A

novel

family

of

diversified

immunoregulatory

receptors

in

teleosts

is

homologous

to

both

mammalian

Fc

receptors

and

molecules

encoded

within

the

leukocyte

receptor

complex

Three

novel

and

closely

related

leukocyte

immune-type

receptors

-LRB-

IpLITR

-RRB-

have

been

identified

in

channel

catfish

-LRB-

Ictalurus

punctatus

-RRB-

These

receptors

belong

to

a

large

polymorphic

and

polygenic

subset

of

the

Ig

superfamily

with

members

located

on

at

least

three

independently

segregating

loci

Like

mammalian

and

avian

innate

immune

regulatory

receptors

IpLITRs

have

both

putative

inhibitory

and

stimulatory

forms

with

multiple

types

coexpressed

in

various

lymphoid

tissues

and

clonal

leukocyte

cell

lines

IpLITRs

have

an

unusual

and

novel

relationship

to

mammalian

and

avian

innate

immune

receptors

the

membrane

distal

Ig

domains

of

an

individual

IpLITR

are

related

to

fragment

crystallizable

receptors

-LRB-

FcRs

-RRB-

and

FcR-like

proteins

whereas

the

membrane

proximal

Ig

domains

are

related

to

several

leukocyte

receptor

complex

encoded

receptors

This

unique

composition

of

Ig

domains

within

individual

receptors

supports

the

hypothesis

that

functionally

and

genomically

distinct

immune

receptor

families

found

in

tetrapods

may

have

evolved

from

such

ancestral

genes

by

duplication

and

recombination

events

Furthermore

the

discovery

of

a

large

heterogeneous

family

of

immunoregulatory

receptors

in

teleosts

reminiscent

of

amphibian

avian

and

mammalian

Ig-like

receptors

suggests

that

complex

innate

immune

receptor

networks

have

been

conserved

during

vertebrate

evolution

Introduction

Immune

receptors

that

bind

similar

ligands

often

form

clusters

that

are

encoded

on

distinct

chromosomes

in

the

human

genome

For

example

receptors

that

bind

to

the

Fc

portion

of

Ig

-LSB-

fragment

crystallizable

receptors

-LRB-

FcRs

-RRB-

-RSB-

are

found

on

chromosome

and

consist

of

the

high-affinity

IgG

and

IgE

receptors

-LRB-

FcγRI

and

FcεRI

-RRB-

and

the

related

low

or

moderate-affinity

Ig

receptors

-LRB-

FcγRII

FcγRIII

and

FcγRIV

-RRB-

-LRB-

Hullet

et

al.

Ravetch

and

Kinet

Hullet

and

Hogarth

Daeron

Falk

et

al.

Falk

and

Ravetch

-RRB-

Alternatively

immune

receptors

involved

in

the

recognition

of

major

histocompatability

class

-LRB-

MHC

I

-RRB-

antigens

such

as

killer

cell

Ig-like

receptors

-LRB-

KIRs

-RRB-

and

leukocyte

Ig-like

receptors

-LRB-

LILRs

-RRB-

are

clustered

together

on

chromosome

in

a

region

known

as

the

leukocyte

receptor

complex

-LRB-

LRC

-RRB-

-LRB-

Kremer

et

al.

Wagtmann

et

al.

Wende

et

al.

Martin

et

al.

-RRB-

Recently

comparative

studies

have

suggested

that

FcRs

and

LRC-encoded

receptors

although

genomically

and

functionally

distinct

appeared

to

have

evolved

from

a

common

ancestor

This

contention

is

supported

in

part

by

recent

attempts

to

find

LRC

regions

in

other

species

Mouse

chromosome

is

syntenic

to

the

human

LRC

and

encodes

the

paired

Ig-like

receptors

-LRB-

PIRs

-RRB-

that

were

originally

discovered

in

an

attempt

to

find

a

murine

IgA

receptor

ortholog

-LRB-

Kubagawa

et

al.

Hayami

et

al.

-RRB-

Subsequently

searches

for

PIR

relatives

resulted

in

the

identification

of

the

chicken

Ig-like

receptors

-LRB-

CHIRs

-RRB-

in

birds

-LRB-

Dennis

et

al.

-RRB-

representing

a

multigene

family

encoded

within

an

avian

LRC-like

region

-LRB-

Nikolaidis

et

al.

Viertlboeck

et

al.

-RRB-

More

importantly

CHIR

Ig

domain

sequences

are

not

only

related

to

the

mammalian

PIRs

and

KIRs

but

also

to

various

FcR

Ig

domains

-LRB-

Dennis

et

al.

-RRB-

This

previously

unrecognized

relationship

was

supported

by

phylogenetic

and

comparative

structural

analysis

and

was

the

first

evidence

suggesting

that

certain

genomically

and

functionally

diverse

mammalian

and

avian

immune

receptor

families

probably

evolved

from

a

common

ancestor

-LRB-

Dennis

et

al.

Nikolaidis

et

al.

-RRB-

Compared

to

mammals

and

birds

bony

fish

are

among

the

most

phylogenetically

distant

vertebrates

with

characteristic

innate

and

adaptive

immune

systems

that

have

mammalian-like

B

and

T

cells

-LRB-

Yoder

-RRB-

Furthermore

due

to

the

establishment

of

clonal

lymphoid

cell

lines

catfish

are

known

to

also

possess

natural

killer

-LRB-

NK

-RRB-

like

cells

capable

of

killing

xenogeneic

targets

by

an

antibody-dependent

cell-mediated

cytotoxicity

-LRB-

ADCC

-RRB-

process

-LRB-

Shen

et

al.

-RRB-

This

functional

evidence

suggested

that

catfish

NK

cells

possess

a

putative

FcμR

-LRB-

Shen

et

al.

-RRB-

Additionally

catfish

NK

cells

can

kill

allogeneic

targets

in

an

ADCC-independent

manner

indicating

that

target

recognition

may

also

involve

KIR

and/or

LILR-like

receptors

-LRB-

Shen

et

al.

-RRB-

However

to

date

FcR

and/or

KIR/LILR

homologs

have

not

been

described

in

ectothermic

vertebrates

making

it

difficult

to

elucidate

the

evolutionary

history

of

these

important

receptor

families

Large

families

of

putative

activatory

and

inhibitory

novel

immune-type

receptors

-LRB-

NITRs

-RRB-

also

belonging

to

the

Ig

superfamily

-LRB-

IgSF

-RRB-

have

been

described

in

teleosts

Pufferfish

-LRB-

Spheroides

nephelus

-RRB-

zebrafish

-LRB-

Danio

rerio

-RRB-

catfish

and

rainbow

trout

-LRB-

Oncorhynchus

mykiss

-RRB-

all

express

NITRs

-LRB-

Strong

et

al.

Yoder

et

al.

Hawke

et

al.

-RRB-

Because

some

NITRs

are

also

expressed

by

catfish

NK

cells

-LRB-

Hawke

et

al.

-RRB-

it

has

been

suggested

that

these

receptors

may

represent

the

functional

orthologs

of

the

mammalian

KIRs

and/or

LILRs

-LRB-

Yoder

et

al.

Hawke

et

al.

-RRB-

However

NITRs

do

not

exhibit

any

sequence

identity

or

phylogenetic

relationship

to

such

receptors

-LRB-

Yoder

et

al.

-RRB-

and

have

only

been

identified

in

bony

fishes

Herein

is

described

a

novel

set

of

IgSF

receptors

present

in

the

channel

catfish

and

possibly

other

teleosts

that

are

composed

of

Ig

domains

related

to

functionally

diverse

immunoreceptor

families

present

in

mammals

Detailed

sequence

analysis

supports

that

individual

catfish

leukocyte

immune-type

receptors

-LRB-

IpLITRs

-RRB-

contain

a

unique

composition

of

Ig

domains

sharing

homology

with

both

FcRs

and

receptors

encoded

within

the

LRC

Thus

IpLITRs

provide

further

evidence

supporting

the

common

evolutionary

origins

of

genomically

and

functionally

distinct

immunoregulatory

receptor

families

present

in

mammals

Materials

and

methods

Experimental

animals

and

cell

lines

Catfish

-LRB-

kg

-RRB-

were

obtained

from

a

commercial

source

-LRB-

ConAgra

Isola

MS

USA

-RRB-

and

maintained

in

individual

tanks

as

described

previously

-LRB-

van

Ginkel

et

al.

-RRB-

Catfish

peripheral

blood

leukocytes

-LRB-

PBL

-RRB-

were

isolated

from

heparinized

blood

by

centrifugation

on

a

cushion

of

Ficoll-Hypaque

-LRB-

Lymphoprep

Accurate

Chemical

Westbury

NY

USA

-RRB-

as

described

previously

-LRB-

Miller

et

al.

-RRB-

Catfish

cells

lines

were

grown

at

°C

in

medium

consisting

of

equal

parts

AIM-V

and

-LRB-

Life

Technologies

Gaithersburg

MD

USA

-RRB-

adjusted

to

catfish

tonicity

with

-LRB-

v/v

-RRB-

deionized

water

and

supplemented

with

μg/ml

U/ml

penicillin

μg/ml

streptomycin

μg/ml

gentamicin

uM

and

heat-inactivated

pooled

normal

catfish

serum

-LRB-

Miller

et

al.

b

-RRB-

is

a

cloned

autonomous

B

cell

line

generated

from

an

outbred

catfish

by

mitogen

stimulation

-LRB-

Wilson

et

al.

-RRB-

is

an

automonous

T

cell

line

derived

from

a

gynogenetic

catfish

-LRB-

Hogan

et

al.

-RRB-

whereas

is

a

macrophage

cell

line

derived

from

a

different

outbred

fish

-LRB-

Miller

et

al.

-RRB-

and

are

clonal

nonautonomous

antigen-dependent

cytotoxic

T

cell

lines

developed

from

an

alloantigen

immunized

outbred

fish

-LRB-

Stuge

et

al.

-RRB-

is

a

clonal

nonautonmous

NK-cell

line

developed

from

an

alloantigen-stimulated

culture

of

PBL

from

a

naive

outbred

fish

-LRB-

Shen

et

al.

-RRB-

The

nonautonomous

cytotoxic

T

cell

and

NK

cell

lines

require

weekly

restimulation

with

irradiated

allogeneic

cells

for

continuous

proliferation

The

channel

catfish

ovary

fibroblast

cell

line

-LRB-

CCO

Browser

and

Plumb

-RRB-

was

provided

by

Dr.

V.G.

Chinchar

-LRB-

University

of

Mississippi

Medical

Center

-RRB-

CCO

cells

were

maintained

in

Dulbecco

's

modified

Eagle

's

medium

-LRB-

Sigma-Aldrich

St.

Louis

MO

USA

-RRB-

supplemented

with

FCS

Channel

catfish

LITRs

and

sequence

analysis

In

a

search

for

IgSF

receptors

expressed

by

alloantigen

stimulated

catfish

PBL

and

various

catfish

clonal

cell

lines

several

expressed

sequence

tag

-LRB-

EST

-RRB-

libraries

were

analyzed

Subsequently

and

cDNAs

were

identified

by

single

pass

sequencing

of

a

cDNA

library

generated

from

a

catfish

mixed

lymphocyte

culture

-LRB-

MLC

-RRB-

enriched

in

NK-like

cells

-LRB-

Shen

et

al.

Stuge

et

al.

-RRB-

was

obtained

from

the

catfish

macrophage

cell

line

-LRB-

Miller

et

al.

-RRB-

cDNA

library

The

cDNAs

were

sequenced

on

both

strands

using

universal

forward

and

reverse

primers

and

gene-specific

primers

Nucleotide

and

amino

acid

sequences

were

analyzed

using

DNASTAR

software

-LRB-

Madison

WI

USA

-RRB-

and

aligned

using

CLUSTALW

-LRB-

Thompson

et

al.

-RRB-

Neighbor-joining

-LRB-

NJ

-RRB-

trees

with

pairwise

gap

deletions

were

drawn

using

MEGA

-LRB-

Kumar

et

al.

-RRB-

Maximum

parsimony

and

maximum

likelihood

trees

were

drawn

with

essentially

the

same

results

-LRB-

data

not

shown

-RRB-

Similarity

searches

were

performed

with

the

extracellular

Ig

domains

of

and

using

position-specific

iterative

-LRB-

PSI

-RRB-

and

Basic

Local

Alignment

Search

Tool

-LRB-

BLAST

-RRB-

analysis

-LRB-

Altschul

et

al.

-RRB-

against

the

National

Center

for

Biotechnology

Information

-LRB-

NCBI

-RRB-

nonredundant

database

SOSUI

-LRB-

signal

-RRB-

beta

version

Simple

Modular

Architecture

Research

Tool

-LRB-

SMART

-RRB-

-LRB-

Letunic

et

al.

-RRB-

Pfam

databases

-LRB-

Bateman

et

al.

-RRB-

and

the

three-dimensional

position-specific

scoring

matrix

server

-LRB-

Kelly

et

al.

-RRB-

were

used

for

predictions

of

signal

peptides

Ig

domains

transmembrane

-LRB-

TM

-RRB-

segments

and

secondary

structure

Sequence

decorations

were

performed

using

GeneDoc

-LRB-

-RRB-

Southern

and

Northern

blot

analyses

Genomic

DNA

was

prepared

from

erythrocytes

of

catfish

siblings

These

siblings

were

representatives

of

a

family

obtained

from

the

Catfish

Genetics

Research

Unit

United

States

Department

of

Agriculture

Agricultural

Research

Service

-LRB-

Stoneville

MS

USA

designated

family

-RRB-

The

sibling

catfish

used

were

and

respectively

-LRB-

Quiniou

et

al.

-RRB-

The

DNA

-LRB-

μg

-RRB-

was

digested

to

completion

with

or

EcoRI

separated

on

agarose

gels

and

transferred

by

capillary

action

onto

Hybond-N

membranes

-LRB-

Amersham

Pharmacia

Biotech

Arlington

Heights

IL

USA

-RRB-

using

standard

techniques

Hybridizations

were

performed

in

Rapid-hyb

buffer

-LRB-

Amersham

Pharmacia

Biotech

-RRB-

at

°C

and

membranes

were

washed

at

high

stringency

-LRB-

°C

with

saline-sodium

citrate

sodium

dodecyl

sulfate

-RRB-

Specific

probes

-LRB-

Table

-RRB-

were

amplified

by

PCR

using

IDPol

DNA

polymerase

-LRB-

ID

Labs

Biotechnology

London

Ontario

Canada

-RRB-

according

to

the

manufacturer

's

recommended

protocol

Typical

parameters

were

min

°C

followed

by

cycles

of

°C

s

°C

s

°C

min

then

extension

at

°C

for

min

Probes

were

random

primed

labeled

with

-LSB-

-RSB-

deoxycytidine

triphosphate

using

a

Megaprime

labeling

kit

-LRB-

Amersham

Pharmacia

Biotech

-RRB-

Table

PCR

primers

used

for

generating

Ig

domain-specific

probes

and

gene-specific

primers

used

in

RT-PCR

experimentsPrimer

GTCGGACAAGGTCAAGTTCTG

GGCTTTTGGTCTCTCTATCAC

AATCCTGATGAACAGGTGTACC

GTGTTTACAGTGGTAGAAACC

AGTCGTGAAGGAGCTGTACTGA

TTCACTGCCAGAATGATGGTCAC

CTGAGGGACATCCTCTGACCTT

GTGTTTACAGTGGTAGAAACC

GTCGGACAAGGTCAAGTTCTG

CTGCAGACATGATGAACTTCT

GTCGGACAAGCTCAAGAGTTT

TGTGTAGTAGAGTGGGTTTCC

GTCGGACAAGGTCAAGTTCTG

GCTTTTGGGTGACTCTCCTCT

GACTGCCACACTGCTCACATTG

TTAGTTACTCAGCAGCTTTCTTCCGSP

gene-specific

primers

Total

RNA

from

catfish

PBL

and

various

tissues

was

prepared

using

RNA-Bee

-LRB-

Tel-test

Friendswood

TX

USA

-RRB-

μg

of

each

sample

were

electrophoresed

on

formaldehyde-agarose

gels

and

transferred

by

capillary

action

onto

Hybond-N

-LRB-

Amersham

Pharmacia

Biotech

-RRB-

hybridized

and

washed

as

above

Reverse

transcription

-LRB-

RT

-RRB-

PCR

Total

RNA

from

PBL

MLC

various

tissues

and

catfish

clonal

cell

lines

were

prepared

as

described

above

and

treated

with

DNase

I

-LRB-

Invitrogen

Life

Technologies

Carlsbad

CA

USA

-RRB-

according

to

the

manufacturer

's

recommended

protocol

One

μg

RNA

was

reverse

transcribed

using

an

oligo-T

primer

and

units

of

Superscript

III

reverse

transcription

-LRB-

RT

-RRB-

-LRB-

Invitrogen

Life

Technologies

-RRB-

RT-PCR

was

performed

using

specific

primers

for

IpLITR

and

the

housekeeping

gene

elongation

factor

alpha

-LRB-

Table

-RRB-

Typical

parameters

were

min

°C

followed

by

cycles

of

°C

s

°C

s

°C

min

then

extension

at

°C

for

min

PCR

products

were

visualized

on

Tris-acetate-EDTA

agarose

gels

PCR

products

were

also

cloned

into

-LRB-

Invitrogen

Life

Technologies

-RRB-

and

verified

by

sequencing

The

Genbank

accession

number

for

is

Results

Catfish

LITR

sequences

In

a

search

for

IgSF

receptors

expressed

by

alloantigen-stimulated

catfish

PBL

and

various

catfish

clonal

cell

lines

several

EST

libraries

were

analyzed

Three

IpLITR

sequences

were

subsequently

identified

that

encoded

type

I

TM

proteins

with

extracellular

Ig

domains

which

were

predicted

using

SMART

-LRB-

see

Electronic

Supplementary

Material

-RRB-

Even

though

these

sequences

vary

in

length

they

are

highly

similar

-LRB-

Fig.

b

-RRB-

All

three

receptors

have

identical

signal

peptides

and

and

have

almost

identically

encoded

Ig

domains

and

-LRB-

Fig.

-RRB-

Comparatively

and

are

and

identical

at

the

amino

acid

level

to

their

and

counterparts

-LRB-

Fig.

-RRB-

encodes

a

amino

acid

extracellular

region

consisting

of

four

Ig

domains

a

acid

TM

segment

and

a

acid

cytoplasmic

tail

-LRB-

CYT

-RRB-

The

CYT

contains

two

immunotyrosine-based

inhibition

motifs

-LRB-

ITIMs

-RRB-

centered

at

and

and

an

ITIM-like

motif

-LRB-

SEYTTE

-RRB-

centered

at

-LRB-

Fig.

-RRB-

-LRB-

Daeron

and

Vivier

Ravetch

and

Lanier

Billadeau

and

Leibson

-RRB-

An

overlapping

immunotyrosine-based

switch

motif

-LRB-

ITSM

TVYSQL

-RRB-

centered

at

is

also

present

in

the

CYT

of

-LRB-

Shlapatska

et

al.

-RRB-

In

contrast

the

smaller

and

the

longer

transcripts

encode

for

molecules

with

identical

acid

TMs

containing

a

lysine

residue

and

very

similar

charged

CYTs

-LRB-

Fig.

-RRB-

Overall

the

individual

IpLITRs

are

composed

of

a

membrane

distal

to

membrane

proximal

ordering

of

Ig

domains

each

containing

similar

and

However

the

membrane

proximal

domains

vary

i.e.

and

are

only

and

identical

respectively

to

all

other

IpLITR

Ig

domains

and

is

only

identical

to

other

IpLITR

domains

-LRB-

Fig.

-RRB-

Fig.

amino

acid

sequence

domain

comparisons

and

schematic

representation

of

and

a

Alignment

of

the

extracellular

and

b

TM/CYT

regions

of

and

Signal

peptide

-LRB-

SP

-RRB-

and

immunoglobulin

-LRB-

Ig

-RRB-

domains

are

labeled

cysteine

residues

predicted

to

be

involved

in

intrachain

disulfide

bonds

are

marked

with

asterisks

gray

shaded

residues

represent

differences

from

in

a

and

differences

from

in

b.

TMs

are

underlined

ITIM-like

motifs

are

boxed

an

overlapping

ITSM

within

the

CYT

of

is

indicated

by

a

bracket

and

TM

charged

residues

are

shaded

black

and

marked

-LRB-

-RRB-

c

Phylogenetic

analysis

of

Ig

domains

in

IpLITRs

NJ

trees

with

pairwise

gap

deletions

were

drawn

using

MEGA

-LRB-

Kumar

et

al.

-RRB-

with

bootstrap

replications

and

bootstrap

values

are

shown

Branch

lengths

were

measured

in

terms

of

amino

acid

substitutions

and

a

scale

bar

are

shown

below

the

trees

The

predicted

SP

Ig

domains

TM

and

CYT

are

indicated

ITIM-like

motifs

are

shown

as

boxes

N-linked

glycosylation

sites

are

marked

as

ballpoint

lines

Individual

IpLITR

domains

are

shaded

according

to

their

relatedness

between

IpLITRs

and

percent

amino

acid

identity

with

Ig

domains

indicated

to

the

left

of

and

and

and

are

identical

to

all

Ig

domains

IpLITRs

belong

to

polymorphic

and

polygenic

gene

families

Sibling

restriction

fragment

length

polymorphism

-LRB-

RFLP

-RRB-

patterns

using

domain-specific

probes

for

the

various

IpLITRs

show

a

large

number

of

hybridizing

bands

with

each

probe

used

as

well

as

differences

in

the

banding

patterns

among

siblings

-LRB-

Fig.

-RRB-

These

findings

indicate

that

the

IpLITR

gene

complex

is

both

polygenic

and

polymorphic

However

it

is

presently

unknown

whether

these

polymorphic

differences

are

due

to

variability

within

the

exons

encoding

these

receptors

and/or

within

the

introns

Some

of

the

high-intensity

bands

found

in

each

of

the

Southern

blots

may

reflect

multiple

IpLITR

copies

further

increasing

the

possible

number

of

genes

Importantly

the

Southern

blot

segregation

patterns

show

that

there

is

more

than

one

IpLITR

locus

For

example

among

the

sibling

fish

used

there

are

at

least

nine

perhaps

more

different

RFLP

patterns

for

IpLITR

-LRB-

Fig.

-RRB-

If

these

genes

were

closely

linked

then

only

four

RFLP

patterns

-LRB-

barring

cross-over

events

-RRB-

would

be

observed

as

shown

for

the

MHC

class

IIβ

RFLP

patterns

-LRB-

Fig.

-RRB-

When

all

Southern

blots

are

compared

at

least

three

distinct

linkage

groups

-LRB-

marked

by

and

-RRB-

are

observed

indicating

the

presence

of

multiple

independently

segregating

loci

and

of

these

are

linked

to

the

MHC

II

RFLP

-LRB-

Fig.

d

-RRB-

Similar

results

were

also

obtained

using

the

probe

-LRB-

data

not

shown

-RRB-

Mining

of

the

zebrafish

genome

for

IpLITR

relatives

-LSB-

whole

genome

shotgun

database

of

the

NCBI

-LRB-

Sanger

Genomic

Institute

Cambridge

UK

-RRB-

-RSB-

further

supports

that

these

receptors

are

present

on

different

chromosomes

because

five

contigs

were

identified

with

IpLITR-like

homologs

on

zebrafish

chromosomes

and

-LRB-

Table

-RRB-

Fig.

blot

analyses

Genomic

DNA

from

sibling

catfish

was

digested

separated

transferred

to

nylon

membranes

and

hybridized

with

a

MHCIIβ

b

c

and

d

probes

Letters

a

i

indicate

different

segregation

patterns

-LRB-

b

e

and

h

represent

variant

segregation

patterns

that

may

be

the

result

of

recombination

-RRB-

Representative

RFLP

bands

illustrating

different

linkage

groups

are

indicated

by

and

Kilobase

size

markers

are

indicated

to

the

left

of

each

blotTable

of

IpLITR-like

genes

in

the

zebrafish

genomeAccession

Search

-LRB-

-RRB-

of

the

zebrafish

whole

genome

shotgun

database

of

the

NCBI

-LRB-

Sanger

Genomic

Institute

-RRB-

IpLITR

expression

Northern

blot

analyses

using

a

cross

hybridizing

probe

revealed

that

LITR

message

is

highly

expressed

in

the

hematopoietic

pronephros

and

mesonephros

with

predominant

transcript

sizes

of

and

Kb

-LRB-

Fig.

-RRB-

Low

levels

of

IpLITR

expression

were

detected

in

the

spleen

with

little

to

no

message

detected

in

the

heart

liver

gill

or

muscle

The

use

of

the

more

specific

probe

revealed

message

expression

not

only

in

the

pronephros

and

mesonephros

but

also

in

the

spleen

gill

and

perhaps

heart

with

transcripts

of

and

Kb

-LRB-

Fig.

-RRB-

RNA

load

levels

for

each

of

the

tissues

tested

were

similar

based

upon

hybridization

with

a

catfish

probe

RT-PCR

using

primers

specific

for

the

putative

inhibitory

and

the

putative

activating

as

representative

IpLITRs

was

also

used

to

assess

tissue

expression

As

shown

in

Fig.

and

are

in

most

cases

coordinately

expressed

in

a

variety

of

different

tissues

and

confirm

the

Northern

blot

data

In

addition

multiple

PCR

products

were

amplified

using

the

and

specific

primers

which

is

reminiscent

of

the

multiple

transcripts

observed

following

RNA

blotting

Because

the

predicted

sizes

of

PCR

products

for

the

prototype

and

are

and

bp

respectively

at

least

two

additional

products

-LRB-

and

bp

-RRB-

and

one

additional

product

-LRB-

bp

-RRB-

were

amplified

-LRB-

Fig.

-RRB-

Fig.

blot

and

RT-PCR

analyses

of

IpLITR

tissue

expression

Catfish

LITR

tissue

expression

was

performed

by

Northern

blot

analysis

using

a

an

probe

and

b

an

probe

Total

RNA

from

pronephros

-LRB-

head

kidney

-RRB-

mesonephros

-LRB-

trunk

kidney

-RRB-

spleen

heart

liver

gill

and

muscle

were

examined

Kilobase

markers

are

on

the

left

margin

and

arrows

indicate

the

major

hybridizing

bands

observed

RNA

integrity

and

load

levels

were

determined

by

hybridization

using

a

catfish

probe

as

a

housekeeping

gene

c

RT-PCR

analyses

of

and

in

various

catfish

tissues

The

sizes

of

the

IpLITR

bands

verified

by

sequencing

are

indicated

at

the

right

margin

and

base

pair

sizes

are

at

the

left

margin

To

further

examine

the

expression

of

and

RT-PCR

was

performed

using

RNA

from

catfish

clonal

macrophage

B

T

and

NK-like

cell

lines

as

well

as

a

MLC

-LRB-

Fig.

-RRB-

RT-PCR

products

of

and

bp

were

obtained

using

primers

and

these

products

were

detected

variably

among

the

cell

lines

examined

It

was

also

found

that

three

RT-PCR

products

were

also

variably

amplified

from

the

different

catfish

leukocyte

cell

lines

using

primers

the

expected

product

a

product

-LRB-

previously

observed

in

tissues

see

Fig.

-RRB-

and

an

additional

product

-LRB-

Fig.

-RRB-

In

contrast

no

IpLITR

message

was

detected

in

the

fibroblast

CCO

cell

line

-LRB-

data

not

shown

-RRB-

indicating

that

these

genes

are

primarily

expressed

in

cells

of

immune

function

Fig.

RT-PCR

analyses

of

and

expression

in

various

catfish

clonal

cell

lines

and

a

polyclonal

MLC

Total

RNA

was

obtained

from

the

catfish

macrophages

B

cells

and

nonautonomous

cytotoxic

T

cells

autonomous

T

cells

a

MLC

and

NK-like

cells

RT-PCR

was

performed

using

primers

specific

for

and

The

sizes

of

the

IpLITR

bands

verified

by

sequencing

are

indicated

at

the

right

margin

and

base

pair

sizes

are

at

the

left

margin

b

Schematic

representation

of

IpLITR-types

identified

by

sequencing

of

RT-PCR

products

obtained

using

and

primers

The

originally

identified

prototype

IpLITRs

are

boxed

and

arrows

indicate

the

relative

positions

of

the

primer

pairs

used

in

RT-PCR

reactions

Sizes

in

base

pairs

corresponding

to

the

bands

observed

in

a

are

indicated

above

each

schematic

The

predicted

SP

Ig

domains

TM

and

CYT

are

indicated

ITIM-like

motifs

are

shown

as

hatched

boxes

and

noncanonical

immunotyrosine-based

activation

motif-like

motifs

shown

as

white

boxes

within

the

CYT

Individual

Ig

domains

are

shaded

according

to

relatedness

as

described

for

Fig.

and

percentages

above

each

receptor

represent

percentage

of

amino

acid

identities

of

the

predicted

extracellular

region

of

the

IpLITR-like

sequences

when

compared

with

the

prototype

IpLITRs

The

sequencing

of

the

various

and

RT-PCR

products

from

all

sources

-LRB-

both

tissues

and

cell

lines

-RRB-

resulted

in

the

identification

of

multiple

different

IpLITR-related

sequences

-LRB-

Stafford

et

al.

manuscript

in

preparation

-RRB-

Schematic

diagrams

of

representative

and

variant

forms

are

shown

in

Fig.

These

highly

related

and

sequences

differed

in

the

number

of

Ig-like

domains

present

and

in

the

case

of

some

also

differed

in

the

length

of

the

CYT

Homology

between

IpLITRs

LRC

and

FcR

genes

When

the

extracellular

region

of

was

used

in

BLASTP

analysis

various

adhesion

molecules

FcRs

FcR-like

proteins

-LRB-

FCRLs

-RRB-

LILRs

and

KIR

gene

family

members

were

identified

with

significant

homology

-LRB-

i.e.

E

values

-RRB-

albeit

with

moderate

amino

acid

identities

ranging

from

-LRB-

Table

-RRB-

Examples

include

the

adhesion

molecules

and

carcinoembryonic

antigen-related

cell

adhesion

molecule

Xenopus

leukocyte

FCRLs

XFL

and

FcγRIIB

When

the

Ig

domains

of

were

used

for

screening

most

matches

were

members

of

the

FcR

gene

family

e.g.

FcγRI

FcγRII

FcγRIII

and

FcεRIα

from

humans

primates

and

rodents

as

well

as

various

FCRL

proteins

-LRB-

Table

-RRB-

Searches

using

the

extracellular

region

of

were

essentially

the

same

as

the

BLASTP

results

and

PSI-BLAST

analysis

further

reinforced

the

evolutionary

relationships

between

IpLITRs

and

mammalian

FcRs

FCRLs

KIRs

and

LILRs

as

well

as

avian

CHIRs

-LRB-

data

not

shown

-RRB-

Table

IpLITR

BLASTP

resultsReceptor

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

FcγRIIGuinea

-LRB-

-RRB-

-LRB-

-RRB-

MouseAAS91578606e-83FcγRIMacaqueAAL92095591e-7

-LRB-

-RRB-

HumanNP_112571574e-71q21FcγRIαHumanCAI12557575e-71q21FcγRIIBMouseAAH19758569e-71XFL1

FcεRIαHumanAAH05912538e-61q23FREB2HumanAAX11390538e-61q23.3

Similarity

search

-LRB-

BLASTP

-RRB-

of

the

NCBI

's

nonredundant

protein

database

using

the

four

Ig

domains

of

and

the

three

Ig

domains

of

carcinoembryonic

antigen-related

cell

adhesion

molecule

XFLXenopus

leukocyte

FCRL

Phylogenetic

analyses

and

alignments

The

IpLITR

and

Ig

domains

are

related

to

those

of

mammalian

FcRs

and

FCRLs

-LRB-

Fig.

-RRB-

in

that

these

domains

clustered

with

the

FcR/FCRLs

and

away

from

catfish

NITRs

and

from

representative

LRC

members

in

a

phylogenetic

tree

In

contrast

to

the

sequence

analyses

the

membrane

proximal

and

sequences

of

and

resulted

in

a

significant

shift

within

the

phylogenetic

tree

IpLITRs

now

clustered

with

the

LRC-encoded

genes

-LRB-

Fig.

-RRB-

The

LRC

relationship

is

supported

by

high

bootstrap

values

when

comparing

and

and

sequences

with

and

sequences

of

representative

LILRs

and

KIRs

as

well

as

with

and

sequences

of

and

CHIRs

The

and

sequences

of

are

related

to

the

and

domains

of

the

FcR

family

-LRB-

Fig.

-RRB-

Thus

is

the

best

example

of

the

unique

domain

structure

of

these

teleost

receptors

as

it

encodes

for

combinations

of

Ig

domains

that

are

phylogenetically

related

to

the

FcRs

and

FCRLs

-LRB-

and

and

-RRB-

as

well

as

LRC

encoded

receptors

-LRB-

and

-RRB-

Fig.

analyses

reveal

unique

Ig

domain

composition

within

individual

IpLITRs

The

Ig

domains

compared

are

indicated

by

gray

shading

in

the

schematics

to

the

left

of

each

phylogenetic

tree

a

Comparisons

of

IpLITR

representative

FcR

and

FCRL

and

human

LRC

and

sequences

b

Comparisons

of

and

with

representative

FcR

FCRL

KIR

LILR

and

and

sequences

as

well

as

and

and

sequences

c

Comparison

of

and

with

and

of

representative

mammalian

FcRs

FCRLs

and

LILRs/KIRs

The

accession

numbers

for

the

various

Ig

domain

sequences

used

are

human

-LRB-

hu

-RRB-

FcγRI

-LRB-

-RRB-

huFcγRII

-LRB-

-RRB-

huFcγRIII

-LRB-

-RRB-

huFcεRI

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

huSIRP

-LRB-

-RRB-

human

-LRB-

-RRB-

mouse

-LRB-

mo

-RRB-

FcγRI

-LRB-

-RRB-

moFcγRII

-LRB-

-RRB-

moFcγRIII

-LRB-

-RRB-

moFcεRI

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

rat

-LRB-

ra

-RRB-

FcγRIII

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

guinea

pig

-LRB-

gp

-RRB-

FcγRII

-LRB-

-RRB-

macaque

-LRB-

mac

-RRB-

FcγRI

-LRB-

-RRB-

bovine

-LRB-

bo

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

boSIRP

-LRB-

-RRB-

chimpanzee

-LRB-

cp

-RRB-

-LRB-

-RRB-

orangutan

-LRB-

or

-RRB-

KIR

-LRB-

-RRB-

monkey

-LRB-

mk

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

catfish

-LRB-

Ip

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

For

all

analyses

SIRPs

and/or

IpNITRs

were

included

as

outgroups

to

root

the

trees

NJ

trees

with

pairwise

gap

deletions

were

drawn

using

MEGA

-LRB-

Kumar

et

al.

-RRB-

with

bootstrap

replications

and

bootstrap

values

are

shown

Branch

lengths

were

measured

in

terms

of

amino

acid

substitutions

and

scale

bars

are

shown

below

the

trees

Subsequently

the

and

Ig

domain

sequences

of

-LRB-

as

a

representative

-RRB-

were

aligned

with

various

mouse

and

human

FcRs

and

three

representative

human

FCRLs

A

majority

of

the

predicted

β-strands

in

is

present

in

similar

positions

as

the

β-strands

found

in

FcR/FCRL

family

members

-LRB-

Fig.

-RRB-

indicating

a

predicted

degree

of

secondary

structural

conservation

between

IpLITRs

and

these

mammalian

immune

receptors

In

most

cases

the

predicted

β-strands

were

present

in

regions

of

high

amino

acid

identity/similarity

however

of

the

FcR

residues

known

to

contact

the

Fc

portion

of

Ig

were

present

-LRB-

Fig.

-RRB-

The

of

a

domain

specific

to

this

IpLITR

type

appears

to

be

related

to

the

membrane

distal

domain

-LRB-

-RRB-

of

several

CHIRs

-LRB-

Fig.

-RRB-

and

alignment

of

with

representative

CHIR

indicated

a

degree

of

amino

acid

identity/conservation

between

these

Ig

domains

-LRB-

Fig.

-RRB-

To

reinforce

the

unique

nature

of

domains

present

within

this

receptor

did

not

cluster

with

the

CHIRs

but

was

related

to

mammalian

FcR

domains

-LRB-

Fig.

-RRB-

Fig.

contains

Ig

domains

related

to

both

FcRs/FCRLs

and

CHIRs

a

Amino

acid

alignment

of

and

sequences

with

representative

mammalian

FcRs

and

FCRLs

Accession

numbers

are

as

in

Fig.

Gray

shading

residues

similar/identical

to

Boxed

residues

contacts

for

Ig

Fc

in

huFcεRI

Black

and

gray

arrows

represent

the

predicted

β-strands

for

and

huFcεRI

respectively

Hatched

boxes

indicate

conserved

cyteines

and

dashes

represent

gaps

b

Phylogenetic

analysis

of

and

sequences

-LRB-

gray

shaded

-RRB-

compared

with

representative

mammalian

FcR

sequences

-LRB-

as

in

Fig.

-RRB-

and

sequences

of

representative

CHIRs

The

accession

numbers

for

the

various

Ig

domain

sequences

used

are

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

CHIR

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

NJ

trees

with

pairwise

gap

deletions

were

drawn

using

MEGA

-LRB-

Kumar

et

al.

-RRB-

with

bootstrap

replications

and

bootstrap

values

are

shown

Branch

lengths

were

measured

in

terms

of

amino

acid

substitutions

and

scale

bars

are

shown

below

the

trees

c

Alignment

of

and

CHIR

Gray

shading

indicates

residues

that

are

similar/identical

to

black

and

gray

arrows

represent

predicted

β-strands

for

and

respectively

dotted

lines

indicate

predicted

helices

and

hatched

boxes

indicate

conserved

cyteines

Gaps

in

alignment

are

indicated

by

dashes

Attempts

to

model

IpLITR

Ig

domains

were

unsuccessful

as

no

sufficient

modeling

templates

were

identified

using

SWISS-Model

However

similarity

searches

against

the

NCBI

's

protein

data

bank

-LRB-

PDB

-RRB-

indicated

a

significant

match

between

and

of

IpLITRs

and

the

Ig

domains

of

human

FcεRIα

-LRB-

E

value

-RRB-

Conversely

the

membrane

proximal

and

Ig

domains

of

and

had

significant

alignment

scores

-LRB-

E

value

-RRB-

with

the

first

two

Ig

domains

of

the

human

natural

cytotoxicity

receptor

Taken

together

these

PDB

search

results

further

support

that

IpLITRs

and

Ig

domain

sequences

are

related

to

mammalian

FcRs

and

FCRLs

and

that

the

and

Ig

domains

are

related

to

LRC

encoded

receptors

Mining

of

the

zebrafish

genome

identified

five

contigs

-LRB-

Table

-RRB-

containing

sequences

encoding

IpLITR-like

proteins

predicted

to

contain

several

Ig

domains

and

in

some

cases

signal

peptides

TM

segments

and

CYTs

with

signaling

motifs

-LRB-

Fig.

-RRB-

These

findings

indicate

that

IpLITR-like

molecules

are

not

unique

to

the

catfish

but

are

likely

common

in

other

teleost

species

Because

was

identified

as

the

closest

match

from

the

NCBI

's

database

to

the

zebrafish

-LRB-

Zf

-RRB-

LITR-like

proteins

it

was

used

for

further

comparisons

Phylogenetic

analysis

of

the

various

Ig

domain

sequences

indicated

that

the

ZfLITR-like

proteins

were

closely

related

to

those

of

IpLITRs

-LRB-

Fig.

-RRB-

In

most

cases

the

membrane

proximal

domains

of

ZfLITRs

are

highly

related

to

their

counterparts

with

amino

acid

identity

-LRB-

Fig.

-RRB-

However

one

of

the

ZfLITRs

-LRB-

-RRB-

contains

two

Ig

domains

similar

to

in

the

membrane

distal

region

This

zebrafish

receptor

also

contains

three

Ig

domains

that

are

phylogentically

related

-LRB-

Fig.

-RRB-

with

high

amino

acid

identity

-LRB-

-RRB-

to

-LRB-

Fig.

-RRB-

The

other

ZfLITR-like

proteins

are

also

composed

of

combinations

of

Ig

domains

that

are

related

to

Ig

domains

with

varying

degrees

of

amino

acid

identities

-LRB-

e.g.

-RRB-

However

of

the

predicted

ZfLITR-like

molecules

contained

membrane

distal

Ig

domains

that

were

related

to

IpLITR

and

which

are

shared

among

all

the

IpLITRs

identified

to

date

This

suggests

either

that

LITR

membrane

distal

domains

may

have

coevolved

with

distinct

ligands

within

individual

fish

species

or

that

ZfLITRs

with

and

domains

similar

to

IpLITRs

have

yet

to

be

identified

It

should

be

noted

however

that

these

zebrafish

domains

were

obtained

from

genomic

sequence

and

that

they

do

not

accurately

represent

the

precise

organization

of

the

Ig

domains

likely

to

be

present

in

encoded

ZfLITRs

which

will

only

be

determined

from

cDNA

sequences

Nonetheless

the

genomic

segments

are

useful

for

domain-to-domain

comparisons

and

reinforced

the

presence

of

IpLITR-like

sequences

in

another

teleost

species

Fig.

of

zebrafish

LITR-like

proteins

a

Phylogenetic

analysis

of

ZfLITR

and

Ig

domains

Ig

domains

were

predicted

using

SMART

-LRB-

Letunic

et

al.

-RRB-

and

the

amino

acid

sequences

aligned

using

CLUSTALW

-LRB-

Thompson

et

al.

-RRB-

NJ

trees

with

pairwise

gap

deletions

were

drawn

using

MEGA

-LRB-

Kumar

et

al.

-RRB-

Only

branch

values

are

shown

and

the

major

Ig

domain

groups

are

designated

by

brackets

and

shaded

according

to

their

relatedness

with

Ig

domain

b

Schematic

representation

of

ZfLITR-like

proteins

compared

with

Domains

are

shaded

according

to

their

phylogenetic

relationship

with

domains

Hatched

bars

on

and

Zf

indicate

signal

peptides

TM

segments

are

solid

black

lines

and

ITIMs

are

shown

as

gray

boxes

for

Amino

acid

identity

vs

corresponding

Ig

domains

are

indicated

as

percentage

value

above

each

domain

and

only

and

were

related

to

the

Ig

domains

of

the

representative

ZfLITR

analyzed

as

indicated

by

the

dashed

box

Predicted

size

in

amino

acids

for

each

molecule

is

indicated

on

the

left

Discussion

The

novel

IpLITRs

described

herein

represent

a

polymorphic

and

polygenic

gene

complex

that

are

primarily

expressed

in

hematopoietic

tissues

The

polymorphic

nature

of

this

gene

complex

was

indicated

by

RFLP

analysis

which

revealed

differences

in

where

various

restriction

enzymes

cut

the

genomic

DNA

among

the

sibling

catfish

However

it

should

be

noted

that

only

detailed

sequence

analyses

of

these

IpLITR

genes

in

a

number

of

individuals

would

be

required

to

determine

if

any

polymorphic

differences

are

also

found

within

the

coding

regions

In

addition

segregation

analyses

suggested

that

IpLITR

genes

are

encoded

within

multiple

independently

segregating

but

homologous

loci

similar

to

the

situation

seen

with

the

ZfLITR

homologs

identified

by

genome

mining

These

paralagous

relationships

strongly

suggest

that

IpLITRs

are

products

of

multiple

gene

duplication

and

translocation

events

from

a

common

ancestoral

gene

Whether

or

not

the

receptors

encoded

by

the

different

IpLITR

loci

are

functionally

redundant

remains

to

be

determined

However

IpLITRs

are

related

to

functionally

and

genomically

distinct

mammalian

FcRs

and

LRC

encoded

receptors

suggesting

that

such

paralogous

loci

could

have

evolved

to

give

rise

to

an

array

of

immunoregulatory

receptors

with

different

functions

The

coordinate

expression

of

IpLITRs

by

myeloid

and

lymphoid

cell

lines

in

combination

with

their

potential

activating

and

inhibitory

signaling

capabilities

suggests

that

they

play

an

important

role

in

immune

cell

function

-LRB-

s

-RRB-

This

possible

importance

of

IpLITRs

in

immune

responses

is

highlighted

by

the

RT-PCR

expression

studies

where

a

large

number

of

highly

related

but

unique

IpLITR

sequences

were

identified

These

variant

IpLITR

sequences

differed

in

the

number

of

Ig

domains

present

and

in

some

cases

in

the

length

of

the

CYT

Therefore

in

addition

to

the

prototype

and

described

here

other

IpLITR

family

members

are

coexpressed

by

catfish

myeloid

and

lymphoid

cells

Whether

or

not

some

of

the

variously

sized

transcripts

represent

messages

from

different

IpLITR

genes

or

are

the

result

of

alternative

splicing

is

unknown

However

the

large

number

of

unique

IpLITRs

found

expressed

suggests

that

IpLITRs

represent

a

complex

immune

receptor

family

that

can

potentially

generate

a

large

number

of

IpLITR

types

The

prototypical

and

encode

receptors

with

four

three

and

six

Ig

domains

respectively

Pairwise

comparisons

and

phylogenetic

analysis

of

these

domains

suggest

a

high

degree

of

conservation

and

a

conserved

membrane

distal

to

membrane

proximal

ordering

Each

of

these

three

receptors

has

very

similar

and

sequences

and

and

have

almost

identical

and

However

variation

does

occur

between

predicted

membrane

proximal

Ig

domains

The

sequence

is

unique

to

this

receptor

and

and

are

not

similar

to

any

other

of

the

prototypical

IpLITR

Ig

domain

sequences

Identification

of

LITR-like

sequences

on

different

zebrafish

chromosomes

and

comparisons

with

IpLITRs

supports

the

notion

that

these

polymorphic

and

polygenic

immune

receptors

are

not

unique

to

catfish

Like

IpLITRs

the

predicted

ZfLITRs

are

also

composed

of

unique

combinations

of

Ig

domains

Such

differences

in

the

Ig

domain

compositions

of

IpLITRs

and

ZfLITRs

may

influence

their

ligand

binding

capabilities

formation

of

homo

or

heterodimers

and

overall

immunomodulatory

functions

has

a

long

CYT

with

two

consensus

ITIMs

and

a

third

ITIM-like

motif

whereas

both

and

have

shorter

CYTs

devoid

of

recognizable

signaling

motifs

However

these

latter

two

receptors

each

contain

a

lysine

residue

within

their

TM

segments

indicating

that

associations

with

an

adaptor

molecule

-LRB-

s

-RRB-

would

be

necessary

for

them

to

initiate

intracellular

signaling

-LRB-

Daeron

and

Vivier

Ravetch

and

Lanier

Billadeau

and

Leibson

-RRB-

Within

the

CYT

of

IpLITRI

was

also

an

overlapping

ITSM

suggesting

that

multiple

motifs

-LRB-

ITIMs

and

ITSM

-RRB-

within

the

CYT

of

this

receptor

may

participate

in

downstream

signaling

cascades

through

recruitment

of

various

adaptor

molecules

-LRB-

Sidorenko

and

Clark

-RRB-

Similarly

predicted

ZfLITR

family

members

were

shown

to

either

contain

long

CYTs

with

ITIM

motifs

or

short

CYTs

with

charged

TM

segments

Genes

for

immunotyrosine-based-activation-motif-bearing

adaptor

molecules

-LRB-

e.g.

FcRγ

chain

and

DAP

-RRB-

have

been

identified

in

several

ectothermic

vertebrates

including

frogs

and

fish

-LRB-

Guselnikov

et

al.

b

-RRB-

Hence

it

seems

likely

that

and

would

each

associate

with

such

molecules

and

function

in

a

similar

manner

as

mammalian

receptors

with

charged

TM

segments

Thus

the

paired

expression

pattern

of

IpLITRs

with

activatory

and

inhibitory

signaling

potential

is

very

similar

to

that

shown

for

several

mammalian

and

avian

families

of

immune

receptors

including

PIRs

LILRs

KIRs

FcRs

and

CHIRs

Similarity

searches

for

IpLITR

relatives

revealed

a

striking

dichotomy

The

majority

of

matches

were

members

of

two

distinct

mammalian

receptor

families

While

appeared

to

be

more

closely

related

to

FcRs

and

FCRLs

and

exhibiting

very

similar

sequences

were

related

to

both

FcR/FCRLs

and

LRC-encoded

receptors

Thus

it

is

postulated

that

the

and

domains

common

to

all

IpLITRs

are

evolutionarily

more

closely

related

to

domains

encoded

by

the

mammalian

FcR

and

FCRLs

a

contention

supported

by

the

retrieval

of

the

human

FcεRI

alpha

chain

as

the

most

similar

structure

from

a

PDB

search

Phylogenetic

analyses

also

supported

this

hypothesis

because

the

IpLITR

and

clustered

with

members

of

the

FcR

and

FCRL

receptor

families

and

away

from

representative

LRC

gene

family

members

Although

the

of

IpLITRs

are

phylogenetically

related

to

FcR

and

FcRLs

there

is

no

evidence

to

support

that

IpLITRs

bind

Ig

It

appears

more

likely

that

the

relatedness

of

these

membrane

distal

Ig

domains

as

also

demonstrated

for

FCRLs

PIRs

and

CHIRs

are

only

evident

at

the

phylogenetic

level

and

that

they

are

not

functional

homologs

Furthermore

during

the

preparation

of

this

manuscript

a

bona

fide

Ig-binding

catfish

FcR

homolog

which

is

not

a

member

of

the

IpLITR

receptor

family

was

identified

and

characterized

-LRB-

Stafford

et

al.

-RRB-

In

contrast

to

the

and

domains

and

and

Ig

domains

are

related

to

several

members

of

the

LRC

-LRB-

human

chromosome

-RRB-

As

above

these

two

domains

clustered

with

one

family

of

receptors

-LRB-

the

LRC

-RRB-

and

not

the

other

-LRB-

i.e.

FcR/FcRLs

-RRB-

The

identification

of

as

a

structural

relative

from

the

PDB

also

supports

this

notion

Interestingly

and

of

are

related

to

FcR

and

FCRLs

but

their

membrane

proximal

location

suggests

that

they

are

unlikely

to

be

involved

in

ligand

recognition

-LRB-

i.e.

Ig

-RRB-

Finally

the

third

domain

of

clustered

with

the

Ig

domains

of

several

representative

CHIRs

Taken

together

IpLITR

extracellular

domain

sequences

appear

reminiscent

of

both

FcR/FCRLs

and

members

of

the

mammalian

and

avian

LRC

implying

that

catfish

LITRs

are

distant

relatives

to

each

of

these

genomically

and

functionally

diverse

receptor

gene

families

At

present

the

functional

significance

of

these

novel

teleost

immune

receptors

is

unknown

Recently

key

insights

into

the

evolution

of

the

FcR

and

LRC

gene

families

and

their

potential

common

origin

has

been

presented

-LRB-

-RRB-

human

chromosomes

and

are

believed

to

be

in

paralogous

regions

of

the

genome

due

to

an

entire

genome

duplication

event

-LRB-

Kasahara

Shiina

et

al.

-RRB-

-LRB-

-RRB-

attempts

at

cloning

rodent

FcαR

-LRB-

-RRB-

orthologs

identified

the

PIR

gene

family

located

on

a

mouse

chromosome

which

is

syntenic

to

the

human

LRC

-LRB-

Kubagawa

et

al.

Hayami

et

al.

-RRB-

-LRB-

-RRB-

the

subsequent

search

for

distant

PIR

relatives

resulted

in

the

discovery

of

the

CHIRs

providing

the

first

molecular

evidence

for

a

common

ancestor

of

LRC

and

FcR

gene

families

-LRB-

Dennis

et

al.

-RRB-

-LRB-

-RRB-

sequencing

of

genomic

segments

encoding

the

CHIR

gene

family

identified

the

chicken

equivalent

of

an

LRC

that

appears

to

have

also

been

expanded

by

gene

duplication

events

-LRB-

Nikolaidis

et

al.

-RRB-

and

-LRB-

-RRB-

the

identification

of

Ig-like

domains

in

chickens

Xenopus

and

bony

fish

that

are

related

to

Ig

domains

of

mammalian

FcRs

and

members

of

the

LRC

-LRB-

Nikolaidis

et

al.

-RRB-

Taken

together

these

findings

suggest

that

immune

receptors

encoded

by

human

chromosome

and

most

likely

evolved

from

a

common

ancestor

that

has

since

undergone

multiple

rounds

of

duplication

and

homologous

recombination

events

Although

there

is

functional

evidence

for

the

existence

of

FcR

or

KIR/LILR-like

homologs

in

ectothermic

vertebrates

-LRB-

Coosemans

and

Hadji-Azimi

Shen

et

al.

-RRB-

their

genetic

identification

has

not

been

previously

reported

and

the

inability

to

clone

these

receptors

has

made

it

difficult

to

elucidate

their

evolutionary

history

The

recent

discovery

of

mammalian

FCRL

genes

located

on

chromosome

however

has

expanded

the

view

of

immune

receptor

evolution

-LRB-

Davis

et

al.

Hatzivassiliou

et

al.

Miller

et

al.

Guselnikov

et

al.

-RRB-

For

example

and

not

only

encode

and

Ig

domains

homologous

to

the

corresponding

domains

of

FcγRI

but

also

contain

variable

numbers

of

unique

membrane

proximal

Ig

domains

-LRB-

Davis

et

al.

Hatzivassiliou

et

al.

-RRB-

Homology

searches

demonstrated

that

the

FCRLs

also

termed

IFGP

-LRB-

for

IgSF

FcR

and

-RRB-

exhibited

a

chimeric

Ig

domain

compositions

and

consequently

these

receptors

were

proposed

to

be

a

phylogenetic

link

between

the

FcRs

rat

NK

cell-specific

antigen

and

adhesion

molecules

-LRB-

Guselnikov

et

al.

-RRB-

This

feature

of

similar

Ig

domains

encountered

in

homologous

but

functionally

diverse

receptors

is

also

a

striking

feature

of

the

IpLITRs

and

supports

the

hypothesis

that

certain

mammalian

immune

receptor

families

-LRB-

i.e.

FcRs

and

KIRs

-RRB-

have

evolved

from

a

common

ancestor

Notably

although

the

IpLITRs

and

domains

are

phylogenetically

and

structurally

similar

to

the

mammalian

FcRs

and

FCRLs

the

IpLITR

ligand

-LRB-

s

-RRB-

is

unknown

This

is

similar

to

the

situation

with

mammalian

FCRL

molecules

whose

membrane

distal

Ig

domains

are

clearly

related

to

the

classic

FcRs

but

are

unlikely

to

be

functional

homologs

because

they

do

not

bind

Ig

-LRB-

Hatzivassiliou

et

al.

Davis

et

al.

-RRB-

Therefore

although

certain

Ig

domains

encoded

by

several

receptor

gene

families

identified

in

humans

rodents

birds

and

fish

-LRB-

i.e.

FCRLs

PIRs

CHIRs

and

IpLITRs

respectively

-RRB-

are

related

to

FcRs

they

appear

to

have

evolved

to

recognize

different

ligands

Recently

it

was

shown

that

rodent

PIRs

recognized

MHC

class

and

-LRB-

Takai

-RRB-

raising

the

possibility

that

related

immunoregulatory

receptors

in

other

species

-LRB-

i.e.

CHIRs

and

IpLITRs

-RRB-

could

also

be

involved

in

MHC

recognition

In

summary

the

discovery

of

individual

catfish

receptors

that

encode

Ig

domains

related

to

both

FcR

and

LRC

gene

families

provides

further

support

for

the

common

origin

of

these

important

mammalian

immune

receptors

Identification

of

LITR-like

sequences

in

the

zebrafish

genome

also

demonstrates

that

these

receptors

are

likely

found

in

all

teleosts

Annotation

of

these

genomic

segments

will

be

necessary

to

determine

if

zebrafish

LITR

homologs

exhibit

conserved

genomic

features

and

syntenic

genomic

location

with

mammalian

and/or

Thus

the

discovery

of

IpLITRs

is

an

important

step

towards

further

understanding

the

common

origin

and

evolutionary

history

of

the

different

families

of

mammalian

immunoregulatory

receptors

The

investigation

of

the

functional

significance

of

these

novel

teleost

receptors

is

underway

and

will

be

aided

by

the

availability

of

channel

catfish

cell

lines

that

coordinately

express

IpLITRs

and

the

eventual

production

of

IpLITR

specific

antibodies

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Electronic

Supplementary

Figure

kb

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

molecular immunology
inhibitory receptors
innate immunity


An integrated approach to determine left atrial volume , mass and function in hypertrophic cardiomyopathy by two-dimensional echocardiography Methods The study included 25 hypertrophic cardiomyopathy -LRB- HCM -RRB- patients -LRB- 15 non-obstructive and 10 obstructive -RRB- and 25 controls for assessment of left atrial -LRB- LA -RRB- volume , mass and function by two-dimensional echocardiography . Measurement included mean LA diameter -LRB- LAD -RRB- , LA mass = -LCB- -LRB- mean LAD + anterior LA wall + posterior LA wall -RRB- 3 − mean LAD3 -RCB- × 0.8 + 0.6 , LA volume = -LSB- -LRB- 8/3 π L · A1 · A2 -RRB- , where L is LA length , A1 and A2 are LA area in 4-chambers and 2-chambers , respectively -RSB- including maximum -LRB- Vmax -RRB- , minimum -LRB- Vmin -RRB- , and pre-atrial contraction -LRB- Vpre-A -RRB- , total atrial stroke volume -LRB- TA-SV -RRB- , TA emptying fraction -LRB- TA-EF -RRB- , active atrial SV -LRB- AA-SV -RRB- , AA-EF , passive atrial SV -LRB- PA-SV -RRB- , PA-EF , atrial expansion index -LRB- AEI -RRB- , and LA kinetic energy -LRB- LA-KE -RRB- = 1/2 × AA-SV × P × V2 . Introduction Hypertrophic cardiomyopathy -LRB- HCM -RRB- is a relatively common form of genetic heart disease affecting approximately 1 in 500 in the general population -LSB- 1 , 2 -RSB- . The pathophysiologic appearance of HCM is complex and includes dynamic left ventricular outflow tract -LRB- LVOT -RRB- obstruction , mitral regurgitation , diastolic dysfunction , myocardial ischemia and cardiac arrhythmia -LSB- 3 -RSB- . Diastolic dysfunction is more common than systolic dysfunction in HCM due to marked left ventricular hypertrophy , interstitial fibrosis and myocardial ischemia . These factors contribute to elevated left atrial -LRB- LA -RRB- and pulmonary vascular pressures -LSB- 4 -RSB- . LA modulates left ventricular filling through three components , a phase of reservoir component or expansion during systole , a conduit phase during diastole and an active contractile component during late diastole -LSB- 5 -RSB- . The active LA contraction has an important role in patients with reduced left ventricular compliance as it is compensating the decreased early filling -LSB- 6 , 7 -RSB- . In HCM , stiffness of LA increases and this affects its reservoir function and may in turn affects the cardiac output -LSB- 8 -RSB- . LA enlargement is a common findings in HCM and associated with increased morbidity and mortality -LSB- 9 , 10 -RSB- . The present study aimed to assess LA size , mass and function in HCM patients compared with normal subjects . Patients and methods A retrospective study included 25 patients -LRB- 80 % males , mean age 38 ± 15 years -RRB- with an established diagnosis of HCM -LSB- 11 -RSB- and good two-dimensional echocardiography -LRB- 2DE -RRB- image quality . According to the type of HCM , patients were classified into two groups the non-obstructive group included 15 patients with resting LVOT gradient < 30 mm Hg , and the obstructive group included 10 patients with resting LVOT gradient ≥ 30 mm Hg . A group of 25 normal age-matched adults -LRB- mean age 35 ± 16 years -RRB- served as control subjects . None of them had evidence of cardiovascular disease by clinical examination , resting ECG and echocardiographic examination . 2DE was undertaken for HCM patients and control subjects with lying in the left lateral decubitus using both apical and parasternal views . 2DE studies were performed using a 3.5 MHz transducer and a commercially available ultrasound system -LRB- Philips Sonos 7500 , Best , The Netherlands -RRB- . The following measures were obtained . Left ventricular -LRB- LV -RRB- function LV fractional shortening and ejection fraction were selected as a marker for systolic function . LV end-diastolic -LRB- LV-EDD -RRB- and end-systolic -LRB- LV-ESD -RRB- dimensions were measured using M-mode from parasternal long axis view and thus LV fractional shortening were calculated by the traditional formula : Fractional shortening -LRB- % -RRB- = -LSB- LV-EDD − LV-ESD -RSB- / LV-EDD % . LV end-diastolic -LRB- LV-EDV -RRB- and end-systolic -LRB- LV-ESV -RRB- volumes were measured by 2D biplane modified Simpson 's method and then ejection fraction was calculated by the formula : Ejection fraction = -LSB- LV-EDV − LV-ESV -RSB- / LV-EDV . Transmitral E/A ratio was defined by pulsed wave Doppler and used as a marker of LV diastolic function . Mitral regurgitation Mitral regurgitation was defined by colour Doppler and graded according to the maximum regurgitant jet area as mild -LRB- jet area < 4 cm2 -RRB- , moderate -LRB- jet area 4 -- 8 cm2 -RRB- , and severe -LRB- jet area > 8 cm2 -RRB- -LSB- 12 -RSB- LA diameter -LRB- LAD -RRB- Maximum LAD was measured at three planes : antero-posterior from parasternal long axis view , medial-lateral and superior-inferior from apical four-chamber view . Then mean LAD -LRB- mean LAD -RRB- was defined as the average of the three LAD -LRB- See Fig. 1 -RRB- Fig. 1Measurement of maximum left atrial diameter -LRB- LAD -RRB- at three planes : -LRB- A -RRB- antero-posterior LAD from parasternal long axis view , -LRB- B -RRB- medial-lateral LAD and -LRB- c -RRB- superior-inferior LAD from apical four-chamber view LA mass LA mass was calculated with the same formula applied for left ventricular mass -LSB- 13 -RSB- -LCB- -LRB- mean LAD + anterior LA wall + posterior LA wall -RRB- 3 − mean LAD3 -RCB- × 0.8 + 0.6 . Thickness of anterior and posterior LA walls was measured from parasternal long axis view . Zooming was used to discriminate between posterior LA wall and pericardium -LRB- See Fig. 2 -RRB- Fig. 2M-mode obtained from parasternal long axis view -LRB- A -RRB- . Zooming was used to measure anterior left atrial wall -LRB- B -RRB- and posterior left atrial LA volume -LRB- LAV -RRB- LAV was assessed according to the formula -LSB- 6 -RSB- 8/3 π L × A1 × A2 where -LRB- L -RRB- is the LA longitudinal axis and -LRB- A1 -RRB- is LA area at apical four-chamber and -LRB- A2 -RRB- at apical two-chamber views . -LRB- L -RRB- was defined as the perpendicular line from mid point of the mitral valve plane to the tip of LA apex -LRB- See Fig. 3 -RRB- . LA area was obtained by manual tracing of LA endocardial border excluding LA appendage and the pulmonary veins when visualized . The superior border of atrial outline was a straight line connecting both sides of the leaflet base attachment points . LAV was calculated at three phases of ventricular cardiac cycle : maximum LAV -LRB- Vmax -RRB- at the end-systole just before mitral valve opening , minimum LAV -LRB- Vmin -RRB- at end-diastole just before mitral valve closure , and LAV before atrial contraction -LRB- VPre-A -RRB- : the last frame before mitral valve reopening . From the three LAV , the following measurements were selected as indices of LA function and calculated according to previous studies -LSB- 14 , 15 -RSB- : Fig. 3Calculation of LAV using 2DE by manual tracing of LA endocardial border at apical four-chamber -LRB- A1 -RRB- and apical two chamber views -LRB- A2 -RRB- , L is the long axis , then apply the formula. : 8/3 π L × A1 × A2 -LRB- 1 -RRB- Total Atrial Stroke Volume -LRB- TA-SV -RRB- defined as Vmax − Vmin , 2 -RRB- Total Atrial Emptying Fraction -LRB- TA-EF -RRB- defined as TA-SV/Vmax × 100 % , 3 -RRB- Active Atrial Stroke Volume -LRB- AA-SV -RRB- defined as VPre A − Vmin , 4 -RRB- Active Atrial Emptying Fraction -LRB- AA-EF -RRB- defined as AA-SV/VPre A × 100 % , 5 -RRB- Atrial Expansion Index -LRB- AEI -RRB- defined as TA-SV/Vmin × 100 % , 6 -RRB- Passive Atrial Stroke Volume -LRB- PA-SV -RRB- defined as Vmax − VPre A , and 7 -RRB- Passive Atrial Emptying Fraction -LRB- PA-EF -RRB- defined as -LRB- Vmax − VPre A -RRB- / Vmax × 100 % . LA kinetic energy -LRB- LA-KE -RRB- LA-KE -LSB- 16 -RSB- was calculated according to the formula 1/2 × AA-SV × P × V2 , where P = 1.06 g cm − 3 -LRB- blood density -RRB- , and -LRB- V -RRB- is the peak velocity of transmitral A wave was measured by pulsed wave Doppler . To characterize the three phases of LA activity , PA-SV and PA-EF were defined as indices for LA conduit function , AA-SV , AA-EF , and LA-KE for LA pump function , and AEI for LA reservoir function . Statistical analysis All values were expressed as mean ± SD . Paired t-test was used for comparison between all HCM patients and controls . Independent sample t-test was used for comparison between both HCM subgroups and between each HCM subgroup and controls . The difference in the values between groups was considered significant if P value < 0.05 . The statistical package used was SPSS version 12.1 . Results Baseline criteria -LRB- See Table 1 -RRB- Both HCM patient group and controls were comparable with respect to age and sex distribution . All patients and controls were in sinus rhythm -LRB- mean heart rate 72 ± 13 beat per minute -RRB- and had normal LV systolic function . All HCM patients had type I diastolic dysfunction -LRB- increased A velocity , with an E/A ratio < 1 -RRB- -LSB- 17 -RSB- . Twenty patients -LRB- 80 % -RRB- were under medications -LRB- 10 patients under Verapamil , six under β-blockers , four under Amiodarone -RRB- . LV-ESD and LV-ESV were significantly smaller in HCM patients than controls , while LV-EDD and LV-EDV showed no difference . LV-FS and LV-EF were significantly higher in HCM patients than controls -LRB- P = 0.001 -RRB- . HCM patients had a higher mean transmitral peak A velocity -LRB- 61.5 ± 20.3 vs. 39.7 ± 9.9 cm/s , P < 0.01 -RRB- . The prevalence and severity of mitral regurgitation were significantly higher in HCM patients compared to controls as 80 % of HCM patients had mitral regurgitation -LRB- 60 % mild and 20 % moderate to severe -RRB- , while 20 % of controls had mild mitral regurgitation -LRB- P < 0.0001 -RRB- . Both HCM patient subgroups -LRB- obstructive and non-obstructive -RRB- showed no significant differences in LV dimensions , volumes and function . Also , no significant difference in the prevalence and severity of mitral regurgitation was present . Table 1Baseline clinical and echocardiographic data of HCM patients and controlsHCM patients N = 25Normal control n = 25P valueAge -LRB- yr -RRB- 38 ± 15 35 ± 15 Male gender -LRB- % -RRB- 24 -LRB- 80 % -RRB- 18 -LRB- 60 % -RRB- Left ventricular FS -LRB- % -RRB- 38.4 ± 9.630.3 ± 5.40.03 Left ventricular EF -LRB- % -RRB- 71.1 ± 17.757.4 ± 1.00.001 Peak A velocity -LRB- cm/s -RRB- 61.5 ± 20.339.7 ± 9.90.01 Mitral regurgitation No5 -LRB- 20 % -RRB- 24 -LRB- 80 % -RRB- 0.0001 Mild15 -LRB- 60 % -RRB- 6 -LRB- 20 % -RRB- Moderate-severe5 -LRB- 20 % -RRB- 0 -LRB- 0 % -RRB- Antero-posterior LAD -LRB- mm -RRB- 42.8 ± 5.834.1 ± 4.20.0001Medial-lateral LAD -LRB- mm -RRB- 45.6 ± 9.9 36.2 ± 3.80.001Superior-inferior LAD -LRB- mm -RRB- 60.8 ± 9.044.8 ± 6.70.0001 Mean LAD -LRB- mm -RRB- 49.7 ± 7.238.4 ± 4.00.0001 Anterior left atrial wall -LRB- mm -RRB- 3.6 ± 1.03.5 ± 1.0 NSPosterior left atrial wall -LRB- mm -RRB- 3.5 ± 1.23.1 ± 1.1 NSLeft atrial mass -LRB- g -RRB- 89.8 ± 37.247.8 ± 18.10.0001 Abbreviations : FS fractional shortening , EF ejection fraction , and LAD left atrial diameter LA size and mass -LRB- Table 1 -RRB- The maximum LAD at the three planes and the mean LAD were significantly larger in HCM patients than controls -LRB- P < 0.001 -RRB- . Thickness of anterior and posterior LA walls showed no significant differences between HCM patients and controls . LA mass was significantly higher in HCM patients than controls -LRB- 89.8 ± 37.2 vs. 32.3 ± 12.0 g , P < 0.0001 -RRB- . No significant differences were found between HCM patient subgroups in the mean LAD , wall thickness and LA mass . LAV and total LA function -LRB- Table 2 -RRB- LAV at the three phases of cardiac cycle -LRB- Vmax , Vmin , and VPre-A -RRB- was significantly higher in both HCM patient subgroups than control group . TA-SV , TA-EF showed no significant differences between both HCM patient subgroups and control group . Vmax was well correlated with the mean LAD -LRB- r = 0.89 , P < 0.0001 -RRB- . Table 2Left atrial mass and function in non-obstructive HCM and obstructive HCM patientsNormal control n = 25 Non-obstructive HCM n = 15Obstructive HCM n = 10 P value *** Mean LAD -LRB- mm -RRB- 38.4 ± 4.049.6 ± 7.649.9 ± 7.00.00010.0001 LA mass -LRB- g -RRB- 47.8 ± 18.186.8 ± 41.193.9 ± 32.50.0020.001 V max -LRB- ml -RRB- 38.2 ± 10.764.7 ± 26.765.9 ± 28.10.0020.009 V min -LRB- ml -RRB- 17.4 ± 7.632.7 ± 22.137.1 ± 19.70.020.008 V pre A -LRB- ml -RRB- 24.9 ± 7.744.7 ± 24.748.5 ± 21.50.0080.005TA-SV -LRB- ml -RRB- 20.5 ± 6.932.0 ± 8.928.8 ± 13.1NSNSTA-EF -LRB- % -RRB- 54.7 ± 14.253.6 ± 13.845.4 ± 12.8NSNSAA-SV -LRB- ml -RRB- 7.3 ± 4.012.0 ± 6.211.4 ± 6.50.0010.001AA-EF -LRB- % -RRB- 31.4 ± 17.829.7 ± 13.525.4 ± 12.8NSNSLA-KE -LRB- kdynes.cm -RRB- 11.9 ± 7.418.3 ± 12.532.5 ± 23.3a0.030.001PA-SV -LRB- ml -RRB- 13.4 ± 5.720.0 ± 8.817.5 ± 9.80.02NSPA-EF -LRB- % -RRB- 33.9 ± 10.134.0 ± 14.827.1 ± 7.0 NS0 .03 AEI -LRB- % -RRB- 141.7 ± 74.0134.8 ± 74.491.1 ± 39.7 NS0 .02 Abbreviations : see text * P value between non-obstructive HCM patients and controls ** P value between obstructive HCM patients and controlsaP value < 0.001 between obstructive and non-obstructive HCM patients LA pump function AA-EF showed no significant differences between both HCM patient subgroups and control group , while AA-SV was significantly higher in both HCM patient subgroups than control group -LRB- 12.0 ± 6.2 ml , 11.4 ± 6.5 ml vs. 7.3 ± 4.0 ml , P < 0.001 -RRB- . No significant differences were found between HCM patient subgroups in AA-EF and AA-SV . LA-KE was significantly higher in HCM patients than controls -LRB- 24.3 ± 18.9 vs. 11.9 ± 7.4 kdynes cm , P = 0.002 -RRB- . LA-KE was significantly higher in obstructive HCM patients than non-obstructive patients -LRB- 32.5 ± 23.3 vs. 18.3 ± 12.5 kdynes cm , P < 0.001 -RRB- . LA conduit function PA-SV was significantly increased in HCM patients than controls -LRB- 18.9 ± 9.2 vs. 13.4 ± 5.7 ml , P = 0.01 -RRB- . PA-SV was significantly higher in non-obstructive HCM patients than controls -LRB- 20.0 ± 8.8 vs. 13.4 ± 5.7 ml , P = 0.02 -RRB- , while in obstructive HCM patients ; it was comparable to controls -LRB- 17.5 ± 9.8 vs. 13.4 ± 5.7 ml -RRB- . PA-EF showed no significant difference between HCM patients and controls . PA-EF was significantly lower in obstructive HCM patients than controls -LRB- 27.1 ± 7.0 vs. 33.9 ± 10.1 , P = 0.02 -RRB- , while in non-obstructive HCM patients ; it was comparable to controls -LRB- 34.0 ± 14.8 vs. 33.9 ± 10.1 -RRB- . LA reservoir function AEI showed no significant difference between HCM patients and controls . AEI was comparable in non-obstructive HCM patients and controls -LRB- 134.8 ± 74.4 % vs. 141.7 ± 74.0 % , P = NS -RRB- but it was significantly lower in obstructive HCM patients than controls -LRB- 91.1 ± 39.7 % vs. 141.7 ± 74.0 % , P = 0.02 -RRB- . Discussion In the present study , LA size was increased in HCM patients as assessed by LA dimension at three directions -LRB- antero-posterior , medial-lateral and superior-inferior -RRB- and by LAV at three phases of cardiac cycle . Thus , LA mass was increased in HCM patients compared to normal subjects . According to Frank -- Starling mechanism , LA pump function in HCM was increased to help ventricular filling as shown by increased AA-SV and LA-KE . LA reservoir and conduit functions were decreased in obstructive HCM patients as shown by decreased AEI and PA-EF . The clinical importance of this study is the close relationship of LA dimension and function with the development of serious complications as atrial fibrillation , cerebral infarction , and progressive heart failure in HCM as shown in previous studies -LSB- 18 -- 20 -RSB- as well as adverse outcome after myectomy -LSB- 21 -RSB- The previous studies describing LA changes in HCM identified increased LAV -LSB- 22 , 23 -RSB- . LA responds to the increased LAV by linear increase of LAD as shown in the present study . The nonlinear correlation between the increased LAD and increased LAV in previous studies -LSB- 24 -RSB- may be due to unidimensional M-mode assessment of LA size while , in the present study , the LA size was relied on 2D multidimensional assessment and the mean LA dimension was comparable to previous studies -LSB- 10 , 25 -RSB- . The increased left ventricular wall thickness and elevated filling pressure in HCM frequently lead to development of abnormal diastolic function . In response to this overload , LA contractility increases to maintain adequate left ventricular filling . In accordance with previous studies -LSB- 26 , 27 -RSB- , AA-SV and LA-KE was significantly increased in our HCM patients than controls . Other studies reported no increase in LA contractility -LSB- 28 , 29 -RSB- . The discrepancy between studies describing LA contractility in HCM is due to difference in hemodynamic conditions , atrio-ventricular interaction and left ventricular systolic function . In HCM patients , the rate of left ventricular relaxation deteriorates due to markedly thickened and non-compliant left ventricle , which led to deterioration of LA conduit function . This was expressed by reduction in PA-EF in our HCM patients compared to controls . LA reservoir function is determined by LA relaxation and mitral annulus displacement during left ventricular contraction -LSB- 30 -RSB- . In accordance with previous studies -LSB- 8 -RSB- , AEI was significantly reduced in obstructive HCM patients , which indicates impaired LA relaxation . AEI showed no difference between our non-obstructive HCM patients and controls indicating preserved LA reservoir function . Study limitation The study had some limitations . The sample size of HCM patients -LRB- 25 patients -RRB- is small but due to low prevalence of HCM -LRB- 0.2 % -RRB- . The assessment of LA mass is a new idea not discussed before by any imaging modalities . Of course it needs validation by autopsy or magnetic resonance imaging but at least it can pave a way for thinking about in further studies either by the same or another formula . Assessment of diastolic dysfunction was relied on transmitral flow by pulsed wave Doppler and other parameters by tissue Doppler imaging were not available . There are no data about prognostic value of atrial remodelling as a predictor for development of atrial fibrillation because it need long time follow up for large sample of patients . However data from previous studies could be relied on -LSB- 18 , 19 -RSB- . Conclusion Hypertrophic cardiomyopathy is associated with dilated LA and increased LAV that reflects the severity of LA overload . Higher AA-SV and LA-KE in HCM patients especially obstructive type indicates augmented LA pump function to overcome the high left ventricular filling pressure caused by severe left ventricular hypertrophy . Lower PA-EF and AEI in obstructive HCM patients indicate impaired LA reservoir and conduit functions . These findings may have clinical implication for the follow up of HCM patients

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

hypertrophic cardiomyopathy
left atrial volume