Note: Descriptions are shown in the official language in which they were submitted.
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Method For Identifying Substances Which Positively Influence Inflammatory
Conditions Of Chronic Inflammatory Airway Diseases
Introduction
The present invention belongs to the field of modulation of inflammatory
processes,
in particular of inflammatory airway diseases, in which macrophages play an
important role. The inflammatory processes can be modulated according to the
invention by influencing the function of receptors on macrophages, which
receptors
io are identified to be involved in the inflammatory process.
Inflammatory processes involve a cascade of reactions. A wide variety of
factors are
involved in inflammatory processes leaving a single treatment to avoid said
factors
unsuccessful. This is in particular true for inflammatory processes of the
airways, like
the chronic inflammatory airway diseases.
Chronic inflammatory airway diseases include Chronic Bronchitis and Chronic
Obstructive Pulmonary Disease (COPD). For example, COPD is a complex disease
encompassing symptoms of several disorders: chronic bronchitis which is
characterized by cough and mucus hypersecretion, small airway disease,
including
inflammation and peribronchial fibrosis, and emphysema. COPD is characterized
by
an accelerated and irreversible decline of lung function. The major risk
factor for
developing COPD is continuous cigarette smoking. Since only about 20% of all
smokers are inflicted with COPD, a genetic predisposition is also likely to
contribute
to the disease.
The initial events in the early onset of COPD are inflammatory, affecting
small and
large airways. An irritation caused by cigarette smoking attracts macrophages
and
neutrophils the number of which is increased in the sputum of smokers.
Perpetual
smoking leads to an ongoing inflammatory response in the lung by releasing
mediators from macrophages, neutrophils and epithelial cells that recruit
inflammatory cells to sites of the injury. So far there is no therapy
available to reverse
the course of COPD. Smoking cessation may reduce the decline of lung function.
Only a few drugs provide some relief for patients. Longlasting ¾2-agonists and
anticholinergics are applied to achieve a transient bronchodilatation. A
variety of
antagonists for inflammatory events are under investigation like, LTB4-, IL-8-
, TNFa-
inhibitors.
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Chronic inflammatory airway diseases can be attributed to activated
inflammatory
immune cells, e.g. macrophages. There is a need for modulating the function of
macrophages in order to eliminate a basis for inflammatory processes.
Description Of The Invention
In the present invention it was found that macrophages involved in an
inflammatory
process, preferably in a chronic inflammatory airway disease, more preferably
in
chronic bronchitis or COPD, show a pattern of differentially expressed nucleic
acid
sequence and protein expression which differs from the pattern of gene
expression
lo of macrophages from healthy donors or donors in an irritated status, which
latter do
contain macrophages in an activated status. Therefore, macrophages show
different
activation levels under different inflammatory conditions and it is shown in
the
present invention that macrophages in an hyperactive status are involved in an
inflammatory process, preferably in a chronic inflammatory airway disease,
more
preferably in chronic bronchitis or COPD. The present invention provides for
the
inhibition of the hyperactivation or the reduction of the hyperactive status
of a
macrophage by allowing the identification of substances which modulate
receptors
involved in the hyperactivation or maintaining the hyperactive status.
2o The invention is based on the identification of a differentially expressed
nucleic acid
sequence or protein which is involved in causing the induction and/or
maintenance of
the hyperactive status of macrophages involved in an inflammatory process,
preferably in a chronic inflammatory airway disease, more preferably in
chronic
bronchitis or COPD. Such differentially expressed nucleic acid sequence or
protein is
in the following named differentially expressed nucleic acid sequence or
protein of
the invention respectively. In particular, the present invention teaches a
link between
phenotypic changes in macrophages due to differentially expressed nucleic acid
sequence and protein expression pattern and involvement of macrophages in
inflammatory processes and, thus, provides a basis for a variety of
applications. For
3o example, the present invention provides a method and a test system for
determining
the expression level of a macrophage protein or differentially expressed
nucleic acid
sequence of the invention and thereby provides e.g. for methods for diagnosis
or
monitoring of inflammatory processes with involvement of hyperactivated
macrophages in mammalian, preferably human beings, especially such beings
suffering from an inflammatory process, preferably in a chronic inflammatory
airway
disease, more preferably in chronic bronchitis or COPD. The invention also
relates to
a method for identifying a substance by means of a differentially expressed
nucleic
acid sequence or protein of the invention processes, which substance
modulates, i.e.
acts as an inhibitor or activator on the said differentially expressed nucleic
acid
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sequence or protein of the invention and thereby positively
influences chronic inflammatory processes by inhibition of
the hyperactivation or reduction of the hyperactive status
of macrophages, and thereby allows treatment of mammals,
preferably human beings, suffering from a said disease. The
invention also relates to a method for selectively
modulating such a differentially expressed nucleic acid
sequence or protein of the invention in a macrophage
comprising administering a substance determined to be a
modulator of said protein or differentially expressed
nucleic acid sequence. The present invention includes the
use of said substances for treating beings in need of a
treatment of an inflammatory process, preferably a chronic
inflammatory airway disease, more preferably chronic
bronchitis or COPD.
One aspect of the invention relates to a method
for determining whether a substance inhibits or reduces an
inflammatory process in which a macrophage is in a
hyperactivated status due to a differentially expressed
macrophage surface receptor, comprising: (a) applying said
substance to a test system which generates a measurable
read-out upon modulation of said macrophage surface receptor
or macrophage surface receptor function, wherein said
macrophage surface receptor is a FPRL-1 receptor comprising
SEQ ID NO:2; and (b) comparing the level of the read-out of
the test system to a control level, wherein a difference in
levels indicates the substance is an inhibitor or an
activator of said macrophage surface receptor; and wherein
the inhibitor of the macrophage surface receptor which is
expressed on a higher level in said hyperactivated
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3a
macrophage or the activator of the macrophage surface
receptor which is expressed on a lower level in said
hyperactivated macrophage indicates the substance inhibits
or reduces said hyperactivated status of said macrophage.
For the present invention in a first step
differentially expressed nucleic acid sequences and proteins
are identified which have a different expression pattern in
a hyperactivated macrophage compared to a macrophage which
is not hyperactivated. For the sake of conciseness this
description deals particularly with investigation of
macrophages involved in COPD, however, equivalent results
may be observed with samples from patients suffering from
other chronic inflammatory airway diseases, e.g. chronic
bronchitis. The investigation of the different expression
pattern leads to the identification of a series of
differentially expressed nucleic acid sequences in
macrophages, differentially expressed in dependency on the
activation status of a macrophage involved in an
inflammatory process, as exemplified in the Examples
hereinbelow.
Briefly, such a differentially expressed nucleic
acid sequence is identified by comparative expression
profiling experiments using a cell or cellular extract from
a hyperactivated macrophage, i.e. for example from the site
of inflammation in a COPD and from the corresponding site of
control being not suffering from said disease, however,
suffering from an irritated condition like cigarette smoke
exposure.
A differentially expressed nucleic acid sequence
or protein of the invention can easily be detected by such a
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3b
method because amongst the differentially expressed
macrophage genes a class of differentially expressed nucleic
acid sequences can be identified which encodes a class of
macrophage surface receptors which is characterized in that
it is expressed at a lower or higher level than the control
level in a macrophage which is not hyperactivated. Such a
macrophage surface receptor of the invention is hereinafter
named ILM receptor. However, the invention does not only
concern a naturally occurring ILM receptor, but also
includes within the meaning of ILM receptor a receptor which
is functionally equivalent to, i.e. which shares the binding
capacities and the cellular function with an ILM receptor.
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An example for an ILM receptor according to the present invention is a FPRL-1
receptor type receptor including FPRL-1 receptor (SEQ ID NO. 2). The term
"receptor type receptor" used in context with the present invention, e.g. FPRL-
1
receptor type receptor, is a receptor which is "functionally equivalent" to,
i.e. which
shares the binding capacities and the cellular function with, the respective
receptor,
e.g. FPRL-1 receptor of Seq. ID NO. 2; the term also encompasses variants,
mutants
or fragments of a naturally occuring receptor, e.g. FPRL-1 receptor. or
naturally
occuring receptor type receptor, e.g. FPRL-1 receptor type receptor, which
variants,
mutants or fragments are functionally equivalent to the receptor, e.g. FPRL-1
receptor.
Further examples for ILM receptors are HM74 receptor type receptor including
HM74
receptor (SEQ ID NO. 21); AICL receptor type receptor including AICL receptor
(SEQ ID NO. 6) ); ILT1 receptor type receptor including ILT1 receptor (SEQ ID
NO.
12); SHPS-1 receptor type receptor including SHPS-1 receptor (SEQ ID NO. 4);
KDEL receptor 1 type receptor including KDEL receptor 1(SEQ ID NO. 8); and CSF-
1 receptor type receptor including CSF-1 receptor (SEQ ID NO. 10). Preferred
is the
respective receptor shown in the sequence listing or a variant, mutant or
fragment
thereof having the same function, even more preferred is the respective
receptor
shown in the sequence listing under SEQ ID NO. 21, 6, 12, 4, 8, 10. In even
more
preferred embodiments the receptors are encoded by the nucleic acid sequences
having the SEQ ID NOs 20, 5, 11, 3, 7 or 9, respectively.
A preferred embodiment of an ILM receptor in context with the present
invention is a
FPRL-1 receptor type receptor. The term FPRL-1 receptor type receptor
accordingly
also encompasses variants, mutants or fragments, of naturally occuring FPRL-1
receptor or FPRL-1 receptor type receptors, which variants, mutants or
fragments
are functionally equivalent to the FPRL-1 receptor. An even more preferred
3o embodiment in context with the description of the embodiments of the
present
invention is the FPRL-1 receptor of Seq. ID NO. 2 or a variant, mutant or
fragment
thereof having the same function, even more preferred is the FPRL-1 receptor
of
Seq.1D NO 2. ln a most preferred embodiment, the FPRL-1 receptor is encoded by
the nucleic acid sequence shown in SEQ ID NO. 1.
According to the present invention, the function of an ILM receptor expressed
at a
lower level than the control level is preferably activated in order to inhibit
hyperactivation or reduce a hyperactivated status of a macrophage, whereby the
function of an ILM receptor which is expressed at a higher level than the
control level
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is preferably inhibited in order to inhibit hyperactivation or reduce a
hyperactivated
status of a macrophage. Function of a receptor in context with the present
invention
is any function of a receptor of the invention which is capable of influencing
the
inflammatory processes. For example, a receptor of the invention is mediating
5 inflammation in that it is activated by a ligand (every substance which has
the
capacity to bind to said receptor at least one of its domains exposed on the
cell
surface) and leads to a intracellular signal involved in inflammatory
processes.
In one embodiment the present invention concerns a method for determining a
substance to be an activator or inhibitor of an ILM receptor characterized in
that the
receptor is deregulated preferably overexpressed or downregulated in a
macrophage
involved in a chronic inflammatory airway disease and which receptor plays a
role in
mediating inflammation. A method according to the invention comprises the
application of a substance of interest to a test system which generates a
measurable
read-out upon modulation of the ILM receptor or of an ILM receptor function. A
test
system useful for performing such method of the invention comprises a cell or
a cell-
free system. For example, in one embodiment according to the invention the
system
is designed in order to allow the testing of substances acting on the
expression level
of the differentially expressed nucleic acid sequence, in another embodiment
the
system allows the testing of substances directly interacting with the receptor
or
interfering with the binding of the receptor with a natural or an artificial
but
appropriate ligand. The latter system comprises a receptor of the invention in
a way
that a substance which should be tested can physically contact said receptor
and
which direct interaction leads to a measurable read-out indicative for the
change of
receptor function.
A method according to the invention comprising a cellular system can be, for
example, a method in which a MonoMac6 or a THP-1 cell is used wherein said
cell is
stimulated with phorbol 12-myristate 13-acetate and with a substance selected
from
a group consisting of LPS and smoke.
The present invention also provides a test system for determining whether a
substance is an activator or an inhibitor according to the invention of an ILM
receptor
function according to the invention, characterized in that the receptor is
involved in a
chronic inflammatory airway disease and which receptor plays a role in
mediating
inflammation, comprising at least an ILM receptor or an expression vector
capable of
expressing an ILM receptor in a cell or a host cell transformed with an
expression
vector capable of expressing an ILM receptor.
For performing a method for determining whether a substance is an activator or
an
inhibitor of receptor function of the present invention cells as well as cell-
free
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systems can be used. Test systems for performing the method can be, for
example,
designed and built up by using elements and methods well known in the art. For
example, cell-free systems may include, for example, cellular compartments or
vesicles comprising a receptor of the invention. Suitable cellular systems
include, for
example, a suitable prokaryotic cell or eukaryotic cell, i.e. such comprising
a
respective receptor of the invention. A cell suitable for performing a said
method of
the invention may be obtained by recombinant techniques, i.e. after
transformation or
transfection with a vector suitable for expression of the desired receptor of
the
invention, or may be a cell line or a cell isolated from a natural source
expressing the
1o desired receptor of the invention. A test system according to the invention
comprising
a cellular system can also be, for example, a test system in which a MonoMac6
or a
THP-1 cell is used wherein said cell is stimulated with phorbol 12-myristate
13-
acetate and with a substance selected from a group consisting of LPS and
smoke. A
test system according to the invention may include a natural or artificial
ligand of the
receptor if desirable or necessary for testing whether a substance of interest
is an
inhibitor or activator of a receptor of the invention.
A test method according to the invention comprises measuring a read-out, i.e.
a
phenotypic change in the test system, for example, if a cellular system is
used a
phenotypic change of the cell. Such change may be a change in a naturally
occurring or artificial response of the cell to receptor activation or
inhibition, e.g. as
detailed in the Examples hereinbelow.
A test method according to the invention can on the one hand be useful for
high
throughput testing suitable for determining whether a substance is an
inhibitor or
activator of the invention, but also e.g. for secondary testing or validation
of a hit or
lead substance identified in high throughput testing.
The present invention also concerns a substance identified in a method
according to
the invention to be an inhibitor or activator of a receptor of the invention.
A
substance of the present invention is any compound which is capable of
activating or
preferably inhibiting a function of a receptor according the invention. An
example of a
way to activate or inhibit a function of a receptor is by influencing the
expression
level of said receptor. Another example of a way to activate or inhibit a
function of a
receptor is to apply a substance directly binding the receptor and thereby
activating
or blocking functional domains of said receptor, which can be done reversibly
or
irreversibly, depending on the nature of the substance applied.
,
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Accordingly, a substance useful for activating or inhibiting receptor function
include
substances acting on the expression of differentially expressed nucleic acid
sequence, but also such acting on the receptor itself. Therefore, according to
the
invention the meaning of the term a substance of the invention includes but is
not
limited to nucleic acid sequences coding for the gene of a receptor of the
invention or
a fragment or variant thereof and being capable of influencing the gene
expression
level, e.g. nucleic acid molecules suitable as antisense nucleic acid,
ribozyme, or for
triple helix formation. Another substance of the invention is e.g. an antibody
or an
organic or inorganic compound directly binding to or interfering with the
binding of an
1o appropriate ligand with a receptor of the invention and thereby affecting
its function.
In a further aspect, the present invention relates to a method for determining
an
expression level of an ILM receptor differentially expressed nucleic acid
sequence or
protein according to the invention comprising determining the level of said
ILM
receptor in a macrophage according to the invention. Such a method can be
used,
for example, for testing whether a substance is capable of influencing
differentially
expressed nucleic acid sequence expression levels in a method outlined above
for
determining whether a substance is an activator or inhibitor. A method for
determining an expression level of an ILM receptor differentially expressed
nucleic
2o acid sequence or protein can, however, also be used for testing the
activation status
of a macrophage, e.g. for diagnostic purposes or for investigation of the
success of
treatment of a disease which is caused by the hyperactivated macrophage.
Accordingly, the invention also relates to a method for diagnosis of a chronic
inflammatory disease or monitoring of such disease, e.g. monitoring success in
treating beings in need of treatment of such disease, comprising determining
the
level of the receptor expressed in a macrophage according to the invention.
Said
macrophage is preferably a mammalian, more preferably a human cell.
Accordingly,
macrophages of the present invention are preferably obtainable from the site
of
inflammation in a mammal and more preferably from a site of inflammation in a
3o human being.
A method for determining expression levels of a receptor according to the
invention
can depending on the purpose of determining the expression level be performed
by
known procedures such as measuring the concentration of respective RNA
transcripts via hybridization techniques or via reporter gene driven assays
such as
luciferase assays or by measuring the protein concentration of said receptor
using
respective antibodies to verify the identity of said protein.
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The present invention relates to the use of a substance according to the
invention for
the treatment of a chronic inflammatory airways disease according to the
invention.
Another embodiment of the present invention relates to a pharmaceutical
composition comprising at least one of the substances according to the
invention
determined to be an activator or an inhibitor using the method for determining
whether the substance is an activator or an inhibitor according to the
invention
characterized in that the respective receptor according to the invention is
overexpressed in a macrophage according to the invention involved in a chronic
inflammatory airway disease according to the invention. The composition may be
To manufactured in a manner that is itself known, e.g. by means of
conventional mixing,
dissolving, granulating, dragee-making, levigating, powdering, emulsifying,
encapsulating, entrapping or lyophilizing processes.
In order to use substances activating or inhibiting according to the invention
as drugs
for treatment of chronic inflammatory airway diseases, the substances can be
tested
in animal models for example an animal suffering form an inflammatory airway
disorder or a transgenic animal expressing a receptor according to the
invention.
Toxicity and therapeutic efficacy of a substance according to the invention
can be
determined by standard pharmaceutical procedures, which include conducting
cell
culture and animal experiments to determine the IC50, LD, and ED50. The data
obtained are used for determining the animal or more preferred the human dose
range, which will also depend on the dosage form (tablets, capsules, aerosol
sprays
ampules, etc.) and the administration route (for example transdermat, oral,
buccal,
nasal, enteral, parenteral, inhalative, intratracheal, or rectal).
A pharmaceutical composition containing a least one substance according to the
invention as an active ingredient can be formulated in conventional manner.
Methods
for making such formulations can be found in manuals, e.g. "Remington
Pharmaceufiicai Science". Examples for ingredients that are useful for
formulating at
3o least orie substance according to the present invention are also found in
WO
99118193.
In a further aspect the invention teaches a method for treating a chronic
inflammatory
airway disease according to the invention which method comprises administering
to
a being preferably to a human being in need of such treatment a suitable
amount of
a pharmaceutical composition comprising at least one substance determined to
be
an activator or inhibitor according to a method for determining whether a
substance
is an activator or an inhibitor according to the invention of an ILM receptor
according
to the invention characterized in that the receptor is overexpressed in a
macrophage
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according to the invention and plays a role in mediating inflammation involved
in a
chronic inflammatory airway disease according to the invention.
In an other embodiment the invention relates to a method for selectively
modulating
ILM receptor concentration in a macrophage, comprising administering a
substance
determined to be an activator or inhibitor of a receptor according to the
invention.
The following examples are meant to illustrate the present invention, however,
shall
not be construed as limitation. However, the Examples describe most preferred
embodiments of the invention.
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Examples
Example 1: Comparative Expression Profiling and FPLR-1 Cloning
5 The following is an illustration of how comparative expression profiling can
be
performed in order to identify receptors according to the present invention.
1.1. Selection of Patients
Three groups of subjects are studied: healthy non-smokers, healthy smokers and
10 patients with COPD.
In order to assess lung function subjects have to perform spirometry. A simple
calculation based on age and height is used to characterise the results. COPD
subjects are included if their FEV, % predicted is <70%. Healthy smokers are
age
and smoking history matched with the COPD subjects but have normal lung
function.
Healthy non-smokers have normal lung function and have never smoked. The
latter
group has a methacholine challenge to exclude asthma. This technique requires
increasing doses of methacholine to be given to the subject, with spirometry
between
each dose. When the FEV, falls 20% the test is stopped and the PC20 is
calculated.
This is the dose of methacholine causing a 20% fall in FEV, and we will
require a
value of >32 as evidence of absence of asthma. All subjects have skin prick
tests to
common allergens and are required to have negative results. This excludes
atopic
individuals. The clinical history of the subjects is monitored and examined in
order to
exclude concomitant disease.
1.2. BAL (bronchoalveolar lavage) Procedure
Subjects are sedated with midazolam prior to the BAL. Local anaesthetic spray
is
used to anaesthetize the back of the throat. A 7mm Olympus bronchoscope is
used.
The lavaged area is the right middle lobe. 250 ml of sterile saline is
instilled and
immediately aspirated. The resulting aspirate contains macrophages.
1.3. BAL Processing
BAL is filtered through sterile gauze to remove debris. The cells are washed
twice in
HBSS, resuspended in 1 ml HBSS (Hank's Balanced Salt Solution) and counted.
The
macrophages are spun to a pellet using 15 mi Falcon blue-cap polypropylen,
resuspended in Trizol reagent (Gibco BRL Life Technologies) at a concentration
of 1
ml Trizol reagent per 10 million cells and then frozen at -70 C.
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1.4. Differential Gene Expression Analysis
Total RNA is extracted from macrophage samples obtained according to Example
1.3. Cell suspensions in Trizol are homogenized through pipetting and
incubated at
room temperature for 5 minutes. 200 I chloroform per ml Trizol is added, the
mixture
carefully mixed for 15 seconds and incubated for 3 more minutes at room
temperature. The samples are spun at 10000g for 15 minutes at 4 C. The upper
phase is transferred into a new reaction tube and the RNA is precipitated by
adding
0.5 ml isopropanol per ml Trizol for 10 minutes at room temperature. Then, the
precipitate is pelleted by using a microcentifuge for 10 minutes at 4 C with
10000g,
the pellet is washed twice with 75% ethanol, air dried and resuspended in DEPC-
HZO.
An RNA cleanup with Qiagen RNeasy Total RNA isolation kit (Qiagen) is
performed
in order to improve the purity of the RNA. The purity of the RNA is determined
by
agarose gelelectrophoresis and the concentration is measured by UV absorption
at
260 nm.
5 g of each RNA is used for cDNA synthesis. First and second strand synthesis
are
performed with the SuperScript Choice system (Gibco BRL Life Technologies). In
a
total volume of 11 l RNA and 1 I of 100 M T7-(dt)24 primer, sequence shown
in
SEQ ID NO. 13, are heated up to 70 C for 10 minutes and then cooled down on
ice
for 2 minutes. First strand buffer to a final concentration of lx, DTT to a
concentration of 10 mM and a dNTP mix to a final concentration of 0.5 mM are
added to a total volume of 18 I. The reaction mix is incubated at 42 C for 2
minutes
and 2 l of Superscript 11 reverse transcriptase (200 Ul l) are added. For
second
strand synthesis 130 l of a mix containing 1.15x second strand buffer, 230 M
dNTPs, 10 U E.coli DNA ligase (10U/ l), E.coli DNA polymerase (10 U/ I), RNase
H
(2U/ l) is added to the reaction of the first strand synthesis and carefully
mixed with a
pipette. Second strand synthesis is performed at 16 C for 2 hours, then 2 I
of T4
DNA polymerase (5 U/ I) are added, incubated for 5 minutes at 16 C and the
reaction is stopped by adding 10 l 0.5 M EDTA.
Prior to cRNA synthesis the double stranded cDNA is purified. The cDNA is
mixed
with an equal volume of phenol:chloroform:isoamylalcohol (25:24:1) and spun
through the gel matrix of phase lock gels (Eppendorf) in a microcentrifuge in
order to
separate the cDNA from unbound nucleotides. The aqueous phase is precipitated
with ammoniumacetate and ethanol. Subsequently, the cDNA is used for in vitro
transcription. cRNA synthesis is performed with the ENZO BioArray High Yield
RNA
Transcript Labeling Kit according to manufacturer's protocol (ENZO
Diagnostics).
Briefly, the cDNA is incubated with lx HY reaction buffer, lx biotin labeled
ribonucleotides, lx DTT, 1 x RNase Inhibitor Mix and 1 x T7 RNA Polymerase in
a
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total volume of 40 l for 5 hours at 37 C. Then, the reaction mix is purified
via
RNeasy columns (Qiagen), the cRNA precipitated with ammonium acetate and
ethanol and finally resuspended in DEPC-treated water. The concentration is
determined via UV spectrometry at 260 nm. The remaining cRNA is incubated with
1 x fragmentation buffer (5x fragmentation buffer: 200 mM Tris acetate, pH
8.1, 500
mM KOAc, 150 mM MgOAc) at 94 C for 35 minutes.
For hybridization of the DNA chip 15 g of cRNA is used, mixed with 50 pM
biotin-
labeled control B2 oligonucleotide, sequence shown SEQ ID NO. 14, lx cRNA
cocktail, 0.1 mg/mI herring sperm DNA, 0.5 mg/mI acetylated BSA, lx MES (2-[N-
morpholino]-ethanesulfonic acid) hybridization buffer in a total volume of 300
I. The
hybridization mixture is heated up to 99 C for 5 minutes, cooled down to 45 C
for 10
minutes and 200 l of the mix are used to fill the probe array. The
hybridization is
performed at 45 C at 60 rpm for 16 hours.
After the hybridization the hybridization mix on the chip is replaced by 300
I non-
stringent wash buffer (100 mM MES, 100 mM NaCi, 0.01 % Tween 20). The chip is
inserted into an Affymetrix Fluidics station and washing and staining is
performed
according to the EukGE-WS2 protocol. The staining solution per chip consists
of 600
l 1x stain buffer (100 mM MES, 1 M NaCI, 0.05% Tween 20), 2 mg/mI BSA, 10
g/ml SAPE (streptavidin phycoerythrin) (Dianova), the antibody solution
consists of
lx stain buffer, 2 mg/mi BSA, 0.1 mg/mI goat IgG, 3 g/mi biotinylated
antibody.
After the washing and staining procedure the chips are scanned on the HP Gene
Array Scanner (Hewlett Packard).
Data Analysis is performed by pairwise comparisons between chips hybridized
with
RNA isolated from COPD smokers and chips hybridized with RNA isolated from
healthy smokers.
One of the different expressed nucleic acid sequences identified is coding for
FPRL-
1(formyl peptide receptor like-1) receptor (also named LXA4R, HM63, FPR2,
FPRH2, FMLP-R-II, Lipoxin A4 receptor); see Seq ID NOs. 1 and 2. It belongs to
the
chemoattractant peptide receptor family including receptors for fMLP (N-formyl-
methionyl-leucyl-phenylalanine), IL-8 or C5a. These receptors show a seven-
transmembrane helix motif and signal through heterotrimeric G-proteins. FPRL-1
receptor was identified as the high-affinity receptor for lipoxin A4 (LXA4)
(Murphy et
al. 1992).
Alveolar macrophages have been shown to produce lipoxins, which are
synthesized
by 15-lipoxygenase (Kim 1988). Lipoxin A4 (LXA4) stimulates chemotaxis,
adherence
and calcium release in monocytes. In neutrophils, though, LXA4 inhibits
chemotaxis
and adhesion, and downregulates transmigration through epithelial cells
(Maddox
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and Serhan 1996). LXA4 was found elevated in BALs from patients with asthma
(Lee
et al. 1990, Serhan 1999). In particular, it was found to cause a dose-
dependent
contraction of human bronchi (Christie et al. 1992). LXA4 is considered to be
a
generic modulator of inflammation in the lung.
1.5. FPRL-1 receptor Overexpressed in COPD Macrophages
FPRL-1 receptor is consistently found upregulated (66.7%) in COPD smokers
compared to healthy smokers. This is demonstrated by calculated "fold change"
values from 42 pairwise comparisons and by average difference ("avg diff")
values
(Table 1, 2). Relative expression levels for non-smokers and healthy smokers
are
similar and elevated levels are restricted to patients with COPD. Therefore,
COPD-
specific effects cause the upregulation.
Table 1: Expression pattern for FPRL-1 receptor: fold change calculation for
42
pairwise comparisons between COPD and healthy smokers. Only values higher than
2fold and lower than -2fold are considered as deregulated. Thus FPRL-1
receptor
was 28 times upregulated and 14 times not regulated.
fold change comparison fold change comparison fold change compariso
2.7 39vs2 2.9 5vs2 3.3 1 vs2
4.6 39vs37 3.6 5vs37 5.5 1 vs37
2 39vs43 1.4 5vs43 1.4 1 vs43
3.1 39vs56 3 5vs56 3.9 1 vs56
4.1 39vs57 3.2 5vs57 5.3 1 vs57
2.9 39vs58 3 5vs58 3.6 1 vs58
2.2 39vs62 2.7 5vs62 2.7 1 vs62
1.3 44vs2 2.7 6vs2 1.4 3vs2
2.7 44vs37 4.1 6vs37 2.9 3vs37
-1.9 44vs43 1.1 6vs43 -1.7 3vs43
1.5 44vs56 3.2 6vs56 1.7 3vs56
2 44vs57 3.5 6vs57 2.3 3vs57
1.4 44vs58 2.9 6vs58 1.5 3vs58
1.1 44vs62 2.2 6vs62 1.2 3vs62
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Table 2: Expression levels of FPRL-1 receptor: "avg difr' values, a relative
indicator
of the intensity of the hybridisation signal on the chip, for each patient are
listed; OS
means obstructed smoker, HS healthy smoker, NS non-smoker
OS avg diff HS avg diff NS avg diff
P 1 1276.7 P 2 490.4 P 48149 248.2
P 3 553.6 P 37 52.1 P 50/52 565.7
P 5 1710.2 P 43 940 P 54/61 142.4
P 6 1046.9 P 56 327.1
P 39 1025.2 P 57 238.7
P 44 507.1 P 58 358.2
P62 469.6
mean + 1020.0 410.9 318.8
std. dev. 452.5 276.3 220.3
Median 1036.1 327.1 248.2
P value for comparisons between COPD smokers and healthy smokers: 0.02
Chip data for FPRL-1 receptor are confirmed by TaqMan analysis (Perkin Elmer
Applied Biosystems) for three COPD and two healthy smokers. Fold changes
obtained by TaqMan very much resemble the data from the gene chips (Table 3).
Table 3: Upregulation of FPRL-1 receptor in COPD smokers determined by gene
chips and TaqMan.
Fold change determination for FPRL-1 receptor by chip data in six comparisons
between COPD smokers and healthy smokers is validated by analysis of the same
samples by TaqMan and the relative upregulation is calculated with GAPDH as a
housekeeping gene.
compariso chip TaqMan comparison chip TaqMan
1 vs2 3.3 4.1 1 vs37 5.5 4.6
3vs2 1.4 2.2 3vs37 2.9 2.5
39vs2 2.7 6.0 39vs37 4.6 6.8
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Another differentially expressed nucleic acid sequence identified codes for
HM74
receptor, see SEQ ID NOs. 20 and 21, which belongs to the family of G-protein-
coupled receptors. HM74 receptor was cloned from a human monocytic library
(Nomura et al. 1993). To date, the ligand has not been identified. HM74
receptor is
5 consistently found upregulated (54.8%) in COPD smokers compared to healthy
smokers. This is demonstrated by calculated õfold change" values (Table 5)
from 42
pairwise comparisons and by õavg diff ` values (Table 6).
10 Table 5. Expression pattern for HM74 receptor: fold change calculation for
42
pairwise comparisons between COPD and healthy smokers. Only values higher than
2fold and lower than -2fold are considered as deregulated. Thus, HM74 receptor
was 23 times upregulated and 17 times not regulated
fold chang comparison fold change comparison fold chang omparison
1.2 39vs2 4.5 5vs2 -1.2 1 vs2
4.7 39vs37 13.8 5vs37 2.8 1 vs37
-2.1 39vs43 2.5 5vs43 -2.2 1 vs43
2.9 39vs56 8.6 5vs56 1.8 1 vs56
2.6 39vs57 8.9 5vs57 1.6 1 vs57
2.6 39vs58 7.7 5vs58 1.6 1 vs58
2.4 39vs62 8.5 5vs62 1.5 1 vs62
2.8 44vs2 1 6vs2 -1.1 3vs2
8.8 44vs37 3.5 6vs37 3 3vs37
1.5 44vs43 -1.7 6vs43 -2 3vs43
5.5 44vs56 2.2 6vs56 1.9 3vs56
5.4 44vs57 2 6vs57 1.7 3vs57
4.9 44vs58 1.9 6vs58 1.7 3vs58
5.2 44vs62 1.9 6vs62 1.7 3vs62
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Table 6: Expression levels of HM74 receptor: "avg difP' values, a relative
indicator of
the intensity of the hybridisation signal on the chip, for each patient are
listed; OS
means obstructed smoker, HS healthy smoker, NS non-smoker
OS avg diff HS avg diff NS avg diff
P 1 3233 P 2 3916.3 P 48/49 1690.7
P 3 3474.5 P 37 1154.5 P 50/52 4176.4
P 5 17671 P 43 5770.5 P 54/61 3504.8
P 6 4094.2 P 56 1860.2
P 39 4201.3 P 57 1639.8
P 44 11068.5 P 58 2080.2
P62 1721.6
mean + 7290.4 2591.9 3124.0
std. dev. 5879.0 1652.5 1285.9
median 4147.8 2243.6 3504.8
Chip data for HM74 receptor are confirmed by TaqMan analysis for three COPD
and
two healthy smokers. Fold changes obtained by TaqMan very much resemble the
data from the gene chips (Table 7).
Table 7: Upregulation of HM74 receptor in COPD smokers determined by gene
chips
and TaqMan.
Fold change determination for HM74 receptor by chip data in six comparisons
between COPD smokers and healthy smokers is validated by analysis of the same
samples by TaqMan and the relative upregulation is calculated with GAPDH as a
housekeeping gene.
comparisor chip TaqMan comparison chip TaqMan
1 vs2 0.8 2.3 1 vs37 2.8 4.5
3vs2 0.9 0.8 3vs37 3.0 1.4
39vs2 1.2 1.4 39vs37 4.7 2.6
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Another differentially expressed nucleic acid sequence identified codes for
AICL
receptor (activation-induced C-type lectin), see SEQ ID NOs. 5 and 6., which
is a
type II membrane protein that recognizes and binds N-acetyl-galactosamin or -
glucosamin moieties of plasma glycoproteins (Oda et al. 1988). It is expressed
in
lymphoid tissues and in hematopoetic cells as well as in NK and T cells. Its
expression is induced during lymphocyte activation and after stimulation with
PMA
(Hamann et al. 1997). Since homologues of AICL receptor are involved in signal
transmission in lymphocytes and in lymphocyte proliferation, it is tempting to
assume
that AICL receptor also participates in these processes (Hamann et al. 1993).
lo AICL receptor is consistently found upregulated (66.7%) in COPD smokers
compared to healthy smokers. This is demonstrated by calculated õfold change"
values (Table 8) from 42 pairwise comparisons and by õavg diff" values (Table
9).
The p value for the comparisons between COPD smokers and healthy smokers was
0.01.
Table 8. Expression pattern for AICL receptor: fold change calculation for 42
pairwise
comparisons between COPD and healthy smokers. Only values higher than 2fold
and lower than -2fold are considered as deregulated. Thus, AICL receptor was
28
times upregulated and 14 times not regulated
fold chang comparison fold change comparison fold change omparison
1 39vs2 1.5 5vs2 -1.3 1 vs2
1.9 39vs37 2.8 5vs37 1.4 1 vs37
-1.4 39vs43 2.4 5vs43 1.3 1 vs43
3.3 39vs56 5 5vs56 2.7 1 vs56
6.9 39vs57 10 5vs57 5.3 1 vs57
3.1 39vs58 4.5 5vs58 2.3 1 vs58
3.3 39vs62 5.1 5vs62 2.7 1 vs62
1.4 44vs2 -1.4 6vs2 -1.5 3vs2
2.6 44vs37 1.2 6vs37 1.2 3vs37
2.3 44vs43 1.1 6vs43 1.1 3vs43
4.2 44vs56 2.3 6vs56 2.3 3vs56
9.6 44vs57 4.5 6vs57 4.5 3vs57
4.3 44vs58 2 6vs58 2 3vs58
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Table 9: Expression levels of AICL receptor: "avg diff' values, a relative
indicator of
the intensity of the hybridisation signal on the chip, for each patient are
listed; OS
means obstructed smoker, HS healthy smoker, NS non-smoker
OS avg diff HS avg diff NS avg diff
P 1 3415.3 P 2 4984.2 P 48/49 748.4
P 3 3412.9 P 37 2388.6 P 50/52 1726.5
P 5 6585.8 P 43 2722.5 P 54/61 1087.9
P 6 3444.7 P 56 1121.1
P 39 4548.4 P 57 656.1
P 44 6291.5 P 58 1476.0
P 62 1113.1
mean + 4622.4 2065.9 1187.5
std. dev. 1474.3 1482.0 496.6
median 3996.6 1476.0 1087.9
Another differentially expressed nucleic acid sequence identified codes for
ILTI
receptor (immunoglobulin-like transcript 1), see SEQ ID NOs. 11 and 12. ILTI
receptor belongs to the Ig superfamily receptors that is related to a subset
of
lo activating receptors similar to NK cell receptors for MHC class I
molecules. ILT1
receptor is a 69 kDa glycosylated transmembrane receptor which is mainly
expressed in lung and liver and in monocytes, granulocytes, macrophages, and
dendritic cells (Samaridis and Colonna 1997). Upon crosslinking with
antibodies ILT1
receptor interacts with the y-chain of the Fc receptor (FcsRly) (Nakajima et
al. 1999)
ILT1 receptor is found consistently upregulated (59.5%) in COPD smokers
compared
to healthy smokers. This is demonstrated by õavg difr" values (Table 10). The
p value
for the comparisons between COPD smokers and healthy smokers was 0.01.
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Table 10: Expression levels of ILT1 receptor: "avg difP" values for each
patient are
listed as well as mean and median values for the three groups of subjects; OS
means obstructed smoker, HS healthy smoker, NS non-smoker
OS avgdiff HS avg diff NS avg diff
P 1 493.5 P 2 412.3 P 48/49 519.7
P 3 1186.0 P 37 457.2 P 50/52 645.0
P 5 1097.1 P 43 382.6 P 54/61 491.2
P 6 1387.6 P 56 180.5
P 39 513.5 P 57 367.8
P 44 1374.5 P 58 720.8
P 62 279.1
mean + 1008.8 400.0 552.0
std. dev. 406.8 168.6 81.8
median 1141.6 382.6 519.7
Another differentially expressed nucleic acid sequence identified codes for
SHPS-1
receptor (SIRP-alphal, MYD1, MFR), see SEQ ID NOs. 3 and 4, which is known to
be highly expressed in macrophages (Fujioka et al. 1996, Kharitonenkov et al.
1997,
Brooke et al. 1998). SHPS-1 receptor is a transmembrane glycoprotein belonging
to
immunoglobulin superfamily. It contains three extracellular Ig-like domains, a
cytoplasmic tail with a potential tyrosine phosphorylation site and an
immunoreceptor
tyrosine-based inhibitory motif (ITIM). Tyrosine phosphorylation of SHPS-1
receptor
occurs upon activation of receptor tyrosine kinases and leads to an
association with
SHP-1 (in macrophages) and SHP-2 (in non-hematopoetic cells) (Veillette et al.
1998). Moreover, other proteins have been found to associate with the
intracytoplasmic domain of SHPS-1 receptor, and it is therefore tempting to
assume
that SHPS-1 receptor acts as a scaffolding protein.
SHPS-1 receptor is consistently found downregulated (73.8%) in COPD smokers
compared to healthy smokers. This is demonstrated by calculated õfold change"
values (Table 11) from 42 pairwise comparisons and by õavg diff" values (Table
12).
The p value for the comparisons between COPD smokers and healthy smokers is
0.005.
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Table 11. Expression pattern for SHPS-1 receptor: fold change calculation for
42
pairwise comparisons between COPD and healthy smokers. Only values higher than
2fold and lower than -2fold are considered as deregulated. Thus, SHPS-1
receptor
is 29 times downregulated and 13 times not regulated
5
old chang comparison fold change comparison fold chang comparison
-1.3 39vs2 -3.4 5vs2 1.3 1 vs2
-2.8 39vs37 -6.8 5vs37 -1.7 1 vs37
-1.6 39vs43 -8.4 5vs43 -2.1 1 vs43
-3.0 39vs56 -7.1 5vs56 -1.8 1 vs56
-5.6 39vs57 -13.2 5vs57 -3.4 1 vs57
-5.4 39vs58 -12.6 5vs58 -3.2 1 vs58
-3.1 39vs62 -7.5 5vs62 -1.9 1vs62
1.4 44vs2 -2.1 6vs2 -1.1 3vs2
-1.5 44vs37 -4.5 6vs37 -2.3 3vs37
-1.8 44vs43 -5.6 6vs43 -2.9 3vs43
-1.6 44vs56 -4.7 6vs56 -2.4 3vs56
-2.6 44vs57 -8.9 6vs57 -4.6 3vs57
-2.5 44vs58 -8.5 6vs58 -4.4 3vs58
-1.7 44vs62 -4.9 6vs62 -2.5 3vs62
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Table 12: Expression levels of SHPS-1 receptor: "avg diff' values for each
patient
are listed as well as mean and median values for the three groups of subjects;
OS
means obstructed smoker, HS healthy smoker, NS non-smoker
OS avg diff HS avg diff NS avg diff
P 1 1837.8 P 2 1442.6 P 48/49 4979.9
P 3 1361.1 P 37 3115.0 P 50/52 1120.5
P 5 291.1 P 43 3897.3 P 54/61 2090.6
P 6 696.3 P 56 3280.8
P 39 1105.4 P 57 6220.7
P 44 2466.0 P 58 5928.9
P 62 3431.7
mean + 1293.0 3902.4 2730.3
std. dev. 783.9 1671.3 2007.7
median 1233.4 3431.7 2090.6 '
Another differentially expressed nucleic acid sequence identified codes for
KDEL
receptor 1, see SEQ ID NOs. 7 and 8, which is a receptor that has important
functions in protein folding and assembly in the endoplasmic reticulum. It
recognizes
lo soluble proteins with the amino acid sequence K-D-E-L and retrieves these
proteins
after binding to the endoplasmic reticulum (Townsley et al. 1993). KDEL
receptor 1
may be involved in the regulation of protein transport in the Golgi complex.
Upon
binding of a ligand the KDEL receptor dimerizes and interacts with ARF GAP
(GTPase-activating protein for the ADP-ribosylation factor) (Aoe et al 1997).
It is consistently found downregulated (71.4%) in COPD smokers compared to
healthy smokers. This is shown by õavg diff' values (Table 13). The p value
for the
comparisons between COPD smokers and healthy smokers is 0.003.
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Table 13: Expression levels of KDEL receptor 1: "avg difP"values for each
patient are
listed as well as mean and median values for the three groups of subjects; OS
means obstructed smoker, HS healthy smoker, NS non-smoker
OS avg diff HS avg diff NS avg diff
P 1 877.6 P 2 930.6 P 48/49 1532.9
P 3 1227.2 P 37 2151.4 P 50/52 786.4
P 5 870.6 P 43 1628.6 P 54/61 1571.5
P 6 1188.6 P 56 2232.9
P 39 1404.5 P 57 2295.1
P 44 798.1 P 58 2364.1
P 62 2092.0
mean + 1061.1 1956.4 1296.9
std. dev. 245.3 512.1 ~- 442.6
median 1033.1 2151.4 1532.9
Another differentially expressed nucleic acid sequence identified codes for
the
macrophage colony-stimulating factor-1 receptor precursor (CSF-1 receptor, c-
fms);
see SEQ ID NOs. 9 and 10. The CSF-1 receptor belongs to the subfamily of
receptor
tyrosine kinases. Activation of the CSF-1 receptor results in complex
formation of
lo multiple proteins, e.g. CSF-1 receptor, Shc, P13K, Grb2, Cbl, SHP-1, Src.
Moreover,
ligand binding also triggers rapid tyrosine phosphorylation of a plethora of
cytoplasmic proteins like Cbl, STAT3, STAT5a, STAT5b, p85P13K, SHP-1, Vav and
proteins involved in cytoskeletal organization (Yeung et al. 1998). CSF-1
receptor
regulates survival, proliferation, differentiation and morphology of
mononuclear
phagocytes (Hampe et al. 1989).
CSF-1 receptor is consistently found downregulated (45.2%) in COPD smokers
compared to healthy smokers. This is shown by õavg diff` values (Table 14).
The p
value for the comparisons between COPD smokers and healthy smokers is 0.002.
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23 Table 14: Expression levels of CSF-1 receptor: "avg diff' values for each
patient are
listed as well as mean and median values for the three groups of subjects; OS
means obstructed smoker, HS healthy smoker, NS non-smoker
OS avg diff HS avg diff NS avg diff
P 1 1136.0 P 2 2591.4 P 48149 2967.7
P 3 2262.5 P 37 3070.6 P 50/52 2041.6
P 5 829.5 P 43 2799.2 P 54/61 2376.4
P 6 1720.3 P 56 3293.1
P 39 1860.7 P 57 3703.4
P 44 1334.1 P 58 1904.9
P 62 2144.5
mean + 1523.9 2786.7 2461.9
std. dev. 522.7 633.2 468.9
median 1527.2 2799.2 2376.4
1.6. Use of TagMan Analysis for Validation of DNA-Chip Data and Diagnosis
io mRNA-expression profiles obtained by DNA-chips are validated by TaqMan
analysis
with the same RNA preparations. Moreover, the method is also applied to
determine
mRNA-levels for FPRL-1 receptor in cultured cell lines and in cells isolated
from
human beings in order to monitor the progress of the disease.
Total RNA isolated from U937-cells that were treated for 3 days with 10 nM
retinoic
acid is used in order to optimize of reaction conditions for determining the
mRNA-
levels of FPRL-1 receptor and setting standard curves for FPRL-1 receptor and
GAPDH (glyceraidehyde-3-phosphate dehydrogenase) as a housekeeping gene.
Quantification of FPRL-1 receptor is done with the following primers: Forward
primer
(FP) see SEQ ID NO. 17, Reverse primer (RP) see SEQ ID NO. 18 and TaqMan
probe (TP) see SEQ ID NO.19 labeled with reporter dye FAM at the 5' end and
quencher dye TAMRA at the 3' end. For determining mRNA-levels for GAPDH a
predeveloped kit "TaqMan GAPDH Control Reagents" (P/N 402869) from Perkin
Elmer Applied Biosystems is used. The GAPDH probe is labeled with JOE as the
reporter dye and TAMRA as the quencher dye. RT-PCR reactions are performed
with the "TaqMan EZ RT-PCR Core Reagents" (P/N N808-0236) kit from Perkin
Elmer Applied Biosystems. Standard curves for FPRL-1 receptor and GAPDH are
performed with increasing concentrations of RNA from U937 cells treated with 1
M
retinoic acid ranging from 0, 5, 10, 25, 50 to 100 ng per assay. Reaction
mixes
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contain 1 x TaqMan EZ-buffer, 3 mM Mn(Oac)Z, 300 M dATP, dCTP, dGTP, and 600
M dUTP, 2.5 U rTth DNA polymerase, 0.25 U AmpErase UNG in a total volume of
25 l. For analysis of FPRL-1 receptor reaction mixes include 300 nM of FP and
RP
and 100 nM of TP. The primer concentrations for determining GAPDH levels are
200
nM for each primer and 100 nM for the GAPDH Taqman probe. In order to
determine
mRNA levels for FPRL-1 receptor and GAPDH in human subjects and cell lines 16
to
50 ng RNA per reaction are used. All samples are run in triplicate. The
reactions are
performed with "MicroAmp Optical 96-well reaction plates" sealed with
"MicroAmp
Optical Caps" (Perkin Elmer Applied Biosystems) in an ABI PRISM 7700 Sequence
Detection System (Perkin Elmer Applied Biosystems). The PCR conditions are 2
minutes at 50 C, 30 minutes at 60 C, 5 minutes at 95 C, followed by 40 cycles
of 20
seconds at 94 C and 1 minute at 59 C. Data analysis is done either by
determining
the mRNA levels for FPRL-1 receptor and GAPDH according to the standard curves
or by directly relating CT values for FPRL-1 receptor to CT values for GAPDH.
The
latter can be done for these genes since the efficiencies for both reactions
are
around 95%. The same method is used for investigating mRNA levels isolated
from
COPD patients in order to diagnose the disease or, after treatment of patients
with
their putative active drugs to monitor the success of the treatment.
The other receptors mentioned in example 1.5 are investigated accordingly by
using
the respective appropriate primers.
1.7. Cell Systems
Human monocytic/macrophage cell lines HL-60, U937, THP-1, and MonoMac 6 are
used as cellular model systems. Cells are grown in RPMI 1640 media containing
10% FCS supplemented with 100 U/mi penicillin, 100 g/mi streptomycin, 2 mM
glutamine, and lx non-essential amino acids. The media for MonoMac6 cells also
includes 5 mi/I OPI media supplement (Sigma). MonoMac6 cells are exclusively
cultured in 24-well plates. Cells are maintained in a humidified atmosphere
with 5%
CO2at 37 C and tested regularly for contamination by mycoplasma.
3o Differentiation is achieved by adding 10 nM PMA (phorbol 12 myristate-13
acetate)
to the media.
1.8. Cloning of FPRL-1 receptor
FPRL-1 receptor is cloned from a total RNA extracted from U937 cells that were
treated with I M retinoic acid for three days. 5 gg RNA is reverse
transcribed into
cDNA with-5 ng oligo(dt)18 primer, 1x first strand buffer, 10 mM DTT, 0.5 mM
dNTPs
and 2 U Superscript II (Gibco BRL Life Technologies) at 42 C for 50 minutes.
Then,
the reaction is terminated at 70 C for 15 minutes and the cDNA concentration
is
determined by UV-spectrophotometry. For amplification of FPRL-1 receptor 100
ng
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of the cDNA and 10 pmol of sequence-specific primers for FPRL-1 receptor
(forward
primer attB1; see SEQ ID NO. 15 and reverse primer attB2 ; see SEQ ID NO. 16)
are
used for PCR. Reaction conditions are: 2 minutes of 94 C, 35 cycles with 30
seconds at 94 C, 30 seconds at 53 C, 90 seconds at 72 C, followed by 7 minutes
at
5 72 C with Taq DNA-polymerase. The reaction mix is separated on a 2% agarose
gel,
a band of about 1000bp is cut out and purified with the QIAEX II extraction
kit
(Qiagen). The concentration of the purified band is determined and about 120
ng are
incubated with 300 ng of pDONR201, the donor vector of the Gateway system
(Gibco BRL Life Technologies), Ix BP clonase reaction buffer, BP clonase
enzyme
10 mix in a total volume of 20 l for 60 minutes at 25 C. Then, reactions are
incubated
with 2 l of proteinase K and incubated for 10 minutes at 37 C. The reaction
mix is
then electroporated into competent DB3.1 cells and plated on Kanamycin-
containing
plates. Clones are verified by sequencing. A clone, designated pDONR-HM63
carrying the nucleic acid sequence shown in SEQ ID No. I is used for further
15 experiments.
The other receptors mentioned in example 1.5 are cloned using analogous
methods.
1.9. Transfection of FPRL-1 receptor
2o The vector containing FPRL-1 receptor described under 1.8 is used to
transfer the
cDNA for FPRL-1 receptor to the expression vector pcDNA3.1(+)/attR that
contains
the "attR1" and "attR2" recombination sites of the Gateway cloning system
(Gibco
BRL Life Technologies) where FPRL-1 receptor is expressed under the control of
the
CMV promoter. 150 ng of the "entry vector" pDONR-HM63 is mixed with 150 ng of
25 the "destination vector" pcDNA3.1(+)/attR, 4 l of the LR Clonase enzyme
mix, 4 l
LR Clonase reaction buffer, added up with TE (Tris/EDTA) to 20 l and
incubated at
25 C for 60 minutes. Then, 2 l of proteinase K solution is added and
incubated for
10 minutes at 37 C. 1 l of the reaction mix is transformed into 50 l DH5a by
a heat-
shock of 30 seconds at 42 C after incubating cells with DNA for 30 minutes on
ice.
3o After heat-shock of the cells 450 l of S.O.C. is added and cells are
incubated at
37 C for 60 minutes. Cells (100 i) are plated on LB plates containing 100
g/ml
ampicillin and incubated over night.
A colony that contains pcDNA3.1(+)/attR with FPRL-1 receptor as an insert is
designated pcDNA/FPRL1 and used for transfection studies.
Cell clones containing vectors obtained in 1.8 carrying nucleic acid sequences
coding for the other receptors described 1.5 are prepared using analogous
methods.
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Example 2: Cellular Systems and Phenotypic Effects of FPRL-1 receptor
Analogous methods as described herein in example 2 for FPRL-1 receptor are
also
performed using the other receptors described in 1.5.
2.1. Cell Systems
Human monocytic/macrophage cell lines HL-60, U937, THP-1, and MonoMac6 are
used as cellular model systems. Cells are grown in RPMI 1640 media containing
10% FCS supplemented with 100 U/mI penicillin, 100 g/mi streptomycin, 2 mM
glutamine, and 1x non-essential amino acids. The media for MonoMac6 also
includes 5 mI/I OPI media supplement (Sigma). MonoMac6 cells are exclusively
cultured in 24-well plates. AII cells are maintained in a humidified
atmosphere with
5% CO2 at 37 C and tested regularly for contamination by mycoplasma.
Differentiation is achieved by adding 10 nM PMA (phorbol 12 myristate-1 3
acetate)
to the media.
Phenotypic effects of FPRL-1 receptor (2.2.-2.9.)
2o 2.2. Ligand Binding Assay
300 ml cell culture is harvested with EDTA solution, the suspension is used to
spin
down the cells at 110-220 x g, resuspended in 10 mM Tris/HCI, pH 7.4, 2.5 mM
CaCi2, 1.2 mM MgC12, 40 g/mi bacitracin, 4 g/mi leupeptin, 4 g/mi
chymostatin,
10 pg/mi pefabloc, 2 pM phosphoamidon and 0,1 mg/mI bovine serum albumin
(BSA Fraktion V, BI Bioproducts) and diluted to 2 x 106 cells/mi.
0.5 ml aliquots are incubated with 0.3 nM 3"-IipoxinA4 (specific activity -10
Ci/mmol)
or in the presence of increasing concentrations of untritiated lipoxin A4 (3-
300 nM)
for 30 minutes at 4 C. The incubation is terminated by harvesting the cells by
a Cell-
Harvester (Skatron) with GF/B filters, washed three times with 3 ml chilled
buffer
consisting of 50 mM Tris/HCI, 100 mM NaCI, 10 mM MgCl21 pH 7.4 and the filter-
pieces transferred in vials. 2 ml scintillation cocktail is added and the
radioactivity
determined with a scintillation counter (LKB). Non-specific binding is
determined in
the presence of 100 nM unlabeled IipoxinA4. A series of peptides and low
molecular
weight compounds, including the peptide ligand MMK-1 (Klein et al. 1998), is
used in
a concentration range of 0.5 to 300 nM under the same reaction conditions in
order
to displace tritiated lipoxin A4.
The bound radioactivity (on the filter pieces) is estimated with a counter,
the values
are recorded on-line and fitted to a model. IC50 values for any substance to
block
binding of 3"-Iipoxin A4 are calculated.
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2.3. Ca2+-Release Determined by FLIPR-Assay
FLIPR-assay (Fluorometric Imaging Plate Reader) with FPRL-1 receptor is
performed with different CHO cell lines that constitutively expresse the G-
protein a-
subunit a16 or the chimeric G-proteins Gqi5 or Gqo5 (these are two Ga(q)
chimeras
harboring the last five residues of Ga(i) or Ga(o)) and FPRL-1 receptor. The
cell lines
CHO/Galpha16 (CHO/Galpha16), CHO/GalphaGqi5 and CHOGalphaGqo5
(Boehringer Ingelheim) that constitutively expresse Ga16, Gqi5 or Gqo5
aretransfected with the FPRL-1 receptor expression vector. The cell lines are
cultured in Ham's F12 media (Bio Whittaker) with 10% FCS (fetal calf serum), 2
mM
io glutamine, 200 ng/ml hygromycin, 100 U/mI penicillin and 100 g/mi
streptomycin in
a humidified atmosphere with 5% CO2 at 37 C. 3-7x105 cells are seeded in a 60
mm
petri dish and grown over night. Cells that are grown to a confluency of 50-
80% are
used for transfection. 6 l FuGene6 (Roche Biochemicals) is added to 100 l of
culture media without serum and equilibrated for 5 minutes at room
temperature.
Then, 2 g of purified pcDNA/FPRL-1 receptor is added to the prediluted
FuGene6
solution, gently mixed, and further incubated at room temperature for 15
minutes.
The media is aspirated from the cells and 4 ml of fresh media is added to the
cells.
The FuGene6/DNA solution is added dropwise to the cells and distributed evenly
by
swirling of the media. After 48 hours the media is aspirated and replaced by
Ham's
2o F12 media, 10% FCS, 2 mM glutamine, 200 ng/ml hygromycin, 100 U/mI
penicillin,100 g/mi streptomycin, and 200 g/ml G418. During the following
five days
the media is replaced daily until dead cells and debris is washed out until
single
colonies of cells are visible. Single colonies are isolated by separation with
cloning
cylinders and releasing them from the surface by addition of 100 l of 1x
trypsin/EDTA. Cells are transferred from the cloning cylinders to 4 ml of
media and
plated in 6 well-plates. Single clones are expanded and the expression of FPRL-
1
receptor in several clones is tested via ligand binding assay (2.2.). The cell
clone
denoted CHO/Galpha16/FPRL-1 receptor, CHO/GalphaGqi5/FPRL-1 receptor, or
CHOGalphaGqo5/FPRL-1 receptor with the highest expression of FPRL-1 receptor
is used for measuring of intracytoplasmic Ca2+ via FLIPR (Molecular Devices).
Cells (CHO/Galphal 6/FPRL-1 receptor, CHO/GalphaGqi5/FPRL-1 receptor, or
CHOGalphaGqo5/FPRL-1 receptor) are seeded in 384-blackwell plates (Corning~
with 2500-5000 cells per well in a volume of 40 l and grown over night in a
humidified atmosphere with 5% CO2 at 37 C. As a negative control CHO/Galpha16,
CHO/GalphaGqi5 or CHOGaIphaGqo5 cells are used. Then, 40 l of a Fluo-4
(Molecular Probes) staining solution is added to each well in order to label
the cells
with Fluo-4 at a final concentration of 2 M. The Fluo-4 staining solution is
composed
of 10.5 ml cell culture media described above, 420 l Probenicid solution
(1.42 g
Probenicid (Sigma), 10 ml 1 M NaOH, 10 ml Hanks buffer), 42 l Fluo-4 stock
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solution (50 g Fluo-4, 23 l DMSO, 23 l Pluronic F-127 (20% in DMSO)
(Molecular
Probes), and 420 l 1 M HEPES. After 45 minutes incubation in a humidified
atmosphere with 5% CO2 at 37 C wells are washed with a EMBLA-washer (4 wash
steps, program 03) using 2000 ml Hanks buffer containing 20 ml Probenicid
solution
as a wash solution and leaving 25 I wash buffer in each well. Then FLIPR is
set to
10000 counts for stained wells and a difference of 1:5 between unstained and
stained wells. Then, 25 l lipoxin A4 and a series of ligands, peptids, and
low
molecular weight compounds, including the peptide ligand MMK-1 is added to the
wells in increasing concentrations (0.5 - 300 nM) diluted in Hanks'
buffer/0.1% BSA.
Substances according to the invention are tested in increasing concentrations
(0.5 -
300 nM) to compete with lipoxin A4 (50 nM) in order to determine their
antagonistic
potential. Fluorescence is recorded starting with the addition of the ligand
every
second for 60 seconds and every 5 seconds for a further 60 seconds.
2.4. Production and Release of Cytokines or Matrix Metalloproteases
Cells of monocytic/macrophage cell lines are treated with lipoxin A4 at cell
densities
between 2.5 and 5 x 105 cells/mI. Cells are harvested after 0, 1, 3, 6, 12,
24, 48, and
72 hours, the supernatant frozen for further investigation, cells are washed
with PBS,
2o and resuspended in 400 ml of RLT buffer (from Qiagen RNeasy Total RNA
Isolation
Kit) with 143 mM p-mercaptoethanol, the DNA sheared with a 20 g needle for at
least
5 times and stored at -70 C.
Total RNAs are isolated with the Qiagen RNeasy Total RNA Isolation Kit
(Qiagen)
according to the manufacturer's protocol. Purified RNA is used for TaqMan
analysis.
The expression levels of cytokines TNFa, IL-1 p, IL-8, IL-6, and human matrix
metalloproteases, MMP-1, MMP-7, MMP-9, MMP-12, are measured using
appropriate primer sequences.
2.4.1. Detection of Secreted Cytokines
Proteins in the supernatants of the cultured and stimulated cells are
precipitated by
adding TCA (tricholoracetic acid) to a final concentration of 10%.
Precipitates are
washed twice with 80% ethanol and pellets are resuspended in 50 mM Tris/HCI,
pH
7.4, 10 mM MgCi21 1 mM EDTA. Protein concentration is determined via the
Bradford
method and 50 g of each sample are loaded on 12% SDS polyacrylamide gels.
Gels are blotted onto PVDF-membranes, blocked for 1 hour in 5% BSA- in TBST,
and
incubated for 1 hour with commercially available antibodies against human TNFa
(tumor necrosis factor a) IL-1 R(interleukin-1 p), IL-8 (interleukin 8), and
IL-6
(interleukin 6). After washing with TBST blots are incubated with anti-human
IgG
conjugated to horseradish-peroxidase, washed again and developed with ECL
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chemiluminescence kit (Amersham). Intensity of the bands are visualised with
BioMax X-ray films (Kodak) and quantified by densitometry.
2.4.2. Detection and Activity of Secreted Matrix Metalloproteases
The procedure is identical to the one described in 2.4.1. Antibodies used for
Western
blotting are against human MMP-1, MMP-7, MMP-9, and MMP-12.
Protease activity is determined with a fluorescent substrate. Supernatants
isolated
from stimulated and unstimulated cells (described above) are incubated in a
total
volume of 50 l with 1 M of the substrate (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu
(EDANS)-Ala-Lys-NH2 (Novabiochem)) for 5 minutes at room temperature. Positive
controls are performed with 125 ng purified MMP-12 per reaction. Protease
activity is
determined by fluorometry with an excitation at 320 nm and an emission at 405
nm.
In an alternative assay to determine proteolytic activity and cell migration a
chemotaxis chamber is used. In the wells of the upper part of the chamber
cells (105
cells per well) are plated on filters coated with an 8 m layer of Matrigel
(Becton
Dickinson). In the lower compartment chemoattractants like lipoxin A4 (100
nM),
MCP-1 (monocyte chemotactic protein 1) (10 ng/ml) are added to the media.
After
five days filters are removed, cells on the undersurface that have traversed
the
Matrigel are fixed with methanol, stained with the Diff-Quik staining kit
(Dade
Behring) and counted in three high power fields (400x) by light microscopy.
2.5. Chemotaxis Assay
In order to determine chemotaxis a 48 well chemotaxis (Boyden) chamber
(Neuroprobe) is used. Cells are starved for 24 hours in RPMI media without
FCS.
Chemoattractants, (50 ng/ml IL-8, 10 ng/ml MCP-1, 10 nM lipoxin A4, 10 nM MMK-
1
peptide (2.3.)) are diluted in RPMI media without FCS and 30 l is placed in
the wells
of the lower compartment. The upper compartment is separated from the lower
compartment by a polycarbonate filter (pore size 8 m). 50 I cell suspension
(5
x104) are placed in the well of the upper compartment. The chamber is
incubated for
5 hours at 37 C in a humidified atmosphere with 5% CO2. Then the filter is
removed,
cells on the upper side are scraped off, cells on the downside are fixed for 5
minutes
in methanol and stained with the Diff-Quik staining set (Dade Behring).
Migrated cells
are counted in three high-power fields (400x) by light microscopy.
2.6. Adherence Assay
Cells are harvested, washed in PBS and resuspended (4x106/ml) in PBS and 1 M
BCECF ((2`-7`-bis-(carboxethyl)-5(6`)-carboxyfluorescein acetoxymethyl) ester,
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Calbiochem) and incubated for 20 minutes at 37 C. Cells are washed in PBS and
resuspended (3.3x 106/ml) in PBS containing 0.1 % BSA. 3x105 cells (90 l) are
added to each well of a 96-well flat bottom plate coated with laminin (Becton
Dickinson) and allowed to settle for 10 minutes. 10 I of agonist (100 nM
lipoxin A4
5 plus lipoxin A4 antagonist) are added and plates are incubated for 20
minutes at
37 C. Then, cells are washed with PBS containing 0.1 % BSA and adherent cells
are
solubilized with 100 l of 0.025 M NaOH and 0.1% SDS. Quantification is
performed
by fluorescence measurement.
10 2.7. Phagocytosis
Cell suspensions (2.5x104 cells/ml) are seeded in 6-well plates with 5 ml of
U937 or
THP-1 or in 24-well plates with 2 ml of MonoMac6 and incubated for 1 hour at
37 C
in a humidified atmosphere with 5% CO2 in the presence of agonists (100 nM
lipoxin
A4, 50 nM MMK-1 peptide (2.3.)) and low molecular weight compounds according
to
15 the invention in order to antagonize agonistic effects. 40 l of a
dispersed
suspension of heat-inactivated Saccharomyces boulardii (20 yeast/cell) are
added to
each well. Cells are incubated for three more hours, washed twice with PBS and
cytocentrifuged. The cytospin preparations are stained with May-Grunwald-
Giemsa
and phagocytosed particles are counted by light microsopy.
25
Example 3: Cell Culture Model for Macrophages isolated from COPD patients
Analogous methods as describes here in example 3 for FPRL-1 receptor are also
performed using receptors described in 1.5.
As a cell culture model for macrophages isolated from COPD patients we select
the
monocytic cell lines MonoMac6 and THP-1. In order to mimic a hyperactivated
status
of these cell lines, cells are treated with PMA. Cells are exposed to further
stimuli
that are to mimic a condition that is similar to the situation in COPD. These
stimuli
are exposure to smoke or to LPS.
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Expression of FPRL-1 after stimulation of MonoMac6 cells with PMA, smoke,
and LPS
MonoMac6 cells are cultivated in 24-well plates in RPMI 1640 media,
supplemented
with 10% FCS (low endotoxin), 2 mM glutamine, lx non-essential amino acids,
200
U/ml penicillin, 200 pg/m{ streptomycin, and 5 ml OPI media supplement
(Sigma).
Cells are grown to a density of 600000 cells per well (2 ml media) and
stimulated
with 10 nM PMA (phorbol 12-myristate 13-acetate) (Sigma), or 20 ng/ml LPS
(lipopolysaccharides from Salmonella minnesota Re595) (Sigma). For smoke
exposure, cells are incubated in media enriched with smoke for 10 minutes at
37 C,
5% CO2 at a density of 1 x 106 cells/mi.
Enrichment of RPMI 1640 media with smoke is performed with the smoke of two
cigarettes. The smoke of the cigarettes is pulled into a 50 ml syringe (about
20
volumes of a 50-m1 syringe per cigarette) and then perfused into 100 ml of
RPMI
1640 media without supplements. Afterwards, the pH of the smoke-enriched media
is
adjusted to 7.4 and the media is sterilized through a 0.2 pm filter before
use. After
the exposure with smoke cells are washed at least twice with RPMI 1640 in
order to
remove residual smoke particles. Then cells are seeded in 24-well plates with
400000-600000 cells per well filled with 2 ml of fresh RPMI 1640 media
including the
supplements mentioned above.
THP-1 cells are grown in 75 cm2 flasks in RPMI 1640 Glutamax supplemented with
10% FCS (low endotoxin), 200 U/mI penicillin, 200 iag/mi streptomycin. Cells
are
treated with 10 nM PMA for 48 hours at 37 C, 5% CO2 in order to differentiate
the
cells to a macrophage-like cell type.Then, media is replaced by new PMA-free
cultivation media with the addition of 20 ng/ml LPS.
Both cell types are cultivated at 37 C, 5% CO2 in a humidified atmosphere and
cells
are harvested at various time points in order to monitor time-dependent
effects. Cells
are spun down and washed with PP-1- rQ-----)ended in 400 l of RLT buffer
(Qiagen
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RNeasy Total RNA Isolation Kit) with 143 mM p-mercaptoethanol, the DNA is
sheared with a 20 g needle for at least 5 times and stored at -70 C.
Total RNAs are isolated with the Qiagen RNeasy Total RNA Isolation Kit
(Qiagen)
according to the manufacturer's protocol. Purified RNA is digested with RNase-
free
DNase (Qiagen) and used for TaqMan analysis.
TaqMan analysis
Taqman analysis is used to determine mRNA-levels for FPRL-1 in cell lines
after
treatment with and without various stimuli at different time points. Total RNA
isolated
from U937 cells that were treated for 3 days with 10 nM retinoic acid is used
in order
to optimize of reaction conditions for determining the mRNA-levels of FPRL-1
and
setting standard curves for FPRL-1 and GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) as a housekeeping gene. Quantification of FPRL-1 is done with
the
following primers: Forward primer (rhHM63 668(-)FP, SEQ ID NO. 22), Reverse
primer (hHM63 525(+)RP, SEQ ID NO. 23) and TaqMan probe (rhHM63 629(-)TP,
SEQ ID NO. 24) labeled with reporter dye FAM at the 5' end and quencher dye
TAMRA at the 3' end. The mRNA-levels for GAPDH are determined with a
predeveloped kit for GAPDH "TaqMan GAPDH Control Reagents" (P/N 402869) from
PE Applied Biosystems. The GAPDH probe is labeled with JOE as the reporter dye
and TAMRA as the quencher dye. RT-PCR reactions are performed with the
"TaqMan EZ RT-PCR Core Reagents" (P/N N808-0236) kit from Perkin Elmer.
Standard curves for FPRL-1 and GAPDH are performed with increasing
concentrations of RNA from U937 cells treated with 1 M retinoic acid ranging
from
0, 5, 10, 25, 50 to 100 ng per assay. Reaction mixes contain 1 x TaqMan EZ-
buffer, 3
mM Mn(Oac)Z, 300 M dATP, dCTP, dGTP, and 600 M dUTP, 2.5 U rTth DNA
polymerase, 0.25 U AmpErase UNG in a total volume of 25 l. For analysis of
FPRL-
1 reaction mixes include 300 nM of rhHM63 668(-)FP and hHM63 525(+)RP and 100
nM of rhHM63 629(-)TP. The primer concentrations for determining GAPDH levels
are 200 nM for each primer and 100 nM for the Taqman probe. In order to
determine
mRNA levels for FPRL-1 and GAPDH in human subjects and cell lines 16 to 50 ng
RNA per reaction are used. All samples are run in triplicate. The reactions
are
performed with "MicroAmp Optical Qr--~~11=11 reaction plates" sealed with
"MicroAmp
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33.
Optical Caps" (PE Applied Biosystems) in an ABI PRISM 7700 Sequence Detection
System (PE Applied Biosystems). The PCR conditions are 2 minutes at 50 C, 30
minutes at 60 C, 5 minutes at 95 C, followed by 40 cycles of 20 seconds at 94
C and
1 minute at 59 C. Data analysis is done either by determining the mRNA levels
for
FPRL-1 and GAPDH according to the standard curves or by directly relating CT
values for FPRL-1 to CT values for GAPDH. The latter procedure can be applied
for
these genes since the efficiencies for both reactions are in good agreement
with
each other (around 95%).
Tab. 1: Expression of FPRL-1 in MonoMac6 cells after stimulation with 10 nM
PMA
t (h) ng FPRL-1 mRNA/ ng GAPDH mRNA
0 0.00
1 0.00
3 0.00
12 0.00
24 0.00
48 0.43
72 0.01
Tab. 2: Expression of FPRL-1 in MonoMac6 cells after differentiation with 10
nM
PMA and stimulation with 20 ng/ml LPS
t (h) ng FPRL-1 mRNA/ ng GAPDH mRNA
0 0.00
1 0.00
3 0.00
12 1.27
24 2.19
48 2.90
72 1.27
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Tab. 3: Expression of FPRL-1 in MonoMac6 cells after differentiation with 10
nM
PMA and stimulation with smoke
t (h) Fold induction of FPRL-1
0 1.00
1 0.02
3 0.14
6 4.44
12 9.90
25 9.35
48 8.73
Tab. 4: Expression of FPRL-1 in THP-1 cells after differentiation with PMA and
stimulation with LPS
t (h) Fold induction of FPRL-1
0 1.00
1 0.23
3 1.81
9 15.77
24 0.82
48 1.59
In order to examine the effects of ligands for FPRL-1, MonoMac6 cells are
seeded at
a density of 250000 cells/mI in 24-well plates (with 2 ml per well), grown for
24 hours
at 37 C, 5% CO2 in a humidified atmosphere before stimulation with 200 nM
lipoxin
A4 (Biomol), W-peptide (1 pM) (synthesized by Metabion, Martinsried), and LPS
(Sigma) as a positive control. Cells are harvested at different time points,
and total
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RNA is isolated as described above using the Qiagen RNeasy Total RNA Isolation
Kit (Qiagen).
The sequence of the W-peptide (Baek et al. 1996, J. Biol. Chem 271, 8170-8175)
is
W-K-Y-M-V-m.
5 The RNA is used for Taqman analysis in order to monitor the expression of
inflammatory markers like TNFa, IL-8, and MMP-12.
Tab. 5: Expression of TNFa in MonoMac 6 cells after stimulation with lipoxin
A4 and
W-peptide
Fold Induction
Fold Induction
t (h) W-peptide (1 pM)
Lipoxin A4 (200 nM)
0 1.00 1.00
3 2.43 1.03
Tab. 6: Expression of IL-8 in MonoMac 6 cells after stimulation with lipoxin
A4 and
W-peptide
Fold Induction
Fold Induction
t (h) W-peptide (1 pM)
Lipoxin A4 (200 nM)
0 1.00 1.00
3 1.99 1.54
Tab. 7: Expression of MMP-12 in MonoMac 6 cells after stimulation with lipoxin
A4
and W-peptide
Fold Induction
Fold Induction
t (h) W-peptide (1 pM)
Lipoxin A4 (200 nM)
0 1.00 1.00
3 1.42 1.51
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Since an increased invasion of macrophages in peripheral airways of COPD
patients
can be observed, we tested the chemotactic ability of MonoMac6 cells which
serve
as a cell culture model for alveolar macrophages. Chemotaxis of MonoMac6 is
determined by administering different ligands for FPRL-1.
MonoMac6 cells are treated with PMA for 24-30 hours in order to induce an
activation state of the cells. Cells are harvested, washed twice with RPMI
1640
without supplements, and seeded at a density of 500000 cells/well (24-well
plate) in
the presence of 10 nM PMA. After 24-30 hours cells are released from the
substratum by repeated rinsing with a pipet, spun down, counted and adjusted
to a
density of 1 x 106 cells/mi of RPMI 1640 media without supplements but in the
presence of 10 nM PMA. Chemotaxis is performed in a 48-well chemotaxis chamber
(Neuroprobe Inc.) and polycarbonate membranes with a pore size of 8 pm
(Neuroprobe Inc.). The lower wells of the chamber are filled with 28 ial of
different
concentrations of lipoxin A4, W-peptide, MCP-1 as a positive control, and RPMI
1640
media without supplements (including 10 nM PMA) as a negative control. The
lower
wells are covered with the polycarbonate membrane and the upper compartments
of
the chamber are filled with 50 pl of the cell suspension (50000 cells per
well). After 4
2o hours of migration at 37 C, 5% CO2 the cells on the upper part of the
membrane are
scraped off and the cells attached at the lower part of the membrane are
stained with
the Diff Quik Staining Set (Dade Behring) according to the manufacturer's
protocol.
Stained cells are counted in 6 to 8 high power fields at a magnification of
250x with a
light microscope. The migration index represents the fold increase in the
number of
cells migrated in response to the chemoattractant over control medium.
Tab. 8: Migration of MonoMac6 cells in resonse to lipoxin A4, W-peptide, and
MCP-1
Stimulus Migration Index
MCP-1 (20 ng/ml) 2.59
Lipoxin A4 (1 pM) 1.68
Lipoxin A4 (100 nM) 1.31
Lipoxin A4 (10 nM) 0.86
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W-peptide (1 pM) 2.46
W-peptide (100 nM) 1.23
W-peptide (10 nM) 0.95
The above examples as well as a cell of each of the above cell culture models
are
used for determining whether a substance is an inhibitor or an activator of an
ILM-
receptor of the invention which is deregulated in a macrophage according to
the
invention by adding a substance to be tested and subsequent measuring of a
respective above described effect.
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Literature (FPRL-1 receptor, HM74 receptor):
Kim, S.J. (1988). Biochem. Biophys. Res. Commun. 150, 870-876.
Lee, T.H., Crea, A.E.G., Gant, V., Spur, B.W., Marron, B.E., Nicolaou, K.C.,
Reardon, E., Brezinski, M., and Serhan, C. (1990). Am. Rev. Respir. Dis. 141,
1453-
1458.
Bao, L., Gerard, N.P., Eddy, R.L., Shows, T., and Gerard, C. (1991). Genomics
13,
437-440.
Christie, P.E., Spur, B.W., and Lee, T.H. (1992). Am Rev. Respir. Dis. 145,
1281-
1284.
Ye, R.D., Cavanagh, S.L., Quehenberger, 0., Prossnitz, E.R., and Cochrane,
C.G.
(1992). Biochem. Biophys. Res. Commun. 184, 582-589.
Perez, H.D., Holmes, R., Kelly, E., McClary, J., and Andrews, W.H. (1992).
Gene,
118, 303-304.
Murphy, P.M., Ozcelik, T., Kenney, R.T., Tiffany, H.L., McDermott, D., and
Francke,
U. (1992). J. Biol. Chem. 267, 7637-7643.
Nomura, H., Nielsen, B.W., and Matsushima, K. (1993). lnternat. Immunol. 5,
1239-
1249.
Levy, B.D., Romano, M., Chapman, H.A., Reilly, J.J., Drazen, J., and Serhan,
C.N.
(1993). J. Clin. Invest. 92, 1572-1579.
Durstin, M., Gao, J.-L., Tiffany, H.L., McDermott, D., and Murphy, P.M.
(1994).
Biochem. Biophys. Res. Commun. 201, 174-179.
Maddox, J.F. and Serhan, C.N. (1996). J. Exp. Med. 183, 137-146.
Maddox, J.F., Hachicha, M., Takano, T., Petasis, N.A., Fokin, V.V., and
Serhan, C.N.
(1997). J. Biol. Chem. 272, 6972-6978.
Klein, C., Paul, J.l., Sauve, K., Schmidt, M.M., Arcangeli, L., Ransom, J.,
Truelheart,
J., Manfredi, J.P., Broach, J.R., and Murphy, A.J. (1998). Nature Biotech. 16,
1334-
1337.
Serhan, J.N. (1999). 133-149 in Lipoxygenases and Their Metabolites, ed. Nigam
and Pace-Asciak, Plenum Press, New York.
Literature (AICL receptor):
Oda, S., Sato, M., Toyoshima, S., and Osawa, T. (1988). J. Biochem. 104, 600-
605.
Hamann, J. Fiebig, H., and Strauss, M. (1993). J. Immunol. 150, 4920-4927.
Hamann, J., Montgomery, K.T., Lau, S., Kucherlapati, R., and van Lier, R.A.W.
(1997). Immunogenetics 45, 295-300.
Literature (ILTI receptor):
Samaridis, J., and Colonna, M. (1997). Eur. J. Immunol. 27, 660-665.
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
39
Nakajima, H., Samaridis, J., Angman, L., and Colonna, M. (1999). J. Immunol.
162,
5-8.
Literature (SHPS-1 receptor):
Fujioka, Y. et al. (1996). Mol. Cell. Biol. 16, 6887-6899.
Kharitonenkov, A., Chen, Z., Sures, I., Wang, H., Schilling, J., and Ullrich,
A. (1997).
Nature 386, 181.186.
Brooke, G.P., Parsons, K.R., and Howard, C.J. (1998). Eur. J. Immunol. 28, 1-
11.
Veillette, A., Thibaudeau, E., and Latour, S. (1998). J. Biol. Chem. 273,
22719-
lo 22728.
Literature (KDEL receptor 1):
Townsley, F.M., Wilson, D.W., and Pelham, D.R. (1993). EMBO J. 12,2821-2829.
Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P.J., and Hsu, V.W.
(1997).
EMBO J. 16, 7305-7316.
Literature (CSF-1 receptor):
Hampe, A., Shamoon, B.M., Gobet, M., Sherr, C.J., and Galibert, F. (1989).
Oncogene Res. 4, 9-17.
Yeung, Y.-G., Wang, Y., Einstein, D.B., Lee, P.S.W., and Stanley, E.R. (1998).
J.
Biol. Chem. 273, 17128-17137.
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1
SEQUENCE LISTING
<110> Boehringer Ingelheim Pharma KG
<120> Method for identifying substances which positively
influence inflammatory conditions of chronic
inflammatory airway diseases
<130> 082OOn
<140>
<141>
<150> UK 0021484.1
<151> 2000-09-01
<160> 24
<170> PatentIn Ver. 2.1
<210> 1
<211> 1910
<212> DNA
<213> Homo sapiens
<400> 1
gaaaaggagc ttagctgctg gtgctgctgg caagatggaa accaacttct ccactcctct 60
gaatgaatat gaagaagtgt cctatgagtc tgctggctac actgttctgc ggatcctccc 120
attggtggtg cttggggtca cctttgtcct cggggtcctg ggcaatgggc ttgtgatctg 180
ggtggctgga ttccggatga cacgcacagt caccaccatc tgttacctga acctggccct 240
ggctgacttt tctttcacgg ccacattacc attcctcatt gtctccatgg ccatgggaga 300
aaaatggcct tttggctggt tcctgtgtaa gttaattcac atcgtggtgg acatcaacct 360
ctttggaagt gtcttcttga ttggtttcat tgcactggac cgctgcattt gtgtcctgca 420
tccagtctgg gcccagaacc accgcactgt gagtctggcc atgaaggtga tcgtcggacc 480
ttggattctt gctctagtcc ttaccttgcc agttttcctc tttttgacta cagtaactat 540
tccaaatggg gacacatact gtactttcaa ctttgcatcc tggggtggca cccctgagga 600
gaggctgaag gtggccatta ccatgctgac agccagaggg attatccggt ttgtcattgg 660
ctttagcttg ccgatgtcca ttgttgccat ctgctatggg ctcattgcag ccaagatcca 720
caaaaagggc atgattaaat ccagccgtcc cttacgggtc ctcactgctg tggtggcttc 780
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
2
tttcttcatc tgttggtttc cctttcaact ggttgccctt ctgggcaccg tctggctcaa 840
agagatgttg ttctatggca agtacaaaat cattgacatc ctggttaacc caacgagctc 900
cctggccttc ttcaacagct gcctcaaccc catgctttac gtctttgtgg gccaagactt 960
ccgagagaga ctgatccact ccctgcccac cagtctggag agggccctgt ctgaggactc 1020
agccccaact aatgacacgg ctgccaattc tgcttcacct cctgcagaga ctgagttaca 1080
ggcaatgtga ggatggggtc agggatattt tgagttctgt tcatcctacc ctaatgccag 1140
ttccagcttc atctaccctt gagtcatatt gaggcattca aggatgcaca gctcaagtat 1200
ttattcagga aaaatgcttt tgtgtccctg atttggggct aagaaataga cagtcaggct 1260
actaaaatat tagtgttatt ttttgttttt tgacttctgc ctataccctg gggtaagtgg 1320
agttgggaaa tacaagaaga gaaagaccgg tggggatttg taagacttag atgagatagt 1380
gcataataag gggaagactt taaagtataa agtaaaatgt ttgctgtagg ttttttatag 1440
ctattaaaaa aaatcagatt atggaagttt tcttctattt ttagtttgct aagagttttc 1500
tgtttctttt tcttacatca tgagtggact ttgcatttta tcaaatgcat tttctacatg 1560
tattaagatg gtcatattat tcttcttctt ttatgtaaat cattataaat aatgttcatt 1620
aagttctgaa tgttaaacta ctcttgaatt cctggaataa accacactta gtcctgatgt 1680
actttaaata tttatatctc acaggagttg gttagaattt ctgtgtttat gtttatatac 1740
tgttatttca ctttttctac tatccttgct aagttttcat agaaaataag gaacaaagag 1800
aaacttgtaa tggtctctga aaaggaattg agaagtaatt cctctgattc tgttttctgg 1860
tgttatatct ttattaaata ttcagaaaaa ttcaccagtg aaaaaaaaaa 1910
<210> 2
<211> 351
<212> PRT
<213> Homo sapiens
<400> 2
Met Glu Thr Asn Phe Ser Thr Pro Leu Asn Glu Tyr Glu Glu Val Ser
1 5 10 15
Tyr Glu Ser Ala Gly Tyr Thr Val Leu Arg Ile Leu Pro Leu Val Val
20 25 30
Leu Gly Val Thr Phe Val Leu Gly Val Leu Gly Asn Gly Leu Val Ile
35 40 45 =
Trp Val Ala Gly Phe Arg Met Thr Arg Thr Val Thr Thr Ile Cys Tyr
50 55 60
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
3
Leu Asn Leu Ala Leu Ala Asp Phe Ser Phe Thr Ala Thr Leu Pro Phe
65 70 75 80
Leu Ile Val Ser Met Ala Met Gly Glu Lys Trp Pro Phe Gly Trp Phe
85 90 95
Leu Cys Lys Leu Ile His Ile Val Val Asp Ile Asn Leu Phe Gly Ser
100 105 110
Val Phe Leu Ile Gly Phe Ile Ala Leu Asp Arg Cys Ile Cys Val Leu
115 120 125
His Pro Val Trp Ala Gln Asn His Arg Thr Val Ser Leu Ala Met Lys
130 135 140
Val Ile Val Gly Pro Trp Ile Leu Ala Leu Val Leu Thr Leu Pro Val
145 150 155 160
Phe Leu Phe Leu Thr Thr Val Thr Ile Pro Asn Gly Asp Thr Tyr Cys
165 170 175
Thr Phe Asn Phe Ala Ser Trp Gly Gly Thr Pro Glu Glu Arg Leu Lys
180 185 190
Val Ala Ile Thr Met Leu Thr Ala Arg Gly Ile Ile Arg Phe Val Ile
195 200 205
Gly Phe Ser Leu Pro Met Ser Ile Val Ala Ile Cys Tyr Gly Leu Ile
210 215 220
Ala Ala Lys Ile His Lys Lys Gly Met Ile Lys Ser Ser Arg Pro Leu
225 230 235 240
Arg Val Leu Thr Ala Val Val Ala Ser Phe Phe Ile Cys Trp Phe Pro
245 250 255
Phe Gln Leu Val Ala Leu Leu Gly Thr Val Trp Leu Lys Glu Met Leu
260 265 270
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
4
Phe Tyr Gly Lys Tyr Lys Ile Ile Asp Ile Leu Val Asn Pro Thr Ser
275 280 285
Ser Leu Ala Phe Phe Asn Ser Cys Leu Asn Pro Met Leu Tyr Val Phe
290 295 300
Val Gly Gin Asp Phe Arg Glu Arg Leu Ile His Ser Leu Pro Thr Ser
305 310 315 320
Leu Glu Arg Ala Leu Ser Glu Asp Ser Ala Pro Thr Asn Asp Thr Ala
325 330 335
Ala Asn Ser Ala Ser Pro Pro Ala Glu Thr Glu Leu Gln Ala Met
340 345 350
<210> 3
<211> 2433
<212> DNA
<213> Homo sapiens
<400> 3
cagccgcggc ccatggagcc cgccggcccg gcccccggcc gcctcgggcc gctgctctgc 60
ctgctgctcg ccgcgtcctg cgcctggtca ggagtggcgg gtgaggagga gctgcaggtg 120
attcagcctg acaagtccgt atcagttgca gctggagagt cggccattct gcactgcact 180
gtgacctccc tgatccctgt ggggcccatc cagtggttca gaggagctgg accagcccgg 240
gaattaatct acaatcaaaa agaaggccac ttcccccggg taacaactgt ttcagagtcc 300
acaaagagag aaaacatgga cttttccatc agcatcagta acatcacccc agcagatgcc 360
ggcacctact actgtgtgaa gttccggaaa gggagccctg acacggagtt taagtctgga 420
gcaggcactg agctgtctgt gcgtgccaaa ccctctgccc ccgtggtatc gggccctgcg 480
gcgagggcca cacctcagca cacagtgagc ttcacctgcg agtcccacgg cttctcaccc 540
agagacatca ccctgaaatg gttcaaaaat gggaatgagc tctcagactt ccagaccaac 600
gtggaccccg taggagagag cgtgtcctac agcatccaca gcacagccaa ggtggtgctg 660
acccgcgagg acgttcactc tcaagtcatc tgcgaggtgg cccacgtcac cttgcagggg 720
gaccctcttc gtgggactgc caacttgtct gagaccatcc gagttccacc caccttggag 780
gttactcaac agcccgtgag ggcagagaac caggtgaatg tcacctgcca ggtgaggaag 840
ttctaccccc agagactaca gctgacctgg ttggagaatg gaaacgtgtc ccggacagaa 900
acggcctcaa ccgttacaga gaacaaggat ggtacctaca.actggatgag ctggctcctg 960
gtgaatgtat ctgcccacag ggatgatgtg aagctcacct gccaggtgga gcatgacggg 1020
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
cagccagcgg tcagcaaaag ccatgacctg aaggtctcag cccacccgaa ggagcagggc 1080
tcaaataccg ccgctgagaa cactggatct aatgaacgga acatctatat tgtggtgggt 1140
gtggtgtgca ccttgctggt ggccctactg atggcggccc tctacctcgt ccgaatcaga 1200
cagaagaaag cccagggctc cacttcttct acaaggttgc atgagcccga gaagaatgcc 1260
5 agagaaataa cacaggacac aaatgatatc acatatgcag acctgaacct gcccaagggg 1320
aagaagcctg ctccccaggc tgcggagccc aacaaccaca cggagtatgc cagcattcag 1380
accagcccgc agcccgcgtc ggaggacacc ctcacctatg ctgacctgga catggtccac 1440
ctcaaccgga cccccaagca gccggccccc aagcctgagc cgtccttctc agagtacgcc 1500
agcgtccagg tcccgaggaa gtgaatggga ccgtggtttg ctctagcacc catctctacg 1560
cgctttcttg tcccacaggg agccgccgtg atgagcacag ccaacccagt tcccggaggg 1620
ctggggcggt gcaggctctg ggacccaggg gccagggtgg ctcttctctc cccacccctc 1680
cttggctctc cagcacttcc tgggcagcca cggccccctc ccccaacatt gccacacacc 1740
tggaggctga cgttgccaaa ccagccaggg aaccaacctg ggaagtggcc agaactgcct 1800
ggggtccaag aactcttgtg cctccgtcca tcaccatgtg ggttttgaag accctcgact 1860
gcctccccga tgctccgaag cctgatcttc cagggtgggg aggagaaaat cccacctccc 1920
ctgacctcca ccacctccac caccaccacc accaccacca ccaccactac caccaccacc 1980
caactggggc tagagtgggg aagatttccc ctttagatca aactgcccct tccatggaaa 2040
agctggaaaa aaactctgga acccatatcc aggcttggtg aggttgctgc caacagtcct 2100
ggcctccccc atccctaggc aaagagccat gagtcctgga ggaggagagg acccctccca 2160
aaggactgga agcaaaaccc tctgcttcct tgggtccctc caagactccc tggggcccaa 2220
ctgtgttgct ccacccggac ccatctctcc cttctagacc tgagcttgcc cctccagcta 2280
gcactaagca acatctcgct gtaagcgcct gtaaattact gtgaaatgtg aaacgtgcaa 2340
tcttgaaact gaggtgttag aaaacttgat ctgtggtgtt ttgttttgtt ttttttctta 2400
aaacaacagc aacgtgaaaa aaaaaaaaaa aaa 2433
<210> 4
<211> 503
<212> PRT
<213> Homo sapiens
<400> 4
Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys
1 5 10 15
Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu
20 25 30
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
6
Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly
35 40 45
Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly
50 55 60
Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr
65 70 75 80
Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser
85 90 95
Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr
100 105 110
Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser
115 120 125
Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg
130 135 140
Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr
145 150 155 160
Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro
165 170 175
Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp
180 185 190
Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile
195 200 205
His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln
210 215 220
Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg
225 230 235 240
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
7
Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu
245 250 255
Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys
260 265 270
Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu
275 280 285
Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn
290 295 300
Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser
305 310 315 320
Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Giy
325 330 335
Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His Pro
340 345 350
Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn Glu
355 360 365
Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val Ala
370 375 380
Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys Ala
385 390 395 400
Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn Ala
405 410 415
Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu Asn
420 425 4=30
Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn Asn
435 440 445
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
8
His Thr Glu Tyr Ala Ser I1e Gln Thr Ser Pro Gln Pro Ala Ser Glu
450 455 460
Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg Thr
465 470 475 480
Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr Ala
485 490 495
Ser Val Gln Val Pro Arg Lys
500
<210> 5
<211> 759
<212> DNA
<213> Homo sapiens
<400> 5
ctgtgctgta aaaacaagag taacattttt atattaaagt taaataaagt tacaactttg 60
aagagagttt ctgcaagaca tgacacaaag ctgctagcag aaaatcaaaa cgctgattaa 120
aagaagcacg gtatgatgac caaacataaa aagtgtttta taattgttgg tgttttaata 180
acaactaata ttattactct gatagttaaa ctaactcgag attctcagag tttatgcccc 240
tatgattgga ttggtttcca aaacaaatgc tattatttct ctaaagaaga aggagattgg 300
aattcaagta aatacaactg ttccactcaa catgccgacc taactataat tgacaacata 360
gaagaaatga attttcttag gcggtataaa tgcagttctg atcactggat tggactgaag 420
atggcaaaaa atcgaacagg acaatgggta catggagcta catttaccaa atcgtttggc 480
atgagaggga gtgaaggatg tgcctacctc agcgatgatg gtgcagcaac agctagatgt 540
tacaccgaaa gaaaatggat ttgcaggaaa agaatacact aagttaatgt ctaagataat 600
ggggaaaata gaaaataaca ttattaagtg taaaaccagc aaagtacttt tttaattaaa 660
caaagttcga gttttgtacc tgtctggtta attctgctta cgtgtcaggc tacacataaa 720
agccacttca aagattggca aaaaaaaaaa aaaaaaaaa 759
<210> 6
<211> 149
<212> PRT
<213> Homo sapiens
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
9
<400> 6
Met Met Thr Lys His Lys Lys Cys Phe Ile Ile Val Gly Val Leu Ile
1 5 10 15
Thr Thr Asn Ile Ile Thr Leu Ile Val Lys Leu Thr Arg Asp Ser Gln
20 25 30
Ser Leu Cys Pro Tyr Asp Trp Ile Gly Phe Gln Asn Lys Cys Tyr Tyr
35 40 45
Phe Ser Lys Glu Glu Gly Asp Trp Asn Ser Ser Lys Tyr Asn Cys Ser
50 55 60
Thr Gln His Ala Asp Leu Thr Ile Ile Asp Asn Ile Glu Glu Met Asn
65 70 75 80
Phe Leu Arg Arg Tyr Lys Cys Ser Ser Asp His Trp Ile Gly Leu Lys
85 90 95
Met Ala Lys Asn Arg Thr Gly Gln Trp Val His Gly Ala Thr Phe Thr
100 105 110
Lys Ser Phe Gly Met Arg Gly Ser Glu Gly Cys Ala Tyr Leu Ser Asp
115 120 125
Asp Gly Ala Ala Thr Ala Arg Cys Tyr Thr Glu Arg Lys Trp Ile Cys
130 135 140
Arg Lys Arg Ile His
145
<210> 7
<211> 1086
<212> DNA
<213> Homo sapiens
<400> 7
ctaaaggtcc cctccccgga gcggagcgca cctagggtcc ctcttccgtc cccccagccc 60
agctacccgt tcagaccagc agcctcgggg ggcacccccc gccagcctgc ctccctcccg 120
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
ctcagccctg ccagggttcc ccagccatga atctcttccg attcctggga gacctctccc 180
acctcctcgc catcatcttg ctactgctca aaatctggaa gtcccgctcg tgcgccggaa 240
tttcagggaa gagccaggtc ctgtttgctg tggtgttcac tgcccgatat ctggacctct 300
tcaccaacta catctcactc tacaacacgt gtatgaaggt ggtctacata gcctgctcct 360
5 tcaccacggt ctggttgatt tatagcaagt tcaaagctac ttacgatggg aaccatgaca 420
cgttcagagt ggagttcctg gtcgttccca cagccattct ggcgttcctg gtcaatcatg 480
acttcacccc tctggagatc ctctggacct tctccatcta cctggagtca gtggccatct 540
tgccgcagct gttcatggtg agcaagaccg gcgaggcgga gaccatcacc agccactact 600
tgtttgcgct aggcgtttac cgcacgctct atctcttcaa ctggatctgg cgctaccatt 660
10 tcgagggctt cttcgacctc atcgccattg tggcaggcct ggtccagaca gtcctctact 720
gcgatttctt ctacctctat atcaccaaag tcctaaaggg gaagaagttg agtttgccgg 780
catagccccg gtcctctcca tctctctcct cggcagcagc gggaggcaga ggaaggcggc 840
agaagatgaa gagctttccc atccaggggt gactttttta agaacccacc tcttgtgctc 900
cccatcccgc ctcctgccgg gtttcagggg gacagtggag gatccaggtc ttggggagct 960
caggacttgg gctgtttgta gttttttgcc ttttagacaa gaaaaaaaaa tctttccact 1020
ctttagtttt tgattctgat gactcgtttt ttcttctact ctgtggcccc aaattttata 1080
aagtga 1086
<210> 8
<211> 212
<212> PRT
<213> Homo sapiens
<400> 8
Met Asn Leu Phe Arg Phe Leu Gly Asp Leu Ser His Leu Leu Ala Ile
1 5 10 15
Ile Leu Leu Leu Leu Lys Ile Trp Lys Ser Arg Ser Cys Ala Gly Ile
20 25 30
Ser Gly Lys Ser Gln Val Leu Phe Ala Val Va1 Phe Thr Ala Arg Tyr
40 45
35 Leu Asp Leu Phe Thr Asn Tyr Ile Ser Leu Tyr Asn Thr Cys Met Lys
50 55 60
Val Val Tyr Ile Ala Cys Ser Phe Thr Thr Val Trp Leu Ile Tyr Ser
65 70 75 80
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
11
Lys Phe Lys Ala Thr Tyr Asp Gly Asn His Asp Thr Phe Arg Val Glu
85 90 95
Phe Leu Val Ile Pro Thr Ala Ile Leu Ala Phe Leu Val Asn His Asp
100 105 110
Phe Thr Pro Leu Glu Ile Leu Trp Thr Phe Ser Ile Tyr Leu Glu Ser
115 120 125
Val Ala Ile Leu Pro Gln Leu Phe Met Val Ser Lys Thr Gly Glu Ala
130 135 140
Glu Thr Ile Thr Ser His Tyr Leu Phe Ala Leu Gly Val Tyr Arg Thr
145 150 155 160
Leu Tyr Leu Phe Asn Trp Ile Trp Arg Tyr His Phe Glu Gly Phe Phe
165 170 175
Asp Leu Ile Ala Ile Val Ala Gly Leu Val Gln Thr Val Leu Tyr Cys
180 185 190
Asp Phe Phe Tyr Leu Tyr Ile Thr Lys Val Leu Lys Gly Lys Lys Leu
195 200 205
Ser Leu Pro Ala
210 -
<210> 9
<211> 3992
<212> DNA
<213> Homo sapiens
<400> 9
ggcttcagga agggcagaca gagtgtccaa aagcgtgaga gcacgaagtg aggagaaggt 60
ggagaagaga gaagaggaag aggaagagga agagaggaag cggagggaac tgcggccagg 120
ctaaaagggg aagaagagga tcagcccaag gaggaggaag aggaaaacaa gacaaacagc 180
cagtgcagag gagaggaacg tgtgtccagt gtcccgatcc ctgcggagct agtagctgag 240
agctctgtgc cctgggcacc ttgcagccct gcacctgcct gccacttccc caccgaggcc 300
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
12
atgggcccag gagttctgct gctcctgctg gtggccacag cttggcatgg tcagggaatc 360
ccagtgatag agcccagtgt ccccgagctg gtcgtgaagc caggagcaac ggtgaccttg 420
cgatgtgtgg gcaatggcag cgtggaatgg gatggccccg catcacctca ctggaccctg 480
tactctgatg gctccagcag catcctcagc accaacaacg ctaccttcca aaacacgggg 540
acctatcgct gcactgagcc tggagacccc ctgggaggca gcgccgccat ccacctctat 600
gtcaaagacc ctgcccggcc ctggaacgtg ctagcacagg aggtggtcgt gttcgaggac 660
caggacgcac tactgccctg tctgctcaca gacccggtgc tggaagcagg cgtctcgctg 720
gtgcgtgtgc gtggccggcc cctcatgcgc cacaccaact actccttctc gccctggcat 780
ggcttcacca tccacagggc caagttcatt cagagccagg actatcaatg cagtgccctg 840
atgggtggca ggaaggtgat gtccatcagc atccggctga aagtgcagaa agtcatccca 900
gggcccccag ccttgacact ggtgcctgca gagctggtgc ggattcgagg ggaggctgcc 960
cagatcgtgt gctcagccag cagcgttgat gttaactttg atgtcttcct ccaacacaac 1020
aacactaagc tcgcaatccc tcaacaatct gactttcata ataaccgtta ccaaaaagtc 1080
ctgaccctca acctcgatca agtagatttc caacatgccg gcaactactc ctgcgtggcc 1140
agcaacgtgc agggcaagca ctccacctcc atgttcttcc gggtggtaga gagtgcctac 1200
ttgaacttga gctctgagca gaacctcatc caggaggtga ccgtggggga ggggctcaac 1260
ctcaaagtca tggtggaggc ctacccaggc ctgcaaggtt ttaactggac ctacctggga 1320
cccttttctg accaccagcc tgagcccaag cttgctaatg ctaccaccaa ggacacatac 1380
aggcacacct tcaccctctc tctgccccgc ctgaagccct ctgaggctgg ccgctactcc 1440
ttcctggcca gaaacccagg aggctggaga gctctgacgt ttgagctcac ccttcgatac 1500
cccccagagg taagcgtcat atggacattc atcaacggct ctggcaccct tttgtgtgct 1560
gcctctgggt acccccagcc caacgtgaca tggctgcagt gcagtggcca cactgatagg 1620
tgtgatgagg cccaagtgct gcaggtctgg gatgacccat accctgaggt cctgagccag 1680
gagcccttcc acaaggtgac ggtgcagagc ctgctgactg ttgagacctt agagcacaac 1740
caaacctacg agtgcagggc ccacaacagc gtggggagtg gctcctgggc cttcataccc 1800
atctctgcag gagcccacac gcatcccccg gatgagttcc tcttcacacc agtggtggtc 1860
gcctgcatgt ccatcatggc cttgctgctg ctgctgctcc tgctgctatt gtacaagtat 1920
aagcagaagc ccaagtacca ggtccgctgg aagatcatcg agagctatga gggcaacagt 1980
tatactttca tcgaccccac gcagctgcct tacaacgaga agtgggagtt cccccggaac 2040
aacctgcagt ttggtaagac cctcggagct ggagcctttg ggaaggtggt ggaggccacg 2100
gcctttggtc tgggcaagga ggatgctgtc ctgaaggtgg ctgtgaagat gctgaagtcc 2160
acggcccatg ctgatgagaa ggaggccctc atgtccgagc tgaagatcat gagccacctg 2220
ggccagcacg agaacatcgt caaccttctg ggagcctgta cccatggagg ccctgtactg 2280
gtcatcacgg agtactgttg ctatggcgac ctgctcaact ttctgcgaag gaaggctgag 2340
gccatgctgg gacccagcct gagccccggc caggaccccg agggaggcgt cgactataag 2400
aacatccacc tcgagaagaa atatgtccgc agggacagtg gcttctccag ccagggtgtg 2460
gacacctatg tggagatgag gcctgtctcc acttcttcaa atgactcctt ctctgagcaa 2520
gacctggaca aggaggatgg acggcccctg gagctccggg acctgcttca cttctccagc 2580
caagtagccc agggcatggc cttcctcgct tccaagaatt gcatccaccg ggacgtggca 2640
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
13
gcgcgtaacg tgctgttgac caatggtcat gtggccaaga ttggggactt cgggctggct 2700
agggacatca tgaatgactc caactacatt gtcaagggca atgcccgcct gcctgtgaag 2760
tggatggccc cagagagcat ctttgactgt gtctacacgg ttcagagcga cgtctggtcc 2820
tatggcatcc tcctctggga gatcttctca cttgggctga atccctaccc tggcatcctg 2880
gtgaacagca agttctataa actggtgaag gatggatacc aaatggccca gcctgcattt 2940
gccccaaaga atatatacag catcatgcag gcctgctggg ccttggagcc cacccacaga 3000
cccaccttcc agcagatctg ctccttcctt caggagcagg cccaagagga caggagagag 3060
cgggactata ccaatctgcc gagcagcagc agaagcggtg gcagcggcag cagcagcagt 3120
gagctggagg aggagagctc tagtgagcac ctgacctgct gcgagcaagg ggatatcgcc 3180
cagcccttgc tgcagcccaa caactatcag ttctgctgag gagttgacga cagggagtac 3240
cactctcccc tcctccaaac ttcaactcct ccatggatgg ggcgacacgg ggagaacata 3300
caaactctgc cttcggtcat ttcactcaac agctcggccc agctctgaaa cttgggaagg 3360
tgagggattc aggggaggtc agaggatccc acttcctgag catgggccat cactgccagt 3420
caggggctgg gggctgagcc ctcacccccc gcctccccta ctgttctcat ggtgttggcc 3480
tcgtgtttgc tatgccaact agtagaacct tctttcctaa tccccttatc ttcatggaaa 3540
tggactgact ttatgcctat gaagtcccca ggagctacac tgatactgag aaaaccaggc 3600
tctttggggc tagacagact ggcagagagt gagatctccc tctctgagag gagcagcaga 3660
tgctcacaga ccacactcag ctcaggcccc ttggagcagg atggctcctc taagaatctc 3720
acaggacctc ttagtctctg ccctatacgc cgccttcact ccacagcctc acccctccca 3780
cccccatact ggtactgctg taatgagcca agtggcagct aaaagttggg ggtgttctgc 3840
ccagtcccgt cattctgggc tagaaggcag gggaccttgg cattggctgg ccacaccaag 3900
caggaagcac aaactccccc aagctgactc atcctaacta acagtcacgc cgtgggatgt 3960
ctctgtccac attaaactaa cagcattaat gc 3992
<210> 10
<211> 972
<212> PRT
<213> Homo sapiens
<400> 10
Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala Trp His
1 5 10 15
Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val
20 25 30
Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val
35 40 45
CA 02418506 2003-02-05
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14
Glu Trp Asp Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly
50 55 60
Ser Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly
65 70 75 80
Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala
85 90 95
Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala
100 105 110
Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu
115 120 125
Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg
130 135 140
Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His
145 150 155 160
Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln
165 170 175
Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg
180 185 190
Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val
195 200 205
Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys
210 215 220
Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn
225 230 235 240
Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg
245 250 255
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His
260 265 270
Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser
5 275 280 285
Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser
290 295 300
10 Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn
305 310 315 320
Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp
325 330 335
Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala
340 345 350
Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser_Leu
355 360 365
Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg
370 375 380
Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr
385 390 395 400
Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr
405 410 415
Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu
420 425 430
Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln
435 440 445 =
Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His
450 455 460
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
16
Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn
465 470 475 480
Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp
485 490 495
Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu
500 505 510
Phe Leu Phe Thr Pro Val Val Val Ala Cys Met Ser Ile Met Ala Leu
515 520 525
Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr Lys Tyr Lys Gln Lys Pro
530 535 540
Lys Tyr Gln Val Arg Trp Lys Ile Ile Glu Ser Tyr Glu Giy Asn Ser
545 550 555 560
Tyr Thr Phe Ile Asp Pro Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu
565 570 575
Phe Pro Arg Asn Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala
. 580 585 590
Phe Gly Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp
595 600 605
Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala
610 615 620
Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser His Leu
625 630 635 640
Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr His Gly
645 650 655
Gly Pro Val Leu Val Ile Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu
660 665 670
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
'17
Asn Phe Leu Arg Arg Lys Ala Glu Ala Met Leu Gly Pro Ser Leu Ser
675 680 685
Pro Gly Gln Asp Pro Glu Gly Gly Val Asp Tyr Lys Asn Ile His Leu
690 695 700
Glu Lys Lys Tyr Val Arg Arg Asp Ser Gly Phe Ser Ser Gin Gly Val
705 710 715 720
Asp Thr Tyr Val Glu Met Arg Pro Vai Ser Thr Ser Ser Asn Asp Ser
725 730 735
Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu Leu
740 745 750
Arg Asp Leu Leu His Phe Ser Ser G1n Va1 Ala Gln Gly Met Ala Phe
755 760 765
Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val Ala Ala Arg Asn Val
770 775 780
Leu Leu Thr Asn Gly His Val Ala Lys Ile Gly Asp Phe Gly Leu Ala
785 790 795 800
Arg Asp Ile Met Asn Asp Ser Asn Tyr Ile Val Lys Gly Asn Ala Arg
805 810 815
Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr
820 825 830
Thr Val Gln Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile
835 840 845
Phe Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys
850 855 860
Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala Phe
865 870 875 880
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
18
Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys Trp Ala Leu Glu
885 890 895
Pro Thr His Arg Pro Thr Phe Gln Gln Ile Cys Ser Phe Leu Gln Glu
900 905 910
Gln Ala Gln Glu Asp Arg Arg Glu Arg Asp Tyr Thr Asn Leu Pro Ser
915 920 925
Ser Ser Arg Ser Gly Gly Ser Gly Ser Ser Ser Ser Glu Leu Glu Glu
930 935 940
Glu Ser Ser Ser Glu His Leu Thr Cys Cys Glu Gln Gly Asp Ile Ala
945 950 955 960
Gln Pro Leu Leu Gln Pro Asn Asn Tyr Gln Phe Cys
965 970
<210> 11
<211> 1696
<212> DNA
<213> Homo sapiens
<400> 11
ccgagtgtcc acaccctgtg cgtctctctg tcctgccagc actgagggct catccatccg 60
cagagcaggg cagtgggagg agacgccatg acccccatcc tcacggtcct gatctgtctc 120
gggctgagtc tgggccccag gacccacgtg caggcagggc acctccccaa gcccaccctc 180
tgggctgagc caggctctgt gatcatccag ggaagtcctg tgaccctcag gtgtcagggg 240
agccttcagg ctgaggagta ccatctatat agggaaaaca aatcagcatc ctgggttaga 300
cggatacaag agcctgggaa gaatggccag ttccccatcc catccatcac ctgggaacac 360
gcagggcggt atcactgtca gtactacagc cacaatcact catcagagta cagtgacccc 420
ctggagctgg tggtgacagg agcctacagc aaacccaccc tctcagctct gcccagccct 480
gtggtgacct taggagggaa cgtgaccctc cagtgtgtct cacaggtggc atttgacggc 540
ttcattctgt gtaaggaagg agaagatgaa cacccacaac gcctgaactc ccattcccat 600
gcccgtgggt ggtcctgggc catcttctcc gtgggccccg tgagcccgag tcgcaggtgg 660
tcgtacaggt gctatgctta tgactcgaac tctccctatg tgtggtctct acccagtgat 720
ctcctggagc tcctggtccc aggtgtttct aagaagccat cactctcagt gcagccaggt 780
cctatggtgg cccctgggga gagcctgacc ctccagtgtg tctctgatgt cggctacgac 840
CA 02418506 2003-02-05
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19
agatttgttc tgtataagga gggagaacgt gacttcctcc agcgccctgg ttggcagccc 900
caggctgggc tctcccaggc.caacttcacc ctgggccctg tgagcccctc ccacgggggc 960
cagtacagat gctacagtgc acacaacctc tcctccgagt ggtcggcccc cagtgacccc 1020
ctggacatcc tgatcacagg acagttctat gacagaccct ctctctcggt gcagccggtc 1080
cccacagtag ccccaggaaa gaacgtgacc ctgctgtgtc agtcacgggg gcagttccac 1140
actttccttc tgaccaagga gggggcaggc catcccccac tgcatctgag atcagagcac 1200
caagctcagc agaaccaggc tgaattccgc atgggtcctg tgacctcagc ccacgtgggg 1260
acctacagat gctacagctc actcagctcc aacccctacc tgctgtctct ccccagtgac 1320
cccctggagc tcgtggtctc agcatcccta ggccaacacc cccaggatta cacagtggag 1380
aatctcatcc gcatgggtgt ggctggcttg gtcctggtgg tcctcgggat tctgctattt 1440
gaggctcagc acagccagag aagcctacaa gatgcagccg ggaggtgaac agcagagagg 1500
acaatgcata cttcagcgtg gtggagcctc agggacagat ctgatgatcc caggaggctc 1560
tggaggacaa tctaggacct acattatctg gactgtatgc tggtcatttc tagagacagc 1620
aatcaatatt tgagtgtaag gaaactgtct ggggtgattc ctagaagatc attaaactgt 1680
ggtacatttt tttgtc 1696
<210> 12
<211> 466
<212> PRT
<213> Homo sapiens
<400> 12
Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly
1 5 10 15
Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp
20 25 30
Ala Glu Pro Gly Ser Val Ile Ile Gln Gly Ser Pro Val Thr Leu Arg
40 45
Cys Gln Gly Ser Leu Gln Ala Glu Glu Tyr His Leu Tyr Arg Glu Asn
35 50 55 60
Lys Ser Ala Ser Trp Val Arg Arg Ile Gln Glu Pro Gly Lys Asn Gly
65 70 75 80
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr His
85 90 95
Cys Gln Tyr Tyr Ser His Asn His Ser Ser Glu Tyr Ser Asp Pro Leu
5 100 105 110
Glu Leu Val Val Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu
115 120 125
10 Pro Ser Pro Val Val Thr Leu Gly Gly Asn Val Thr Leu Gln Cys Val
130 135 140
Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp
145 150 155 160
Glu His Pro Gln Arg Leu Asn Ser His Ser His Ala Arg Gly Trp Ser
165 170 175
Trp Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg Trp Ser
180 185 190
Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Val Trp Ser Leu
195 200 205
Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys Pro
210 215 220
Ser Leu Ser Val Gln Pro Gly Pro Met Val Ala Pro Gly Glu Ser Leu
225 230 235 240
Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu Tyr
245 250 255
Lys Glu Gly Glu Arg Asp.Phe Leu Gln Arg Pro Gly Trp Gln Pro Gln
260 265 270
Ala Gly Leu Ser Gin Ala Asn Phe Thr Leu Gly Pro Val Ser Pro Ser
275 280 285
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
21
His Gly Gly Gln Tyr Arg Cys Tyr Ser Ala His Asn Leu Ser Ser Glu
290 295 300
Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln Phe
305 310 315 320
Tyr Asp Arg Pro Ser Leu Ser Val Gln Pro Val Pro Thr Val Ala Pro
325 330 335
Gly Lys Asn Val Thr Leu Leu Cys Gln Ser Arg Gly Gln Phe His Thr
340 345 350
Phe Leu Leu Thr Lys Glu Gly Ala Gly His Pro Pro Leu His Leu Arg
355 360 365
Ser Glu His Gln Ala Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro
370 375 380
Val Thr Ser Ala His Val Gly Thr Tyr Arg Cys Tyr Ser Ser Leu Ser
385 390 395 400
Ser Asn Pro Tyr Leu Leu Ser Leu Pro Ser Asp Pro Leu Glu Leu Val
405 410 415
Val Ser Ala Ser Leu Gly Gln His Pro Gln Asp Tyr Thr Val Glu Asn
420 425 430
Leu Ile Arg Met Gly Val Ala Gly Leu Val Leu Val Val Leu Gly Ile
435 440 445
Leu Leu Phe Giu Ala Gln His Ser Gln Arg Ser Leu Gln Asp Ala Ala
450 455 460
Gly Arg
465
<210> 13
<211> 63
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 13
ggccagtgaa ttgtaatacg actcactata gggaggcggt tttttttttt tttttttttt 60
ttt 63
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 14
gtcgtcaaga tgctaccgtt cagga 25
<210> 15
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 15
ggggacaagt ttgtacaaaa aagcaggcta tggaaaccaa cttctcca 48
<210> 16
<211> 53
<212> DNA
<213> Artificial Sequence
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
23
<220>
<223> Description of Artificial Sequence: Primer
<400> 16
ggggaccact ttgtacaaga aagctgggtt cacattgcct gtaactcagt ctc 53
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 17
agcccatagc agatggcaac 20
<210> 18
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 18
tgtactttca actttgcatc ctgg 24
<210> 19
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
24
<400> 19
aagccaatga caaaccggat aatccctc 28
<210> 20
<211> 2051
<212> DNA
<213> Homo sapiens
<400> 20
cgccactttg ctggagcatt cactaggcga ggcgctccat cggactcact agccgcactc 60
atgaatcggc accatctgca ggatcacttt ctggaaatag acaagaagaa ctgctgtgtg 120
ttccgagatg acttcattgc caaggtgttg ccgccggtgt tggggctgga gtttatcttt 180
gggcttctgg gcaatggcct tgccctgtgg attttctgtt tccacctcaa gtcctggaaa 240
tccagccgga ttttcctgtt caacctggca gtagctgact ttctactgat catctgcctg 300
ccgttcgtga tggactacta tgtgcggcgt tcagactgga actttgggga catcccttgc 360
cggctggtgc tcttcatgtt tgccatgaac cgccagggca gcatcatctt cctcacggtg 420
gtggcggtag acaggtattt ccgggtggtc catccccacc acgccctgaa caagatctcc 480
aattggacag cagccatcat ctcttgcctt ctgtggggca tcactgttgg cctaacagtc 540
cacctcctga agaagaagtt gctgatccag aatggccctg caaatgtgtg catcagcttc 600
agcatctgcc ataccttccg gtggcacgaa gctatgttcc tcctggagtt cctcctgccc 660
ctgggcatca tcctgttctg ctcagccaga attatctgga gcctgcggca gagacaaatg 720
gaccggcatg ccaagatcaa gagagccatc accttcatca tggtggtggc catcgtcttt 780
gtcatctgct tccttcccag cgtggttgtg cggatccgca tcttctggct cctgcacact 840
tcgggcacgc agaattgtga agtgtaccgc tcggtggacc tggcgttctt tatcactctc 900
agcttcacct acatgaacag catgctggac cccgtggtgt actacttctc cagcccatcc 960
tttcccaact tcttctccac tttgatcaac cgctgcctcc agaggaagat gacaggtgag 1020
ccagataata accgcagcac gagcgtcgag ctcacagggg accccaacaa aaccagaggc 1080
gctccagagg cgttaatggc caactccggt gagccatgga gcccctctta tctgggccca 1140
acctcaaata accattccaa gaagggacat tgtcaccaag aaccagcatc tctggagaaa 1200
cagttgggct gttgcatcga gtaatgtcac tggactcggc ctaaggtttc ctggaacttc 1260
cagattcaga gaatctgatt tagggaaact gtggcagatg agtgggagac tggttgcaag 1320
gtgtgaccac aggaatcctg gaggaacaga gagtaaagct tctaggcatc tgaaacttgc 1380
ttcatctctg acgctcgcag gactgaagat gggcaaattg taggcgtttc tgctgagcag 1440
agttggagcc agagatctac ttgtgacttg ttggccttct tcccacatct gcctcagact 1500
ggggggggct cagctcctcg ggtgatatct agcctgcttg tgagctctag cagggataag 1560
gagagctgag attggaggga attgtgttgc tcctggagga agcccaggca tcattaaaca 1620
agccagtagg tcacctggct tccgtggacc aattcatctt tcagacaagc tttagagaaa 1680
tggactcagg gaagagactc acatgctttg gttagtatct gtgtttccgg tgggtgtaat 1740
aggggattag ccccagaagg gactgagcta aacagtgtta ttatgggaaa ggaaatggca 1800
CA 02418506 2003-02-05
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ttgctgcttt caaccagcga ctaatgcaat ccattcctct cttgtttata gtaatctaag 1860
ggttgagcag ttaaaacggc ttcaggatag aaagctgttt cccacctgtt tcgttttacc 1920
attaaaaggg aaacgtgcct ctgccccacg ggtagagggg gtgcacgttc ctcctggttc 1980
cttcgcttgt gtttctgtac ttaccaaaaa tctaccactt caataaattt tgataggaga 2040
5 caaaaaaaaa a 2051
<210> 21
<211> 387
10 <212> PRT
<213> Homo sapiens
<400> 21
Met Asn Arg His His Leu Gln Asp His Phe Leu Glu Ile Asp Lys Lys
15 1 5 10 15
Asn Cys Cys Val Phe Arg Asp Asp Phe Ile Ala Lys Val Leu Pro Pro
20 25 30
20 Val Leu Gly Leu Glu Phe Ile Phe Gly Leu Leu Gly Asn Gly Leu Ala
40 45
Leu Trp Ile Phe Cys Phe His Leu Lys Ser Trp Lys Ser Ser Arg Ile
50 55 60
Phe Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu Ile Ile Cys Leu
65 70 75 80
Pro Phe Val Met Asp Tyr Tyr Val Arg Arg Ser Asp Trp Asn Phe Gly
85 90 95
Asp Ile Pro Cys Arg Leu Val Leu Phe Met Phe Ala Met Asn Arg Gln
100 105 110
Gly Ser Ile Ile Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg
115 120 125
Val Val His Pro His His Ala Leu Asn Lys Ile Ser Asn Trp Thr Ala
130 135 140
CA 02418506 2003-02-05
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26
Ala Ile Ile Ser Cys Leu Leu Trp Gly Ile Thr Val Gly Leu Thr Val
145 150 155 160
His Leu Leu Lys Lys Lys Leu Leu Ile Gln Asn Gly Pro Ala Asn Val
165 170 175
Cys Ile Ser Phe Ser Ile Cys His Thr Phe Arg Trp His Glu Ala Met
180 185 190
Phe Leu Leu Glu Phe Leu Leu Pro Leu Gly Ile Ile Leu Phe Cys Ser
195 200 205
Ala Arg Ile Ile Trp Ser Leu Arg Gln Arg Gln Met Asp Arg His Ala
210 215 220
Lys Ile Lys Arg Ala Ile Thr Phe Ile Met Val Val Ala Ile Vai Phe
225 230 235 240
Val Ile Cys Phe Leu Pro Ser Val Val Val Arg Ile Arg Ile Phe Trp
245 250 255
Leu Leu His Thr Ser Gly Thr Gln Asn Cys Glu Val Tyr Arg Ser Val
260 265 270
Asp Leu Ala Phe Phe Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met
275 280 285
Leu Asp Pro Val Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Asn Phe
290 295 300
Phe Ser Thr Leu Ile Asn Arg Cys Leu Gln Arg Lys Met Thr Gly Glu
305 310 315 320
Pro Asp Asn Asn Arg Ser Thr Ser Val Glu Leu Thr Gly Asp Pro Asn
325 330 335
Lys Thr Arg Gly Ala Pro Glu Ala Leu Met Ala Asn Ser Gly Glu Pro
340 345 350
CA 02418506 2003-02-05
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27
Trp Ser Pro Ser Tyr Leu Gly Pro Thr Ser Asn Asn His Ser Lys Lys
355 360 365
Gly His Cys His Gln Glu Pro Ala Ser Leu Glu Lys Gln Leu Gly Cys
370 375 380
Cys Ile Glu
385
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 22
agcccatagc agatggcaac 20
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 23
tgtactttca actttgcatc ctgg 24
<210> 24
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
CA 02418506 2003-02-05
WO 02/18938 PCT/EP01/09727
28
<400> 24
aagccaatga caaaccggat aatccctc 28
