SUGAR-FREE GLYCOBIOLOGY
SUGAR-FREE
“Unusual” Sugar Chain
BASIC SCIENCE “It”-- is a carboxylate
Make lots of ” It” Make an “It” antibody
?sugar chain structure
?Who binds to “ it”
? Who binds these inflammatory proteins RAGE
Inflammatory proteins
INFLAMMATION
Expansion, homing and activation of CD4CD45RBhi naive T cells
CD4CD45RBhi(naïve) Mesenteric lymph nodes
Priming and clonal expansion
Involves interaction with DCs Recognized by addressin molecules on
vascular endothelium of
intestine and home to lamina propria
IL12
CD80/86-CD28 Acquire gut homing molecules
CD40-CD154 α4β7, CCR9 Lamina Propria
CD134L-CD134
Th1 CD40-CD154
CD80/86-CD28
CD134L-CD134
MIF
leukocyte Proinflammatory
recruitment Cytokines
from blood and TNFα
macrophages
Inflammation IFNγ
IL-12, IL-23 Apoptotic
NO pathways
Expansion, homing and activation of CD4CD45RBhi naive T cells
CD4CD45RBhi(naïve) Mesenteric lymph nodes
Priming and clonal expansion
Involves interaction with DCs Recognized by addressin molecules on
vascular endothelium of
intestine and home to lamina propria
IL12
CD80/86-CD28 Acquire gut homing molecules
CD40-CD154 α4β7, CCR9 Lamina Propria
CD134L-CD134
Th1 CD40-CD154
CD80/86-CD28
CD134L-CD134
MIF
leukocyte Proinflammatory
recruitment Cytokines
from blood and TNFα
macrophages
Inflammation IFNγ
IL-12, IL-23 Apoptotic
NO pathways
1600
Secretion of cytokines from
LPS-activated RAW264.7
IL-12 p40
IL12p40 pg/ml
1200 murine macrophages
800
400
0
160
TNFα
TNFα ng/106 cells
120
80
40
0
2000 IL-10
IL-10 pg/ml
1600
1200
800
400
0
untreated LPS LPS+GB3.1 LPS+control Ab
mAbGB3.1 inhibits LPS-induced TNFα and IL23p19 gene expression
in murine macrophages
0.7
0.6 TNF α
0.5
0.4
0.3
0.2
0.1
0
untreated LPS treated LPS + LPS +
GB3.1 control Ab
IL-23p19
0.6
0.5
0.4
0.3
0.2
0.1
0.0
[-LPS] [+LPS] [+LPS/GB3] [+LPS/cAb]
GB3.1 augments cell death of activated macrophages
120
unactivated cultures
% of total cells in100 -LPS
80
LPS Activated macrophages
60 +LPS
40 +LPS/cAb
20
0
+LPS/+GB3.1
0 20 40 60 80
Time after LPS activation (h)
120
untreated cultures
% of total cells in
100
80
60 Unactivated macrophages
40
20
0
0 20 40 60 80
Time of treatment (h)
mAbGB3.1 inhibits onset of colitis
• GB3.1 reduces CD4+ T cell accumulation specifically in
colon tissues
• GB3.1 does not induce significant apoptosis of
unactivated or activated T-cells
• GB3.1 inhibited recruitment of monocytes into
inflammed areas or caused apoptosis of recruited
macrophages
• No upregulation of MAdCAM expression
• Proinflammatory cytokine production is reduced
• Signaling pathways involved?
p65 [LPS activation] NF-kappaB p65
p65 [IFN/LPS]
0.28
0.24
0.2
0.16
0.12
0.08
0.04
0
[unactivated] [activated] [activated/GB3.1] [activated/cAb]
NF-kappaB p65 is enhanced in colon of Crohn’s patients
Did GB3.1 block NF-κB in treated mice cells?
NF-kB levels in colonic lamina propria cells
cAb Tx
1.4
1.2
1
0.8 p65
GB3 Tx
0.6 Normal RelB
0.4
0.2
0
1 2 3 4 5 6
mouse #
WILL GB3.1 REVERSE COLITIS?
early Day 9 Day21
Leithhauser et al
Lab Invest 81, 1339, 2001
Clinical Symptoms
Reconstituted, Untreated/cAb treated
Loss of weight, loss of hair, soft stools, ill
Reconstituted, GB3.1 treated
Minimal weight loss, no hair loss, normal stools, healthy
Weight loss curves
120
110
100
AJ0944 (control B)
AJ3524 (Control A)
AJ2527 (GB3.1)
90
80
70
0 10 20 30 40 50
Days post transfer
mAbGB3.1 treatment reverses colitis in the emerging phase of disease
inflammation: proximal colon
7
6
5
4
3
2
1
0
no cell transfer cell transfer/untreated cell transfer/cAb cell transfer/GB3.1
treated treated
Treatment
mAbGB3.1 treatment reverses colitis in the emerging phase of disease
inflammation: distal colon
8
7
6
5
4
3
2
1
0
no cell transfer cell transfer/untreated cell transfer/cAb cell transfer/GB3.1
treated treated
Treatment
mAbGB3.1
treatment reverses No cell transfer
colitis in the emerging
phase of disease
Reconstituted/
untreated
Reconstituted/
cAb Treated
Reconstituted/
mAbGB3.1
treated
CONCLUSIONS
ANTICARBOXYLATE ANTIBODY GB3.1 BLOCKS THE ONSET OF COLITIS
THE ANTIBODY ALSO SEEMS TO REVERSES ESTABLISHED COLITIS
EFFECTS ARE PROBABLY MEDIATED BY APOPTOSIS OF ACTIVATED MACROPHAGE
GB3.1 MAY BE A POTENTIAL TREATMENT FOR IBD
In this collaboration between the Freeze and Kronenberg labs
Geetha Srikrishna pioneered these studies and contributed most of the data