PLoS

One

Published

online

Sep

doi

PMCID

Ethical

and

Scientific

Considerations

Regarding

Animal

Testing

and

Research

Hope

R

Ferdowsian

and

Nancy

Beck

Catriona

J

MacCallum

Editor

Author

information

Copyright

and

License

information

This

article

has

been

cited

by

other

articles

in

PMC

In

William

Russell

and

Rex

Burch

published

the

seminal

book

The

Principles

of

Humane

Experimental

Technique

which

emphasized

r

eduction

r

efinement

and

r

eplacement

of

animal

use

principles

which

have

since

been

referred

to

as

the

Rs

These

principles

encouraged

researchers

to

work

to

reduce

the

number

of

animals

used

in

experiments

to

the

minimum

considered

necessary

refine

or

limit

the

pain

and

distress

to

which

animals

are

exposed

and

replace

the

use

of

animals

with

nonanimal

alternatives

when

possible

Despite

the

attention

brought

to

this

issue

by

Russell

and

Burch

and

since

the

number

of

animals

used

in

research

and

testing

has

continued

to

increase

raising

serious

ethical

and

scientific

issues

Further

while

the

Rs

capture

crucially

important

concepts

they

Animals

Research

and

Alternatives

Measuring

Progress

Years

Later

which

aims

to

spur

ethical

and

scientific

advancement

Introduction

One

of

the

most

influential

attempts

to

examine

and

affect

the

use

of

animals

in

research

can

be

traced

back

with

the

publication

of

The

Principles

of

Humane

Experimental

Technique

William

Russell

and

Rex

Burch

published

this

seminal

book

in

response

to

marked

growth

in

medical

and

veterinary

research

and

the

concomitant

increase

in

the

numbers

of

animals

used

Russell

and

Burchs

text

emphasized

r

eduction

r

efinement

and

r

eplacement

of

animal

use

principles

which

have

since

been

referred

to

as

the

Rs

These

principles

encouraged

researchers

to

work

to

reduce

the

number

of

animals

used

in

experiments

to

the

minimum

considered

necessary

refine

or

limit

the

pain

and

distress

to

which

animals

are

exposed

and

replace

the

use

of

animals

with

nonanimal

alternatives

when

possible

Despite

the

attention

brought

to

this

issue

by

Russell

and

Burch

the

number

of

animals

used

in

research

and

testing

has

continued

to

increase

Recent

estimates

suggest

that

at

least

million

animals

are

used

each

year

worldwide

However

this

is

likely

an

underestimate

and

it

is

impossible

to

accurately

quantify

the

number

of

animals

used

in

or

for

experimentation

Full

reporting

of

all

animal

use

is

not

required

or

made

public

in

most

countries

Nevertheless

based

on

available

information

it

is

clear

that

the

number

of

animals

used

in

research

has

not

significantly

declined

over

the

past

several

decades

The

Rs

serve

as

the

cornerstone

for

current

animal

research

guidelines

but

questions

remain

about

the

adequacy

of

existing

guidelines

and

whether

researchers

review

boards

and

funders

have

fully

and

adequately

implemented

the

Rs

Further

while

the

Rs

capture

crucially

important

concepts

they

do

not

adequately

reflect

the

substantial

developments

in

our

new

knowledge

about

the

cognitive

and

emotional

capabilities

of

animals

an

updated

understanding

of

the

harms

inherent

in

animal

research

and

the

changing

cultural

perspectives

about

the

place

of

animals

in

society

In

addition

serious

questions

have

been

raised

about

the

effectiveness

of

animal

testing

and

research

in

predicting

anticipated

outcomes

In

August

the

Georgetown

University

Kennedy

Institute

of

Ethics

the

Johns

Hopkins

University

Center

for

Alternatives

to

Animal

Testing

the

Institute

for

In

Vitro

Sciences

The

George

Washington

University

and

the

Physicians

Committee

for

Responsible

Medicine

jointly

held

a

two

day

multidisciplinary

international

conference

in

Washington

DC

to

address

the

scientific

legal

and

political

opportunities

and

challenges

to

implementing

alternatives

to

animal

research

This

twoday

symposium

aimed

to

advance

the

study

of

the

ethical

and

scientific

issues

surrounding

the

use

of

animals

in

testing

and

research

with

particular

emphasis

on

the

adequacy

of

current

protections

and

the

promise

and

This

Collection

combines

some

papers

that

were

written

following

this

conference

with

an

aim

to

highlight

relevant

progress

and

research

This

Overview

provides

a

brief

summary

of

the

ethical

and

scientific

considerations

regarding

the

use

of

animals

in

research

and

testing

some

of

which

are

highlighted

in

the

accompanying

Collection

Go

to

Analysis

and

Discussion

Ethical

Considerations

and

Advances

in

the

Understanding

of

Animal

Cognition

Apprehension

around

burgeoning

medical

research

in

the

late

and

the

first

half

of

the

th

century

sparked

concerns

over

the

use

of

humans

and

animals

in

research

Suspicions

around

the

use

of

humans

were

deepened

with

the

revelation

of

several

exploitive

research

projects

including

a

series

of

medical

experiments

on

large

numbers

of

prisoners

by

the

Nazi

German

regime

during

World

War

II

and

the

Tuskegee

syphilis

study

These

abuses

served

as

the

impetus

for

the

establishment

of

the

Nuremberg

Code

Declaration

of

Helsinki

and

the

National

Commission

for

the

Protection

of

Human

Subjects

of

Biomedical

and

Behavioral

Research

and

the

resulting

Belmont

Report

Today

these

guidelines

provide

a

platform

for

the

protection

of

human

research

subjects

including

the

principles

of

respect

beneficence

and

justice

as

well

as

special

protections

for

vulnerable

populations

Laws

to

protect

animals

in

research

have

also

been

established

The

British

Parliament

passed

the

first

set

of

protections

for

animals

in

with

the

Cruelty

to

Animals

Act

Approximately

ninety

years

later

the

US

adopted

regulations

for

animals

used

in

research

with

the

passage

of

the

Laboratory

Animal

Welfare

Act

of

Subsequent

national

and

international

laws

and

guidelines

have

provided

basic

protections

but

there

are

some

significant

inconsistencies

among

current

regulations

For

example

the

US

Animal

Welfare

Act

excludes

purposebred

birds

rats

or

mice

which

comprise

more

than

of

animals

used

in

research

In

contrast

certain

dogs

and

cats

have

received

special

attention

and

protections

Whereas

the

US

Animal

Welfare

Act

excludes

birds

rats

and

mice

the

US

guidelines

overseeing

research

conducted

with

federal

funding

includes

protections

for

all

vertebrates

The

lack

of

consistency

is

further

illustrated

by

the

US

Government

Principles

for

the

Utilization

and

Care

of

Vertebrate

Animals

Used

in

Testing

Research

and

Training

which

stress

compliance

with

the

US

Animal

Welfare

Act

and

other

applicable

Federal

laws

guidelines

and

policies

While

strides

have

been

made

in

the

protection

of

both

human

and

animal

research

subjects

the

nature

of

these

protections

is

markedly

different

Human

research

protections

emphasize

specific

principles

aimed

at

protecting

the

interests

of

individuals

and

populations

sometimes

to

the

detriment

of

the

scientific

question

This

differs

significantly

from

animal

research

guidelines

where

the

importance

of

the

scientific

question

being

researched

commonly

takes

precedence

over

the

interests

of

individual

animals

Although

scientists

and

ethicists

have

published

numerous

articles

relevant

to

the

ethics

of

animal

research

current

animal

research

guidelines

do

not

articulate

the

rationale

for

the

central

differences

between

The

concept

of

costs

to

individual

animals

can

be

further

examined

through

the

growing

body

of

research

on

animal

emotion

and

cognition

Studies

published

in

the

last

few

decades

have

dramatically

increased

our

understanding

of

animal

sentience

suggesting

that

animals

potential

for

experiencing

harm

is

greater

than

has

been

appreciated

and

that

current

protections

need

to

be

reconsidered

It

is

now

widely

acknowledged

by

scientists

and

ethicists

that

animals

can

experience

pain

and

distress

Potential

causes

of

harm

include

invasive

procedures

disease

and

deprivation

of

basic

physiological

needs

Other

sources

of

harm

for

many

animals

include

social

deprivation

and

loss

of

the

ability

to

fulfill

natural

behaviors

among

other

factors

Numerous

studies

have

demonstrated

that

even

in

response

to

gentle

handling

animals

can

show

marked

changes

in

physiological

and

hormonal

markers

of

stress

Although

pain

and

suffering

are

subjective

experiences

studies

from

multiple

disciplines

provide

objective

evidence

of

animals

abilities

to

experience

pain

Animals

demonstrate

coordinated

responses

to

pain

and

many

emotional

states

that

are

similar

to

those

exhibited

by

humans

Animals

share

genetic

neuroanatomical

and

physiological

similarities

with

humans

and

many

animals

express

pain

in

ways

similar

to

humans

Animals

also

share

similarities

with

humans

in

genetic

developmental

and

environmental

risk

factors

for

psychopathology

For

example

fear

operates

in

a

less

organized

subcortical

neural

circuit

than

pain

and

it

has

been

described

in

a

wide

variety

of

species

More

complex

markers

of

psychological

distress

have

also

been

described

in

animals

Varying

forms

of

depression

have

been

repeatedly

reported

in

animals

including

nonhuman

primates

dogs

pigs

cats

birds

and

rodents

among

others

Anxiety

disorders

such

as

posttraumatic

stress

disorder

have

been

described

in

animals

including

chimpanzees

and

elephants

In

addition

to

the

capacity

to

experience

physical

and

psychological

pain

or

distress

animals

also

display

many

languagelike

abilities

complex

problemsolving

skills

tool

related

cognition

and

pleasureseeking

with

empathy

and

selfawareness

also

suggested

by

some

research

Play

behavior

an

indicator

of

pleasure

is

widespread

in

mammals

and

has

also

been

described

in

birds

Behavior

suggestive

of

play

has

been

observed

in

other

taxa

including

reptiles

fishes

and

cephalopods

Selfawareness

assessed

through

mirror

selfrecognition

has

been

reported

for

chimpanzees

and

other

great

apes

magpies

and

some

cetaceans

More

recent

studies

have

shown

that

crows

are

capable

of

creating

and

using

tools

that

require

access

to

episodiclike

memory

formation

and

retrieval

These

findings

suggest

that

crows

and

related

species

display

evidence

of

causal

reasoning

flexible

learning

strategies

imagination

and

prospection

similar

to

findings

in

great

apes

These

findings

also

challenge

our

assumptions

about

species

similarities

and

differences

and

their

relevance

in

solving

ethical

dilemmas

regarding

the

use

of

animals

in

research

Predictive

Value

of

Animal

Data

and

the

Impact

of

Technical

Innovations

on

Animal

Use

In

the

last

decade

concerns

have

mounted

about

how

relevant

animal

experiments

are

to

human

health

outcomes

Several

papers

have

examined

the

concordance

between

animal

and

human

data

demonstrating

that

findings

in

animals

were

not

reliably

replicated

in

human

clinical

research

Recent

systematic

reviews

of

treatments

for

various

clinical

conditions

demonstrated

that

animal

studies

have

been

poorly

predictive

of

human

outcomes

in

the

fields

of

neurology

and

vascular

disease

among

others

These

reviews

have

raised

questions

about

whether

human

diseases

inflicted

upon

animals

sufficiently

mimic

the

disease

processes

and

treatment

responses

seen

in

humans

The

value

of

animal

use

for

predicting

human

outcomes

has

also

been

questioned

in

the

regulatory

toxicology

field

which

relies

on

a

codified

set

of

highly

standardized

animal

experiments

for

assessing

various

types

of

toxicity

Despite

serious

shortcomings

for

many

of

these

assays

most

of

which

are

to

years

old

the

field

has

been

slow

to

adopt

newer

methods

The

year

marked

a

turning

point

in

the

toxicology

field

with

publication

of

a

landmark

report

by

the

US

National

Research

Council

NRC

highlighting

the

need

to

embrace

in

vitro

and

computational

methods

in

order

to

obtain

data

that

more

accurately

predicts

toxic

effects

in

humans

The

report

Toxicity

Testing

in

the

st

Century

A

Vision

and

a

Strategy

was

commissioned

by

the

US

Environmental

Protection

Agency

partially

due

to

the

recognition

of

weaknesses

in

existing

approaches

to

toxicity

testing

The

NRC

vision

calls

for

a

shift

away

from

animal

use

in

chemical

testing

toward

computational

models

and

highthroughput

and

highcontent

in

vitro

methods

The

report

emphasized

that

these

methods

can

provide

more

predictive

data

more

quickly

and

affordably

than

traditional

in

vivo

methods

Subsequently

published

articles

address

the

implementation

of

this

vision

for

improving

the

current

system

of

chemical

testing

and

assessment

While

a

sea

change

is

underway

in

regulatory

toxicology

there

has

been

much

less

dialogue

surrounding

the

replacement

of

animals

in

research

despite

the

fact

that

far

more

animals

are

used

in

basic

and

applied

research

than

in

regulatory

toxicology

The

use

of

animals

in

research

is

inherently

more

difficult

to

approach

systematically

because

research

questions

are

much

more

diverse

and

less

proscribed

than

in

regulatory

toxicology

Because

researchers

often

use

very

specialized

assays

and

systems

to

address

their

hypotheses

replacement

of

animals

in

this

area

is

a

more

individualized

endeavour

Researchers

and

oversight

boards

have

to

evaluate

the

relevance

of

the

research

question

and

whether

the

tools

of

modern

molecular

and

cell

biology

genetics

biochemistry

and

computational

biology

can

be

used

in

lieu

of

animals

While

of

these

tools

on

their

own

are

capable

of

replicating

a

whole

organism

they

do

provide

a

mechanistic

understanding

of

molecular

events

It

is

important

for

researchers

and

reviewers

to

assess

differences

in

the

clinical

presentation

and

manifestation

of

Examples

of

existing

and

promising

nonanimal

methods

have

been

reviewed

recently

by

Langley

and

colleagues

who

highlighted

advances

in

fields

including

orthodontics

neurology

immunology

infectious

diseases

pulmonology

endocrine

and

metabolism

cardiology

and

obstetrics

Many

researchers

have

also

begun

to

rely

solely

on

human

data

and

cell

and

tissue

assays

to

address

large

areas

of

therapeutic

research

and

development

In

the

area

of

vaccine

testing

and

development

a

surrogate

invitro

human

immune

system

has

been

developed

to

help

predict

an

individuals

immune

response

to

a

particular

drug

or

vaccine

This

system

includes

a

blooddonor

base

of

hundreds

of

individuals

from

diverse

populations

and

offers

many

benefits

including

predictive

highthroughput

in

vitro

immunology

to

assess

novel

drug

and

vaccine

candidates

measurement

of

immune

responses

in

diverse

human

populations

faster

cycle

time

for

discovery

better

selection

of

drug

candidates

for

clinical

evaluation

and

reductions

in

the

time

and

costs

to

bring

drugs

and

vaccines

to

the

market

In

the

case

of

vaccines

this

system

can

be

used

at

every

stage

including

in

vitro

disease

models

antigen

selection

and

adjuvant

effects

safety

testing

clinical

trials

manufacturing

and

potency

assays

When

compared

with

data

from

animal

experiments

this

system

has

produced

more

accurate

preclinical

data

The

examples

above

illustrate

how

innovative

applications

of

technology

can

generate

data

more

meaningful

to

humans

and

reduce

or

replace

animal

use

but

advances

in

medicine

may

also

require

novel

approaches

to

setting

research

priorities

The

Dr

Susan

Love

Research

Foundation

which

focuses

on

eradicating

breast

cancer

has

challenged

research

scientists

to

move

from

animal

research

to

breast

cancer

prevention

research

involving

women

If

researchers

could

better

understand

the

factors

that

increase

the

risk

for

breast

cancer

as

well

as

methods

for

effective

prevention

fewer

women

would

require

treatment

for

breast

cancer

Whereas

animal

research

is

largely

investigatorinitiated

this

model

Similarly

any

approach

that

emphasizes

evidencebased

prevention

would

provide

benefits

to

both

animals

and

humans

Resource

limitations

might

require

a

strategic

approach

that

emphasizes

diseases

with

the

greatest

public

health

threats

which

increasingly

fall

within

the

scope

of

preventable

diseases

Conclusion

It

is

clear

that

there

have

been

many

scientific

and

ethical

advances

since

the

first

publication

of

Russell

and

Burchs

book

However

some

in

the

scientific

community

are

beginning

to

question

how

well

data

from

animals

translates

into

germane

knowledge

and

treatment

of

human

conditions

Efforts

to

objectively

evaluate

the

value

of

animal

research

for

understanding

and

treating

human

disease

are

particularly

relevant

in

the

modern

era

considering

the

availability

of

increasingly

sophisticated

technologies

to

address

research

questions

Ethical

objections

to

the

use

of

animals

have

been

publically

voiced

for

more

than

a

century

well

before

there

was

a

firm

scientific

understanding

of

animal

emotion

and

cognition

Now

a

better

understanding

of

animals

capacity

for

pain

and

suffering

is

prompting

many

to

take

a

closer

look

at

the

human

use

of

animals

Articles

in

the

accompanying

Collection

only

briefly

touch

on

the

many

scientific

and

ethical

issues

surrounding

the

use

of

animals

in

testing

and

research

While

it

is

important

to

acknowledge

limitations

to

nonanimal

methods

remain

recent

developments

demonstrate

that

these

limitations

should

be

viewed

as

rousing

challenges

rather

than

insurmountable

obstacles

Although

discussion

of

these

issues

can

be

difficult

progress

is

most

likely

to

occur

through

an

ethically

consistent

evidencebased

approach

This

collection

aims

to

spur

further

steps

forward

toward

a

more

coherent

ethical

framework

for

scientific

advancement

Go

to

Acknowledgments

The

authors

thank

the

conference

speakers

and

participants

for

their

participation

Go

to

Footnotes

Competing

Interests

HRF

and

NB

are

employed

by

Physicians

Committee

for

Responsible

Medicine

which

is

a

nongovernmental

organization

which

promotes

higher

ethical

standards

in

research

and

alternatives

to

the

use

of

animals

in

research

education

and

training

Physicians

Committee

for

Responsible

Medicine

is

a

nonprofit

organization

and

the

authors

adhered

to

PLoS

ONE

policies

on

sharing

data

and

materials

Funding

The

authors

are

grateful

to

the

National

Science

Foundation

grant

and

the

Arcus

Foundation

grant

for

the

financial

support

for

the

corresponding

conference

Animals

Research

and

Alternatives

Measuring

Progress

Years

Later

The

funders

had

no

role

in

study

design

data

collection

and

analysis

decision

to

publish

or

preparation

of

the

manuscript

Go

to

References

Russell

WMS

Burch

RL

The

principles

of

humane

experimental

technique

London

Methuen

Taylor

K

Gordon

N

Langley

G

Higgins

W

Estimates

for

worldwide

laboratory

animal

use

in

Altern

Lab

Anim

PubMed

Ibrahim

DM

Reduce

refine

replace

the

failure

of

the

three

Rs

and

the

future

of

animal

experimentation

University

of

Chicago

Legal

Forum

Arizona

Legal

Studies

Discussion

Paper

No

Available

Accessed

Jan

Rusche

B

The

Rs

and

animal

welfare

conflict

or

way

forward

ALTEX

PubMed

Hackam

DG

Redelmeier

DA

Translation

of

research

evidence

from

animals

to

humans

JAMA

PubMed

Horrobin

DF

Modern

biomedical

research

an

internally

selfconsistent

universe

with

little

contact

with

medical

reality

Nat

Rev

Drug

Discov

PubMed

Perel

P

Roberts

I

Sena

E

Wheble

P

Briscoe

C

et

al

Comparison

of

treatment

effects

between

animal

experiments

and

clinical

trials

systematic

review

BMJ

Available

Accessed

Jan

PMC

free

article

PubMed

Ioannidis

JPA

Evolution

and

translation

of

research

findings

from

bench

to

where

PLoS

Clin

Trials

Available

Accessed

Jan

PMC

free

article

PubMed

Pound

P

Ebrahim

S

Sandercock

P

Bracken

MB

Roberts

I

Where

is

the

evidence

that

animal

research

benefits

humans

BMJ

Available

Accessed

Jan

PMC

free

article

PubMed

Macleod

M

What

can

systematic

review

and

metaanalysis

tell

us

about

the

experimental

data

supporting

stroke

drug

development

Int

J

Neuroprot

Neuroregener

Garber

K

Realistic

rodents

Debate

grows

over

new

mouse

models

of

cancer

J

Natl

Cancer

Inst

PubMed

Matthews

RAJ

Medical

progress

depends

on

animal

models

doesnt

it

J

R

Soc

Med

PMC

free

article

PubMed

Grass

GM

Sinko

PJ

Effect

of

diverse

datasets

on

the

predictive

capability

of

ADME

models

in

drug

discovery

DDT

Gossel

PP

William

Henry

Welch

and

the

antivivisection

legislation

in

the

District

of

Columbia

J

Hist

Med

Allied

Sci

PubMed

Lederer

SE

Subjected

to

science

Baltimore

The

Johns

Hopkins

University

Press

United

States

Nuremberg

code

Trials

of

War

Criminals

before

the

Nuremberg

Military

Tribunals

under

Control

Council

Law

No

Washington

DC

US

Government

Printing

Office

Available

httpohsrodnihgovguidelinesnuremberghtml

Accessed

Jan

World

Medical

Association

Declaration

of

Helsinki

th

WMA

General

Assembly

Helsinki

Finland

Available

httphistorynihgovresearchdownloadshelsinkipdf

Accessed

Jan

National

Commission

for

the

Protection

of

Human

Subjects

of

Biomedical

and

Behavioral

Research

The

Belmont

Report

Washington

DC

US

Department

of

Health

Education

and

Welfare

Available

httpohsrodnihgovguidelinesbelmonthtml

Accessed

Jan

Parliament

of

the

United

Kingdom

Cruelty

to

Animals

Act

Available

Accessed

Jan

Animal

Welfare

Act

USC

Rollin

BE

The

regulation

of

animal

research

and

the

emergence

of

animal

ethics

a

conceptual

history

Theor

Med

Bioeth

PubMed

Institute

of

Laboratory

Animal

Resources

Commission

on

Life

Sciences

National

Research

Council

Guide

for

the

care

and

use

of

laboratory

animals

Washington

DC

National

Academy

Press

Office

of

Laboratory

Animal

Welfare

Public

Health

Service

policy

on

humane

care

and

use

of

laboratory

animals

Available

httpgrantsnihgovgrantsolawreferencesphspolhtmPublicHealthServicePolicyonHumaneCareandUseofLaboratory

Accessed

Jan

Office

of

Laboratory

Animal

Welfare

US

Government

principles

for

the

utilization

and

care

of

vertebrate

animals

used

in

testing

research

and

training

Available

Accessed

Jan

PubMed

Gregory

NG

Physiology

and

behavior

of

animal

suffering

Oxford

UK

Blackwell

Science

McMillan

FD

editor

Mental

Health

and

WellBeing

in

Animals

Oxford

UK

Blackwell

Publishing

Professional

Institute

of

Laboratory

Animal

Resources

Commission

on

Life

Sciences

National

Research

Council

Recognition

and

alleviation

of

pain

and

distress

in

laboratory

animals

Washington

DC

National

Academy

Press

Posner

LP

Introduction

Pain

and

distress

in

fish

a

review

of

the

evidence

ILAR

J

PubMed

Sneddon

LU

Pain

perception

in

fish

indicators

and

endpoints

ILAR

J

PubMed

Balcombe

JP

Barnard

N

Sandusky

C

Laboratory

routines

cause

animal

stress

Contemp

Top

Lab

Anim

Sci

PubMed

Panksepp

J

Affective

neuroscience

the

foundations

of

human

and

animal

emotions

Oxford

Oxford

University

Press

Koob

GF

Ehlers

CL

Kupfers

DJ

editors

Animal

Models

of

Depression

Boston

MA

Birkhäuser

Rush

DK

Mineka

S

Suomi

SJ

The

effects

of

control

and

lack

of

control

on

active

and

passive

avoidance

in

rhesus

monkeys

Behav

Res

Ther

PubMed

Seligman

MEP

Learned

helplessness

Annu

Review

Med

PubMed

Bradshaw

GA

Capaldo

T

Lindner

L

Grow

G

Building

an

inner

sanctuary

traumainduced

symptoms

in

nonhuman

great

apes

Journal

of

Trauma

and

Dissociation

PubMed

Bradshaw

GA

Schore

AN

Brown

JL

Poole

JH

Moss

CJ

Concept

elephant

breakdown

Nature

PubMed

Bradshaw

GA

Schore

AN

How

elephants

are

opening

doors

Developmental

neuroethology

attachment

and

social

context

Ethology

Boysen

ST

Himes

GT

Current

issues

and

emerging

theories

in

animal

cognition

Annu

Rev

Psychol

PubMed

Shettleworth

SJ

Cognition

evolution

and

behavior

Oxford

UK

Oxford

University

Press

deWaal

F

The

age

of

empathy

natures

lessons

for

a

kinder

society

New

York

NY

Random

House

Inc

Hauser

MD

Chomsky

N

Fitch

T

The

faculty

of

language

what

is

it

who

has

it

and

how

did

it

evolve

Science

PubMed

Langford

DJ

Crager

SE

Shehzad

Z

Smith

SB

Sotocinal

SG

et

al

Social

modulation

of

pain

as

evidence

for

empathy

in

mice

Science

PubMed

Balcombe

J

Animal

pleasure

and

its

moral

significance

Applied

Animal

Behavior

Science

Burghardt

GM

The

genesis

of

animal

play

testing

the

limits

Cambridge

UK

MIT

Press

Ortega

JC

Bekoff

M

Avian

play

comparative

evolutionary

and

developmental

trends

Auk

Gamble

JR

Cristol

DA

Dropcatch

behaviour

is

play

in

herring

gulls

Larus

argentatus

Animal

Behaviour

Emery

NJ

Clayton

NS

The

mentality

of

crows

convergent

evolution

of

intelligence

in

corvids

and

apes

Science

PubMed

OCollins

VE

Macleod

MR

Donnan

GA

Horky

LL

van

der

Worp

BH

et

al

experimental

treatments

in

acute

stroke

Ann

Neurol

PubMed

Committee

on

Toxicity

Testing

and

Assessment

of

Environmental

Agents

National

Research

Council

Toxicity

testing

in

the

century

a

vision

and

a

strategy

Washington

DC

National

Academy

Press

Hartung

T

Toxicology

for

the

st

century

Nature

PubMed

Andersen

ME

Krewski

D

Toxicity

testing

in

the

st

century

bringing

the

vision

to

life

Toxicol

Sci

PubMed

Langley

G

Evans

T

Holgate

ST

Jones

A

Replacing

animal

experiments

choices

chances

and

challenges

Bio

Essays

PubMed

Higbee

RG

Byers

AM

Dhir

V

Drake

D

Fahlenkamp

HG

et

al

An

immunologic

model

for

rapid

vaccine

assessment

a

clinical

trial

in

a

test

tube

Altern

Lab

Anim

PubMed

Byers

AM

Tapia

TM

Sassano

ER

Wittman

V

In

vitro

antibody

response

to

tetanus

in

the

MIMIC

system

is

a

representative

measure

of

vaccine

immunogenicity

Biologicals

PubMed

Dr

Susan

Love

Research

Foundation

National

Cancer

Institute

Cancer

Biomedical

Informatics

Grid

Health

of

Women

Study

Available

httpcabigcancergovactioncollaborationshowstudy

Accessed

Jan

Beauchamp

TL

Orlans

FB

Dresser

R

Morton

DB

Gluck

JP

The

Human

Use

of

Animals

Case

Studies

in

Ethical

Choice

nd

ed

New

York

NY

Oxford

University

Press

Articles

from

PLoS

ONE

are

provided

here

courtesy

of

Public

Library

of

Science

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


J Indian Soc Periodontol v154 OctDec 2011 PMC3283947 J Indian Soc Periodontol 2011 OctDec 154 425428 doi 1041030972124X92587 PMCID PMC3283947 Oral malignant melanoma A silent killer Ashvini Padhye and Jovita Dsouza Author information Article notes Copyright and License information This article has been cited by other articles in PMC Abstract Oral malignant melanomas are extremely rare lesions and occur commonly in the maxillary gingiva more frequently on the palate with fewer incidences in the mandibular gingiva Though these lesions are biologically aggressive they often go unnoticed since they are clinically asymptomatic in the early stages and usually present merely as a hyperpigmented patch on the gingival surface These lesions if diagnosed at an early in situ stage are potentially curable and definitely have a better prognosis but unfortunately as they are clinically asymptomatic it results in delayed diagnosis thus making the prognosis extremely poor This paper presents the case of Keywords Gingiva hyperpigmentation oral malignant melanoma pigmented lesions Go to INTRODUCTION Melanoma is a malignant tumor comprising of melanocytes in which cells are derived from the neural crest that constitute the melanin pigment in the basal and suprabasal layers of the epithelium Although most melanomas arise in the skin they may also arise from mucosal surfaces 1 2 Primary malignant melanoma has been described in virtually all sites and organ systems to where neural crest cells migrate 3 Although malignant melanoma is the third most common skin malignancy yet it comprises of only 3 to 5 of all cutaneous malignancies 4 Malignant melanomas of the oral cavity are extremely rare accounting for 028 of all malignant melanomas 5 6 The relative incidence of malignant melanoma among mucosal neoplasms of the head and neck is 007 as reviewed by Hormia and Vuori 7 It is characterized by proliferation of malignant melanocytes along the junction between the epithelial and connective tissues as well as within the connective tissue The most common site is the palate which accounts for about 40 of cases followed by the buccal gingiva which accounts for one third of the cases 8 The maxillary gingiva is more commonly involved as compared to the mandibular gingiva 9 Other oral sites include the buccal mucosa floor of the mouth tongue and lips When encountered melanoma of the oral cavity is more frequently found in males than in females while the reverse is true in cutaneous melanomas Although oral malignant melanomas are rare they tend to be more aggressive than their cutaneous counterparts and often prove to be fatal The prognosis for patients with oral melanoma is much worse than for those with cutaneous lesions and the overall fiveyear survival rate is about 1538 2 10 In this paper we present a rare and unusual case of a 42yearold male patient who presented with hyperpigmented gingiva Go to CASE REPORT A 42yearold male patient came to the Department of Periodontology and Oral Implantology with the chief complaint of patches of darkened gums in comparison to the adjacent pink gums The increased pigmentation was not associated with any symptoms and the patients concern was purely aesthetic He noticed small isolated patches of hyperpigmentation on the buccal mucosa of the mandibular anterior region two years ago There was a rapid spread of the hyperpigmented lesions to form larger patches involving a considerably large portion of the mandibular arch The patient found these to be unsightly and aesthetically unpleasant and hence sought dental On clinical examination the hyperpigmented lesion was present only on the mandibular gingiva involving the marginal interdental and attached gingiva from the distal aspect of 32 to the distal aspect of 44 Figure 1 The lesion also involved the lingual aspect of the attached gingiva from the distal aspect of 32 to distal aspect of 44 Figure 2 There was also an isolated patch of pigmentation seen on the attached gingiva associated with a missing 46 Figure 3 Figure 1 Dispersed patches of hyperpigmented gingiva on the labial aspect of the mandibular anterior region Figure 2 Dispersed patches of hyperpigmented gingiva on the lingual aspect of the mandibular anterior region Figure 3 Isolated patch of pigmentation on the mandibular right gingiva The lesion was blackishblue in color with irregular raised borders clearly demarcated from the adjacent clinically normal gingiva and confined within the limits of the attached gingiva The surface appeared wrinkled proliferative and granular There was no evidence of bleeding from the surface Figure 4 Figure 4 Surface appearance Wrinkled proliferative and granular On palpation the lesion was fibrotic and nontender The regional lymphnodes were not palpable Based on the history and clinical examination a differential diagnosis of Pigmented nevi Melanoachanthoma and oral malignant melanoma was derived upon Reports of routine blood investigations were normal Radiographs did not reveal any significant abnormal findings An incisional biopsy was performed and sent for histopathological investigation The report revealed sheets of melanin containing pleomorphic cells in the connective tissue confirming the diagnosis of Oral Malignant Melanoma Figure 5 Figure 5 Histopathology Sheets of melanin containing pleomorphic cells in the connective tissue The patient was referred to an Oral and maxillofacial surgeon and an Oncosurgeon for further treatment The required therapy was initiated immediately but unfortunately due to the extremely aggressive and invasive nature of the tumor the patient succumbed to death within four months of the therapy Go to DISCUSSION Oral malignant melanomas are extremely rare lesions accounting for approximately 2 of all melanomas with only a few reported cases in literature 11 13 Although mucosal melanoma was first described by Weber in 1895 the etiology of this disease is still obscure Cigarette smoking denture irritation and alcohol consumption are some of the suggested risk factors but their correlation is still unsubstantiated 14 Tobacco and formaldehyde exposures have also been suggested as causative agents for intraoral melanomas 15 In the present case the patient was not exposed to any of these factors and hence the possible etiological factor for this patient is still unclear Onset of malignant melanomas is usually between the ages of 40 to 70 years mean 55 years 16 17 The lesion is three times more common in men than in women In the oral cavity malignant melanoma almost exclusively occurs in the palate and maxillary gingiva with an incidence of 80 and 914 respectively 18 19 There are very few reported cases of malignant melanoma on the mandibular gingiva 20 22 Our patient is one of those rare cases as the site of occurrence of the lesion is the mandibular gingiva The patient had noticed appearance of the pigmented lesion about two years ago and it didnt seem to proliferate at a rapid rate then Most primary oral melanomas appear as new lesions from apparently normal mucosa whereas about 3050 are preceded by oral pigmentations for several months or even years Some of the premelanoma lesions include mucosal melanosis and a variety of melanocytic nevi 23 24 Oral melanosis has been suggested as a predisposing factor for the development of oral melanoma in 30 to 73 of patients 25 However although the incidence of melanosis in India is low there in a higher occurrence of primary oral melanoma thereby contraindicating this hypothesis 26 Primary oral melanoma is initially asymptomatic and progresses unnoticed by patients contributing to delay in diagnosis Generally recognition of the lesion occurs when there is a breakdown of the overlying epithelium or hemorrhage 27 It is a known fact that early diagnosis and treatment of cutaneous melanomas is imperative in order to reduce mortality If the lesions are diagnosed in an initial stage where the cancer cells are limited to the epidermis layer of the skin the melanoma is nearly 100 curable by excision Malignant melanomas can range from mucosal pink through brown and blue to black lesions with or without ulcerations They could be flat or elevated with an erythematous border around it Oral melanoma may appear in various forms including pigmented macule pigmented nodule or a large pigmented exophytic lesion or an amelanotic variant of any of these three forms Lopez et al identified five types of oral malignant melanoma OMM on the basis of clinical appearance Pigmented nodular type nonpigmented nodular type pigmented macular type pigmented mixedtype and nonpigmented mixed type 28 This case could be identified as the pigmented nodular type of OMM The mucosal melanomas can show two principal patterns An in situ pattern in which the neoplasm is limited to the epithelium and epithelialconnective tissue interface junctional and an invasive pattern in which the neoplasm is found within the supporting connective tissue Greene et al 29 suggested the following criteria for a lesion to be considered as primary malignant melanoma of the oral cavity 1 demonstration of malignant melanoma both histologically and clinically 2 the presence of junctional activity and 3 the inability to demonstrate any other primary site Based on these criteria this case could be considered as a primary oral malignant melanoma This tumor is characterized by marked aggressive and invasive behavior that manifests by both local and distant metastases to sites such as lungs liver brain and bones 30 Lymphatic metastasis at the time of diagnosis seems to be a crucial prognostic factor for oral melanomas The American Joint Committee on Cancer AJC does not have published guidelines for the staging of oral malignant melanomas yet probably due to the rare occurrence of this lesion Surgery remains the preferred treatment along with chemotherapy radiotherapy and immunotherapy It has been shown that recent surgical excision followed by immunochemotherapy has reduced or prevented the recurrence of the lesion but there are only a few reported cases in literature 31 Extensive research is still required on this front In the cases where metastasis has already occurred the disease is considered as classically incurable surgery being considered only for palliative care 30 and other treatment modalities like radiotherapy and chemotherapy could be considered under the palliative care The prognosis of oral melanoma is poor with a fiveyear survival of 055 of cases The median survival for all oral mucosal melanomas is slightly more than two years from the time of diagnosis 32 Primary mucosal melanomas are exceedingly rare and biologically aggressive malignancies Tumors diagnosed early essentially during the in situ stage are potentially curable and have a better prognosis We therefore lay emphasis and reiterate on the importance of maintaining a high index of suspicion and early detection and diagnosis for any pigmented lesions especially if they occur in highrisk sites like the palate or gingiva Diagnosis must be based on thorough detailed history and valid histologic evidence Go to CONCLUSION Oral malignant melanoma though an extremely rare malignancy is potentially very aggressive and a rapidly invasive tumor Clinically these tumors are very silent and asymptomatic in their appearance tending to be misleading Thus the importance of early detection and diagnosis which can be life saving can not be overemphasised Go to Footnotes Source of Support Nil Conflict of Interest None declared Go to REFERENCES 1 Boulaadas M Benazzou S Mourtada F Sefiani S Nazih N Essakalli L et al Primary oral malignant melanoma J Craniofac Surg 2007 18 105961 PubMed 2 Ebenezer J Malignant melanoma of the oral cavity Indian J Dent Res 2006 17 946 PubMed 3 Pandey M Abraham EK Mathew A Ahamed IM Primary malignant melanoma of the upper aerodigestive tract Int J Oral Maxillofac Surg 1999 28 459 PubMed 4 Boring CC Squires TS Tong T Montgomery S Cancer statistics 1994 CA Cancer J Clin 1994 44 726 PubMed 5 Pliskin ME Mastrangelo MJ Brown AM Cister RP Metastatic melanoma of the maxilla presenting as a gingival swelling Oral Surg Oral Med Oral Pathol 1976 41 1014 PubMed 6 Hoffman S Yohn J Robinson W Norris D Melanoma 1Clinical characteristics Hosp Prac 1994 29 4750 PubMed 7 Hormia M Vuori EE Mucosal melanoma of the head and neck J Laryngol 1969 83 34959 PubMed 8 Hicks MJ Flaitz CM Oral mucosal melanoma Epidemiology and pathobiology Oral Oncol 2000 36 15269 PubMed 9 Shafer WG Hine MK Levy BM 4th Ed Philadelphia WB Saunders 1983 A Textbook of Oral Pathology pp 1335 10 Doval DC Rao CR Saitha KS Vigayakumar M Misra S Mani K et al Malignant melanoma of the oral cavity Report of 14 cases from a regional cancer centre Eur J Surg Oncol 1996 22 2459 PubMed 11 Ardekian L Rosen DJ Peled M Rachmiel A Machtei EE el Naaj IA et al Primary gingival malignant melanoma Report of 3 cases J Periodontol 2000 71 11720 PubMed 12 Robertson GR DeFiebre BK Firtell DN Primary malignant melanoma of the mouth J Oral Surg 1979 37 34952 PubMed 13 Chaudhry AP Hamped A Gorlin KJ Primary malignant melanoma of the oral cavity a review of 105 cases Cancer 1958 11 9238 PubMed 14 Freedman DM Sigurdson A Doody MM Rao RS Linet MS Risk of melanoma in relation to smoking alcohol intake and other factors in a large occupational cohort Cancer Causes Control 2003 14 84757 PubMed 15 Holmstrom M Lund VJ Malignant melanoma of the nasal cavity after occupational exposure to formaldehyde Br J Ind Med 1991 48 911 PMC free article PubMed 16 Smyth AG WardBooth RP Avery BS To EW Malignant melanoma of the oral cavity an increasing clinical diagnosis Br J Oral Maxillofac Surg 1993 31 2305 PubMed 17 Rapini RP Golitz LE Greer RO Jr Krekorian EA Poulson T Primary malignant melanoma of the oral cavityA review of 177 cases Cancer 1985 55 154351 PubMed 18 Eneroth CM Malignant Melanoma of the Oral Cavity Int J Surg 1975 4 1917 PubMed 19 Tanaka M Mimura M Ogi K Amagasa T Primary malignant melanoma of the oral cavity Assessment of outcome from the clinical records of 35 patients Int J Oral Maxillofac Surg 2004 33 7615 PubMed 20 Fauzdar S Rao DD Arthanari KK Krishnan G Naikmasur VG Revanappa MM Malignant melanoma of the mandibular gingiva Rare Tumors 2010 2 e25 PMC free article PubMed 21 Lourenço SV Bologna SB Colucci F Neto CF Montenegro FL Nico MM Oral mucosal melanoma of the mandibular gingiva A case report Cutis 2010 86 8993 PubMed 22 Sharon A Ross AS Primary malignant melanoma of the mandibular gingiva Int J Oral Surg 1984 13 5458 PubMed 23 Hatch CL Pigmented lesions of the oral cavity Dent Clin North Am 2005 49 185201 PubMed 24 Kahn MA Weathers DR Hoffman JG Transformation of a benign oral pigmentation to primary oral melanoma Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005 100 4549 PubMed 25 Takagi M Ishikawa G Mori W Primary malignant melanoma of the oral cavity in JapanWith special reference to mucosal melanosis Cancer 1974 34 35870 PubMed 26 Soman CS Sirsat MV Primary malignant melanoma of the oral cavity in Indians Oral Surg Oral Med Oral Pathol 1974 38 42634 PubMed 27 Rowland HN Schnetler JF Primary malignant melanoma arising in the dorsum of the tongue Br J Oral Maxillofac Surg 2003 41 1978 PubMed 28 LopezGraniel CM OchoaCarrilo FJ MeneseGarcia A Malignant melanoma of the oral cavity Diagnosis and treatment experience in a Mexican population Oral Oncol 1999 35 42530 PubMed 29 Greene GW Haynes JW Dozier M Blumberg JM Bernier JL Primary malignant melanoma of the oral mucosa Oral Surg Oral Med Oral Pathol 1953 6 143543 PubMed 30 Martinez EA Alonso FC Siles MS Melanoma of the oral mucosa with cerebral metastasis A clinical case Oral Oncol Extra 2005 41 303 31 Umeda M Komatsubara H Shigeta T Ojima Y Minamikawa T Shibuya Y et al Treatment and prognosis of malignant melanoma of the oral cavity Preoperative surgical procedure increases risk of distant metastasis Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008 106 517 PubMed 32 Chidzonga MM Mahomva L Marimo C MakunikeMutasa R Primary malignant melanoma of the oral mucosa J Oral Maxillofac Surg 2007 65 111720 PubMed Articles from Journal of Indian Society of Periodontology are provided here courtesy of Medknow Publications

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

oral malignant melanoma
mandibular gingiva
hyperpigmented


Journal List Mayo Clin Proc v8712 2012 Dec PMC3547550 Mayo Clin Proc 2012 Dec 8712 e101e104 doi 101016jmayocp201206026 PMCID PMC3547550 18YearOld Woman With Fever Abdominal Pain and Elevated Liver Enzymes Michael E Wilson a Brent T Cengia b and Seth Sweetser c Author information Copyright and License information This article has been cited by other articles in PMC An 18yearold woman from rural Minnesota was admitted to the hospital after presenting to a local emergency department with a 3day history of severe right upper quadrant abdominal pain and a 2week history of headaches fever nausea and vomiting A review of systems was otherwise unrevealing Her only medication was trimethoprimsulfamethoxazole for a recent urinary tract infection She had no other notable medical history She reported rare alcohol use 3 years previously and no recent use of overthecounter medications or dietary supplements smoking illegal drug use or sexual activity Physical examination findings and vital signs were unremarkable aside from a Laboratory studies revealed the following reference ranges shown parenthetically alkaline phosphatase 738 UL 52144 UL aspartate aminotransferase AST 285 UL 843 UL alanine aminotransferase ALT 417 UL 745 UL total bilirubin 23 mgdL 0110 mgdL direct bilirubin 12 mgdL 0003 mgdL and platelet count 101 10 9 L 150450 10 9 L Her hemoglobin level leukocyte count urinalysis results and amylase lipase lactate electrolyte and serum creatinine levels were all within normal limits Results of serologies for acute hepatitis A B and C a heterophile antibody test and a pregnancy test were negative and salicylate and acetaminophen levels were unremarkable 1 Which one of the following possible diagnoses should be confirmed or ruled out immediately a Acute cholangitis b BuddChiari syndrome c Ischemic hepatitis d Medicationinduced hepatitis e West Nile virus infection The patients laboratory studies suggest hepatocellular injury with mild hyperbilirubinemia which can be seen with acute cholangitis caused by choledocholithiasis Acute cholangitis must be considered in every patient with right upper quadrant pain fever and hyperbilirubinemia because it has a high mortality rate if diagnosis is delayed Initial testing would include liver ultrasonography and blood cultures Acute BuddChiari syndrome can present as right upper quadrant pain with biochemical evidence of hepatocellular injury and occurs when an obstruction in the hepatic veins causes hepatic congestion However hepatomegaly and newonset ascites are usually present and fever would be unusual Although the differential The patient underwent abdominal ultrasonography which revealed a normalappearing liver and gallbladder with no intrahepatic or extrahepatic duct dilatation Her spleen was enlarged at 167 cm in the craniocaudal position upper limit of the normal range 1213 cm Doppler ultrasonography showed normal hepatic and portal veins Computed tomography of the abdomen with intravenous contrast was unrevealing aside from mild splenomegaly with no evidence of portal or hepatic vein thrombosis At this point the patient has fever abdominal pain elevated transaminase and bilirubin levels splenomegaly and thrombocytopenia of unclear etiology 2 Which one of the following is the least likely cause of this patients abnormal liver enzyme levels a Autoimmune hepatitis b Acute infectious mononucleosis c Human granulocytic anaplasmosis d Acute alcoholic hepatitis e Acute human immunodeficiency virus HIV infection Autoimmune hepatitis is usually a chronic condition but can present as acute hepatitis Overlap syndromes encompassing autoimmune hepatitis and either primary biliary cirrhosis or primary sclerosing cholangitis can lead to concomitant hyperbilirubinemia Negative results on a heterophile antibody test do not rule out mononucleosis and this and other causes of viral hepatitis must remain in the differential diagnosis Anaplasmosis is caused by a rickettsiallike organism transmitted via the Ixodes scapularis tick It classically presents with elevated transaminase levels leukopenia and thrombocytopenia Acute alcoholic hepatitis typically presents with an ASTALT ratio greater than 2 a normal or minimally elevated ALT level and leukocytosis Liver enzyme including alkaline phosphatase elevations greater than 300 UL and certainly greater than 500 UL suggest another mechanism of hepatocellular injury Thus acute alcoholic hepatitis is unlikely to be the sole cause of hepatitis in this patient Acute HIV infection can rarely present with the acute hepatitis and constitutional symptoms seen in our patient and is an important diagnosis to consider in an unexplained febrile illness The patients fever and abdominal pain persisted without a unifying diagnosis The AST and ALT levels increased to 297 UL and 447 UL respectively Evaluation for autoimmune hepatitis was positive for antismooth muscle antibodies 120 titer and antinuclear antibodies 13 U weakly positive 1129 U and negative for antikidneyliver microsome antibodies and antimitochondrial antibodies Serum evaluations were negative for Anaplasma Ehrlichia Bartonella Babesia Toxoplasma HIV enzyme immunoassay serology West Nile virus Q fever and cytomegalovirus CMV IgG and IgM Ceruloplasmin Creactive protein albumin and ferritin levels were normal as were erythrocyte sedimentation rate prothrombin time and results from serum protein electrophoresis and blood cultures A peripheral blood smear showed atypical lymphocytes Lactate dehydrogenase was elevated at 1247 UL 122222 UL 3 Which one of the following is the most appropriate next step in management a Corticosteroid treatment for autoimmune hepatitis b Confirmatory testing for acute viral hepatitis c Liver biopsy d Endoscopic retrograde cholangiopancreatography e Urine drug screen Autoimmune hepatitis is a clinical diagnosis based primarily on 4 features absence of viral markers presence and level of autoantibodies elevated serum IgG level and histologic features on liver biopsy Our patients mildly elevated antinuclear antibodies and antismooth muscle antibodies do not fit the classic diagnostic criteria for autoimmune hepatitis and empiric treatment should not be initiated without a firm diagnosis Serum evaluation to rule out viral hepatitis should be performed before considering a liver biopsy If evaluation for viral hepatitis yields negative results a liver biopsy would be the next best step and could help to establish a diagnosis An HIV viral load was undetectable ruling out acute HIV infection Results of repeated testing for CMV IgG and IgM were negative thus making CMV hepatitis unlikely Despite negative results on the heterophile antibody test we suspected that the young womans symptom complex could be caused by EpsteinBarr virus EBV infection Additional testing was performed 4 Which one of the following would provide the best evidence to confirm this suspected diagnosis a Atypical lymphocytes on peripheral smear b Repeated heterophile antibody test c IgM antibodies to EBV viral capsid d IgG antibodies to EBV viral capsid and EBV nuclear antigen e EBV DNA polymerase chain reaction Although atypical lymphocytes are detected on peripheral smear in up to 75 of patients with EBV mononucleosis they have a low specificity for diagnostic purposes A heterophile IgM antibody test is the initial diagnostic test of choice but results may be falsely negative within the first 3 weeks after onset of symptoms If heterophile IgM antibodies are present no further testing is indicated If heterophile IgM antibodies are absent and EBV infection is suspected EBVspecific antibody testing is indicated In patients with mononucleosis EBV viral capsid IgM is present at symptom onset and this finding has a high sensitivity and 1 On rare occasions test results for EBV viral capsid IgM can be falsely positive If this is the case a positive EBV viral capsid IgG result 6 weeks after diagnosis may provide further confirmation if needed Our patients test results were positive for EBV viral capsid IgM negative for EBV viral capsid IgG and negative for EBV nuclear antigen IgG consistent with a diagnosis of acute EBV infection 5 Which one of the following is the best management strategy for this patients illness a Intravenous acyclovir if liver enzyme levels continue to increase b Prednisone c Supportive care d No contact sports for the next 7 days e Lowprotein diet for 6 weeks Intravenous acyclovir has been shown to reduce oropharyngeal viral shedding during treatment of acute EBV infection but has not been shown to affect peripheral viremia or the duration or severity of symptoms 2 No clinical benefit has been identified for the use of corticosteroids in acute EBV infection except in cases of specific complications such as acute upper airway obstruction 3 Treatment for EBV infection is supportive and includes symptomatic relief for fever pharyngitis and abdominal pain Although splenomegaly is common splenic rupture is a rare complication and most commonly occurs within 3 weeks of symptom onset Therefore return to contact sports can be considered after a minimum of 3 weeks Lowprotein diets are often used for patients with severe liver disease to reduce nitrogen metabolite and ammonia levels Our patient does not have liver failure and can follow a normal diet The patient received supportive care and was dismissed from the hospital On a followup visit 1 week later her symptoms had resolved except for fatigue and her AST and ALT levels had improved to 123 UL and 267 UL respectively Her thrombocytopenia and hyperbilirubinemia had also completely resolved Go to Discussion EpsteinBarr virus most commonly presents as the clinical syndrome of infectious mononucleosis although the virus has been linked to a number of other disorders such as nasopharyngeal carcinoma Burkitt lymphoma Hodgkin disease oral hairy leukoplakia and lymphoproliferative disorders in patients with immunodeficiency A member of the herpesvirus family EBV infection is spread by contact with oral secretions and symptoms become apparent after an incubation period of 30 to 50 days 4 Although as many as 90 to 95 of all adults are seropositive and shed the virus in saliva EBV infection accounts for less than 2 of all cases of adults presenting with pharyngitis 5 In children mononucleosis often causes nonspecific symptoms or no symptoms at all In adolescents and adults mononucleosis classically presents in more than 50 of cases with the triad of fever pharyngitis and cervical axillary or inguinal lymphadenopathy 4 Fatigue palatal petechiae and abdominal pain are also common 1 Lymphocytosis 50 lymphocytes and 10 or more atypical lymphocytes on peripheral blood smear are present in more than twothirds of patients and can aid in diagnosis 6 On ultrasonography splenomegaly is present in up to 100 of patients although it is rarely detected on physical examination 5 Splenic rupture occurs in 01 of mononucleosis cases almost never occurs later than 3 weeks after symptom onset and has been found to be atraumatic in onehalf of cases 7 Additional less common findings include hematologic abnormalities mild thrombocytopenia hemolytic anemia and disseminated intravascular coagulation genital ulcers myocarditis and acute interstitial nephritis Upper airway obstruction secondary to mucosal edema and pharyngeal lymphoid hyperplasia may occur in 1 of patients Neurologic complications including GuillainBarré syndrome aseptic meningitis encephalitis and cranial nerve palsies are seen in 1 to 5 of patients 45 Elevated liver enzymes occur in 80 to 90 of patients with EBV infections commonly peak 2 to 3 weeks after symptom onset and normalize within 3 to 6 weeks after symptom onset The AST and ALT levels typically do not increase to greater than 5 times the upper limit of normal although elevations higher than 10 times normal and acute liver failure requiring liver transplant have been reported 8 Elevations in alkaline phosphatase and conjugated and unconjugated bilirubin concentrations may also be present 9 The diagnosis of mononucleosis is based on compatible clinical findings the presence of lymphocytosis or atypical lymphocytes and a positive serologic test result Heterophile IgM antibodies are measurable in approximately 75 of patients in the first week of illness and 95 in the third week after symptom onset 5 with a sensitivity of 85 and a specificity of nearly 100 10 If the heterophile antibody test result is negative and there is a high clinical suspicion for mononucleosis the next best test to order is determination of EBVspecific antibodies EpsteinBarr viral capsid IgM antibodies are diagnostic for acute infection 97 sensitivity 94 specificity 11 They are detectable at symptom onset due to a 30 to 50day incubation period and disappear in approximately 3 months EpsteinBarr viral capsid IgG antibodies appear shortly after symptom onset and are present for life indicative of current or prior infection EpsteinBarr virus nuclear antigen IgG antibodies become detectable 6 to 12 weeks after symptom onset and also persist throughout life Treatment is supportive including rest adequate hydration and acetaminophen or nonsteroidal antiinflammatory drugs for fever throat pain and abdominal pain Because of a small risk for splenic rupture patients should abstain from contact sports for a minimum of 3 to 4 weeks after symptom onset and until the patient is asymptomatic 512 The use of corticosteroids or antiviral therapy remains controversial and the subject of ongoing investigation A metaanalysis published in 1999 evaluated 5 randomized controlled trials comparing acyclovir and placebo in the treatment of mild to severe disease and found no clinical benefit with acyclovir 2 A 2006 Cochrane review concluded that there was insufficient evidence to recommend corticosteroid treatment for uncomplicated infectious mononucleosis 3 The diagnosis of EBV infectious mononucleosis should be considered in young patients with elevated transaminase levels hyperbilirubinemia splenomegaly fever and abdominal pain even when the classic findings of pharyngitis and palpable lymphadenopathy are not present The disorder is diagnosed by detection of either heterophile IgM antibodies or EBV viral capsid IgM antibodies Treatment is supportive and the prognosis is excellent Go to Footnotes See end of article for correct answers to questions CORRECT ANSWERS 1 a 2 d 3 b 4 c 5 c Go to References 1 Pitetti RD Laus S Wadowsky RM Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary EpsteinBarr virus infection in children Pediatr Infect Dis J 2003 22 8736739 PubMed 2 Torre D Tambini R Acyclovir for treatment of infectious mononucleosis a metaanalysis Scand J Infect Dis 1999 31 6543547 PubMed 3 Candy B Hotopf M Steroids for symptom control in infectious mononucleosis Cochrane Database Syst Rev 20063 CD004402 PubMed 4 Cohen JI EpsteinBarr virus infection N Engl J Med 2000 343 7481492 PubMed 5 Ebell MH EpsteinBarr virus infectious mononucleosis Am Fam Physician 2004 70 712791287 PubMed 6 Brigden ML Au S Thompson S Brigden S Doyle P Tsaparas Y Infectious mononucleosis in an outpatient population diagnostic utility of 2 automated hematology analyzers and the sensitivity and specificity of Hoaglands criteria in heterophilepositive patients Arch Pathol Lab Med 1999 123 10875881 PubMed 7 Farley DR Zietlow SP Bannon MP Farnell MB Spontaneous rupture of the spleen due to infectious mononucleosis Mayo Clin Proc 1992 67 9846853 PubMed 8 Feranchak AP Tyson RW Narkewicz MR Fulminant EpsteinBarr viral hepatitis orthotopic liver transplantation and review of the literature Liver Transpl Surg 1998 4 6469476 PubMed 9 Kofteridis DP Koulentaki M Valachis A Epstein Barr virus hepatitis Eur J Intern Med 2011 22 17376 PubMed 10 Linderholm M Boman J Juto P Linde A Comparative evaluation of nine kits for rapid diagnosis of infectious mononucleosis and EpsteinBarr virusspecific serology J Clin Microbiol 1994 32 1259261 PMC free article PubMed 11 Bruu AL Hjetland R Holter E Evaluation of 12 commercial tests for detection of EpsteinBarr virusspecific and heterophile antibodies Clin Diagn Lab Immunol 2000 7 3451456 PMC free article PubMed 12 Luzuriaga K Sullivan JL Infectious mononucleosis published correction appears in N Engl J Med 2010363151486 N Engl J Med 2010 362 2119932000 PubMed Articles from Mayo Clinic Proceedings are provided here courtesy of The Mayo Foundation for Medical Education and Research

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

fever
abdominal pain
elevated liver enzymes