Oral Presentations - Federation of Clinical Immunology Societies

S12 Abstracts
Rationale: Pollen hypersensitivity is a major cause of
seasonal airway disease world wide. We recently resolved
the crystal structure and mapped the linear IgE epitopes of
Jun a 1. Heat and chemical denaturation of Jun a 1
reduced the binding of human IgE, suggesting the importance
of conformational epitopes for reactivity. Methods:
To identify conformational IgE epitopes, we developed a
panel of monoclonal antibodies (mAbs) against Jun a 1 and
selected those that were heat sensitive and inhibited the
binding of IgE from patient sera. These mAbs were used to
screen a random peptide, phage display library. After
repeated panning, phage clones were isolated and their
peptide sequences determined. To compare the affinity
and specificity of phage-expressed peptides the binding of
the phage to mAbs and patient IgE, and the ability of the
phage to inhibit binding of Jun a 1 to the mAb were tested
by ELISA. Consensus amino acids from selected phage were
matched with clusters of amino acid residues exposed on
the surface of Jun a 1, using GETAREA (http://www.scsb.
utmb.edu/cgi-bin/get_a_form.tcl) and a program specifically
designed to analyze similarity between the specific
phage residues and surface patches on allergens. Results:
Consensus amino acids from phage clones revealed common
patterns of amino acids which were identical or
similar to compact patches on the 3-D structure of Jun a 1
and represent putative conformational epitopes. Conclusion:
Bioinformatics search tools can play an important role
in identifying the IgE epitopes on allergens with known
structures or reliable 3D models.
doi:10.1016/j.clim.2007.03.203
OR.23 Antibodies Against Human Cytomegalovirus
in the Pathogenesis of Atherosclerosis: A Gene Array
Approach
Antonio Puccetti, Professor of Medicine, Gaslini
Institute-Department of Immunology, Genova, Marzia Dolci,
England Research Fellow, Gaslini Institute-Department of
Immunology, Genova, Dimitri Peterlana, PhD Student,
University of Verona-Department of Internal Medicine,
Verona, Riccardo Navone, Research Fellow, University of
Verona-Department of Internal Medicine, Verona, Caterina
Bason, Research Fellow, University of Verona-Department of
Internal Medicine, Verona, Claudio Lunardi, Professor of
Medicine, University of Verona-Department of Internal
Medicine, Verona
Human cytomegalovirus (hCMV) is involved in the
pathogenesis of atherosclerosis. We have shown in patients
with atherosclerosis that antibodies directed against the
hCMV-derived proteins US28 and UL122 are able to induce
endothelial cell damage and apoptosis of non-stressed
endothelial cells through cross-rection with normally
expressed surface molecules. Our aim was to dissect the
molecular basis of such interaction and to investigate
mechanisms linking innate immunity to atherosclerosis. We
analysed the gene expression profiles in endothelial cells
stimulated with antibodies affinity purified against either
the UL122 or the US28 peptides. Real-time polymerase
chain reaction was used to validate the microarray results.
Supernatant of endothelial cells incubated with antibodies
was analysed for the presence of Heat Shock Protein (HSP)
60 and was used to assess stimulation of toll-like receptor-
4 (TLR4). Antibodies against UL122 and US28 induced the
expression of genes encoding for adhesion molecules,
chemokines, growth factors and molecules involved in the
apoptotic process together with other genes involved in
the initiation and progression of atherosclerosis. HSP60 was
released in the medium of cells incubated with anti-US28
antibodies and was able to engage TLR4. Antibodies
directed against hCMV modulate the expression of genes
coding for molecules involved in the pathogenesis of
atherosclerosis. Moreover, endothelial cells exposed to
such antibodies express HSP60 on the cell surface and
release HSP60 in the medium able to activate TLR4. These
data confirm that hCMV plays a crucial role in mediating
the atherosclerotic process and that HSP60 is an endogenous
ligand for TLR4 signaling.
doi:10.1016/j.clim.2007.03.204
OR.24 Differential Effects of Aire Gene Deficiency
on the Development of Autoimmune
Encephalomyelitis Induced by Two Representative
Myelin Peptides
Asako Tagawa, Research Fellow, National Institute of
Neuroscience, NCNP, Kodaira, Tokyo, Japan, Toshimasa
Aranami, Section chief, Department of Immunology,
National Institute of Neuroscience, NCNP, Kodaira, Tokyo,
Japan, Mitsuru Matsumoto, Director, Division of Molecular
Immunology, Institute for Enzyme Research, University of
Tokushima, Japan, Tokushima, Japan, Takashi Yamamura,
Director, Department of Immunology, National Institute of
Neuroscience, NCNP, Kodaira, Tokyo, Japan
Hazardous autoimmunity is prevented partly by clonal
deletion of autoimmune T cells in the thymus. In the process
referred to as central tolerance, autoimmune regulator
(Aire) gene plays a pivotal role through transcriptional
control of ectopic antigen expression. In fact, thymic
expression of myelin antigens was shown to be involved in
shaping Tcell repertoire that would mediate development of
EAE. Here we asked if Aire gene deficiency might alter
manifestations of EAE induced with representative, encephalitogenic
peptides myelin oligodendrocyte glycoprotein
(MOG)35–55 and proteolipid protein (PLP)178–191 by using
Aire−/− mice. It is thought that MOG is not expressed in the
thymus (JCI 12: 544, 2003), whereas thymic expression of
PLP178–191 was previously proven (Nat Med 6:56, 2000).
First, we assessed recall responses to the peptides in the
immunized mice, and found that sensitized T cells from the
Aire−/− produced a higher amount of IL-17 than from wildtype
(WT) mice regardless of the peptides used for
immunization. Anti-MOG35–55 response was also associated
with a higher IFN-ã production in Aire−/−. However, there
was no significant difference between Aire−/− and WT in
IFN-ã production in response to PLP178–191, which resulted
in an augmented Th17/Th1 ratio in PLP178–191-immunized
mice. PLP178–191 immunization induced more serious EAE in
Aire−/− than WT, whereas such a difference could not be