Download CD40-Ligation in Vivo Promotes Activation and Migration of

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Nedical Research Society
competitivequantitative RT-PCR, we found inmased GR expression
in skeletal muscle in men is associated with resistance to insulinmediated glucose uptake. Using 5'-RACE-PCR (Rapid Amplification
of cDNA ends) we have shown that one mechanism for tissuespecific differences in GR is effects upon one or more multiple
alternate promoters of the GR gene. Further understanding of tissuespecific variations in GR may offer fundamental insights into the
pathophysiology of insulin resistance. Variations in HPA axis
activity may be a key mechanism to explain the effects of early life
events on later cardiovascular disease and offer potential for novel
thempatic strakgies to reduce cardiovascular risk.
Y13
INTERFERON7
MEDIATED
HOST
CELL
RESISTANCE TO CRYPTOSPORIDIIUMPARWMINFECTION
RCG Pollok, MJG Farthing, M Bajaj-Eliott, I Sanderson, V McDonald.
Digestive Diseases Research Centre, St Bartholomew's and Royal
London School of Medicine, London, UK.
Introduction C . p m is a major cause of diarrhoea world-wide.
Interferon y (IFN-y) is known to have a key role in the control of
Cparvum infection. However the mechanism of action of IFN-y is
unclear. We therefore determined the direct action of IFN-y on intestinal
epithelial cells (IECs) in mediating inhibition of infection. We examined
the role of the IFN-y receptor (FN-yR) and subsequent cellular
metabolism in mediating this action.
Methods IEC lines HT29, Caco 2 and H4 were grown on coverslips
with or without IFN-y and infected with parasites. Infection quantified by
both immunofluorescence assay (FA) and Giemsa staining. Parasite
attachment to paraformaldhyde-fixed HT29 cells was quantified by IFA.
IFN-yR expression was determined by immunoblotting. Cellular
signalling was studied using a selective Janus kinase-2 inhibitor
tyrophostin B42.The role of tryptophan and Fe2' in mediating the action
of IFN-y was evaluated by exogenous supplementation.
Results IFN-y inhibited infection in HT29 cells and Cac0-2 cells in a
concentration and time dependent fashion (~%0.001)but failed to inhibit
infection in H4 cells. This difference was reflected in IFN-yR expression
in the cell lines, with no detectable expression in H4 cells. IFN-y
inhibited parasite invasion but not attachment (40.4% f 3.4, ~ ~ 0 . 0 0 3 ) .
Both B42 and ferrous sulphate dose dependently r e d d the inhibitory
action of IFN-y (max inhib. 41.6% f 3.6, 6 . 0 1 ; and 55.Yh f 4.4,
6 . 0 0 1 rapeCtively). Tryptophan supplementationwas ineffective.
Discussion We have demonstrated the key role of enterocyte IFN-yR
expression and Janus kinase activity in mediating the inhibitory effects of
IFN-y. The inhibitory effect was independent of attachment but
dependent on both early host invasion events and cellular Fe2'. We
speculate that inhibition of invasion and modulation of cellular Fez' are
important mechanisms in IFN-y mediated control of C.parvum infection
in Vivo.
y14
THE ROLE OF SONIC HEDGEHOG SIGNALLING IN
EARLY TOOTH DEVELOPMENT
MT COBOURNE*, L SARKAR, S NAYLORt, M SMALLEYt, T
DALEt AND PT SHARPE
Department of Craniofacial Development, Floor 28, GKT Dental
Institute, London SEl 9RT thstitute of Cancer Research, 237
Fulham Road, London SW6 6JB
Sonic hedgehog (Shh) expression is highly localised to the
epithelium at the future sites of tooth development suggesting a role
for this signalling peptide in the initiation of tooth formation.
Analysis of Shh 4-mutant embryos identified only a rudimentary
first branchial arch, with no expression of early gene markers of
odontogenesis. To determine the role of Shh signalling in tooth germ
initiation we have therefore used explant culhlres of mandibular arch
primordia. Here we show that Shh can induce cell proliferation and
that proliferation in the developing tooth germ can be inhibited by
antibodies blocking Shh signalling. Blocking Shh signalling at E10.5
results in down-regulation of Ptc and Gli-1 expression and arrest of
tooth development. Thus, Shh has an essential role in early tooth bud
formation by stimulating localised epithelial cell proliferation.
During the initiation of mammalian tooth development boundaries
that distinguish oral from dental ectoderm must be formed to
correctly position the sites of tooth formation. We describe a
reciprocal relationship between the expression of Wnt- 76 in
presumptive oral ectoderm and Shh in presumptive dental ectoderm
in mouse embryos that mark boundaries between these cells with
different developmental fates. Using a murine retrovirus to
ectopically express Wnr-76 in presumptive dental ectoderm in
mandibular arch explants we show that Shh expression in the
ectoderm and Prc expression in the underlying ectomesenchymeare
down-regulated and that tooth development is subsequently arrested.
This suggests that Wnt-7b acts to repress Shh expression in oral
ectoderm, thus maintaining the boundaries between oral and dental
ectodermal cells. Implantation of beads soaked in Shh protein into
Wnt-76 infected explants resulted in a complete rescue of tooth
development, confirming that the repressive action of Wnt-7b
specifically effects Shh signalling. (Funded by a MRURCS England
Clinical Training Fellowship to MTC).
Y15 CD4O-Ligation In Vivo Promotes Activation and
Migration of Dendritic Cells and Enhances Resistance to
Infection with Mycobacretiurn tuberculosis
Stephen Jolles., Ricardo Tascon, Gerry Klaus*. AM Ager', M. Jo Colston
Divisions of Cellular Immunology* and Mycobactenal Research, The National
Institute for Medical Research, The Ridgeway. Mill Hill, London, NW7 1AA
Adaptive immune responses begin after antigen bearing dendritic
cells (DCs) traffic from peripheral tissues to lymph nodes where
they play a key role in the initiation of specific T-cell responses.
Ligation of CD40 is known to be an important signal in the
activation and migration of Langerhans cells (LC). Furthermore. the
importance of CD40 ligation in the generation of protective immune
responses against infectious agents (Leishmania major, Trypansomu
cruzi) and tumors has been demonstrated in a number of studies.
M.tuberculosis has been shown to use a number of evasion strategies
avoid an effective immune response including impairment of
antigen presentation and downregulation of CD40 ligand.
to
We investigated whether there is an impairment of Langerhans cell
migration in Mtuberculosis infected mice and whether ligation of
CD40 using an anti CD40 MAb could overcome any antigen
processinglpresentation defect in Mtuberculosis infected mice and
result in increased immunity against M.tuberculosis infection.
We have demonstrated that CD40 ligation in vivo results in
Langerhans cell migration from the epidermis with virtual clearing
of LC by day 7 following antLCD40 mAb. Epidermal LC have a
more mature phenotype and dendritic cells accumulate in the
draining nodes of anti-CD40 treated mice.
M.tuberculosis infected mice were treated with either anti-CD40 or
control antibody. The colony counts of h4,tuberculosis in the lungs
and spleens showed a 10-100 fold reduction in viable
M.tuberculosis. This effect is greater than any other immunological
therapy to date (including dendritic cell immunotherapy and DNA
vaccination) and suggests potential new approaches in multidrug
resistant M.tuberculosis and vaccine design.