our study tricuspid annulus diameter -LRB- and area -RRB- could be reliably obtained with RT3DE . Tricuspid annulus measurement is of critical importance in the TV surgical decision-making process if a patient is operated for mitral valve disease and has concomitant TV regurgitation -LSB- 18 , 19 -RSB- . In addition , TV area could be reliably obtained and this may have important implications for the diagnosis of tricuspid stenosis -LSB- 20 , 21 -RSB- . Visualization of commissures and measurement of its width were obtained with difficulty , in particular for the antero-posterior commissure . Commissural width also showed weak interobserver correlation . This may be due to differences in the commissural levels and tissue dropout . For proper assessment of the three commissures , more cut planes with different angles are needed . However , assessment of commissural width may also be a valuable tool for the diagnosis , follow up , and selection of therapeutic strategy of tricuspid stenosis . All our RT3DE measurements were consistent with the measurements described in anatomical studies -LSB- 2 , 3 -RSB- . Our data may potentially take RT3DE a step further into clinical routine -LRB- providing accurate TV measurements -RRB- and may enhance the understanding of TV valve morphology during the cardiac cycle -LRB- Fig. 7 -RRB- . Also , the detailed assessment by RT3DE may affect the therapeutic decision of various TV abnormalities and thus expand the abilities of non-invasive cardiology -LSB- 22 -RSB- . Fig. 7Visualization of the 3 TV leaflets during valve closure -LRB- A -RRB- , at early diastole -LRB- B -RRB- , and at late diastole -LRB- C -RRB- Limitation of study The main limitation of this study is that RT3DE data were not compared with a `` gold standard '' such as magnetic resonance imaging , autopsy or surgical findings . Also , RT3DE images more critically depend on 2DE image quality and could be obtained only in patients with sinus rhythm during hold breath that limits its application to all . The study included patients with narrow age range -LRB- 21 -- 39 years -RRB- , and thus the normal findings in this study is confined to this age group and could not be applied to both extremities -LRB- < 21 and > 39 years -RRB- . Conclusion Three-dimensional imaging of the TV is feasible in a large number of patients . RT3DE may add to functional 2DE data in description of TV anatomy and providing highly reproducible and actual reality -LRB- anatomic and functional -RRB- measurements .

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