U.S. patent application number 10/874015 was filed with the patent office on 2004-12-16 for method for identifying substances which positively influence inflammatory conditions of chronic inflammatory airway diseases.
This patent application is currently assigned to Boehringer Ingelheim Pharma KG. Invention is credited to Jung, Birgit, Kistler, Barbara, Kraut, Norbert, Mueller, Stefan, Quast, Karsten, Seither, Peter, Weith, Andreas.
Application Number | 20040253630 10/874015 |
Document ID | / |
Family ID | 27255867 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253630 |
Kind Code |
A1 |
Jung, Birgit ; et
al. |
December 16, 2004 |
Method for identifying substances which positively influence
inflammatory conditions of chronic inflammatory airway diseases
Abstract
The present invention relates to substances which modulate
receptors involved in inflammatory processes and whose modulated
functions positively influence inflammatory diseases.
Inventors: |
Jung, Birgit; (Schwabenheim,
DE) ; Kraut, Norbert; (Eberhardzell, DE) ;
Mueller, Stefan; (Mainz, DE) ; Kistler, Barbara;
(Pfungstadt, DE) ; Seither, Peter; (Risseg Halde,
DE) ; Quast, Karsten; (Schemmerberg, DE) ;
Weith, Andreas; (Eberhardzell, DE) |
Correspondence
Address: |
BOEHRINGER INGELHEIM CORPORATION
900 RIDGEBURY ROAD
P. O. BOX 368
RIDGEFIELD
CT
06877
US
|
Assignee: |
Boehringer Ingelheim Pharma
KG
Ingelheim
DE
|
Family ID: |
27255867 |
Appl. No.: |
10/874015 |
Filed: |
June 22, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10874015 |
Jun 22, 2004 |
|
|
|
09944807 |
Aug 31, 2001 |
|
|
|
6773895 |
|
|
|
|
60233748 |
Sep 19, 2000 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/7.21 |
Current CPC
Class: |
G01N 2800/12 20130101;
G01N 2500/00 20130101; G01N 33/6893 20130101; C07K 14/705
20130101 |
Class at
Publication: |
435/006 ;
435/007.21 |
International
Class: |
C12Q 001/68; G01N
033/567 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 1, 2001 |
GB |
0021484.1 |
Claims
What is claimed is:
1. A method for determining an expression level of an ILM receptor
comprising: determining the level of ILM receptor expressed in a
macrophage.
2. The method according to claim 1 in which said macrophage is a
mammalian cell.
3. The method according to claim 2 in which said macrophage is a
human cell.
4. The method according to claim 3 in which the analysis is
performed using a macrophage or a part thereof obtainable from the
site of inflammation.
5. The method according to claim 4 in which the analysis is
performed using a macrophage or a part thereof obtainable from the
site of inflammation in a mammal.
6. The method according to claim 5 in which the analysis is
performed using a macrophage or a part thereof obtainable from the
site of inflammation in a human being.
7. The method according to claim 1 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor type
receptor, HM74 receptor type receptor, AICL receptor type receptor,
ILT1 receptor type receptor, SHPS receptor type receptor, KDEL
receptor 1 type receptor, and CSF-1 receptor type receptor.
8. The method according to claim 1 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor (SEQ
ID NO:2), HM74 receptor (SEQ ID NO:21), AICL receptor (SEQ ID
NO:6), ILT1 receptor (SEQ ID NO:12), SHPS-1 receptor (SEQ ID NO:4),
KDEL receptor 1 (SEQ ID NO:8), and CSF-1 receptor (SEQ ID
NO:10).
9. The method according to claim 1 in which said receptor is an
FPRL-1 receptor type receptor.
10. The method according to claim 9 in which the FPRL-1 receptor
type receptor is SEQ ID NO:2 or a variant, mutant, or fragment
thereof having the same function.
11. The method according to claim 1 for diagnosis or monitoring of
a chronic inflammatory airway disease.
12. The method according to claim 11 in which the chronic
inflammatory airway disease is selected from the group consisting
of chronic bronchitis and COPD.
13. The method according to claim 11 in which the macrophage or a
part thereof is obtained from the site of inflammation.
14. The method according to claim 13 in which the macrophage or a
part thereof is obtained from a site of inflammation in a
mammal.
15. The method according to claim 14 in which the mammal is a human
being.
16. A test system kit for determining whether a substance is an
activator or an inhibitor of an ILM receptor function wherein the
receptor is involved in a chronic inflammatory airway disease and
wherein the receptor plays a role in mediating inflammation
comprising at least: a. an ILM receptor, or b. an expression vector
capable of expressing an ILM receptor in a cell, or c. a host cell
transformed with an expression vector capable of expressing an ILM
receptor.
17. The test system kit according to claim 16 in which said
receptor is a receptor selected from the group consisting of FPRL-1
receptor type receptor, HM74 receptor type receptor, AICL receptor
type receptor, ILT1 receptor type receptor, SHPS receptor type
receptor, KDEL receptor 1 type receptor, and CSF-1 receptor type
receptor.
18. The test system kit according to claim 16 in which said
receptor is a receptor selected from the group consisting of FPRL-1
receptor (SEQ ID NO:2), HM74 receptor (SEQ ID NO:21), AICL receptor
(SEQ ID NO:6), ILT1 receptor (SEQ ID NO:12), SHPS-1 receptor (SEQ
ID NO:4), KDEL receptor 1 (SEQ ID NO:8), and CSF-1 receptor (SEQ ID
NO:10).
19. The test system kit according to claim 16 in which said
receptor is a FPRL-1 receptor type receptor.
20. The test system kit according to claim 19 in which the FPRL-1
receptor type receptor is SEQ ID NO 2 or a variant, mutant, or
fragment thereof having the same function.
21. The test system kit according to claim 20 comprising a cell
expressing an ILM receptor.
22. The test system kit according to claim 21 in which the cell is
a MonoMac6 or a THP-1 cell, wherein said cell is stimulated with
phorbol 12-myristate 13-acetate and with a substance selected from
a group consisting of LPS and smoke.
23. A substance determined to be an activator or inhibitor of an
ILM receptor.
24. The substance according to claim 23 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor type
receptor, HM74 receptor type receptor, AICL receptor type receptor,
ILT1 receptor type receptor, SHPS receptor type receptor, KDEL
receptor 1 type receptor, and CSF-1 receptor type receptor.
25. The substance according to claim 23 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor (SEQ
ID NO:2), HM74 receptor (SEQ ID NO:21), AICL receptor (SEQ ID
NO:6), ILT1 receptor (SEQ ID NO:12), SHPS-1 receptor (SEQ ID NO:4),
KDEL receptor 1 (SEQ ID NO:8), and CSF-1 receptor (SEQ ID
NO:10).
26. The substance according to claim 23 in which said receptor is
an FPRL-1 receptor type receptor.
27. The substance according to claim 26 in which the FPRL-1
receptor type receptor is SEQ ID NO:2 or a variant, mutant, or
fragment thereof having the same function.
28. A substance which is an activator or inhibitor of an ILM
receptor for the treatment of a disease.
29. The substance according to claim 28 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor type
receptor, HM74 receptor type receptor, AICL receptor type receptor,
ILT1 receptor type receptor, SHPS receptor type receptor, KDEL
receptor 1 type receptor, and CSF-1 receptor type receptor.
30. The substance according to claim 28 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor (SEQ
ID NO:2), HM74 receptor (SEQ ID NO:21), AICL receptor (SEQ ID
NO:6), ILT1 receptor (SEQ ID NO:12), SHPS-1 receptor (SEQ ID NO:4),
KDEL receptor 1 (SEQ ID NO:8), and CSF-1 receptor (SEQ ID
NO:10).
31. The substance according to claim 28 in which said receptor is
an FPRL-1 receptor type receptor.
32. The substance according to claim 31 in which the FPRL-1
receptor type receptor is SEQ ID NO:2 or a variant, mutant, or
fragment thereof having the same function.
33. The substance according to claim 28 in which said disease is a
chronic inflammatory airway disease.
34. The substance according to claim 33 in which said chronic
inflammatory airway disease is selected from the group consisting
of chronic bronchitis and COPD.
35. A pharmaceutical composition comprising at least one substance
determined to be an activator or inhibitor of an ILM receptor; and
a pharmaceutically acceptable carrier.
36. The pharmaceutical composition according to claim 35 in which
said receptor is a receptor selected from the group consisting of
FPRL-1 receptor type receptor, HM74 receptor type receptor, AICL
receptor type receptor, ILT1 receptor type receptor, SHPS receptor
type receptor, KDEL receptor 1 type receptor, and CSF-1 receptor
type receptor.
37. The pharmaceutical composition according to claim 35 in which
said receptor is a receptor selected from the group consisting of
FPRL-1 receptor (SEQ ID NO:2), HM74 receptor (SEQ ID NO:21), AICL
receptor (SEQ ID NO:6), ILT1 receptor (SEQ ID NO:12), SHPS-1
receptor (SEQ ID NO:4), KDEL receptor 1 (SEQ ID NO:8), and CSF-1
receptor (SEQ ID NO:10).
38. The pharmaceutical composition according to claim 35 in which
said receptor is an FPRL-1 receptor type receptor.
39. The pharmaceutical composition according to claim 38 in which
the FPRL-1 receptor type receptor is SEQ ID NO:2 or a variant,
mutant, or fragment thereof having the same function.
40. A method for treating a chronic inflammatory airway disease
comprising administering to a 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 of an ILM
receptor.
41. The method according to claim 40 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor type
receptor, HM74 receptor type receptor, AICL receptor type receptor,
ILT1 receptor type receptor, SHPS receptor type receptor, KDEL
receptor 1 type receptor, and CSF-1 receptor type receptor.
42. The method according to claim 40 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor (SEQ
ID NO:2), HM74 receptor (SEQ ID NO:21), AICL receptor (SEQ ID
NO:6), ILT1 receptor (SEQ ID NO:12), SHPS-1 receptor (SEQ ID NO:4),
KDEL receptor 1 (SEQ ID NO:8), and CSF-1 receptor (SEQ ID
NO:10).
43. The method according to claim 40 in which said receptor is an
FPRL-1 receptor type receptor.
44. The method according to claim 43 in which the FPRL-1 receptor
type receptor is SEQ ID NO:2 or a variant, mutant, or fragment
thereof having the same function.
45. The method according to claim 40 wherein the being is a
mammal.
46. The method according to claim 45 wherein the being is a human
being.
47. The method according to claim 40 for treating a chronic
inflammatory airway disease selected from the group consisting of
chronic bronchitis and COPD.
48. A method for selectively modulating an ILM receptor in a
macrophage comprising administering a substance determined to be an
activator or inhibitor of an ILM receptor.
49. The method according to claim 48 in which the macrophage is
involved in a chronic inflammatory airway disease.
50. The method according to claim 49 in which the chronic
inflammatory airway disease is selected from the group consisting
of chronic bronchitis and COPD
51. The method according to claim 48 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor type
receptor, HM74 receptor type receptor, AICL receptor type receptor,
ILT1 receptor type receptor, SHPS receptor type receptor, KDEL
receptor 1 type receptor, and CSF-1 receptor type receptor.
52. The method according to claim 48 in which said receptor is a
receptor selected from the group consisting of FPRL-1 receptor (SEQ
ID NO:2), HM74 receptor (SEQ ID NO:21), AICL receptor (SEQ ID
NO:6), ILT1 receptor (SEQ ID NO:12), SHPS-1 receptor (SEQ ID NO:4),
KDEL receptor 1 (SEQ ID NO:8), and CSF-1 receptor (SEQ ID
NO:10).
53. The method according to claim 48 in which said receptor is an
FPRL-1 receptor type receptor.
54. The method according to claim 53 in which the FPRL-1 receptor
type receptor is SEQ ID NO:2 or a variant, mutant, or fragment
thereof having the same function.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/944,807, filed Aug. 31, 2001, which claims, as does the
present application, priority to U.S. provisional application Ser.
No. 60/233,748, filed Sep. 19, 2000, the disclosures of both of
which are incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 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 are identified to be involved in the inflammatory
process.
[0003] 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.
[0004] 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.
[0005] 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
.beta.-agonists and anticholinergics are applied to achieve a
transient bronchodilatation. A variety of antagonists for
inflammatory events are under investigation like, LTB.sub.4-,
IL-8-, TNF.alpha.-inhibitors.
[0006] 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.
SUMMARY OF THE INVENTION
[0007] The present invention relates to substances which modulate
receptors involved in inflammatory processes and whose modulated
functions positively influence inflammatory diseases.
DESCRIPTION OF THE INVENTION
[0008] 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 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 a 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.
[0009] 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 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 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.
[0010] 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.
[0011] 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.
[0012] A differentially expressed nucleic acid sequence or protein
of the invention can easily be detected by such a 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.
[0013] 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.
[0014] 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 NOs: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.
[0015] 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 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 ID
NO:2. In a most preferred embodiment, the FPRL-1 receptor is
encoded by the nucleic acid sequence shown in SEQ ID NO:1.
[0016] 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 is preferably inhibited in order to inhibit hyperactivation
or reduce a hyperactivated status of a macrophage. A 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
mediates inflammation in that it is activated by a ligand (any
substance which has the capacity to bind to said receptor to at
least one of its domains exposed on the cell surface) and leads to
an intracellular signal involved in inflammatory processes.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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 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. 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
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. Test systems of the
invention may be availbale as kits.
[0021] 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.
[0022] 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 which directly binds the receptor,
thereby activating or blocking functional domains of said receptor,
which can be done reversibly or irreversibly, depending on the
nature of the substance applied.
[0023] Accordingly, a substance useful for activating or inhibiting
receptor function includes substances acting on the expression of a
differentially expressed nucleic acid sequence, but also 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
appropriate ligand with a receptor of the invention and thereby
affecting its function.
[0024] 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 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.
[0025] 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 human being.
[0026] 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.
[0027] 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 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.
[0028] 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 from an inflammatory
airway disorder or a transgenic animal expressing a receptor
according to the invention.
[0029] 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 IC.sub.50, LD.sub.50 and ED.sub.50.
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 transdermal, oral, buccal,
nasal, enteral, parenteral, inhalative, intratracheal, or
rectal).
[0030] 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
Pharmaceutical Science". Examples for ingredients that are useful
for formulating at least one substance according to the present
invention are also found in WO 99/18193, which is hereby
incorporated by reference.
[0031] 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 according to the invention and plays
a role in mediating inflammation involved in a chronic inflammatory
airway disease according to the invention.
[0032] In another 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.
[0033] Included herein are exemplified embodiments, which are
intended as illustrations of single aspects of the invention.
Indeed, various modifications of the invention in addition to those
herein will become apparent to those skilled in the art from the
foregoing description and drawings. Such modifications are intended
to fall within the scope of the present invention.
[0034] All publications and patent applications cited herein are
incorporated by reference in their entireties.
EXAMPLES
Example 1
[0035] Comparative Expression Profiling and FPLR-1 Cloning
[0036] The following is an illustration of how comparative
expression profiling can be performed in order to identify
receptors according to the present invention.
[0037] 1.1. Selection of Patients
[0038] Three groups of subjects are studied: healthy non-smokers,
healthy smokers and patients with COPD.
[0039] 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.sub.1 % predicted is less than 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.sub.1 falls 20% the test is stopped and the
PC.sub.20 is calculated. This is the dose of methacholine causing a
20% fall in FEV.sub.1 and we will require a value of greater than
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.
[0040] 1.2. BAL (Bronchoalveolar Lavage) Procedure
[0041] Subjects are sedated with midazolam prior to the BAL. Local
anaesthetic spray is used to anaesthetize the back of the throat. A
7 mm 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.
[0042] 1.3. BAL Processing
[0043] 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 ml Falcon blue-cap polypropylene, 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.degree. C.
[0044] 1.4. Differential Gene Expression Analysis
[0045] 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.mu. 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 10,000 g for 15 minutes
at 4.degree. 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.degree.
C. with 10,000 g, the pellet is washed twice with 75% ethanol, air
dried and resuspended in DEPC-H.sub.2O.
[0046] 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
.mu.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 .mu.l RNA
and 1 .mu.l of 100 .quadrature.M T7-(dt).sub.24 primer, sequence
shown in SEQ ID NO:13, are heated up to 70.degree. C. for 10
minutes and then cooled down on ice for 2 minutes. First strand
buffer to a final concentration of 1.times., 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 .mu.l. The reaction mix is incubated
at 42.degree. C. for 2 minutes and 2 .mu.l of Superscript II
reverse transcriptase (200 U/.mu.l) are added. For second strand
synthesis 130 .mu.l of a mix containing 1.15.times. second strand
buffer, 230 .mu.M dNTPs, 10 U E.coli DNA ligase (10U/.mu.l), E.coli
DNA polymerase (10 U/.mu.l), RNase H (2U/.quadrature.l) is added to
the reaction of the first strand synthesis and carefully mixed with
a pipette. Second strand synthesis is performed at 16.degree. C.
for 2 hours, then 2 .mu.l of T4 DNA polymerase (5 U/.mu.l) are
added, incubated for 5 minutes at 16.degree. C. and the reaction is
stopped by adding 10 .mu.l 0.5 M EDTA.
[0047] 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 1.times. HY reaction buffer,
1.times. biotin labeled ribonucleotides, 1.times. DTT, 1.times.
RNase Inhibitor Mix and 1.times. T7 RNA Polymerase in a total
volume of 40 .mu.l for 5 hours at 37.degree. 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.times. fragmentation buffer (5.times. fragmentation buffer: 200
mM Tris acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) at 94.degree.
C. for 35 minutes.
[0048] For hybridization of the DNA chip 15 .mu.g of cRNA is used,
mixed with 50 pM biotin-labeled control B2 oligonucleotide,
sequence shown SEQ ID NO:14, 1.times. cRNA cocktail, 0.1 mg/ml
herring sperm DNA, 0.5 mg/ml acetylated BSA, 1.times. MES
(2-[N-morpholino]-ethanesulfonic acid) hybridization buffer in a
total volume of 300 .mu.l. The hybridization mixture is heated up
to 99.degree. C. for 5 minutes, cooled down to 45.degree. C. for 10
minutes and 200 .mu.l of the mix are used to fill the probe array.
The hybridization is performed at 45.degree. C. at 60 rpm for 16
hours. After the hybridization the hybridization mix on the chip is
replaced by 300 .mu.l non-stringent wash buffer (100 mM MES, 100 mM
NaCl, 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 .mu.l 1.times. stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween
20), 2 mg/ml BSA, 10 .mu.g/ml SAPE (streptavidin phycoerythrin)
(Dianova), the antibody solution consists of 1.times. stain buffer,
2 mg/ml BSA, 0.1 mg/ml goat IgG, 3 .mu.g/ml biotinylated
antibody.
[0049] After the washing and staining procedure the chips are
scanned on the HP Gene Array Scanner (Hewlett Packard).
[0050] 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
LXA.sub.4R, 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 A.sub.4 (LXA.sub.4) (Murphy, P.
M. et al. 1992, J. Biol. Chem. 267:7637-7643). Alveolar macrophages
have been shown to produce lipoxins, which are synthesized by
15-lipoxygenase (Kim, S. J., 1988, Biochem. Biophys Res. Commun.
150:870-876). Lipoxin A.sub.4 (LXA.sub.4) stimulates chemotaxis,
adherence and calcium release in monocytes. In neutrophils, though,
LXA.sub.4 inhibits chemotaxis and adhesion, and downregulates
transmigration through epithelial cells (Maddox, J. F. and Serhan,
C. N. 1996, J. Exp. Med. 183:137-146). LXA.sub.4 was found elevated
in BALs from patients with asthma (Lee, T. H. et al. 1990, Am. Rev.
Respir. Dis. 141:1453-1458 and Serhan, J. N. 1999, Lipoxygenases
and Their Metabolites, ed. Nigam and Pace-Asciak, Plenum Press, New
York 133-149). In particular, it was found to cause a
dose-dependent contraction of human bronchi (Christie et al. 1992,
Am. Rev. Respir. Dis. 145:1281-1284). LXA.sub.4 is considered to be
a generic modulator of inflammation in the lung.
[0051] 1.5. FPRL-1 Receptor Overexpressed in COPD Macrophages
[0052] 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.
1TABLE 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 fold fold change
comparison change comparison change comparison 2.7 39vs2 2.9 5vs2
3.3 1vs2 4.6 39vs37 3.6 5vs37 5.5 1vs37 2 39vs43 1.4 5vs43 1.4
1vs43 3.1 39vs56 3 5vs56 3.9 1vs56 4.1 39vs57 3.2 5vs57 5.3 1vs57
2.9 39vs58 3 5vs58 3.6 1vs58 2.2 39vs62 2.7 5vs62 2.7 1vs62 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
[0053]
2TABLE 2 Expression levels of FPRL-1 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 1276.7 P 2 490.4 P 48/49 248.2 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 + std.
1020.0 .+-. 452.5 410.9 .+-. 276.3 318.8 .+-. 220.3 dev. Median
1036.1 327.1 248.2 P value for comparisons between COPD smokers and
healthy smokers: 0.02
[0054] 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).
3TABLE 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. Comparison Chip TaqMan Comparison Chip TaqMan
1vs2 3.3 4.1 1vs37 5.5 4.6 3vs2 1.4 2.2 3vs37 2.9 2.5 39vs2 2.7 6.0
39vs37 4.6 6.8
[0055] 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, H. et al. 1993,
Intemat. Immunol. 5:1239-1249). To date, the ligand has not been
identified. HM74 receptor is consistently found upregulated (54.8%)
in COPD smokers compared to healthy smokers. This is demonstrated
by calculated "fold change" values (Table 5) from 42 pairwise
comparisons and by "avg diff" values (Table 6).
4TABLE 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 fold fold change
comparison change comparison change comparison 1.2 39vs2 4.5 5vs2
-1.2 1vs2 4.7 39vs37 13.8 5vs37 2.8 1vs37 -2.1 39vs43 2.5 5vs43
-2.2 1vs43 2.9 39vs56 8.6 5vs56 1.8 1vs56 2.6 39vs57 8.9 5vs57 1.6
1vs57 2.6 39vs58 7.7 5vs58 1.6 1vs58 2.4 39vs62 8.5 5vs62 1.5 1vs62
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
[0056]
5TABLE 6 Expression levels of HM74 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 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 + std. 7290.4 .+-. 5879.0 2591.9 .+-. 1652.5 3124.0 .+-.
1285.9 dev. median 4147.8 2243.6 3504.8
[0057] 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).
6TABLE 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. Comparison Chip TaqMan Comparison Chip TaqMan
1vs2 0.8 2.3 1vs37 2.8 4.5 3vs2 0.9 0.8 3vs37 3.0 1.4 39vs2 1.2 1.4
39vs37 4.7 2.6
[0058] 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, S. et al. 1988,
J. Biochem. 104:600-605). 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, J. et al. 1997, Immunogenetics 45:295-300). 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, J. et al. 1993, Immunol. 150:4920:4927).
[0059] AICL receptor is consistently found upregulated (66.7%) in
COPD smokers compared to healthy smokers. This is demonstrated by
calculated "fold change" values (Table 8) from 42 pairwise
comparisons and by "avg diff" values (Table 9). The p value for the
comparisons between COPD smokers and healthy smokers was 0.01.
7TABLE 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 fold fold change
comparison change comparison change comparison 1 39vs2 1.5 5vs2
-1.3 1vs2 1.9 39vs37 2.8 5vs37 1.4 1vs37 -1.4 39vs43 2.4 5vs43 1.3
1vs43 3.3 39vs56 5 5vs56 2.7 1vs56 6.9 39vs57 10 5vs57 5.3 1vs57
3.1 39vs58 4.5 5vs58 2.3 1vs58 3.3 39vs62 5.1 5vs62 2.7 1vs62 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 4.2 44vs62 2.3 6vs62 2.3
3vs62
[0060]
8TABLE 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 + std. 4622.4 .+-. 1474.3 2065.9 .+-. 1482.0 1187.5 .+-. 496.6
dev. median 3996.6 1476.0 1087.9
[0061] Another differentially expressed nucleic acid sequence
identified codes for ILT1 receptor (immunoglobulin-like transcript
1), see SEQ ID NOs:11 and 12. ILT1 receptor belongs to the Ig
superfamily receptors that is related to a subset of 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, J. and
Colonna, M., 1997, Eur. J. Immunol. 27:660-665). Upon crosslinking
with antibodies ILT1 receptor interacts with the .gamma.-chain of
the Fc receptor (Fc.epsilon.RI.gamma.(Nakajima et al., 1999 J.
Immunol. 162(1):5-8)
[0062] ILT1 receptor is found consistently upregulated (59.5%) in
COPD smokers compared to healthy smokers. This is demonstrated by
"avg diff" values (Table 10). The p value for the comparisons
between COPD smokers and healthy smokers was 0.01.
9TABLE 10 Expression levels of ILT1 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 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 + std. dev. 1008.8
.+-. 406.8 400.0 .+-. 168.6 552.0 .+-. 81.8 median 1141.6 382.6
519.7
[0063] Another differentially expressed nucleic acid sequence
identified codes for SHPS-1 receptor (SIRP-alpha1, MYD1, MFR), see
SEQ ID NOs:3 and 4, which is known to be highly expressed in
macrophages (Fujioka, Y. et al. 1996, Mol. Cell. Biol. 16:6887-6899
and Kharitonenkov, A. et al. 1997, Nature 386:181-186; Brooke, G.
P. et al. 1998, Eur J. Immunol. 28:1:11). 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,
A. et al. 1998, J. Biol. Chem. 273:22719-22728). 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.
[0064] SHPS-1 receptor is consistently found downregulated (73.8%)
in COPD smokers compared to healthy smokers. This is demonstrated
by calculated "fold change" values (Table 11) from 42 pairwise
comparisons and by "avg diff" values (Table 12). The p value for
the comparisons between COPD smokers and healthy smokers is
0.005.
10TABLE 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 fold fold fold change
comparison change comparison change comparison -1.3 39vs2 -3.4 5vs2
1.3 1vs2 -2.8 39vs37 -6.8 5vs37 -1.7 1vs37 -1.6 39vs43 -8.4 5vs43
-2.1 1vs43 -3.0 39vs56 -7.1 5vs56 -1.8 1vs56 -5.6 39vs57 -13.2
5vs57 -3.4 1vs57 -5.4 39vs58 -12.6 5vs58 -3.2 1vs58 -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
[0065]
11TABLE 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 +
std. 1293.0 .+-. 783.9 3902.4 .+-. 1671.3 2730.3 .+-. 2007.7 dev.
median 1233.4 3431.7 2090.6
[0066] 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 soluble
proteins with the amino acid sequence K-D-E-L and retrieves these
proteins after binding to the endoplasmic reticulum (Townsley, F.
M. et al. 1993, EMBO J. 12:2821-2829). 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, T. et al 1997, EMBO J.
16:7305-7316).
[0067] It is consistently found downregulated (71.4%) in COPD
smokers compared to healthy smokers. This is shown by "avg diff"
values (Table 13). The p value for the comparisons between COPD
smokers and healthy smokers is 0.003.
12TABLE 13 Expression levels of KDEL receptor 1: "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
1 877.6 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 +
std. 1061.1 .+-. 245.3 1956.4 .+-. 512.1 1296.9 .+-. 442.6 dev.
median 1033.1 2151.4 1532.9
[0068] 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 multiple proteins, e.g. CSF-1 receptor, Shc,
PI3K, Grb2, Cb1, SHP-1, Src. Moreover, ligand binding also triggers
rapid tyrosine phosphorylation of a plethora of cytoplasmic
proteins like Cb1, STAT3, STAT5a, STAT5b, p85PI3K, SHP-1, Vav and
proteins involved in cytoskeletal organization (Yeung, Y.-G. et al.
1998, J. Biol. Chem. 273:17128-17137). CSF-1 receptor regulates
survival, proliferation, differentiation and morphology of
mononuclear phagocytes (Hampe, A. et al. 1989, Oncogene Res.
4:9-17).
[0069] CSF-1 receptor is consistently found downregulated (45.2%)
in COPD smokers compared to healthy smokers. This is shown by "avg
diff" values (Table 14). The p value for the comparisons between
COPD smokers and healthy smokers is 0.002.
13TABLE 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 48/49 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 +
std. 1523.9 .+-. 522.7 2786.7 .+-. 633.2 2461.9 .+-. 468.9 dev.
median 1527.2 2799.2 2376.4
[0070] 1.6. Use of TaqMan Analysis for Validation of DNA-Chip Data
and Diagnosis
[0071] 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.
[0072] 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
(glyceraldehyde-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 .mu.M retinoic
acid ranging from 0, 5, 10, 25, 50 to 100 ng per assay. Reaction
mixes contain 1.times. TaqMan EZ-buffer, 3 mM Mn(Oac).sub.2, 300
.mu.M dATP, dCTP, dGTP, and 600 .mu.M dUTP, 2.5 U rTth DNA
polymerase, 0.25 U AmpErase UNG in a total volume of 25 .mu.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.degree. C., 30 minutes at 60.degree. C., 5 minutes at 95.degree.
C., followed by 40 cycles of 20 seconds at 94.degree. C. and 1
minute at 59.degree. 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 C.sub.T values for FPRL-1
receptor to C.sub.T 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.
[0073] 1.7. Cell Systems
[0074] 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/ml
penicillin, 100 .mu.g/ml streptomycin, 2 mM glutamine, and 1.times.
non-essential amino acids. The media for MonoMac6 cells also
includes 5 ml/l OPI media supplement (Sigma). MonoMac6 cells are
exclusively cultured in 24-well plates. Cells are maintained in a
humidified atmosphere with 5% CO.sub.2 at 37.degree. C. and tested
regularly for contamination by mycoplasma.
[0075] Differentiation is achieved by adding 10 nM PMA (phorbol 12
myristate-13 acetate) to the media.
[0076] 1.8. Cloning of FPRL-1 Receptor
[0077] FPRL-1 receptor is cloned from a total RNA extracted from
U937 cells that were treated with 1 .mu.M retinoic acid for three
days. 5 .mu.g RNA is reverse transcribed into cDNA with 5 ng
oligo(dt).sub.18 primer, 1.times. first strand buffer, 10 mM DTT,
0.5 mM dNTPs and 2 U Superscript II (Gibco BRL Life Technologies)
at 42.degree. C. for 50 minutes. Then, the reaction is terminated
at 70.degree. C. for 15 minutes and the cDNA concentration is
determined by UV-spectrophotometry. For amplification of FPRL-1
receptor 100 ng of the cDNA and 10 pmol of sequence-specific
primers for FPRL-1 receptor (forward primer attB 1; 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.degree. C., 35 cycles with
30 seconds at 94.degree. C., 30 seconds at 53.degree. C., 90
seconds at 72.degree. C., followed by 7 minutes at 72.degree. C.
with Taq DNA-polymerase. The reaction mix is separated on a 2%
agarose gel, a band of about 1,000 bp 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), 1.times. BP clonase reaction buffer, BP clonase
enzyme mix in a total volume of 20 .mu.l for 60 minutes at
25.degree. C. Then, reactions are incubated with 2 .mu.l of
proteinase K and incubated for 10 minutes at 37.degree. 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:1 is used for further
experiments.
[0078] The other receptors mentioned in example 1.5 are cloned
using analogous methods.
[0079] 1.9. Transfection of FPRL-1 Receptor
[0080] 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 the "destination vector" pcDNA3.1(+)/attR, 4
.mu.l of the LR Clonase enzyme mix, 4 .mu.l LR Clonase reaction
buffer, added up with TE (Tris/EDTA) to 20 .mu.l and incubated at
25.degree. C. for 60 minutes. Then, 2 .mu.l of proteinase K
solution is added and incubated for 10 minutes at 37.degree. C. 1
.mu.l of the reaction mix is transformed into 50 .mu.l
DH5.quadrature. by a heat-shock of 30 seconds at 42.degree. C.
after incubating cells with DNA for 30 minutes on ice. After
heat-shock of the cells 450 .mu.l of S.O.C. is added and cells are
incubated at 37.degree. C. for 60 minutes. Cells (100 .mu.l) are
plated on LB plates containing 100 .mu.g/ml ampicillin and
incubated over night.
[0081] A colony that contains pcDNA3.1(+)/attR with FPRL-1 receptor
as an insert is designated pcDNA/FPRL1 and used for transfection
studies.
[0082] 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.
Example 2
[0083] Cellular Systems and Phenotypic Effects of FPRL-1
Receptor
[0084] Analogous methods as described herein in example 2 for
FPRL-1 receptor are also performed using the other receptors
described in 1.5.
[0085] 2.1. Cell Systems
[0086] 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/ml
penicillin, 100 .mu.g/ml streptomycin, 2 mM glutamine, and 1.times.
non-essential amino acids. The media for MonoMac6 also includes 5
ml/l OPI media supplement (Sigma). MonoMac6 cells are exclusively
cultured in 24-well plates. All cells are maintained in a
humidified atmosphere with 5% CO.sub.2 at 37.degree. C. and tested
regularly for contamination by mycoplasma.
[0087] Differentiation is achieved by adding 10 nM PMA (phorbol 12
myristate-13 acetate) to the media.
[0088] Phenotypic effects of FPRL-1 receptor (2.2.-2.9.)
[0089] 2.2. Ligand Binding Assay
[0090] 300 ml cell culture is harvested with EDTA solution, the
suspension is used to spin down the cells at 110-220.times.g,
resuspended in 10 mM Tris/HCl, pH 7.4, 2.5 mM CaCl.sub.2, 1.2 mM
MgCl.sub.2, 40 .mu.g/ml bacitracin, 4 .mu.g/ml leupeptin, 4
.mu.g/ml chymostatin, 10 .mu.g/ml pefabloc, 2 .mu.M phosphoamidon
and 0.1 mg/ml bovine serum albumin (BSA Fraktion V, BI Bioproducts)
and diluted to 2.times.10 cells/ml.
[0091] 0.5 ml aliquots are incubated with 0.3 nM 3.sup.H-lipoxinA4
(specific activity approximately 10 Ci/mmol) or in the presence of
increasing concentrations of untritiated lipoxin A4 (3-300 nM) for
30 minutes at 4.degree. 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/HCl, 100 mM NaCl, 10 mM MgCl.sub.2, 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 lipoxinA4. A series of peptides and low molecular weight
compounds, including the peptide ligand MMK-1 (Klein, C. et al.
1998, Nature Biotech. 16:1334-1337), 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. IC.sub.50 values for any substance
to block binding of 3.sup.H-lipoxin A4 are calculated.
[0092] 2.3. Ca.sup.2+-Release Determined by FLIPR-Assay
[0093] FLIPR-assay (Fluorometric Imaging Plate Reader) with FPRL-1
receptor is performed with different CHO cell lines that
constitutively expresse the G-protein .alpha.-subunit .alpha.16 or
the chimeric G-proteins Gqi5 or Gqo5 (these are two G.alpha.(q)
chimeras harboring the last five residues of G.alpha.(i) or
G.alpha.(o)) and FPRL-1 receptor. The cell lines CHO/Galpha16
(CHO/Galpha16), CHO/GalphaGqi5 and CHOGalphaGqo5 (Boehringer
Ingelheim) that constitutively express G.alpha.16, 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 glutamine, 200 ng/ml hygromycin, 100 U/ml
penicillin and 100 .mu.g/ml streptomycin in a humidified atmosphere
with 5% CO.sub.2 at 37.degree. C. 3-7.times.10.sup.5 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 .mu.l
FuGene6 (Roche Biochemicals) is added to 100 .mu.l of culture media
without serum and equilibrated for 5 minutes at room temperature.
Then, 2 .mu.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 F12 media, 10% FCS, 2 mM glutamine,
200 ng/ml hygromycin, 100 U/ml penicillin,100 .mu.g/ml
streptomycin, and 200 .mu.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 .mu.L of 1.times. 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 Ca.sup.2+ via
FLIPR (Molecular Devices).
[0094] Cells (CHO/Galpha16/FPRL-1 receptor, CHO/GalphaGqi5/FPRL-1
receptor, or CHOGalphaGqo5/FPRL-1 receptor) are seeded in
384-blackwell plates (Corning) with 2500-5,000 cells per well in a
volume of 40 .mu.l and grown overnight in a humidified atmosphere
with 5% CO.sub.2 at 37.degree. C. As a negative control
CHO/Galpha16, CHO/GalphaGqi5 or CHOGalphaGqo5 cells are used. Then,
40 .mu.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 .mu.M. The Fluo-4 staining solution is composed
of 10.5 ml cell culture media described above, 420 .mu.l Probenicid
solution (1.42 g Probenicid (Sigma), 10 ml 1 M NaOH, 10 ml Hanks
buffer), 42 .mu.l Fluo-4 stock solution (50 .mu.g Fluo-4, 23
.quadrature.l DMSO, 23 .mu.l Pluronic F-127 (20% in DMSO)
(Molecular Probes), and 420 .mu.l 1M HEPES. After 45 minutes
incubation in a humidified atmosphere with 5% CO.sub.2 at
37.degree. C. wells are washed with a EMBLA-washer (4 wash steps,
program 03) using 2,000 ml Hanks buffer containing 20 ml Probenicid
solution as a wash solution and leaving 25 .mu.l wash buffer in
each well. Then FLIPR is set to 10,000 counts for stained wells and
a difference of 1:5 between unstained and stained wells. Then, 25
.mu.l lipoxin A4 and a series of ligands, peptides, 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.
[0095] 2.4. Production and Release of Cytokines or Matrix
Metalloproteases
[0096] Cells of monocytic/macrophage cell lines are treated with
lipoxin A4 at cell densities between 2.5 and 5.times.10.sup.5
cells/ml. 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, and resuspended in 400 ml of RLT buffer (from
Qiagen RNeasy Total RNA Isolation Kit) with 143 mM
.beta.-mercaptoethanol, the DNA sheared with a 20 g needle for at
least 5 times and stored at 70.degree. 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 TNF.alpha.,
IL-1.beta., IL-8, IL-6, and human matrix metalloproteases, MMP-1,
MMP-7, MMP-9, MMP-12, are measured using appropriate primer
sequences.
[0097] 2.4.1. Detection of Secreted Cytokines
[0098] 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/HCl, pH 7.4, 10
mM MgCl.sub.2, 1 mM EDTA. Protein concentration is determined via
the Bradford method and 50 .mu.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 TNF.alpha. (tumor
necrosis factor .alpha.) IL-1.beta. (interleukin-1.beta.), 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
chemiluminescence kit (Amersham). Intensity of the bands are
visualised with BioMax X-ray films (Kodak) and quantified by
densitometry.
[0099] 2.4.2. Detection and Activity of Secreted Matrix
Metalloproteases
[0100] 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.
[0101] 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 .mu.l
with 1 .mu.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.
[0102] 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 (10.sup.5 cells per well) are
plated on filters coated with an 8 .mu.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 (400.times.) by light
microscopy.
[0103] 2.5. Chemotaxis Assay
[0104] 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 .mu.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
.mu.m). 50 .mu.l cell suspension (5.times.10.sup.4) are placed in
the well of the upper compartment. The chamber is incubated for 5
hours at 37.degree. C. in a humidified atmosphere with 5% CO.sub.2.
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 (400.times.) by light
microscopy.
[0105] 2.6. Adherence Assay
[0106] Cells are harvested, washed in PBS and resuspended
(4.times.10.sup.6/ml) in PBS and 1 .mu.M BCECF
((2-7-bis-(carboxethyl)-5(- 6)-carboxyfluorescein acetoxymethyl)
ester, Calbiochem) and incubated for 20 minutes at 37.degree. C.
Cells are washed in PBS and resuspended (3.3.times.10.sup.6/ml) in
PBS containing 0.1% BSA. 3.times.10.sup.5 cells (90 .mu.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
.mu.l of agonist (100 nM lipoxin A4 plus lipoxin A4 antagonist) are
added and plates are incubated for 20 minutes at 37.degree. C.
Then, cells are washed with PBS containing 0.1% BSA and adherent
cells are solubilized with 100 .mu.l of 0.025 M NaOH and 0.1% SDS.
Quantification is performed by fluorescence measurement.
[0107] 2.7. Phagocytosis
[0108] Cell suspensions (2.5.times.10.sup.4 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.degree. C. in a
humidified atmosphere with 5% CO.sub.2 in the presence of agonists
(100 nM lipoxin A4, 50 nM MMK-1 peptide (2.3.)) and low molecular
weight compounds according to the invention in order to antagonize
agonistic effects. 40 .mu.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.
Example 3
[0109] Cell Culture Model for Macrophages Isolated from COPD
Patients
[0110] Analogous methods as described here in Example 3 for FPRL-1
receptor are also performed using receptors described in 1.5.
[0111] 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.
[0112] Expression of FPRL-1 after Stimulation of MonoMac6 Cells
with PMA, Smoke, and LPS
[0113] MonoMac6 cells are cultivated in 24-well plates in RPMI 1640
media, supplemented with 10% FCS (low endotoxin), 2 mM glutamine,
1.times. non-essential amino acids, 200 U/ml penicillin, 200
.mu.g/ml streptomycin, and 5 ml OPI media supplement (Sigma). Cells
are grown to a density of 600,000 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.degree. C., 5%
CO.sub.2 at a density of 1.times.10.sup.6 cells/ml. 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-ml 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 .mu.m 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 400,000-600,000 cells per well
filled with 2 ml of fresh RPMI 1640 media including the supplements
mentioned above.
[0114] THP-1 cells are grown in 75 cm.sup.2 flasks in RPMI 1640
Glutamax supplemented with 10% FCS (low endotoxin), 200 U/ml
penicillin, 200 .mu.g/ml streptomycin. Cells are treated with 10 nM
PMA for 48 hours at 37.degree. C., 5% CO.sub.2 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.
[0115] Both cell types are cultivated at 37.degree. C., 5% CO.sub.2
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 PBS, resuspended in 400 .mu.l of RLT buffer
(Qiagen RNeasy Total RNA Isolation Kit) with 143 mM
.beta.-mercaptoethanol, the DNA is sheared with a 20 g needle for
at least 5 times and stored at -70.degree. C.
[0116] 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.
[0117] TaqMan Analysis
[0118] 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 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 668FP, 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 .mu.M retinoic acid ranging from 0, 5,
10, 25, 50 to 100 ng per assay. Reaction mixes contain 1.times.
TaqMan EZ-buffer, 3 mM Mn(Oac).sub.2, 300 .mu.M dATP, dCTP, dGTP,
and 600 .mu.M dUTP, 2.5 U rTth DNA polymerase, 0.25 U AmpErase UNG
in a total volume of 25 .mu.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 96-well reaction plates" sealed
with "MicroAmp Optical Caps" (PE Applied Biosystems) in an ABI
PRISM 7700 Sequence Detection System (PE Applied Biosystems). The
PCR conditions are 2 minutes at 50.degree. C., 30 minutes at
60.degree. C., 5 minutes at 95.degree. C., followed by 40 cycles of
20 seconds at 94.degree. C. and 1 minute at 59.degree. 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
C.sub.T values for FPRL-1 to C.sub.T 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%).
14TABLE 15 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
[0119]
15TABLE 16 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
[0120]
16TABLE 17 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
[0121]
17TABLE 18 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
[0122] In order to examine the effects of ligands for FPRL-1,
MonoMac6 cells are seeded at a density of 250,000 cells/ml in
24-well plates (with 2 ml per well), grown for 24 hours at
37.degree. C., 5% CO.sub.2 in a humidified atmosphere before
stimulation with 200 nM lipoxin A4 (Biomol), W-peptide (1 .mu.M)
(synthesized by Metabion, Martinsried), and LPS (Sigma) as a
positive control. Cells are harvested at different time points, and
total RNA is isolated as described above using the Qiagen RNeasy
Total RNA Isolation Kit (Qiagen).
[0123] The sequence of the W-peptide (Baek et al. 1996, J. Biol.
Chem 271, 8170-8175) is W-K-Y-M-V-m.
[0124] The RNA is used for Taqman analysis in order to monitor the
expression of inflammatory markers like TNF.alpha., IL-8, and
MMP-12.
18TABLE 19 Expression of TNF.alpha. in MonoMac 6 cells after
stimulation with lipoxin A4 and W-peptide Fold Induction Fold
Induction t (h) Lipoxin A4 (200 nM) W-peptide (1 .mu.M) 0 1.00 1.00
3 2.43 1.03
[0125]
19TABLE 20 Expression of IL-8 in MonoMac 6 cells after stimulation
with lipoxin A4 and W-peptide Fold Induction Fold Induction t (h)
Lipoxin A4 (200 nM) W-peptide (1 .mu.M) 0 1.00 1.00 3 1.99 1.54
[0126]
20TABLE 21 Expression of MMP-12 in MonoMac 6 cells after
stimulation with lipoxin A4 and W-peptide Fold Induction Fold
Induction t (h) Lipoxin A4 (200 nM) W-peptide (1 .mu.M) 0 1.00 1.00
3 1.42 1.51
[0127] 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.
[0128] 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 500,000 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.times.10.sup.6 cells/ml 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 .mu.m (Neuroprobe
Inc.). The lower wells of the chamber are filled with 28 .mu.l 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 .mu.l of the cell suspension
(50,000 cells per well). After 4 hours of migration at 37.degree.
C., 5% CO.sub.2 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 250.times. 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.
21TABLE 22 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 .mu.M) 1.68 Lipoxin A4 (100 nM) 1.31 Lipoxin A4 (10
nM) 0.86 W-peptide (1 .mu.M) 2.46 W-peptide (100 nM) 1.23 W-peptide
(10 nM) 0.95
[0129] 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.
Sequence CWU 1
1
24 1 1910 DNA Homo sapiens 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 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 2 351 PRT Homo sapiens 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 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 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 Gln 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 3 2433 DNA Homo sapiens
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 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 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 4 503 PRT Homo sapiens 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 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 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 Gly 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
425 430 Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn
Asn 435 440 445 His Thr Glu Tyr Ala Ser Ile 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 5 759 DNA Homo sapiens 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 6 149 PRT Homo sapiens 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
7 1086 DNA Homo sapiens 7 ctaaaggtcc cctccccgga gcggagcgca
cctagggtcc ctcttccgtc cccccagccc 60 agctacccgt tcagaccagc
agcctcgggg ggcacccccc gccagcctgc ctccctcccg 120 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 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 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 8 212 PRT Homo sapiens 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 Val Phe Thr Ala
Arg Tyr 35 40 45 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 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 9 3992 DNA
Homo sapiens 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 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
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 10 972 PRT Homo sapiens 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 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 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 275 280 285 Thr
Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser 290 295
300 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 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 Gly 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 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 Gln Gly Val 705 710 715 720 Asp Thr Tyr Val Glu Met Arg Pro Val
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 Gln Val 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 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 11 1696 DNA Homo sapiens 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
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 12 466 PRT Homo sapiens 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 35 40 45 Cys Gln Gly Ser Leu Gln Ala Glu Glu Tyr His
Leu Tyr Arg Glu Asn 50 55 60 Lys Ser Ala Ser Trp Val Arg Arg Ile
Gln Glu Pro Gly Lys Asn Gly 65 70 75 80 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 100 105 110 Glu Leu Val
Val Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu 115 120 125 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 Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Pro
Ser 275 280 285 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 Glu Ala Gln His Ser
Gln Arg Ser Leu Gln Asp Ala Ala 450 455 460 Gly Arg 465 13 63 DNA
Artificial Sequence Description of Artificial Sequence Primer 13
ggccagtgaa ttgtaatacg actcactata gggaggcggt tttttttttt tttttttttt
60 ttt 63 14 25 DNA Artificial Sequence Description of Artificial
Sequence Primer 14 gtcgtcaaga tgctaccgtt cagga 25 15 48 DNA
Artificial Sequence Description of Artificial Sequence Primer 15
ggggacaagt ttgtacaaaa aagcaggcta tggaaaccaa cttctcca 48 16 53 DNA
Artificial Sequence Description of Artificial Sequence Primer 16
ggggaccact ttgtacaaga aagctgggtt cacattgcct gtaactcagt ctc 53 17 20
DNA Artificial Sequence Description of Artificial Sequence Primer
17 agcccatagc agatggcaac 20 18 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 tgtactttca actttgcatc
ctgg 24 19 28 DNA Artificial Sequence Description of Artificial
Sequence Primer 19 aagccaatga caaaccggat aatccctc 28 20 2051 DNA
Homo sapiens 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 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 caaaaaaaaa a 2051
21 387 PRT Homo sapiens 21 Met Asn Arg His His Leu Gln Asp His Phe
Leu Glu Ile Asp Lys Lys 1 5 10 15 Asn Cys Cys Val Phe Arg Asp Asp
Phe Ile Ala Lys Val Leu Pro Pro 20 25 30 Val Leu Gly Leu Glu Phe
Ile Phe Gly Leu Leu Gly Asn Gly Leu Ala 35 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 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 Val
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 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 22 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 22 agcccatagc
agatggcaac 20 23 24 DNA Artificial Sequence Description of
Artificial Sequence Primer 23 tgtactttca actttgcatc ctgg 24 24 28
DNA Artificial Sequence Description of Artificial Sequence Primer
24 aagccaatga caaaccggat aatccctc 28
* * * * *