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Cases and Mortality Rates for Infectious Diseases
Disease
Worldwide
Annual
Deaths
U.S
Annual Cases
All Infectious Diseases
Annual
Deaths
17,000,000
(1995)
n/a
165,750
(1992)2
HIV/AIDS
2,300,000
(1997)
71,293
(1995)
32,655
(1996)
Acute respiratory diseases
4,400,000
(1995)
90,400,000
(1994)
607
(1996)
Foodbourne illnesses
3,100,000
(1995)
6,000,000
(1983)
9,000
(1983)
Malaria
2,000,000
(1995)
n/a
n/a
Tuberculosis
3,100,000
(1995)
22,860
(1995)
1,194
(1996)
Hepatitis B
1,100,000
(1995)
10,805
(1995)
3,811
(1996)
Chlamydia
n/a
4,000,000
(1996)3
n/a
1
Sources include the World Health Organization and the Centers for Disease Control and
Prevention (CDC).
Ghost E. coli
Bacterial Cell Envelopes, LPS, Lipid A partialstructures
Inflammation,
Schock
Immune stimulationadjuvans- activity
Carrier-Targeting
Major immunological questions:
Endotoxic activities, therapeutic window, routes of administration
Routes of activation comparing LPS, uptake by APCs
Stimulation of immune responses
Possibility for packaging, targeting to specific cells
Avanti's New Potent Vaccine Adjuvant
Detoxified Lipid A
Natural (Salmonella Minnesota, R595
monophosphoryl derivative
endotoxically inactive
acting as an adjuvant
inducing tolerance to Salmonella enteritidis
LPS and tumor necrosis factor alpha (TNFalpha)
Structure LPS
Limulus Test, Endotoxicity
EU/mg
S-layer ( B.sphaericus)
S-layer ( B. stear.)
Ghosts (E.coli O26:B6)
Ghosts (S.typhim.)
LPS (E.coli O26:B6)
LPS ( S. ab. equi )
Control
1,E+00
1,E+02
1,E+04
1,E+06
1,E+08
Stimulation of TNFa by bacterial ghosts
in Mo
Endotoxicity does not limit the use of bacterial ghosts as
candidate vaccines.
Bacterial ghosts prepared from Escherichia coli O26:B6 and
Salmonella typhimurium C5 induce dose-dependent antibody
responses against bacterial cells or their corresponding
lipopolysaccharides (LPS) in doses 25 ng kg-1 when
administered intravenously to rabbits.
No significant fever responses in rabbits have been recorded
in doses of < 250 ng kg-1 E. coli O26:B6 ghosts and up to
doses of 250 ng kg-1 S. typhimurium C5 ghosts when
applying test methods recommended by the US
pharmacopoeia.
Vaccine 1997 Feb;15(2):195-202
LPS, CD14
Hypothesis:
transmembrane integrin
glycoproteins e.g.
Mac-1 serve as a
signaling partner for
glycolipid-linked
glycoproteins that lack
trans-membrane and
cytoplasmic domains.
Inhibition of TNFa by anti CD14
TNF[ng/ml]
LPS
FCS aCD14 p<
(ng/ml)
(ug/ml)
10
10
+
-
-
10
10
+
-
0,5
0,5
0,05
10
10
+
-
0,1
0,1
0,02
100
100
+
-
-
100
100
+
-
0,5
0,5
0,02
0,02
100
100
+
-
0,1
0,1
0,05
0,05
-
+/-
-
-
+/-
+/-
0
5
10
15
20
25
Pathways for LPS stimulation
LPS- LBP
?
HSP60?
IL-6, IFNy,...
Tolllike
R-4
CD14
GPI
PLC
CR3
CREB,
ATF1
Janus
?
Kinase
?
MAPK/ p38
ERK
R
MAC
1
JNK
Stat
P
3,1,4
MAPK
NFkB
...,myk,Elk, Mnk
eIF4E
AH,10/2000
Uptake of ghosts in RAW-Mo
GFP labelled Ghost, RAW cells
FISH, GFP mRNA in RAW Mo
Inhibition of LPS stimulated TNFa
Inhibition of Ghost stimulated TNFa
by kinase inhibitors in supernatant
by kinase inhibitors packed inside ghosts
140
120
120
100
SB/G5
% of Control
% of Co
100
LPS
80
60
80
60
40
40
20
20
0
0
1
2
3
4
5
6
7
Co
Ghosts5; 0,5 ul
Ghost/SB
Ghosts/G5
LPS: Co, 0,001; 0,01; 0,1 ug/ml , Co:50-200 pg/ml TNFa, 100%: 5-20 ng/ml TNFa
Ghosts: E. Coli, 5, 0,5 ul; 100 %: 5-20 ng/ml TNFa
SB203580: 5, 10, 20 uM; G5: Protein kinase CK2 inhibitor, 5,20, 50 uM
APCs and T-cells
Quantitative Real Time PCR
( Light Cycler System )
Reverse transcripton reaction
mRNA Isolation:
Quick PrepMicro mRNA Purification Kit
mRNA semiquantification:
Nuleic dotMetricTM Basic Kit (Geno
Technology)
RT- PCR:
LightCyclerTM System (Roche), FastStart DNA Master SYBR
Green I (Roche)
Quantification:
external cDNA standards with known amounts of initial copy
number over a 105 - fold range were coamplified
calculation with LightCycler Quantification Software v3.39
during the log- linear phase of the amplification
ß- actin was used to normalize for inefficiencies in cDNA
synthesis
Amplification in RT- PCR
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
-2
B
24
20
16
dF/dT
Fluorescence
A
12
106
105
104
10
102
8
3
no
templ
ate
4
0
no
template
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Cycle number
-4
71
73
75
77
79
81 83 85
Temperat
ure (°C)
87
89
91
93
IL-12, TNFa mRNA in Ghost- stimulated
THP-1 MO (RT- PCR)
IL12
TNFa
Eif2by
M
Co
3
6
16
16
hrs
Stimulation IL-12 and TNFa in THP1 cells
ELISA
1000
pg/ml
100
TNFa
10
1
0h
IL-12
4h
16h
LPS •
Ghosts o
Stimulation of IL-12 and TNFa in PBM
1000
pg/ml
TNFa
100
IL-12
10
Co
0,001
0,01
0,1
ug/ml
1
10
Immune mediators in DC derived from PBM
( IL4, GMCSF, 5-7 d )
secrete large amounts of inflammatory
cytokines as precursors
high Ag capture capacity at their immature
stage
stimulation of quiescient, B and T
lymphocytes
low levels of antigen to induce strong T cell
response
Adherence of ALEXA labeled ghosts
Stimulation of TNFa in PBM and DC
Stimulation of IL-12 ( p40, p70)
protein in PBM and DC
25000
300
IL-12 p70+p40 (pg/ml)
IL-12 p70+p40 (pg/ml)
350
250
200
150
100
50
0
20000
15000
10000
5000
0
Control
LPS
Ghosts
Control
LPS
Ghosts
Stimulation of IL-12 p40 mRNA
in DC
IL-12 p40 relative mRNA level
60
50
40
30
20
10
0
Control
LPS
Ghosts
Stimulation of IL-18 relative
mRNA in DC
IL-18 relative mRNA level
50
40
30
20
10
0
Control
LPS
Ghosts
Ghosts and DC
What are the mechanisms for the good
activation of IL-12 in APC, especially DC ?
Does this have a clinical value ?
Microbial stimuli that promote
DC maturation
Bacteria
( E. coli, M. tuberculosis, Staph. aureus, Strept. ,...
Protozoa
LPS
Bacterial DNA,
Viral and doubled-stranded RNA
Bacterial heat shock proteins
Activation of DC functions by microbial stimuli.
Migration
Exit of activated DCs from peripheral sites
Entry into the T cell areas of secondary lymphoid tissues
Antigen presentation
Upregulation of antigen presenting molecules (MHC class I and class II, CD1)
Delivery of antigen to the MHC class I pathway
Upregulation of molecules involved in interaction with T lymphocytes
Costimulatory molecules (B7-1, B7-2)
Adhesion molecules (ICAM-1, VLA-4)
Signalling molecules (CD40)
Production of cytokines
Induction of IL-12, TNF, IL-10, IL-6, IFN-/
Recruitment of DC precursors to peripheral sites
Increased transient survival of DCs in the absence of growth signals
Irreversible maturation followed by apoptotic death
Mature DCs and ghosts induce strong T-cell
Pavol KUDELA
responses, MLR
iDCs
mDCs+G
BH
absorbance (A450-A690)
2,25
mDCs
G
BF
1,75
1,25
maturation mix (MM)-TNF-; IL-1; PGE2;
IL-6; GM-CSF; IL-4
MM + bacterial ghosts + GM-CSF + IL-4
0,75
1:10
1:30
1:60
1:100
1:300
Ratio DC : T cell
1:600
1:1000
Induce ghosts differentiation and
activation of DC functions ?
MHC class I and II,
CD11a,b,c, CD50, CD54,
CD58
Activation of T- cells
IL-12, T cell attractant
chemokine
Gut 2000 Jul;47(1):79-87
Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial
cell/leucocyte co-cultures.
Haller D, Bode C, Hammes WP, Pfeifer AM, Schiffrin EJ, Blum S.
Institute of Biological Chemistry and Nutrition Science, University Hohenheim, Germany.
BACKGROUND AND AIM: Intestinal epithelial cells (IEC) are thought to participate in the
mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis
Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced
expression of IL-8, MCP-1, IL-1beta, and TNF-alpha mRNA in the presence of underlying
leucocytes.
Leucocyte sensitised CaCO-2 cells produced TNF-alpha and IL-1beta whereas IL-10 was
exclusively secreted by human peripheral blood mononuclear cells.
CaCO-2 cells alone remained hyporesponsive to the bacterial challenge.
CONCLUSION: The differential recognition of non-pathogenic bacteria by CaCO-2 cells
required the presence of underlying leucocytes. These results strengthen the hypothesis that
bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.
Microbiol Immunol 1999;43(10):925-35
Cytokine secretion by stimulated monocytes depends on the growth phase and heat
treatment of bacteria: a comparative study between lactic acid bacteria and invasive
pathogens.
Haller D, Bode C, Hammes WP.
Hohenheim University, Stuttgart, Germany. [email protected]
The challenge of monocytes with three LAB strains, Listeria monocytogenes or
enterohaemorrhagic Escherichia coli (EHEC) elicited a strain specific, dose-dependent
biphasic TNF-alpha secretion.
LPS exhibited a higher capacity to stimulate monocytes than purified gram positive cell walls
or muramyldipeptide.
In comparison to pathogenic bacteria, the maximal secretory TNF-alpha response (TNFmax)
was up to 2 fold higher with LAB strains.
In general, the amount of bacteria (EDmax) necessary to induce maximal TNF-alpha
secretion (TNFmax) was approximately 1 to 3 log higher for heat killed bacteria when
compared to live bacterial cells illustrating the significant lower potential of heat killed bacteria
to activate monocytes.
Bacterial cell envelopes
for vaccine development
• No endotoxic limitations
• lipid A activation pathways, CD14, p38 MAP kinase
• provide adjuvans activity comparable to lipid A structures
• effective uptake by phagocytes ( others by different mechanisms ?)
• potent inducers of IL-12, ( IL-18 )
• may propagate maturation of dendritic cells
• may enable packaging of systemic toxic compounds and targeting
to specific cells or organs
AH, 10/2000
M. Szostak
Horst Mader
M. Schmalnauer
Gudrun Kohl
Fürst Ladani
Beate Mayr
Margit Weghofer