Cases and Mortality Rates for Infectious Diseases
description
Transcript of Cases and Mortality Rates for Infectious Diseases
DiseaseWorldwide
AnnualDeaths
U.S
Annual Cases Annual Deaths
All Infectious Diseases 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).
Cases and Mortality Rates for Infectious Diseases
Ghost E. coli
Inflammation,
Schock
Immune stimulation- adjuvans- 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
Bacterial Cell Envelopes, LPS, Lipid A partial- structures
Avanti's New Potent Vaccine Adjuvant
Natural (Salmonella Minnesota, R595
monophosphoryl derivative
endotoxically inactive
acting as an adjuvant
inducing tolerance to Salmonella enteritidis LPS and tumor necrosis factor alpha (TNF-alpha)
Detoxified Lipid A
Structure LPS
1,E+00 1,E+02 1,E+04 1,E+06 1,E+08
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
Limulus Test, Endotoxicity
0
2.000
4.000
6.000
8.000
10.000
0,00001 0,0001 0,001 0,01 0,1 1 10 100
[g/ml]
TN
F [
pg
/ml]
LPS
MDPghost
S-layer(B.sph) S-layer(B.st.)
Stimulation of TNFa by bacterial ghostsin Mo
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
Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines.
Hypothesis:
transmembrane integrin glycoproteins e.g.Mac-1 serve as a signaling partner for glycolipid-linked glycoproteins that lack trans-membrane and cytoplasmic domains.
LPS, CD14
0 5 10 15 20 25
TNF[ng/ml] LPS FCS aCD14 p< (ng/ml) (ug/ml)
10 + - 10 - -
10 + 0,5 0,05 10 - 0,5
10 + 0,1 0,02 10 - 0,1
100 + - 100 - -
100 + 0,5 0,02 100 - 0,5 0,02
100 + 0,1 0,05 100 - 0,1 0,05
- +/- -
- +/- +/-
Inhibition of TNFa by anti CD14
CD14GPIToll-
likeR-4
LPS- LBP
Stat3,1,4
HSP60?
NFkB
IL-6, IFNy,...
MAPK/ p38 JNK
ERK MAPK
CR3
MAC1
?
R
Janus
Kinase
CREB,
ATF1
?
PLC?
...,myk,Elk, Mnk
eIF4E AH,10/2000
P
Pathways for LPS stimulation
Uptake of ghosts in RAW-Mo
GFP labelled Ghost, RAW cells
FISH, GFP mRNA in RAW Mo
0
20
40
60
80
100
120
Co Ghosts5; 0,5 ul Ghost/SB Ghosts/G5
% o
f Con
trol
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
0
20
40
60
80
100
120
140
1 2 3 4 5 6 7
% of
Co
Inhibition of LPS stimulated TNFa Inhibition of Ghost stimulated TNFa
by kinase inhibitors in supernatant by kinase inhibitors packed inside ghosts
LPS
SB/G5
APCs and T-cells
mRNA Isolation: Quick PrepMicro mRNA Purification Kit mRNA semiquantification:Nuleic dotMetricTM Basic Kit (Geno Technology)
Reverse transcripton reaction 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
Quantitative Real Time PCR( Light Cycler System )
-4
24
0
4
8
12
16
20
71 73 75 77 79 81 83 85 87 89 91 93
no template
-dF/
dT
Temperature (°C)
B
32
-202468
1012141618202224262830
400 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
106 105
104 103
102
no template
Cycle number
Fluo
resc
ence
A
Amplification in RT- PCR
IL-12
TNFa
Eif2-by
M Co 3 6 16 16 hrs
IL-12, TNFa mRNA in Ghost- stimulated THP-1 MO (RT- PCR)
1
10
100
1000
0h 4h 16h
pg/m
l
TNFa
IL-12LPS
Ghosts o
Stimulation IL-12 and TNFa in THP1 cellsELISA
10
100
1000
Co 0,001 0,01 0,1 1 10
ug/ml
pg/m
l
TNFa
IL-12
Stimulation of IL-12 and TNFa in PBM
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
0
200
400
600
800
1000
1200
1400
Control LPS Ghosts
TN
F-
(p
g/m
l)
0
200
400
600
800
1000
Control LPS Ghosts
TNF-
(pg/
ml)
Stimulation of TNFa in PBM and DC
0
50
100
150
200
250
300
350
Control LPS Ghosts
IL-1
2 p7
0+p4
0 (p
g/m
l)
0
5000
10000
15000
20000
25000
Control LPS Ghosts
IL-1
2 p7
0+p4
0 (p
g/m
l)
Stimulation of IL-12 ( p40, p70) protein in PBM and DC
0
10
20
30
40
50
60
Control LPS Ghosts
IL-1
2 p
40 r
ela
tive
mR
NA
le
ve
l
Stimulation of IL-12 p40 mRNA in DC
0
10
20
30
40
50
Control LPS Ghosts
IL-1
8 r
ela
tiv
e m
RN
A l
ev
el
Stimulation of IL-18 relative mRNA in DC
What are the mechanisms for the good activation of IL-12 in APC, especially DC ?
Does this have a clinical value ?
Ghosts and DC
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
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
Activation of DC functions by microbial stimuli.
0,75
1,25
1,75
2,25
1:10 1:30 1:60 1:100 1:300 1:600 1:1000
Ratio DC : T cell
ab
sorb
an
ce (
A45
0-A
690)
iDCs mDCs
mDCs+G GBH BF
Pavol KUDELA
maturation mix (MM)-TNF-; IL-1; PGE2; IL-6; GM-CSF; IL-4
MM + bacterial ghosts + GM-CSF + IL-4
Mature DCs and ghosts induce strong T-cell responses, MLR
MHC class I and II, CD11a,b,c, CD50,
CD54, CD58
Activation of T- cells
IL-12, T cell attractant chemokine
Induce ghosts differentiation and activation of DC functions ?
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 organsAH, 10/2000
M. Szostak
Horst Mader
M. Schmalnauer
Gudrun Kohl
Fürst Ladani
Beate Mayr
Margit Weghofer