Power Point of the ASM Presentation (May 2009 - TherimuneX

workkinkajouΒιοτεχνολογία

5 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

202 εμφανίσεις

Mitali
Purohit
*, Carol
Artlett
*,
Sihem

Sassi

Gaha
*, James D. Thacker
1*
, Richard F. Rest*


*Department of Microbiology and Immunology, Drexel University College of Medicine

1
TherimuneX Pharmaceuticals, Inc., Doylestown, PA

Characterization of a Unique, Naturally
-
occurring Immunomodulatory
Lipopeptide: 1
-
peptidyl
-
2,3
-
diacylglyceride (PDAG
)

FUNDING
:

This

research

has

been

funded

by

Drexel

University

College

of

Medicine

in

a

cooperative

agreement

with

TherimuneX

Pharmaceuticals,

Inc
.


FUTURE STUDIES



Determine

whether

PDAG

stimulates

neutrophils,

monocytes

or

macrophages

to

express

antibacterial

activities

in

vitro
.




Determine the cellular origin of PDAG and conditions
for its production or release.




Determine what pathways are activated by PDAG in
non
-
immune cells.




Determine if PDAG induces an antimicrobial effect
in
vivo
.


SUMMARY

Modified from NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 4 APRIL 2007



PDAG

Controlled induction of
cytokines and
chemokines

Delayed cell
migration and
recruitment

PDAG PROTECTS IN A MOUSE


PERITONITIS MODEL

Figure

6
:

Swiss

Webster

mice

were

challenged

with

Salmonella

typhimurium

(
5
x
10
3

cfu/mouse)

by

intraperitoneal

injection
.

Mice

were

treated

with

natural

PDAG

in

normal

saline

(
1
.
5

mg

/mouse
;

n=
15
)

or

saline

alone

(n=
15
)

by

subcutaneous

injection

a

day

before

bacterial

challenge
.

Mortality

was

monitored

daily

for

ten

days
.

By

day

10
,

all

mice

in

the

control

group

were

dead

where

as

there

was

20
%

death

in

the

PDAG

treated

group
.

Administration

of

PDAG

did

not

cause

a

cytokine

storm
.

Lymphopenia,

neutrophilia,

and

monocytosis

were

observed

in

the

infected

control

mice,

whereas

circulating

neutrophils

and

monocytes

in

PDAG

treated

mice

were

at

or

near

normal

levels
.

The

bacterial

load

in

the

spleens

of

the

mice

was

50
%

less

in

PDAG

treated

mice

on

day

1

(data

not

shown)
.


INTRODUCTION

PDAG INDUCES CYTOKINE & CHEMOKINE

RELEASE FROM HUMAN BLOOD

Figure

2
:

Fresh

heparinized

human

blood

was

diluted

five
-
fold

with

RPMI

1640

medium

and

incubated

with

dilutions

of

purified

natural

PDAG

for

13

or

24

hrs
.

After

incubation,

samples

were

centrifuged

and

plasma

was

assayed

using

the

Excelarray

Human

Inflammation

kit,

or

Chemotaxis

kit

(Thermo

Fisher

Scientific)
.

PDAG

induced

a

dose

and

time

dependent

release

of

cytokines
.

Figure

4
:

Normal

human

fibroblasts

were

cultured

in

DMEM

with

10
%

FBS

at

37
o
C,

with

or

without

natural

PDAG
.

RNA

was

purified

from

treated

and

control

fibroblasts,

and

assayed

for

the

indicated

cytokines

or

signaling

molecules

by

quantitative

RT

PCR
.

Values

are

normalized

to


-
actin

expression
.

0
1
2
3
4
5
6
7
8
MIP-1a
IL-1a
IL-1b
IL-8
IL-33
IL-18
mRNA


(relative


level
)

Control
PDAG
0
100
200
300
400
500
600
700
MCP-1
NFkB
NALP-3
Caspase-1
IL-6
mRNA (relative level)

Control
PDAG
PDAG INDUCES CYTOKINE RELEASE
FROM HUMAN FIBROBLASTS

0
20
40
60
80
100
120
140
160
IL-6
IL-8
relative m
-
RNA level

Cytokines

Control
PDAG
Figure 5:
PDAG treatment of keratinocytes induces increased expression of IL
-
6
and IL
-
8 as determined by RT
-
PCR. Values are normalized to
β
-
actin.

PDAG INDUCES CYTOKINE RELEASE
FROM HUMAN KERATINOCYTES

Diverse

cells

and

mediators

are

involved

in

innate

immunity
.

We

wanted

to

investigate

the

effects

of

PDAG

on

fibroblasts

and

keratinocytes

which

are

excellent

sources

of

cytokines

and

chemokines

during

inflammatory

response

to

infections
.


To

control

disease

caused

by

emerging

antibiotic
-
resistant

pathogens,

there

is

a

constant

quest

to

develop

novel

therapeutic

agents

and

adjuncts

to

antimicrobials
.

We

have

pursued

a

broad
-
spectrum

strategy

that

involves

boosting

the

innate

immune

system

to

combat

a

broad

range

of

infectious

agents
.

In

the

search

of

such

immunomodulatory

molecules,

we

screened

the

low
-
molecular

weight

inflammatory

proteome

of

mammalian

plasma

and

serum

and

identified

a

unique

lipopeptide



1
-
peptidyl
-
2
,
3
-
diacylglyceride

(PDAG)
.

PDAG

stimulates

the

innate

immune

response

resulting

in

activation

of

tissue

resident

immune

cells,

recruitment

of

phagocytic

cells,

and

clearance

of

pathogens
.

Serum
-
derived

PDAG

stimulates

significant

release

from

whole

human

blood

of

proinflammatory

cytokines

IL
-
6
,

IL
-
8
,

MCP
-
1
,

MIP
-
1
α

and

MIP
-
1
β
.

The

cytokine

profile

induced

by

PDAG

differs

from

that

induced

by

LPS,

indicating

a

lack

of

endotoxin

contamination

of

the

natural

product
.

In

addition,

PDAG

stimulates

isolated

human

macrophages

and

THP
-
1

macrophage
-
like

cells

to

release

IL
-
6

and

IL
-
8

in

a

dose

and

time

dependent

manner
.

Interestingly,

PDAG

also

stimulates

cultured

human

fibroblasts

to

secrete

IL
-
6
.

We

determined

whether

PDAG

activity

was

due

to

the

entire

molecule,

PDAG

peptide

or

DAG
.

Neither

DAG

nor

synthetic

PDAG

peptide

alone

induce

cytokine

release

from

whole

blood
.

PDAG

is

an

exciting

new

and

unique

immunoregulatory

molecule

that

has

a

plethora

of

possible

clinical

uses
.

[This

work

is

funded

by

Drexel

University

College

of

Medicine
.
]

ABSTRACT


The

evolution

and

rapid

spread

of

resistant

microorganisms

are

significant

problems

in

nosocomial

infections

and

are

of

increasing

importance

in

community

acquired

infections
.

There

is

an

urgent

need

for

alternatives

to

antimicrobials

that

can

either

have

direct

microbicidal

activity

or

that

can

exert

their

effects

by

stimulating

the

innate

immune

system
.

Immunomodulatory

peptides,

such

as

the

defensins,

stimulate

a

number

of

effects

on

innate

immune

cells,

including

monocytes,

macrophages,

and

neutrophils

to

act

as

chemoattractants,

promote

histamine

release

from

mast

cells,

and

enhance

phagocytosis

at

the

site

of

infection
.




In

our

investigation

of

the

low
-
molecular

weight

inflammatory

proteome

in

mammalian

plasma

and

serum

we

identified

a

new

immunoactive

lipopeptide

with

the

general

structure

:





wherein

X
1

is

a

linear

peptide

and

X
2

and

X
3

are

arachidonic

and

stearic

acid

respectively
.

The

peptide

is

covalently

linked

to

the

diacylglycerol

by

an

ester

bond

at

the

C
-
terminus

of

the

peptide
.

We

hypothesize

that

1
-
peptidyl
-
2
,
3
-
diacylglycerol,

“PDAG”,

activates

the

innate

immune

cascade,

activates

tissue

resident

immune

cells

at

the

site

of

injury,

recruits

professional

phagocytic

cells

to

local

tissue,

clears

pathogens,

and

abates

disease

progression
.

ISOLATION OF NATURAL PDAG

Dialyze Caprine serum

Reduce dialysate volume by lyophilization

Extract lyophilized fraction with chloroform/ methanol and
remove solids

Prepare purified PDAG by preparative reversed phase HPLC.
PDAG samples are >99% chromatographically pure.

Determine PDAG composition by mass spectrometry and
sequence the PDAG peptide

The PDAG peptide sequence is homologous with an extra
-
cellular loop
of TRPC
-
1, a transmembrane Ca
2+

channel protein.





PDAG IS HOMOLOGOUS TO TRPC1

Figure

1
:

Putative

domain

structure

and

topology

of

TRPC
1
.

Percent

identity

between

TRPC
1

and

at

least

one

other

member

of

the

TRP

family

is

indicated
.

The

red

circle

indicates

the

PDAG

peptide

sequence
.

Wes,

et

al
.

Proc
.

Natl
.

Acad
.

Sci
.

USA

Vol
.

92
,

pp
.

9652
-
9656

PDAG INDUCES IL
-
6 AND IL
-
8
RELEASE FROM MACROPHAGES

Figure

3
:

PMA
-
differentiated

THP
-
1

cells

in

RPMI,

10
%

HI

FBS

and

50

µM

beta
-
mercaptoethanol

were

treated

with

1
:
100

natural

PDAG

for

24

hrs
.

Supernatants

were

assayed

using

the

Excelarray

Human

Inflammation

kit
.

LPS

is

used

as

a

positive

control
.

Similar

results

were

seen

with

human

monocyte

derived

macrophages
.

0
200
400
600
800
1000
1200
IL-6
IL-8
Amount of Cytokine
(pg)

Cytokines

Control
LPS 5ng/ml
PDAG (1:100)
Untreated

PDAG