U.S. patent application number 11/894503 was filed with the patent office on 2008-09-04 for methods for treating inflammation.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. Invention is credited to Kevan Herold, Ira Lamster, Ann Marie Schmidt, David M. Stern, Shi Du Yan.
Application Number | 20080214453 11/894503 |
Document ID | / |
Family ID | 39733558 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080214453 |
Kind Code |
A1 |
Stern; David M. ; et
al. |
September 4, 2008 |
Methods for treating inflammation
Abstract
The present invention provides a method for treating
inflammation in a subject which comprises administering to the
subject soluble receptor for advanced glycation endproduct (sRAGE)
in an amount effective to inhibit binding of advanced glycation
endproducts (AGEs) to RAGE thereby treating inflammation in the
subject. The present invention also provides for a method for
treating inflammation in a subject which comprises administering to
the subject an agent in an amount effective to inhibit the
interaction between receptor for advanced glycation endproduct
(RAGE) and its ligand thereby treating inflammation in the
subject.
Inventors: |
Stern; David M.;
(Cincinnati, OH) ; Herold; Kevan; (Scarsdale,
NY) ; Yan; Shi Du; (Tenafly, NJ) ; Schmidt;
Ann Marie; (Franklin Lakes, NJ) ; Lamster; Ira;
(Wyckoff, NJ) |
Correspondence
Address: |
John P. White;Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
The Trustees of Columbia University
in the City of New York
|
Family ID: |
39733558 |
Appl. No.: |
11/894503 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09872185 |
Jun 1, 2001 |
7258857 |
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11894503 |
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08755235 |
Nov 22, 1996 |
6790443 |
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09872185 |
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08948131 |
Oct 9, 1997 |
6555651 |
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08755235 |
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PCT/US99/23303 |
Oct 6, 1999 |
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08948131 |
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09263312 |
Mar 5, 1999 |
6555340 |
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PCT/US99/23303 |
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09167705 |
Oct 6, 1998 |
7081241 |
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09263312 |
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Current U.S.
Class: |
514/19.3 |
Current CPC
Class: |
C07K 16/18 20130101;
A61K 38/1774 20130101; A61P 29/00 20180101; Y10S 514/825 20130101;
A61K 31/00 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 29/00 20060101 A61P029/00 |
Goverment Interests
[0002] The invention disclosed herein was made with Government
support under Grant Nos. HL21006 and AG00603 from the National
Institutes of Health, U.S. Department of Health and Human Services.
Accordingly, the U.S. Government has certain rights in this
invention.
Claims
1. A method for treating inflammation in a subject which comprises
administering to the subject soluble receptor for advanced
glycation endproduct (sRAGE) (SEQ ID NO:1) in an amount effective
to treat inflammation in the subject.
2. A method for treating inflammation in a subject which comprises
administering to the subject a polypeptide consisting essentially
of the V-domain (SEQ ID NO:2) of receptor for advanced glycation
endproduct (RAGE) in an amount effective to treat inflammation in
the subject.
3. A method for treating inflammation in a subject which comprises
administering to the subject an agent in an amount which inhibits
the interaction between receptor for advanced glycation endproduct
(RAGE) and its ligand thereby treating inflammation in the
subject.
4-25. (canceled)
Description
[0001] This application claims the priority of U.S. Ser. No.
08/755,235, filed Nov. 22, 1996; and U.S. Ser. No. 08/948,131,
filed Oct. 9, 1997; and PCT International Application No.
PCT/US99/23303 which is continuation-in-part of U.S. Ser. No.
09/263,312, filed Mar. 5, 1999 which is a continuation-in-part of
U.S. Ser. No. 09/167,705, filed Oct. 6, 1998, the contents of all
of which are hereby incorporated by reference into this
application.
BACKGROUND OF THE INVENTION
[0003] Throughout this application, various publications are
referenced by author and date. Full citations for these
publications may be found listed alphabetically at the end of each
section of the Experimental Details section of the application. The
disclosures of these publications in their entireties are hereby
incorporated by reference into this application in order to more
fully describe the state of the art as known to those skilled
therein.
SUMMARY OF THE INVENTION
[0004] The present invention provides a method for treating
inflammation in a subject which comprises administering to the
subject soluble receptor for advanced glycation endproduct (sRAGE)
in an amount effective to inhibit binding of advanced glycation
endproducts (AGEs) to RAGE thereby treating inflammation in the
subject.
BRIEF DESCRIPTION OF THE FIGURES
Wound Healing
[0005] FIG. 1. Effect of sRAGE on wound healing in the
genetically-diabetic db+/db+ mouse. A full-thickness 1.5.times.1.5
cm wound was created on the backs of db+/db+ mice or control,
heterozygote db+/m+mice and covered with TEGADERM.RTM.. Diabetic
wounds were treated with either phosphate-buffered saline (PBS)
directly under the TEGADERM.RTM. daily for 7 days commencing on day
3 following surgery or with sRAGE (200 ng). Wound area was measured
at baseline through day 21 by placing a glass slide over the wound
area, tracing the wound area, and placing this information into a
computer in order to calculate the percentage of wound closure as a
function of time. Left axis represents percent wound closure.
[0006] FIG. 2. Administration of sRAGE to the genetically-diabetic
db+/db+mouse improves wound healing: dose-response studies. Wounds
were created as above and treated from days 3 through 9 with sRAGE
(either 2,000, 200, or 20 ng/day) or with phosphate-buffered
saline. At day 10, wound area was measured and compared with
initial wound area as above. Results are presented as fold increase
in percent wound healing compared with mice treated with phosphate
buffered saline (defined as one in figure). All statistical
analyses are shown comparing wound healing in the presence of
different doses of sRAGE vs. treatment of diabetic wounds with
phosphate-buffered saline.
[0007] FIGS. 3A and 3B. AGE-immunoreactive epitopes in the wounds
of diabetic (db+/db+) mice. 1.5.times.1.5 cm full-thickness wounds
created in the backs of diabetic mice (db+/db+ mice; FIG. 3A) and
non-diabetic mice (db+/m+; FIG. 3B) were excised, fixed and
sections stained with affinity-purified anti-AGE IgG.
Magnification: 200.times..
Periodontal Disease
[0008] FIG. 4. Measurements of alveolar bone loss in mice treated
with sRAGE. Statistical analysis=Group I vs. Group II--p=0.002;
Group I vs. Group III--p=0.005; Group III vs. Group IV:
p=0.009.
Delayed Type Hypersensitivity
[0009] FIG. 5. Immunohistochemistry of human kidney (active lupus
nephritis). Kidney tissue from a patient with active lupus
nephritis was obtained, fixed in formalin and paraffin-embedded
sections were prepared. Sections were stained with rabbit anti-RAGE
IgG. Increased expression of RAGE was noted in the podocytes of the
glomerulus.
[0010] FIG. 6. Incubation of HUVECs with EN-RAGE results in
increased cell surface VCAM-1. Human umbilical vein endothelial
cells were cultured in serum-free RPMI 1640 without endothelial
cell growth factor for 24 hrs and then stimulated with EN-RAGE or
bovine serum albumin (BSA); both 10 .mu.g/ml. Where indicated,
cells were pretreated with rabbit anti-human RAGE IgG, nonimmune
rabbit IgG; in certain cases, EN-RAGE was pretreated with the
indicated concentration of soluble RAGE (sRAGE) for 2 hrs prior to
stimulation with EN-RAGE. After eight hrs stimulation with EN-RAGE,
cells were fixed as described above. Cell surface ELISA employing
anti-VCAM-1 IgG was performed. Statistical considerations are shown
in the figure.
[0011] FIG. 7. Incubation of HUVECs with EN-RAGE increases VCAM-1
functional activity: increased binding of Molt-4 cells. Assessment
of functional VCAM-1 activity was determined using
.sup.51Cr-labelled Molt-4 cells (ATCC) as described above. HUVEC
were treated with either BSA (10 .mu.g/ml) or EN-RAGE (5 .mu.g/ml)
for eight hrs. Molt-4 cells (5.times.10.sup.7/ml) were incubated
for 2 hrs in RPMI containing .sup.51Cr (0.1 mCi). At the end of
that time, cells were washed with PBS and then added to the
monolayer of treated HUVEC for one hour. Unbound Molt-4 cells were
removed by washing three times with PBS. Cells were then lysed in
buffer containing triton-X 100 (2%) in order to release Molt-4
cell-bearing radioactivity. Statistical considerations are shown in
the figure.
[0012] FIG. 8. Delayed hypersensitivity model: suppression of
inflammation in the presence of soluble RAGE. CF-1 mice were
sensitized with mBSA; after three weeks, mBSA was injected into the
hind foot pad. Certain mice were treated with the indicated
concentrations of mouse serum albumin, sRAGE or the indicated
F(ab').sub.2 antibody fragments of RAGE or EN-RAGE. Inflammation
score was defined as above (scale; 1-9).
[0013] FIG. 9. Nucleic Acid Sequence of bovine EN-RAGE. The cDNA
for bovine EN-RAGE was cloned and deposited with Genbank at
Accession No. AF 011757. (SEQ ID NO:16).
Collagen-Induced Arthritis
[0014] FIG. 10. Identification of wild-type RAGE and (G82S) and
(S82S) polymorphisms. Genomic DNA was prepared from whole blood of
controls, and subjects with RA. Amplification of exon 3 of the RAGE
gene was performed as described; the resulting PCR products were
digested with Alu 1. Upon agarose gel electrophoresis,
identification of wild-type RAGE (G82G), and mutant RAGE (G82S) or
(S82S) alleles was performed.
[0015] FIGS. 11A-11E. Transfection of CHO cells with mutant RAGE
(82S) confers increased affinity and cellular responsiveness to
EN-RAGE. CHO cells, which endogenously do not express RAGE, were
stably-transfected with pcDNA3.1 vector containing cDNA encoding
wild-type human RAGE or mutant (82S) human RAGE. "Mock" controls
indicated empty vector. FIG. 11A. Immunoblotting. Lysates of
stably-transfected CHO cells were prepared and subjected to
immunoblotting using anti-human RAGE IgG (2 .mu.g/ml). FIGS. 11B-C.
Radioligand binding assays. Purified EN-RAGE was radiolabelled
using .sup.125-I and radioligand binding assays were performed in
96-well tissue culture dishes containing the indicated transfected
CHO cells. Assays were performed in the presence of the indicated
concentration of radiolabelled EN-RAGE.+-.an 50-fold molar excess
of unlabeled EN-RAGE. Elution of bound material was performed in a
solution containing heparin. Equilibrium binding data were analyzed
according to the equation of Klotz and Hunston. Where indicated,
pretreatment with either antibodies (70 .mu.g/ml), human soluble
RAGE or bovine serum albumin (50-fold molar excess) was performed.
The mean .+-.standard deviation (SD) is shown. In FIG. 1C, *
indicates p<0.01 versus respective controls. FIG. 11D.
Activation of p44/p42 MAP kinases. The indicated stably-transfected
CHO cells were incubated with EN-RAGE, 10 .mu.g/ml, for one hr.
Cell lysates were subjected to SDS-PAGE and transfer of the gels'
contents to nitrocellulose. Immunblotting was performed using
anti-phosphorylated p44/p42 MAP kinase (1 .mu.g/ml). Where
indicated, pretreatment with either BSA or sRAGE (50-fold molar
excess), or the indicated IgG (70 .mu.g/ml), for 2 hrs was
performed. Control immunoblotting using antibody to total p44/p42
MAP kinase revealed that there were no differences in levels of
total p44/p42 in each group (not shown). FIG. 11E. Activation of
NF-kB. Nuclear extracts were prepared from the indicated
stably-transfected CHO cells incubated with EN-RAGE, 10 .mu.g/ml,
for 6 hrs, and EMSA was performed. Where indicated, cells were
pretreated with either nonimmune/anti-RAGE IgG (70 .mu.g/ml),
soluble RAGE or BSA (50-fold molar excess) for 2 hrs prior to
incubation with EN-RAGE. In FIGS. 11D-11E, bands were scanned into
a densitometer, and band density was quantified using ImageQuant.
These experiments were performed at least three times, and
representative experiments are shown.
[0016] FIGS. 12A-F. Human peripheral blood mononuclear phagocytes
(Mps) expressing RAGE (G82S) or (S82S) display increased
responsiveness to EN-RAGE. MPs were purified from the blood of
human subjects expressing wild-type RAGE, (G82S), or (S82S). For
these experiments, MPs from 8 wild-type RAGE-bearing subjects, and
8 subjects bearing RAGE (G82S) or (S82S) were employed. FIGS.
12A-B. Activation of p44/p42 MAP kinases. The indicated MPs were
incubated with EN-RAGE, 10 .mu.g/ml, for one hr, or with no
mediator. Cell lysates were prepared and immunoblotting performed
using anti-phosphorylated p44/p42 MAP kinase. Densitometric
analysis was performed and is shown in FIG. 12B. Since multiple
experiments demonstrated that there were no differences in the
extent of cellular activation in MPs bearing (G82S) or (S82S),
these two groups were combined for analyses. Control immunoblotting
using antibody to total p44/p42 MAP kinase revealed that there were
no differences in levels of total p44/p42 in each group. The mean
.+-.SD is shown. FIGS. 12C-D. Generation of TNF-alpha (FIG. 12C)
and IL-6 (FIG. 12D). Human MPs bearing the indicated RAGE alleles
were cultured in the presence of either no mediator, or EN-RAGE, 10
.mu.g/ml, for 14 hrs. Supernatants were retrieved and levels of
TNF-alpha and IL-6 determined by ELISA. The mean .+-.SD is shown.
FIGS. 12E-F. Activity of MMP-9. Human MPs bearing the indicated
RAGE alleles were cultured in the presence of either no mediator,
or EN-RAGE, 10 .mu.g/ml, for 14 hrs. Supernatants were retrieved
and subjected to zymography to assess levels of activated MMP-9.
Bands were scanned into a densitometer and band density was
quantified. The mean .+-.SD is shown. In FIGS. 12B, C, D and F, *
indicates p<0.01 versus baseline. Other statistical comparisons
are indicated.
[0017] FIGS. 13A-J. Induction of arthritis by bovine type II
collagen in dba/1 mice enhances expression of RAGE and EN-RAGEs.
Dba/1 mice were immunized/challenged with bovine type II collagen.
Control mice were not treated. Six weeks after immunization, joint
tissue from the hind feet (FIGS. 13A-H) or stifle joint (FIGS.
13I-J) was prepared for study. FIGS. 13A-H. Histology. In FIGS.
13A-B, tissue was subjected to H&E analysis.
Immunohistochemistry using anti-RAGE IgG (30 .mu.g/ml) (FIGS. 13
C-D); anti-EN-RAGE IgG (3 .mu.g/ml) (FIGS. 13E-F); or rabbit
nonimmune IgG (30 .mu.g/ml) (FIGS. 13G-H) was performed. Scale bar:
300 .mu.m. FIGS. 13I-J. Immunoblotting. Lysates were prepared from
stifle joints and subjected to immunoblotting using anti-RAGE IgG
(4.7 .mu.g/ml) (FIG. 13I); or anti-EN-RAGE IgG (2 .mu.g/ml) (FIG.
13J). For each group, n=3 mice per condition; representative bands
are shown. Densitometric analysis of band intensity was performed,
and the mean .+-.SD is shown.
[0018] FIGS. 14A-F. Blockade of RAGE suppresses development of
arthritis and markers of inflammation in dba/1 mice
immunized/challenged with bovine type II collagen. FIGS. 14A-B,
Clinical scoring. In FIG. 14A, at the indicated time points after
immunization/challenge with bovine type II collagen and treatment
with vehicle, murine serum albumin (MSA) or sRAGE, hind foot pad
thickness was measured with calipers. The mean .+-.standard
deviation (SD) is shown; n=10 mice per group. * indicates
p<0.001. In FIG. 14B, clinical scoring of wrist joint
redness/swelling was performed by a blinded observer 6 weeks after
immunization with bovine type II collagen. The mean .+-.SD is
shown; n=10 mice per group. * indicates p=0.0001. FIGS. 14C-F.
Assessment of markers of inflammation. FIGS. 14C-D. TNF-alpha. In
FIG. 14C, stifle joint tissue of mice with collagen-induced
arthritis was retrieved six weeks after immunization with bovine
type II collagen. Lysates were subjected to immunoblotting using
anti-murine TNF-alpha IgG (1 .mu.g/ml). In FIG. 14D, plasma from
mice with collagen-induced arthritis was subjected to ELISA for
levels of TNF-alpha. In FIG. 14C, 3 mice per group; and in FIG.
14D, 10 mice per group, were employed. The mean .+-.SD is shown. In
FIG. 14C, * indicates p=0.001; and in FIG. 14D, * indicates p=0.03.
FIGS. 14E-F. IL-6 and IL-2. Stifle joint tissue was retrieved from
control mice (clear bars) and mice with collagen-induced arthritis
(black bars). Lysates were prepared and ELISA was performed for
determination of levels of IL-6 (FIG. 14E) and IL-2 (FIG. 14F).
Results are reported as ng/.mu.g tissue. The mean .+-.SD is shown;
n=6 mice per group. In FIG. 14E, * indicates p=0.04.
[0019] FIGS. 15A-D. Blockade of RAGE suppresses generation of MMPs
in dba/1 mice immunized/challenged with bovine type II collagen.
FIGS. 15A-B. Immunoblotting. Stifle joint tissue was retrieved from
control mice (clear bars) and mice with bovine type II
collagen-induced arthritis (black bars) six weeks after initial
immunization. Lysates were prepared and subjected to immunoblotting
using either anti-MMP-2 IgG (FIG. 15A) or anti-MMP-9 IgG (FIG.
15B). The mean .+-.SD is shown; n=3 mice per group. FIGS. 15C-D.
Zymography. Lysates were prepared from stifle joint tissue of
control mice, or mice with collagen-induced arthritis. Zymography
was performed and the results subjected to densitometric analysis.
The mean .+-.SD is shown; n=3 mice per group.
Statistical analyses: MMP-2: * indicates p=0.001 vs control; and
p=0.004 vs sRAGE. MMP-9: * indicates p=0.02 vs control; and p=0.005
vs sRAGE.
[0020] FIGS. 16A-B. Blockade of RAGE suppresses extra-articular
inflammatory responses induced by bovine type II collagen.
[0021] FIG. 16A Ear swelling. Six weeks after immunization with
bovine type II collagen, MSA- and sRAGE-treated mice were injected
with bovine type II collagen (10 .mu.g) into ear tissue. Ear
thickness was measured with calipers by a blinded observer
immediately prior to local injection, and 18 hrs later. The mean
.+-.SD is shown; n=5 mice per group. FIG. 16B. Splenocyte
proliferation. Splenocytes were prepared from the indicated mice at
sacrifice, 6 weeks after immunization. Baseline levels of
splenocyte proliferation, and proliferation in the presence of
bovine type II collagen or PMA (0.1 .mu.g/ml in each case) was
determined. Note that no additional MSA or sRAGE was added to the
culture system. The mean .+-.SD is shown; n=5 mice per group.
Autoimmune Diseases, EAE
[0022] FIG. 17. Life Table analysis of development of diabetes in
NOD.SCID recipients with and without treatment with sRAGE. A single
experiment representative of 3 is presented. Soluble RAGE treatment
reduced the development of diabetes in the NOD.SCID recipients. In
pooled experiments (n=12 in sRAGE treated and n=13 controls, the
incidence of diabetes at 35 days was 17% and 92%, respectively;
p<0.001).
[0023] FIG. 18. Histology of islets in control and sRAGE treated
recipients of splenocytes (H&E staining).
[0024] FIG. 19. Immunostaining for TNF-a and IL-1.beta. in the
islets of sRAGE-treated and control mice. Pancreases from untreated
(control) or sRAGE-treated recipient mice were stained with
antibodies to TNF (a) or IL-1.beta. (b). Expression of these
inflammatory cytokines (dark brown) was reduced in sRAGE-treated
mice and, in the latter, was predominantly in the area of
peri-insulitis.
[0025] FIG. 20. Symptomatic scoring of EAE in B10.PL mice immunized
with MBP-derived peptide and treated with vehicle (mouse serum
albumin; 50 .mu.g/day; fatty acid-free, Sigma) or sRAGE (50
.mu.g/day).
[0026] FIGS. 21A-21D. Histologic analysis (H&E) of spinal cord
from mice immunized with MBP and treated with vehicle (FIG. 21B;
the animal was sacrificed on day 21 with full-blown symptomatic
EAE) or sRAGE (FIG. 21C; the animal was sacrificed on day 35 and
was asymptomatic) as in FIG. 20. FIG. 21A shows normal mouse spinal
cord. FIG. 21D shows quantitation of nuclei in affected areas (this
reflects principally cells in the inflammatory infiltrate).
[0027] FIG. 22. Symptomatic analysis of B10.PL mice infused with
activated 1AE10 cells as described in the text. As indicated, mice
received either anti-RAGE IgG or nonimmune IgG for 15 days (see
text for details experimental protocol).
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides a method for treating
inflammation in a subject which comprises administering to the
subject an amount of soluble receptor for advanced glycation
endproducts (sRAGE) effective to treat inflammation in the
subject.
[0029] The present invention also provides a method for treating
inflammation in a subject which comprises administering to the
subject a therapeutically effective amount of an agent which
inhibits binding of advanced glycation endproducts (AGEs) to any
receptor for advanced glycation endproducts so (RAGE) as to treat
inflammation in the subject. The advanced glycation endproduct
(AGE) may be a pentosidine, a carboxymethyllysine, a
carboxyethyllysine, a pyrallines, an imidizalone, a methylglyoxal,
an ethylglyoxal.
[0030] The present invention also provides a method for treating
arthritis in a human subject which comprises administering to the
subject a therapeutically effective amount of an agent which
inhibits binding of advanced glycation endproducts (AGEs) to any
receptor for advanced glycation endproducts so (RAGE) as to treat
arthritis in the subject.
[0031] Inflammation in the subject may be associated with any one
or more of various conditions or diseases. For example, the
inflammation in the subject may be due to a wound, to periodontal
disease, to delayed-type hypersensitivity, to autoimmune disease,
or to arthritis. The subject may be suffering from an autoimmune
disease. In one embodiment, the subject is suffering from multiple
sclerosis, autoimmune encephalitis, lupus nephritis, or autoimmune
complications from diabetes. The subject may be suffering from
diabetes, for example, type I diabetes. The subject may be
suffering from Behchet's syndrome. The subject may be suffering
from Sjogren's syndrome. The subject may be suffering from colitis,
ulcerative colitis, inflammatory colitis, Crohn's disease or the
like. The subject may be suffering from arthritis which is
osteoarthritis, rheumatoid arthritis, collagen-induced arthritis,
psoriatic arthritis, lupus-induced arthritis, or trauma-induced
arthritis. The subject may be suffering from another overall
condition or disease which includes a manifestation of
inflammation, such as arthritis. In another embodiment, the subject
is suffering from an allergy or is experiencing an allergic
response. In one embodiment, the subject is suffering from asthma.
The subject may be suffering from allergic asthma. In another
embodiment, the subject is suffering from systemic lupus
erythematosus, inflammatory lupus nephritis, septic shock or
endotoxemia.
[0032] In a further embodiment, the subject is suffering from an
autoimmune or inflammatory disorder in which recruitment of
EN-RAGE-containing inflammatory cells occurs. In another
embodiment, the subject is suffering from a bacterial-associated or
other pathogen-associated infection.
[0033] The inflammation to be treated, in one embodiment of the
invention, is caused by the accumulation of the AGEs in certain
tissues dependent upon the ongoing biology in a subject. For
example, the lesions in the blood vessels which can occur in a
subject suffering from diabetes are due to the increased
accumulation of AGEs in the presence of higher sugar in the blood.
Therefore, the administration of the agent as described herein
would reduce the interaction between the AGEs in the blood and the
receptor for AGE, thereby reducing inflammation at that site.
Therefore, the present invention encompasses inflammation which
would occur in a subject at locations where the AGE products
accumulate due to the overriding disease or condition.
[0034] The present invention also provides for a method for
inhibiting periodontal disease in a subject which comprises
administering topically to the subject a pharmaceutical composition
which comprises sRAGE in an amount effective to accelerate wound
healing and thereby inhibit periodontal disease. The pharmaceutical
composition may comprise sRAGE in a toothpaste.
[0035] The present invention provides for a new proinflammatory
cytokine-like molecule (EN-RAGE) (which has some sequence
similarity to the family of calgranulin molecules). EN-RAGE is a
protein located inside of inflammatory cells (such as neutrophils)
and which may be released by such inflammatory cells. EN-RAGE has
biological activity that may be responsible for the propagation and
sustainment of an inflammatory response by interacting with
cellular receptor RAGE.
[0036] The subject on which any of the methods of the invention is
employed may be any mammal, e.g. a human subject, a murine subject,
a bovine subject, a porcine subject, a canine subject, a primate
subject, a feline subject, etc. Preferably, the subject is a human
subject. However, for methods of identifying a compound or agent
which is useful in treating inflammation, preferably, the subject
is a primate, or murine subject.
[0037] The cell may be a eukaryotic cell. The cell may be a cell of
a subject. The subject may be a human. The cell may be a neuronal
cell, an endothelial cell, a glial cell, a microglial cell, a
smooth muscle cell, a somatic cell, a bone marrow cell, a liver
cell, an intestinal cell, a germ cell, a myocyte, a mononuclear
phagocyte, an endothelial cell, a tumor cell, a lymphocyte cell, a
mesangial cell, a retinal epithelial cell, a retinal vascular cell
a ganglion cell or a stem cell. The cell may also be other kinds of
cells not explicitly listed herein. The cell may be any human cell.
The cell may be a normal cell, an activated cell, a neoplastic
cell, a diseased cell or an infected cell.
The Agent
[0038] In accordance with the method of this invention, the agent
may comprise a polypeptide, a peptidomimetic, an organic molecule,
a carbohydrate, a lipid, an antibody or a nucleic acid. The
polypeptide may be synthesized chemically or produced by standard
recombinant DNA methods. In accordance with the method of this
invention, the polypeptide may comprise an advanced glycation
endproduct polypeptide or a portion thereof, a receptor for an
advanced glycation endproduct polypeptide or a portion thereof, a
soluble receptor for advanced glycation endproduct polypeptide
(sRAGE) or a portion thereof. In one embodiment of the invention,
the portion of sRAGE is the V-domain of RAGE, which is the amino
terminal 112 amino acids (not including the leader peptide).
[0039] The sequence of the V-domain of mature human RAGE is the
following:
TABLE-US-00001 (SEQ ID NO:1) Ala Gln Asn Ile Thr Ala Arg Ile Gly
Glu Pro Leu Val Leu Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg
Leu Glu Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser
Pro Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro Asn Gly
Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly Ile Phe Arg Cys
Gln Ala Met Asn Arg Asn Gly Lys Glu Thr Lys Ser Asn Tyr Arg Val Arg
Val Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp Ser Ala Ser Glu Leu
Thr.
[0040] The sequence of mature human RAGE not including the 22 amino
acid leader sequence is:
TABLE-US-00002 (SEQ ID NO:2) Ala Gln Asn Ile Thr Ala Arg Ile Gly
Glu Pro Leu Val Leu Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg
Leu Glu Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser
Pro Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro Asn Gly
Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly Ile Phe Arg Cys
Gln Ala Met Asn Arg Asn Gly Lys Glu Thr Lys Ser Asn Tyr Arg Val Arg
Val Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp Ser Ala Ser Glu Leu
Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys Val Ser Glu Gly Ser Tyr
Pro Ala Gly Thr Leu Ser Trp His Leu Asp Gly Lys Pro Leu Val Pro Asn
Glu Lys Gly Val Ser Val Lys Glu Gln Thr Arg Arg His Pro Glu Thr Gly
Leu Phe Thr Leu Gln Ser Glu Leu Met Val Thr Pro Ala Arg Gly Gly Asp
Pro Arg Pro Thr Phe Ser Cys Ser Phe Ser Pro Gly Leu Pro Arg His Arg
Ala Leu Arg Thr Ala Pro Ile Gln Pro Arg Val Trp Glu Pro Val Pro Leu
Glu Glu Val Gln Leu Val Val Glu Pro Glu Gly Gly Ala Val Ala Pro Gly
Gly Thr Val Thr Leu Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile
His Trp Met Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu
Ile Leu Pro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala
Thr His Ser Ser His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser Ile
Ile Glu Pro Gly Glu Glu Gly Pro Thr Ala Gly Ser Val Gly Gly Ser Gly
Leu Gly Thr Leu Ala Leu Ala Leu Gly Ile Leu Gly Gly Leu Gly Thr
Ala.
[0041] The agent may be a composition which consists essentially of
sRAGE. The agent may be a polypeptide which is fragment of sRAGE,
for example, a fragment which is the V-domain of sRAGE.
[0042] In several embodiments of the present invention, the agent
is a peptide having an amino acid sequence corresponding to the
amino acid sequence of a V-domain of a RAGE or soluble RAGE is
exemplified by the following amino acid sequences:
TABLE-US-00003 (SEQ ID No:3)
A-Q-N-I-T-A-R-I-G-E-P-L-V-L-K-C-K-G-A-P-K-K-P-P-Q- R-L-E-W-K; (SEQ
ID NO:4) G-Q-N-I-T-A-R-I-G-E-P-L-V-L-S-C-K-G-A-P-K-K P-P-Q-
Q-L-E-W-K; (SEQ ID NO:5)
G-Q-N-I-T-A-R-I-G-E-P-L-M-L-S-C-K-A-A-P-K-K-P-T-Q- K-L-E-W-K; (SEQ
ID NO:6) D-Q-N-I-T-A-R-I-G-K-P-L-V-L-N-C-K-G-A-P-K-K-P-P-Q-
Q-L-E-W-K.
[0043] The present invention provides for an isolated peptide
having an amino acid sequence which corresponds to the amino acid
sequence of the first 1-112 amino acids of human RAGE (which is the
V-domain of human RAGE), or which corresponds to amino acids 5-35
of the V-domain of human RAGE, or any other smaller portion of the
V-domain of human RAGE. Representative peptides of the present
invention include but are not limited to peptides having an amino
acid sequence which corresponds to amino acid numbers (2-30),
(5-35), (10-40), (15-45), (20-50), (25-55), (30-60), (30-65),
(10-60), (8-100), 14-75), (24-80), (33-75), (45-110) of human sRAGE
protein.
[0044] The agent or inhibitor of the present invention may comprise
a peptide having an amino acid sequence corresponding to amino acid
numbers 1-30 of the V-domain of sRAGE (soluble receptor for
advanced glycation endproducts). The sRAGE may be human, mouse, rat
or bovine sRAGE.
[0045] The agent may be a peptide, a peptidomimetic, a nucleic acid
or a small molecule. The terms "peptide" and "polypeptide" are used
interchangably throughout. The peptide may be at least a portion of
the sequence from amino acid 1 to amino acid 30 of sRAGE. The
peptide may be a peptide consisting essentially of the amino acid
sequence of SEQ ID NOS: 1, 2, 3, 4, 5, or 6. The peptide may be
smaller than SEQ ID NO:1, retaining amino acid regions necessary to
mimic the binding site of sRAGE. The peptide may comprise amino
acids 1-112 of a RAGE protein (not including the leader sequence),
i.e. the V-domain. The peptide may consist essentially of the
V-domain of a RAGE protein (SEQ ID NO:1).
[0046] The polypeptide may be a peptidomimetic, a synthetic
polypeptide or a polypeptide analog. The polypeptide may be a
non-natural polypeptide which has chirality not found in nature,
i.e. D-amino acids or L-amino acids.
[0047] The polypeptide may be a derivative of a natural
polypeptide, a modified polypeptide, a labelled polypeptide, or a
polypeptide which includes non-natural peptides. The peptidomimetic
may be identified from screening large libraries of different
compounds which are peptidomimetics to determine a compound which
is capable of inhibiting interaction of an amyloid .beta. peptide
with a receptor for advanced glycation endproduct. In another
embodiment, the polypeptide may be labeled with a detectable
moiety. The detectable moiety may be selected from the group
consisting of: a fluorescent label, a digoxigenin, a biotin, an
enzyme, a radioactive atom, a paramagnetic ion, and a
chemiluminescent label.
[0048] In another embodiment, the agent comprises a nucleic acid
molecule which is a ribozyme or an antisense nucleic acid
molecule.
[0049] The agent may comprise a peptide having an amino acid
sequence corresponding to the amino acid sequence of a V-domain of
a sRAGE linked to a second peptide, wherein the second peptide may
be an albumin, a globulin or a peptide chosen from a group of
peptides, wherein each peptide of the group comprises a different
length peptide, and wherein the sequence of each peptide
corresponds to any sequence of amino acids taken from within amino
acid number 31 through amino acid number 281 of the human, bovine,
mouse or rat sRAGE protein.
[0050] The abbreviations used herein for amino acids are those
abbreviations which are conventionally used: A=Ala=Alanine;
R=Arg=Arginine; N=Asn=Asparagine; D=Asp=Aspartic acid;
C=Cys=Cysteine; Q=Gln=Glutamine; E=Glu=Gutamic acid; G=Gly=Glycine;
H=His=Histidine; I=Ile=Isoleucine; L=Leu=Leucine; K=Lys=Lysine;
M=Met=Methionine; F=Phe=Phenyalanine; P=Pro=Proline; S=Ser=Serine;
T=Thr=Threonine; W=Trp=Tryptophan; Y=Tyr=Tyrosine; V=Val=Valine.
The amino acids may be L- or D-amino acids. An amino acid may be
replaced by a synthetic amino acid which is altered so as to
increase the half-life of the peptide or to increase the potency of
the peptide, or to increase the bioavailability of the peptide.
[0051] The peptide or polypeptide of the present invention may
comprise alterations to the sequences provided in SEQ ID NOS:1 to
6. The peptide of the present invention may comprise alterations in
sequence which do not affect the functionality of the peptide in a
negative way, but which may increase the functionality of the
peptide in a position way, e.g. increase the potency of the
peptide. Some examples of such alterations to the human sequence of
the first 30 amino acids (1-30) of the V-domain of sRAGE (SEQ ID
NO:1) are listed hereinbelow as examples: [0052] (a) Substitute
D-alanine for L-alanine in position 6; [0053] (b) Substitute
D-lysine for L-lysine in position 15; [0054] (c) Substitute
D-alanine for L-alanine in position 6 and D-lysine for L-lysine in
position 15; [0055] (d) Omit amino acids 1-5 of the V domain,
making the N-amino end group L-alanine; [0056] (e) Omit amino acids
1-5 of the V domain making the N-amino acid D-alanine; [0057] (f)
Substitute D-lysine for the L-lysine in the amino acid number "30"
position of the V domain of Sequence I.D. No. 1; [0058] (g)
Substitute L-arginine for L-lysine in the 30 position of the V
domain; [0059] (h) Substitute L-arginine for L-lysine in the 30
position of the V domain and add glycine as the carboxyl terminal
group to produce a 31 amino acid peptide; [0060] (i) Substitute
L-arginine for L-lysine in the 30 position of the amino acid
peptide containing the amino acid sequence of 6-30 described for
the V domaine of sRAGE; [0061] (j) Substitute L-arginine for
L-lysine in the 30 position of the amino acid peptide containing
the amino acid sequence of 6-30 described for the V domaine of
sRAGE and add glycine as the carboxyl terminal group to produce a
25 amino acid sequence peptide; [0062] (k) Substitute D-lysine for
L-lysine in the 30 position of the 6-30 amino acid sequence
designated for the V domain; [0063] (l) Substitute D-lysine for
L-lysine in the 30 position of the 6-30 amino acid sequence
designated for the V domaine and add L-alanine at the C-terminal
position of the new 26 amino acid peptide; [0064] (m) Substitute
D-valine for L-valine in the 13 position of the V domaine 30 amino
acid peptide designated 6-30 of the sRAGE V domain; [0065] (n)
Substitute D-valine for L-valine in the 13 position of the 25 amino
acid peptide designated 6-30 of the sRAGE V domain; [0066] (o)
Substitute D-alanine for L-alanine in the 6 position of the 30
amino acid peptide and D-valine for L-valine in the 13 position of
the 30 amino acid of the V domain; [0067] (p) Substitute D-alanine
for L-alanine in the 6 position and D-valine for L-valine in the 13
position of the 25 amino acid peptide designated 6-30 of the V
domain of sRAGE; [0068] (q) the above-listed (a)-(p) peptides
derivatized through the carboxylic acid of position 30 with
albumin, globulins or different length peptides composed of amino
acids contained within positions 31 through 281 of the human,
mouse, rat or bovine sRAGE protein.
[0069] In addition to naturally-occurring forms of polypeptides
derived from sRAGE, the present invention also embraces other
polypeptides such as polypeptide analogs of sRAGE which have the
equivalent funcationality of the peptide of SEQ ID NO:1 or 2 or a
more potent or more positive functionality. Such analogs include
fragments of sRAGE. Following the procedures of the published
application by Alton et al. (WO 83/04053), one can readily design
and manufacture genes coding for microbial expression of
polypeptides having primary conformations which differ from that
herein specified for in terms of the identity or location of one or
more residues (e.g., substitutions, terminal and intermediate
additions and deletions). Alternately, modifications of cDNA and
genomic genes can be readily accomplished by well-known
site-directed mutagenesis techniques and employed to generate
analogs and derivatives of sRAGE polypeptide. Such products share
at least one of the biological properties of sRAGE but may differ
in others. As examples, products of the invention include those
which are foreshortened by e.g., deletions; or those which are more
stable to hydrolysis (and, therefore, may have more pronounced or
longerlasting effects than naturally-occurring); or which have been
altered to delete or to add one or more potential sites for
O-glycosylation and/or N-glycosylation or which have one or more
cysteine residues deleted or replaced by e.g., alanine or serine
residues and are potentially more easily isolated in active form
from microbial systems; or which have one or more tyrosine residues
replaced by phenylalanine and bind more or less readily to target
proteins or to receptors on target cells. Also comprehended are
polypeptide fragments duplicating only a part of the continuous
amino acid sequence or secondary conformations within sRAGE, which
fragments may possess one property of sRAGE and not others. It is
noteworthy that activity is not necessary for any one or more of
the polypeptides of the invention to have therapeutic utility or
utility in other contexts, such as in assays of sRAGE antagonism.
Competitive antagonists may be quite useful in, for example, cases
of overproduction of sRAGE.
[0070] Of applicability to polypeptide analogs of the invention are
reports of the immunological property of synthetic peptides which
substantially duplicate the amino acid sequence in
naturally-occurring proteins, glycoproteins and nucleoproteins.
More specifically, relatively low molecular weight polypeptides
have been shown to participate in immune reactions which are
similar in duration and extent to the immune reactions of
physiologically-significant proteins such as viral antigens,
polypeptide hormones, and the like. Included among the immune
reactions of such polypeptides is the provocation of the formation
of specific antibodies in immunologically-active animals [Lerner et
al., 1981; Ross et al., 1981; Walter et al., 1981; Wong et al.,
1982; Baron et al., 1982; Dressman et al., 1982; and Lerner,
Scientific American, 1983. See also, Kaiser et al., 1984] relating
to biological and immunological properties of synthetic peptides
which approximately share secondary structures of peptide hormones
but may not share their primary structural conformation.
[0071] The polypeptide of the present invention may be a
peptidomimetic compound which may be at least partially unnatural.
The peptidomimetic compound may be a small molecule mimic of a
portion of the amino acid sequence of sRAGE. The compound may have
increased stability, efficacy, potency and bioavailability by
virtue of the mimic. Further, the compound may have decreased
toxicity. The peptidomimetic compound may have enhanced mucosal
intestinal permeability. The compound may be synthetically
prepared. The compound of the present invention may include L-, D-,
DL- or unnatural amino acids, alpha,alpha-disubstituted amino
acids, N-alkyl amino acids, lactic acid (an isoelectronic analog of
alanine). The peptide backbone of the compound may have at least
one bond replaced with PSI-[CH.dbd.CH] (Kempf et al. 1991). The
compound may further include trifluorotyrosine, p-Cl-phenylalanine,
p-Br-phenylalanine, poly-L-propargylglycine, poly-D,L-allyl
glycine, or poly-L-allyl glycine.
[0072] One embodiment of the present invention is a peptidomimetic
compound having the biological activity of preventing accelerated
athersclerosis in a subject wherein the compound has a bond, a
peptide backbone or an amino acid component replaced with a
suitable mimic. Examples of unnatural amino acids which may be
suitable amino acid mimics include .beta.-alanine, L-.alpha.-amino
butyric acid, L-.gamma.-amino butyric acid, L-.alpha.-amino
isobutyric acid, L-.epsilon.-amino caproic acid, 7-amino heptanoic
acid, L-aspartic acid, L-glutamic acid, cysteine
(acetamindomethyl), N-.epsilon.-Boc-N-.alpha.-CBZ-L-lysine,
N-.epsilon.-Boc-N-.alpha.-Fmoc-L-lysine, L-methionine sulfone,
L-norleucine, L-norvaline, N-.alpha.-Boc-N-.delta.CBZ-L-ornithine,
N-.delta.-Boc-N-.alpha.-CBZ-L-ornithine,
Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, Boc-L-thioproline.
(Blondelle, et al. 1994; Pinilla, et al. 1995).
[0073] In accordance with the method of this invention, the agent
may comprise a peptide, a peptidomimetic, an organic molecule, a
carbohydrate, a lipid, an antibody or a nucleic acid. The peptide
of this invention may comprise an advanced glycation endproduct
peptide or a portion thereof, a receptor for advanced glycation
endproduct peptide or a portion thereof, a soluble receptor for
advanced glycation endproduct peptide or a portion thereof. The
peptide of the present invention may comprise any part of the first
112 amino acids of the sRAGE protein. The peptide of the present
invention may comprise the V-domain of a soluble RAGE protein. The
peptide of the present invention may be a smaller portion of the
V-domain of a soluble RAGE protein. The peptide of the present
invention may be a peptide which corresponds to the V-domain of
human RAGE, mouse RAGE, rat RAGE, bovine RAGE or fish RAGE.
[0074] In accordance with the method of this invention, the agent
may be a peptide (polypeptide), a peptidomimetic, an organic
molecule, a carbohydrate, a lipid, an antibody or a nucleic acid.
In the case of polypeptides, the polypeptide may be an advanced
glycation endproduct (AGE) polypeptide or a portion thereof, a
receptor for advanced glycation endproduct polypeptide or a portion
thereof, a soluble receptor for advanced glycation endproduct
polypeptide or a portion thereof, e.g., soluble RAGE, or a
recombinant polypeptide. The polypeptide may be the V-domain of
sRAGE, or amino acids 1-30 of the V-domain of sRAGE. The
polypeptide of this invention may comprise an advanced glycation
endproduct polypeptide or a portion thereof, a receptor for
advanced glycation endproduct polypeptide or a portion thereof, a
soluble receptor for advanced glycation endproduct polypeptide or a
portion thereof. The polypeptide of the present invention may
comprise any part of the first 112 amino acids of the sRAGE
protein. The polypeptide of the present invention may comprise the
V-domain of a soluble RAGE protein. The polypeptide of the present
invention may be a smaller portion of the V-domain of a soluble
RAGE protein. The polypeptide of the present invention may be a
polypeptide which corresponds to the V-domain of human RAGE, mouse
RAGE, rat RAGE, bovine RAGE or fish RAGE. The polypeptide may be
synthesized chemically or produced by standard recombinant DNA
methods. In the case of antibodies, the antibody may be an
anti-RAGE antibody or an anti-RAGE F(ab').sub.2 fragment.
[0075] Of applicability to polypeptide analogs of the invention are
reports of the immunological property of synthetic peptides which
substantially duplicate the amino acid sequence extant in
naturally-occurring proteins, glycoproteins and nucleoproteins.
More specifically, relatively low molecular weight polypeptides
have been shown to participate in immune reactions which are
similar in duration and extent to the immune reactions of
physiologically-significant proteins such as viral antigens,
polypeptide hormones, and the like. Included among the immune
reactions of such polypeptides is the provocation of the formation
of specific antibodies in immunologically-active animals [Lerner et
al., Cell, 23, 309-310 (1981); Ross et al., Nature, 294, 654-658
(1981); Walter et al., Proc. Natl. Acad. Sci. USA, 78, 4882-4886
(1981); Wong et al., Proc. Natl. Sci. USA, 79, 5322-5326 (1982);
Baron et al., Cell, 28, 395-404 (1982); Dressman et al., Nature,
295, 185-160 (1982); and Lerner, Scientific American, 248, 66-74
(1983). See also, Kaiser et al. [Science, 223, 249-255 (1984)]
relating to biological and immunological properties of synthetic
peptides which approximately share secondary structures of peptide
hormones but may not share their primary structural
conformation.
[0076] The present invention also encompasses a pharmaceutical
composition which comprises a therapeutically effective amount of
the peptide having an amino acid sequence corresponding to the
amino acid sequence of a V-domain of a receptor for advanced
glycation endproduct (RAGE) and a pharmaceutically acceptable
carrier. The carrier may be a diluent, an aerosol, a topical
carrier, an aqueous solution, a nonaqueous solution or a solid
carrier. The carrier may be a polymer or a toothpaste. The
pharmaceutical composition may comprise the peptide having an amino
acid sequence corresponding to the amino acid sequence of a
V-domain of a RAGE linked to a second peptide, wherein the second
peptide may be an albumin, a globulin or a peptide chosen from a
group of peptides, wherein each peptide of the group comprises a
different length peptide, and wherein the sequence of each peptide
corresponds to any sequence of amino acids taken from within amino
acid number 31 through amino acid number 281 of the human sRAGE
protein.
[0077] In one embodiment of the invention, the agent consists
essentially of a portion of the peptide consisting of an amino acid
sequence corresponding to the amino acid sequence of a V-domain of
a receptor for advanced glycation endproduct. In one embodiment,
the agent consists of sRAGE. In one embodiment, the agent consists
of the V-domain of sRAGE.
[0078] In one embodiment of the invention, the agent is an
inhibitor of the interaction between RAGE and AGE or RAGE and
another binding partner. The inhibitor comprises a peptide, a
peptidomimetic compound, a nucleic acid molecule, a small molecule,
an organic compound, an inorganic compound, or an antibody or a
fragment thereof. The inhibitor may be the isolated peptide having
an amino acid sequence corresponding to the amino acid sequence of
a V-domain of a receptor for advanced glycation endproduct. In one
embodiment, the inhibitor is capable of specifically binding to the
amyloid-.beta. peptide. In one embodiment, the agent consists
essentially of a peptide having the amino acid sequence
A-Q-N-I-T-A-R-I-G-E-P-L-V-L-K-C-K-G-A-P-K-K-P-P-Q-R-L-E-W-K (SEQ ID
NO:7). In another embodiment, the agent consists essentially of a
peptide consisting of the amino acid sequence A-Q-N-I-T-A-R-I-G-E
(SEQ ID NO:8).
[0079] In one embodiment of the invention, the agent is an
antibody. In accordance with the method of this invention, the
antibody may comprise an anti-RAGE antibody or an anti-RAGE
F(ab').sub.2 fragment. The fragment of the antibody which is useful
in the present invention is that which binds the antigen.
Antibodies may be humanized or chimeric. The antibody may be a
human antibody, a primate antibody, or a murine antibody. The
portion or fragment of the antibody may comprise a complementarity
determining region or a variable region. In one embodiment, the
antibody may be capable of specifically binding to the receptor for
advanced glycation endproduct. The antibody may be a monoclonal
antibody, a polyclonal antibody.
[0080] The agent may be conjugated to a carrier. The peptide or
agent may be linked to an antibody, such as a Fab or a Fc fragment
for specifically targeted delivery. The carrier may be a diluent,
an aerosol, a topical carrier, an aqeuous solution, a nonaqueous
solution or a solid carrier.
[0081] As used herein, the term "suitable pharmaceutically
acceptable carrier" encompasses any of the standard
pharmaceutically accepted carriers, such as phosphate buffered
saline solution, acetate buffered saline solution (a likely vehicle
for parenteral administration), water, emulsions such as an
oil/water emulsion or a triglyceride emulsion, various types of
wetting agents, tablets, coated tablets and capsules. An example of
an acceptable triglyceride emulsion useful in intravenous and
intraperitoneal administration of the compounds is the triglyceride
emulsion commercially known as Intralipid.RTM..
[0082] When administered orally or topically, such agents and
pharmaceutical compositions would be delivered using different
carriers. Typically such carriers contain excipients such as
starch, milk, sugar, certain types of clay, gelatin, stearic acid,
talc, vegetable fats or oils, gums, glycols, or other known
excipients. Such carriers may also include flavor and color
additives or other ingredients. The specific carrier would need to
be selected based upon the desired method of deliver, e.g., PBS
could be used for intravenous or systemic delivery and vegetable
fats, creams, salves, ointments or gels may be used for topical
delivery.
[0083] This invention also provides for pharmaceutical compositions
including therapeutically effective amounts of protein compositions
and/or agents capable of inhibiting the binding of an
amyloid-.beta. peptide with a receptor for advanced glycation
endproduct in the subject of the invention together with suitable
diluents, preservatives, solubilizers, emulsifiers, adjuvants
and/or carriers useful in treatment of neuronal degradation due to
aging, a learning disability, or a neurological disorder. Such
compositions are liquids or lyophilized or otherwise dried
formulations and include diluents of various buffer content (e.g.,
Tris-HCl., acetate, phosphate), pH and ionic strength, additives
such as albumin or gelatin to prevent absorption to surfaces,
detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid
salts), solubilizing agents (e.g., glycerol, polyethylene
glycerol), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol,
parabens), bulking substances or tonicity modifiers (e.g., lactose,
mannitol), covalent attachment of polymers such as polyethylene
glycol to the agent, complexation with metal ions, or incorporation
of the agent into or onto particulate preparations of polymeric
agents such as polylactic acid, polglycolic acid, hydrogels, etc,
or onto liposomes, micro emulsions, micelles, unilamellar or multi
lamellar vesicles, erythrocyte ghosts, or spheroplasts. Such
compositions will influence the physical state, solubility,
stability, rate of in vivo release, and rate of in vivo clearance
of the agent or composition. The choice of compositions will depend
on the physical and chemical properties of the agent capable of
alleviating the symptoms of the cognitive disorder of memory or the
learning disability in the subject.
[0084] The agent of the present invention may be delivered locally
via a capsule which allows sustained release of the agent or the
peptide over a period of time. Controlled or sustained release
compositions include formulation in lipophilic depots (e.g., fatty
acids, waxes, oils). Also comprehended by the invention are
particulate compositions coated with polymers (e.g., poloxamers or
poloxamines) and the agent coupled to antibodies directed against
tissue-specific receptors, ligands or antigens or coupled to
ligands of tissue-specific receptors. Other embodiments of the
compositions of the invention incorporate particulate forms
protective coatings, protease inhibitors or permeation enhancers
for various routes of administration, including parenteral,
pulmonary, nasal and oral.
[0085] Portions of the agent of the invention may be "labeled" by
association with a detectable marker substance (e.g., radiolabeled
with .sup.125I or biotinylated) to provide reagents useful in
detection and quantification of compound or its receptor bearing
cells or its derivatives in solid tissue and fluid samples such as
blood, cerebral spinal fluid or urine.
[0086] When administered, agents (such as a peptide comprising the
V-domain of sRAGE) are often cleared rapidly from the circulation
and may therefore elicit relatively short-lived pharmacological
activity. Consequently, frequent injections of relatively large
doses of bioactive agents may by required to sustain therapeutic
efficacy. Agents modified by the covalent attachment of
water-soluble polymers such as polyethylene glycol, copolymers of
polyethylene glycol and polypropylene glycol, carboxymethyl
cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or
polyproline are known to exhibit substantially longer half-lives in
blood following intravenous injection than do the corresponding
unmodified agents (Abuchowski et al., 1981; Newmark et al., 1982;
and Katre et al., 1987). Such modifications may also increase the
agent's solubility in aqueous solution, eliminate aggregation,
enhance the physical and chemical stability of the agent, and
greatly reduce the immunogenicity and reactivity of the agent. As a
result, the desired in vivo biological activity may be achieved by
the administration of such polymer-agent adducts less frequently or
in lower doses than with the unmodified agent.
[0087] Attachment of polyethylene glycol (PEG) to agents is
particularly useful because PEG has very low toxicity in mammals
(Carpenter et al., 1971). For example, a PEG adduct of adenosine
deaminase was approved in the United States for use in humans for
the treatment of severe combined immunodeficiency syndrome. A
second advantage afforded by the conjugation of PEG is that of
effectively reducing the immunogenicity and antigenicity of
heterologous compounds. For example, a PEG adduct of a human
protein might be useful for the treatment of disease in other
mammalian species without the risk of triggering a severe immune
response. The compound of the present invention capable of
alleviating symptoms of a cognitive disorder of memory or learning
may be delivered in a microencapsulation device so as to reduce or
prevent an host immune response against the agent or against cells
which may produce the compound. The agent of the present invention
may also be delivered microencapsulated in a membrane, such as a
liposome.
[0088] Polymers such as PEG may be conveniently attached to one or
more reactive amino acid residues in a protein such as the
alpha-amino group of the amino terminal amino acid, the epsilon
amino groups of lysine side chains, the sulfhydryl groups of
cysteine side chains, the carboxyl groups of aspartyl and glutamyl
side chains, the alpha-carboxyl group of the carboxy-terminal amino
acid, tyrosine side chains, or to activated derivatives of glycosyl
chains attached to certain asparagine, serine or threonine
residues.
[0089] Numerous activated forms of PEG suitable for direct reaction
with proteins have been described. Useful PEG reagents for reaction
with protein amino groups include active esters of carboxylic acid
or carbonate derivatives, particularly those in which the leaving
groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or
1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing
maleimido or haloacetyl groups are useful reagents for the
modification of protein free sulfhydryl groups. Likewise, PEG
reagents containing amino hydrazine or hydrazide groups are useful
for reaction with aldehydes generated by periodate oxidation of
carbohydrate groups in proteins.
Administration
[0090] The administration of the agent may be effected by
intralesional, intraperitoneal, intramuscular or intravenous
injection; by infusion; or may involve liposome-mediated delivery;
or topical, nasal, oral, anal, ocular or otic delivery. The
administration may comprise subcutaneous, vaginal, sublingual,
uretheral, transdermal, or intrathecal.
[0091] The agent may be delivered hourly, daily, weekly, monthly,
yearly (e.g. in a time release form) or as a one time delivery. The
delivery may be continuous delivery for a period of time, e.g.
intravenous delivery. The agent or pharmaceutical composition of
the present invention may be delivered intercranially or into the
spinal fluid.
[0092] In accordance with the method of this invention, the
therapeutically effective amount may comprise a dose of from about
200 ng/day/kg body weight to about 200,000 ng/day/kg body weight or
from about 50 ng/day/kg to about 500,000 ng/day/kg body weight.
[0093] In the practice of the method administration may comprise
daily, weekly, monthly or hourly administration, the precise
frequency being subject to various variables such as age and
condition of the subject, amount to be administered, half-life of
the agent in the subject, area of the subject to which
administration is desired and the like.
[0094] In connection with the method of this invention, a
therapeutically effective amount of may include dosages which take
into account the size and weight of the subject, the age of the
subject, the severity of the symptom, the surface area of the
wound, the efficacy of the agent, the method of delivery of the
agent and the history of the symptoms in the subject. One of
ordinary skill in the art would be readily able to determine the
exact dosages and exact times of administration based upon such
factors. For example, a therapeutically effective amount may a dose
of from about 200 ng/day/kg body weight to about 200,000 ng/day/kg
body weight. In this regard, it has been shown that 24 micrograms
administered intraperitoneally daily (on days 3-9) to wounded
diabetic mice resulted in greatly reduced inflammation. In this
regard, the dose may also be administered as a single dose or as a
series of doses over a period of time.
[0095] The effective amount of the agent may comprise from about
0.000001 mg/kg body weight to about 100 mg/kg body weight. In one
embodiment, the effective amount may comprise from about 0.001
mg/kg body weight to about 50 mg/kg body weight. In another
embodiment, the effective amount may range from about 0.01 mg/kg
body weight to about 10 mg/kg body weight. The actual effective
amount will be based upon the size of the agent, the
biodegradability of the agent, the bioactivity of the agent and the
bioavailability of the agent. The agent may be delivered topically
in a creme or salve carrier. It may be reapplied as needed based
upon the absorbancy of the carrier to the skin or mucosa or wound.
If the agent does not degrade quickly, is bioavailable and highly
active, a smaller amount will be required to be effective. The
effective amount will be known to one of skill in the art; it will
also be dependent upon the form of the agent, the size of the agent
and the bioactivity of the agent. One of skill in the art could
routinely perform empirical activity tests for a agent to determine
the bioactivity in bioassays and thus determine the effective
amount.
Wound Healing
[0096] One example of inflammation in a subject is that which is
associated with a wound in a subject. One embodiment of treating
inflammation in a subject is improving wound healing in a subject.
The present invention provides a method for improving wound healing
in a subject which comprises administering to the subject a
therapeutically effective amount of an agent which inhibits binding
of advanced glycation endproducts (AGEs) to a receptor for advanced
glycation endproducts (RAGE), over a sufficient period of time in a
sufficient amount so as to improve wound healing in the
subject.
[0097] The present invention provides a method for alleviating
inflammation in a subject which comprises administering a
therapeutically effective amount of an agent which inhibits binding
of advanced glycation endproducts to any receptor for advanced
glycation endproducts so as to treat symptoms of inflammation in
the subject.
[0098] There may be other mechanisms by which soluble RAGE may
improve inflammation in a subject. Soluble RAGE may have other
effects, such as anti-inflammatory effects that are at least in
part, independent of binding up AGE's and interfering with their
ability to activate cellular RAGE.
[0099] The mechanism of reducing inflammation in the subject may be
biochemical in nature or competitive in nature.
[0100] As used herein "AGE" means an advanced glycation endproduct;
"RAGE" means a receptor for an advanced glycation endproduct;
"sRAGE" means a soluble form of a receptor for an advanced
glycation endproducts, such as the extracellular two-thirds of the
RAGE polypeptide.
[0101] In the practice of the methods of the invention a
"therapeutically effective amount" is an amount of an agent which
is capable of inhibiting the binding of AGE to any receptor for
advanced glycation endproduct (RAGE). Accordingly, the effective
amount will vary with the subject being treated, as well as with
the type of inflammation to be treated. For the purposes of this
invention, the methods of administration are to include, but are
not limited to, administration cutaneously, subcutaneously,
intravenously, parenterally, orally, topically, or by aerosol.
[0102] Portions of the agent of the invention may be "labeled" by
association with a detectable marker substance (e.g., radiolabeled
with .sup.125I or biotinylated) to provide reagents useful in
detection and quantification of such agent or its receptor bearing
cells or its derivatives in solid tissue and fluid samples such as
blood, serum, cerebral spinal fluid or urine.
Compositions
[0103] The present invention provides compositions consisting
essentially of an agent which reduces inflammation and a carrier.
The agent may be an inhibitor of the interaction between RAGE and
AGEs. The agent may be an inhibitor of the binding activity of
RAGE.
[0104] When administered, compounds are often cleared rapidly from
the circulation and may therefore elicit relatively short-lived
pharmacological activity. Consequently, frequent injections of
relatively large doses of bioactive compounds may by required to
sustain therapeutic efficacy. Compounds modified by the covalent
attachment of water-soluble polymers such as polyethylene glycol,
copolymers of polyethylene glycol and polypropylene glycol,
carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinylpyrrolidone or polyproline are known to exhibit
substantially longer half-lives in blood following intravenous
injection than do the corresponding unmodified compounds
(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al.,
1987). Such modifications may also increase the compound's
solubility in aqueous solution, eliminate aggregation, enhance the
physical and chemical stability of the compound, and greatly reduce
the immunogenicity and reactivity of the compound. As a result, the
desired in vivo biological activity may be achieved by the
administration of such polymer-compound adducts less frequently or
in lower doses than with the unmodified compound.
[0105] Attachment of polyethylene glycol (PEG) to compounds is
particularly useful because PEG has very low toxicity in mammals
(Carpenter et al., 1971). For example, a PEG adduct of adenosine
deaminase was approved in the United States for use in humans for
the treatment of severe combined immunodeficiency syndrome. A
second advantage afforded by the conjugation of PEG is that of
effectively reducing the immunogenicity and antigenicity of
heterologous compounds. For example, a PEG adduct of a human
protein might be useful for the treatment of disease in other
mammalian species without the risk of triggering a severe immune
response. The compound of the present invention capable of
improving wound healing in a subject may be delivered in a
microencapsulation device so as to reduce or prevent a host immune
response against the compound or against cells which may produce
the compound. The compound of the present invention may also be
delivered microencapsulated in a membrane, such as a liposome.
[0106] Polymers such as PEG may be conveniently attached to one or
more reactive amino acid residues in a protein such as the
alpha-amino group of the amino terminal amino acid, the epsilon
amino groups of lysine side chains, the sulfhydryl groups of
cysteine side chains, the carboxyl groups of aspartyl and glutamyl
side chains, the alpha-carboxyl group of the carboxy-terminal amino
acid, tyrosine side chains, or to activated derivatives of glycosyl
chains attached to certain asparagine, serine or threonine
residues.
[0107] Numerous activated forms of PEG suitable for direct reaction
with proteins have been described. Useful PEG reagents for reaction
with protein amino groups include active esters of carboxylic acid
or carbonate derivatives, particularly those in which the leaving
groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or
1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing
maleimido or haloacetyl groups are useful reagents for the
modification of protein free sulfhydryl groups. Likewise, PEG
reagents containing amino hydrazine or hydrazide groups are useful
for reaction with aldehydes generated by periodate oxidation of
carbohydrate groups in proteins.
[0108] The invention also provides a kit which comprises a
therapeutic amount of an agent, which agent is capable of
inhibiting binding of advanced glycation endproducts to a receptor
for advanced glycation endproducts, over a sufficient period of
time in a sufficient amount so as to treat chronic symptoms of
diabetes in the subject. A kit may include a composition which
includes sRAGE or a portion thereof in a form which is previously
dose regulated and time regulated so that a subject may easily take
such therapeutic at home or away from a clinical setting. The kit
also includes a means for administering the agent to the
subject.
[0109] This invention is illustrated in the Experimental Details
section which follows. These sections are set forth to aid in an
understanding of the invention but are not intended to, and should
not be construed to, limit in any way the invention as set forth in
the claims which follow thereafter.
EXPERIMENTAL DETAILS
Example 1
Treating Wound Healing as an Example of Treating Inflammation
[0110] Improved Wound Healing in Diabetic Mice by Treatment with
the Soluble Receptor for Advanced Glycation Endproducts (sRAGE)
[0111] Ineffective healing of wounds is a serious problem in
diabetes, contributing to increased morbidity (Reynolds, 1985;
Galloway and Shuman, 1963; and Pearl and Kanat, 1988). The
reparative response in wound healing is orchestrated by multiple
cellular elements which work together in many ways, including
infiltration of the lesion by inflammatory effector cells.
Subsequent to this, fibroblastic elements together with
inflammatory effector cells provide antibacterial mechanisms and
promote removal of necrotic tissue, as well as laying down of new
connective tissue. A fundamental disorder of glucose metabolism
might perturb these complex and interactive protective processes.
Previous work has suggested that cellular dysfunction in diabetic
wound healing involves defective neutrophil function (Bagdade et
al., 1978; Nolan et al., 1978; and Mowat and Baum, 1971), delayed
infiltration of the wound with inflammatory cells (Greenhalgh et
al., 1990 and Fahey et al., 1991), decreased production of collagen
(Goodson and Hunt, 1977 and Goodson and Hunt, 1986), and diminished
activity of endogenous growth factors, such as basic fibroblast
growth factor (Giardino et al., 1994), which could provide a basis
for the slower formation of granulation tissue and wound
closure.
[0112] Defective wound healing in diabetes continues to be an
important cause of morbidity in the postoperative period, following
trauma, and in the repair of cutaneous lesions. Advanced Glycation
Endproducts (AGEs) are the result of nonenzymatic
glycation/oxidation of proteins/lipids. Accelerated formation and
accumulation of AGEs in tissues of patients with diabetes has been
linked, in certain situations, to the development of secondary
complications. An important means by which AGEs perturb homeostatic
processes is through their interaction with cellular binding sites;
the best characterized of these is Receptor for AGE or RAGE, an
immunoglobulin superfamily molecule expressed by endothelium,
monocytes, and smooth muscle cells, as well as mesangial cells and
neurons. AGE engagement of RAGE leads to endothelial activation,
with expression of adhesion molecules, enhanced procoagulant
properties, and diminished barrier function; and perturbation of
monocytes, with changes in cell motility and activation, resulting
in expression of proinflammatory cytokines. The interaction of AGEs
with RAGE-bearing cells, especially endothelium and mononuclear
phagocytes, may promote chronic cellular activation thereby
preventing optimal wound healing as reflected by formation of
granulation tissue and new connective tissue. The data herein are
consistent with this concept: using a secondary intention wound
model in diabetic mice, wound closure is enhanced following
administration of soluble(s) RAGE, the extracellular domain of the
receptor. These experiments contribute to a long-term goal and
long-felt need, understanding the contribution of cellular
interactions of AGEs in the pathogenesis of diabetic
complications.
[0113] Poor wound healing in diabetes is likely to be a
manifestation of a basic defect in the host inflammatory-reparative
response, in addition to possible underlying vascular
insufficiency. Exposure of macromolecules to aldose sugars results
in nonenzymatic glycation and oxidation (Baynes, 1991; Sell and
Monnier, 1989; Ruderman et al., 1992; and Vlassara et al., 1994),
initially the reversible early glycation adducts, Schiff bases and
Amadori products, form. Following further complex molecular
rearrangements, the irreversible AGEs come about. The latter
comprise a heterogenous group of structures characterized by
fluorescence, propensity to form cross-links, generation of
reactive oxygen intermediates (ROIs) and interaction with cellular
receptors, the best characterized of which is Receptor for AGE, or
RAGE (Schmidt et al., 1992; Neeper et al., 1992; and Schmidt et
al., 1994a). AGEs accumulated in the tissues in diabetes influence
end-organ function by two general mechanisms: directly, via effects
on tissue architecture, consequent to the formation of cross-links
and trapping of plasma proteins, and indirectly, by interaction
with cellular elements, such as endothelial cells (Ecs),
mononuclear phagocytes (Mps), central to homeostasis as well as the
host response to pathophysiologically relevant stimuli.
[0114] Studies have suggested that the extracellular two-thirds of
the molecule, soluble or sRAGE, appeared to be able to inhibit the
interaction of circulating AGEs with cellular surfaces (Schmidt et
al., 1994b). For example, binding of radiolabelled AGE albumin, a
prototypic ligand developed in the laboratory, to cultured
endothelial cells or peripheral blood-derived mononuclear
phagocytes, was inhibited in the presence of increasing doses of
sRAGE. In vivo, clearance of radiolabelled AGE albumin from the
circulation of a normal mouse after intravenous injection, was
delayed upon treatment with sRAGE. Extrapolation of these findings
was attempted to the setting of wound healing. The goal in these
studies was to assess the role of AGE-RAGE interaction in the
setting of the host response to wounding.
[0115] In order to assess the contribution of AGE-RAGE interaction
to defective wound healing in diabetes, the wound healing response
in diabetic was compared to normal animals, and to determine if
blockade of RAGE would ameliorate wound closure in diabetes. In
these studies, it was found that administration of soluble RAGE
improved wound healing in genetically-diabetic mice. These data
support the hypothesis that RAGE blockade may represent a feasible
target for intervention in diabetic wound healing as well as other
complications of diabetes, such as renal, retinal, neurological,
cardiovascular, cerebrovascular and peripheral vascular diseases.
Diabetic subjects experience increased restenosis and local
problems after angioplasty which suggests that soluble RAGE may be
beneficial in reducing restenosis after balloon/stent injury.
Materials and Methods
[0116] Murine model of diabetes. A genetic model of
insulin-resistant/hyperglycemic diabetes (db+/db+mice) due to an
autosomal recessive trait (chromosome 4) which results in
abnormalities of glucose metabolism and obesity in homozygote mice
was employed. Heterozygote mice (db+/+m) do not develop these
abnormalities, and are employed as controls (Coleman, 1982 and Wyse
and Dulin, 1970). Diabetic animals are hyperglycemic
(glucose>400 mg/dl by age 3 months), and develop abnormalities
similar to human complications, including a defective wound repair.
Life expectancy of homozygote mice is 6-8 months. Wounding studies
began when mice reached 8 weeks of age, as AGEs are present by that
time.
[0117] Model of wound healing. For analysis of wound healing in
diabetes, a secondary intention wound model was employed
(Greenhalgh et al., 1990), as it stimulates, in part, the clinical
situation following breakdown of skin in an ulcerated area. A
full-thickness 1.5.times.1.5 cm wound was created on the back of
the mouse which was subsequently covered by TEGADERM (clear,
plastic closure). The initial area of the wound was measured by
placing a sterile glass slide over the area, and tracing the edges
of the wound. The area was then determined by using a computer
program (NIH Image 157). Serial measurement of the wound dimensions
were made on days 3, 5, 7, 10, 14 and 17. This data, consistent
with those of previous studies (Greenhalgh et al., 1990), showed
significant delay of wound repair in the diabetic mouse especially
within the first 2-3 weeks after creation of the wound. Animals in
each group were sacrificed at days 17 for analysis. Studies began
when mice reached 8-10 weeks of age. In certain experiments, mice
were treated with soluble RAGE (the extracellular two-thirds of the
molecule) under the TEGADERM on days 3 through 9 after the initial
wounding procedure.
Immunohistochemistry for Detection of Advanced Glycation
Endproducts.
[0118] At the time of the wounding procedure, 1.5.times.1.5 cm
wounds were excised, fixed in formalin (10%) and then processed for
immunohistochemistry using affinity-purified anti-AGE IgG (Miyata
et al., 1996).
Results
[0119] In order to understand the role of RAGE in diabetic wound
healing, 1.5.times.1.5 cm wounds were created on the backs of
db+/db+ or db+/m+mice. It was first determined that there was no
statistically-significant difference in original wound area among
the groups of mice receiving the various treatment regimens. When
sRAGE (200 ng/day) was administered under the TEGADERM daily from
days 3 through 9, the wound healing observed in diabetic mice was
significantly enhanced compared with diabetic mice treated with
vehicle (phosphate buffered saline; p<0.05; FIG. 1).
Furthermore, the healing observed in diabetic mice treated with
sRAGE approximated that observed in control, db+/m+mice treated
with vehicle (differences were not statistically significant).
(FIG. 1).
[0120] Consistent with the hypothesis that these findings were due
to receptor-mediated mechanisms, dose-response studies revealed
that there was no enhancement of diabetic wound healing upon
administration of sRAGE, 2,000 ng/day, compared with a daily dose
of 200 ng/day (differences were not significant; FIG. 2). However,
consistent with the studies described herein in diabetic mice,
treatment with either 200 or 2,000 ng/day sRAGE (administered on
days 3 through 9) was significantly superior to treatment of these
mice with phosphate buffered saline when the final wound area was
measured on day ten after creation of the wound (FIG. 2). However,
at a daily dose of sRAGE of 20 ng/day, there was no significant
difference in wound healing in the diabetic mice receiving sRAGE
versus those diabetic mice receiving vehicle. (FIG. 2).
[0121] In order to determine if diabetic wounds were enriched in
AGE-immunoreactive material, immunohistochemistry was performed of
diabetic versus control mice wounds using affinity-purified
anti-AGE IgG. These studies demonstrated that there was a
significant increase in AGE-reactive material in the wound tissue
of the diabetic mice (FIG. 3A) compared with the nondiabetic
control animals (FIG. 3B).
Discussion
[0122] The results of these studies indicate that in diabetic
tissue such as wounds, there is increased deposition/formation of
AGEs. Such AGEs, upon interaction with their cellular receptor
RAGE, result in the generation of a sustained inflammatory
environment in which healing and quiescence of the potent effector
cells and mediators is markedly delayed. It was hypothesized that
interference with AGE-RAGE interaction might result in accelerated
healing. In these studies, it was demonstrated that local
administration of soluble RAGE improved diabetic wound healing in a
dose-dependent manner. The specific mechanisms which underlie the
efficacy of administration of sRAGE is important. It is possible
that administration of sRAGE improves any one of a number of
important steps in physiologic wound healing such as inflammation,
angiogenesis and/or formation and deposition of new granulation
tissue, specifically collagen.
[0123] Taken together, these data suggest that in an AGE-enriched
environment such as that observed in diabetes, interference with
AGE-cellular RAGE interaction might result in amelioration of the
chronic complications of diabetes. Given that RAGE is expressed in
the endothelium and smooth muscle of the vasculature, in mesangial
cells, in certain neural and vascular cells of the retina, and in
certain neurons of both the central and peripheral nervous systems
as well as other cells, it is likely that blockade of cellular RAGE
might result in improved diabetic complications that might
otherwise lead to heart attacks, stroke, peripheral vascular
disease, amputation of the extremities, kidney disease/failure,
blindness, impotence and neuropathy. RAGE is found in monocytes and
macrophages and may be present in other cell types wherein
therapeutic intervention may also be possible. The present studies
support the concept that administration of sRAGE (or other forms of
RAGE blockade; such as recombinant sRAGE, RAGE-based peptides,
anti-RAGE IgG or anti-RAGE F(ab').sub.2) might present a novel form
of therapeutic intervention in this chronic, debilitating
disorder.
REFERENCES FOR EXAMPLE 1
[0124] Bagdade, J. et al. (1978) Impaired granulocyte adherence. A
reversible defect in host defense in patients with poorly
controlled diabetes. Diabetes 27:677-681. [0125] Baynes, J. (1991)
Role of oxidative stress in development of complications in
diabetes. Diabetes 40:405-412. [0126] Coleman, D. (1982)
Diabetes-obesity syndromes in mice. Diabetes 31 (Suppl.):1-6.
[0127] Fahey, T. et al. (1991) Diabetes impairs the late
inflammatory response to wound healing. Surg. Res. 50:308-313.
[0128] Galloway, J. and Shuman, D. (1963) Diabetes and Surgery. Am.
J. Med. 34:177-191. [0129] Giardino, I. et al. (1994) Nonenzymatic
glycosylation in vitro and in bovine endothelial cells after basic
fibroblast growth factor activity. J. Clin. Invest. 94:110-117.
[0130] Goodson, W. and Hunt T. (1977) Studies of wound healing in
experimental diabetes mellitus. J. Surg. Res. 22:221-227. [0131]
Goodson, W. and Hunt T. (1986) Wound collagen accumulation in obese
hyperglycemic mice. Diabetes 35:491-495. [0132] Greenhalgh, D. et
al. (1990) PDGF and FGF stimulate wound healing in the genetically
diabetic mouse. Am. J. Pathol. 136:1235-1246. [0133] Mowat, A. and
Baum, J. (1971) Chemotaxis of polymorphonuclear leukocytes from
patients with diabetes mellitus. NEJM 284:621-627. [0134] Neeper,
M. et al. (1992) Cloning and expression of RAGE: a cell surface
receptor for AGEs. J. Biol. Chem. 267:14998-15004. [0135] Nolan, C.
et al. (1978) Further characterization of the impaired bactericidal
function of granulocytes in patients with poorly controlled
diabetes. Diabetes 27:889-894. [0136] Pearl, S, and Kanat, I.
(1988) Diabetes and healing: a review of the literature. J. Foot
Surg. 27:268-273. [0137] Reynolds, C. (1985) Management of the
diabetic surgical patient. A systematic but flexible plan is the
key. Postgrad. Med. 77:265-279. [0138] Ruderman, N. et al. (1992)
Glucose and diabetic vascular disease. FASEB J. 6:2905-2914. [0139]
Schmidt, A-M et al. (1994a) Cellular receptors for AGEs.
Arterioscler. Thromb. 14:1521-1528. [0140] Schmidt, A-M. et al.
(1994b) RAGE has a central role in vessel wall interactions and
gene activation in response to AGESs. PNAS, USA 91:8807-8811.
[0141] Schmidt, A-M et al. (1992) Isolation and characterization of
binding proteins for AGEs from lung tissue which are present on the
endothelial surface. J. Biol. Chem. 267:14987-14997. [0142] Sell,
D. and Monnier, V. (1989) Structure elucidation of senescence
cross-link from human extracellular matrix. J. Biol. Chem.
264:21597-21602. [0143] Vlassara, H. et al. (1994) Pathogenic
effects of AGEs:biochemical, biologic, and clinical implications
for diabetes and aging. Lab. Invest. 70:138-151. [0144] Wyse, B.
and Dulin, W. (1970) The influence of age and dietary conditions on
diabetes in the Db mouse. Diabetologia 6:268-273.
Example 2
Treating Periodontal Disease as an Example of Treating
Inflammation
[0145] sRAGE Suppresses Accelerated Periodontal Disease in Diabetic
Mice.
[0146] A model of accelerated periodontal disease in diabetic mice
and the effects of sRAGE were studied. Efficacy of sRAGE is shown
in diabetic (streptozotocin C57BL6/J mice). Administration of
soluble RAGE (full-length extracellular form of approximately 40
kDa) inhibits accelerated alveolar bone loss, which is the hallmark
of periodontal disease.
[0147] Intraperitoneal injection of soluble RAGE suppresses bone
loss in diabetic mice (db+/db+).
Administration of Soluble(s) RAGE Suppresses Alveolar Bone Loss in
a Murine Model of Accelerated Periodontal Disease in Diabetes.
[0148] Diabetes was induced in C57BL6/J mice by administration of
streptozotocin. Diabetes was defined as two serial measurements of
serum glucose .gtoreq.300 mg/dl. Alternatively, an equal number of
mice were treated with vehicle for streptozotocin, phosphate
buffered saline. One month after induction of diabetes, mice were
treated every other day for four consecutive days with oral/anal
administration of the human periodontal pathogen, Porphyromonas
gingivalis (Pg) or vehicle, phosphate-buffered saline. Two months
later, mice were sacrificed and decapitated. The mandibles were
isolated and, under a dissecting microscope (Olympus), and using
curved microdissecting forceps (23/4 inches, 0.6 mm wide) and a
scalpel with a No. 15C blade, the lingual gingival tissue from the
posterior area of each quadrant was dissected. Beginning with a
horizontal sulcular incision at the gingival margin of the
posterior teeth, the gingiva was reflected (full thickness) with
the scalpel blade. Vertical release incisions were made and the
tissue was removed (separating the tissue with a horizontal
incision just below the mucogingival junction). Tissue was then
placed in formalin (10%) for further analysis.
[0149] After the above procedures, mandibles were exposed to KOH
(2%) for three days and then mechanically defleshed. The jaws
(exposure of the lingual surfaces of each 1/2 mandible) were then
embedded in lab putty. In order to remove angulation as a variable,
the buccal and lingual cusps of the posterior teeth in the 1/2
mandible were superimposed during embedding and viewed from the
lingual surface prior to photography. The defleshed jaws were
photographed using the magnifying dissecting microscope and
Ektachrome.RTM. 160T film (color slides).
[0150] These slides, at a magnification of 40.times., were then
magnified further 4.times.. Images were then traced onto standard
tracing paper. For each mouse, a total area of the distance between
the cemento-enamel junction (CEJ) and alveolar bone crest (BC) for
a total of 6 posterior teeth was measured by scanning the tracing
into a Macintosh.RTM. computer/scanner and the images analyzed
using the program NIH Image 157.RTM. (along with Adobe
Photoshop.RTM. photography program). Total area (in arbitrary pixel
units) is reported for each mouse (6 teeth) as indicated in FIG. 4.
Statistical analysis was performed using one way analysis of
variance. At two months, a significant 1.55-fold increase in
alveolar bone loss was observed in diabetic mice compared with
nondiabetic controls (see specific data below). Similar results
were observed in db+/db+ mice
(genetically-diabetic/insulin-resistant) one month after infection
with Pg compared with nondiabetic controls (m+/db+).
[0151] In order to test if administration of sRAGE would ameliorate
alveolar bone loss in Pg-treated C57BL6/J mice, certain diabetic
mice were treated with sRAGE (MSR; either 35 .mu.g IP/day for two
months or 3.5 .mu.g IP/day for two months). Control diabetic mice
were treated with equimolar concentrations of mouse serum albumin
(70 .mu.g IP/day for two months). All mice were treated with Pg. At
the end of that time, measurements of alveolar bone loss were made.
The results are as follows:
TABLE-US-00004 Alveolar bone loss Condition (CEJ to alveolar BC)
(I) Diabetic/albumin 6,222 .+-. 406 pixels (SD) (II)
Nondiabetic/albumin 4,018 .+-. 501 pixels (SD) (III)
Diabetic/MSR(35 .mu.g/day) 5,242 .+-. 463 pixels (SD) (IV)
Diabetic/MSR(3.5 .mu.g/day) 6,198 .+-. 427 pixels (SD)
[0152] Many diabetic complications may result from the interaction
of AGE's with RAGE to cause cellular perturbation. AGE acts as a
ligand for the V-domain of RAGE to mediate such cellular
perturbation. This invention provides a method for inhibiting
cellular perturbation in a subject associated with a diabetic
condition which comprises administering to the subject an amount of
an inhibitor of the interation of AGE's with RAGE on the surface of
a cell effective to inhibit the interaction and thereby inhibit the
cellular perturbation in the subject and treat the diabetic
condition.
[0153] AGE (advanced glycation endproducts) are a heterogeneous
group of compounds. A single or specific pathogenic AGE compound
(s) are being identified. Examples of AGEs include but are not
limited to: pentosidine (alone or protein-bound modification);
carboxymethyllysine (alone or protein-bound modification);
carboxyethyllysine (alone or protein-bound modification);
pyrallines (alone or protein-bound modification); methylglyoxal
(alone or protein-bound modification) and ethylglyoxal (alone or
protein-bound modification). One of these AGE's may be a pathogenic
ligand for a specific cellular perturbation due to an interaction
of the AGE with the V-domain of RAGE. This interaction may be a
critical contributory factor in many complications associated with
diabetes. This invention provides for inhibitors of such an
interaction which may be administered to subjects with diabetic
complications.
[0154] Cells which may be acted upon by this binding of AGE's to
RAGE on the cell surface include endothelial cells, vascular smooth
muscle cells, neuronal cells, macrophages, lymphocytes, retinal
vascular cells, retinal neuronal cells, mesangial cells and
connective tissue cells and cells associated with connective tissue
such as cells associated with gingiva and skin. Cells which may be
acted upon by this binding of AGE's to RAGE are not limited to this
list but may include other cells present in a human body. The
present invention provides compounds and compositions which may be
useful in inhibiting this interaction, thereby ameliorating the
cellular perturbation and ultimately the symptoms associated with
diabetes.
[0155] Cellular perturbations in those cells that sRAGE, or other
peptides or agents provided for by the present invention include
but are not limited to: oxidant stress, hyperpermeability, enhanced
expression of adhesion molecules such as Vascular Cell Adhesion
Moleucle--1; enhanced expression of tissue factor; enhanced
macrophage chemotaxis and activation, such as with increased
production of cytokines and growth factors; enhanced migration of
smooth muscle cells, activation of smooth muscle cells, neuronal
oxidant stress and apoptosis. Advanced glycation endproducts (AGE)
are the irreversible result of nonenxymatic glycation and
oxidation. These AGE's form in the connection with a number of
conditions such as: aging, diabetes, inflammation, renal failure,
amyloidoses, and hyperlipidemia. AGE's also form in connection with
other disease states and abnormal conditions which are not
explicitly listed herein but which are encompassed by the present
invention.
Therapeutic Agents Identified through In Vitro Means, are Shown to
be Effective In Vivo for Inhibition of Symptoms Associated with
Diabetic Complications.
[0156] The therapeutic agent identified may be shown to be
effective in wound healing. In wound healing experiments, the
secondary intention wound model in genetically diabetic mice would
be used. The agent (or peptide or pharmaceutical composition) is
applied topically to the wounded area, and wound closure (change in
wound area), epithelialization and other histologic indices (such
as collagen production, extracellular matrix production, fibrin,
etc.) is measured. Each of these measurements are indices of the
effectiveness of the agent on increasing wound healing.
[0157] In periodontal disease, genetically diabetic and
streptozotocin-treated mice are utilized as animal model systems to
examine bone loss after treatment with peptides having the sequence
of Seq I.D. No. 1. Bone loss is measured quantitatively via
histological methods and geometrical area determinations. The
peptide of Seq. ID No. 1, V-domain peptide, agent or pharmaceutical
composition is administered locally (e.g. "painting on" the agent)
and/or systemically. Reduced bone loss is an indication of an
effective agent.
[0158] In accelerated atherosclerosis, streptozotocin-treated apoE
"knock-out" mice on a normal chow diet are employed as animal
models of this disease condition. The agent (or peptide or
pharmaceutical composition) is administered systemically, and
quantitative data is gathered by measuring lesion area in the
animals after treatment. This data gives an indication of the
effectiveness of each agent. The smaller the lesion area as
compared to non-treated controls, the more effective the agent.
[0159] In diabetic impotence, a rat model with
streptozotocin-treated animals is employed in which erections are
monitored following administration of apomorphine. The number and
frequency of erections is measured in the presence and in the
absence of the agent and such data is compared so as to evaluate
the effectiveness of the agent to inhibit symptoms of
impotence.
[0160] In diabetic retinopathy, diabetic rat and mouse models are
used as animal model systems to measure changes in blood flow and
retinal pathology. Again, the agent (or peptide or pharmaceutical
composition) is administered systemically, and quantitative data is
gathered by blood flow and qualitative data is gathered by
examining retinal pathology in the animals after treatment.
[0161] In diabetic nephropathy, diabetic mice and rat models are
employed as animal models of diabetic nephropathy. Changes in
glomerular filtration rate and renal blood flow are measured in
animals given a therapeutic agent and measured in animals given a
placebo. In addition, the appearance of protein in the urine and
histologic changes in glomeruli are determined in each animal. The
effectiveness of the agent is evaluated based upon these
measurements in inhibiting diabetic nephropathy.
[0162] In diabetic neuropathy, genetically diabetic mice are
utilized as an animal model for the determination of the
effectiveness of the agent of the present invention. The mice are
treated with the compound systemically. The mice are then observed
to determine changes in nerve conduction velocity and changes in
the number of myelinated peripheral nerve fibers. Such data
compared with equivalent measurements determined in an untreated
animal will provide an indication of the effectiveness of the agent
of the present invention.
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Example 3
Treating Delayed Type Hypersensitivity as an Example of Treating
Inflammation
[0228] Interaction of EN-RAGE (Extracellular Novel Rage Binding
Protein) with Receptor for AGE (RAGE) Perpetuates Inflammatory
Responses Suppression of Delayed-Type Hypersensitivity Reactions
with Soluble Receptor for Age (sRAGE)
[0229] Expression of RAGE, the Receptor for Advanced Glycation
Endproducts, is increased in the setting of inflammation. Here we
report a new member of the calgranulin family of proinflammatory
cytokines called EN-RAGE (or Extracellular Novel RAGE-binding
protein), which interacts with RAGE on cells such as endothelial
cells, to alter cellular properties in a manner consistent with
perturbation. Administration of soluble RAGE (the extracellular
ligand binding domain of RAGE; sRAGE) or anti-RAGE or anti-EN-RAGE
F(ab').sub.2 fragments markedly attentuated inflammation in a model
of delayed hypersensitivity. These data link RAGE to the
inflammatory response and identify EN-RAGE and RAGE as novel
targets for anti-inflammatory intervention. Soluble RAGE,
furthermore, is thus a prototypic structure for the design of a new
class of anti-inflammatory agents.
[0230] The Receptor for AGE (RAGE) is a member of the
immunoglobulin superfamily of cell-surface molecules (1-2).
Originally identified and characterized as a cellular receptor for
glucose (aldose sugar)-modified proteins, or Advanced Glycation
Endproducts (AGEs) (3-13), RAGE has subsequently been reported to
interact with other ligands, in both settings of normal development
and in Alzheimer's disease (14-16). In normal development, RAGE
interacts with amphoterin, a polypeptide which mediates neurite
outgrowth in cultured embryonic neurons. In those studies, either
anti-RAGE F(ab').sub.2 or soluble RAGE (sRAGE) inhibited neurite
outgrowth on amphoterin-coated matrices, but not on matrices coated
with other substrates such as laminin or poly-1-lysine (3). In
later studies, RAGE was identified as a receptor on neurons and
microglia for amyloid-.beta.-peptide, a polypeptide linked to the
pathogenesis of neuronal toxicity and death in Alzheimer's
disease.
[0231] In unpublished observations from our laboratory, we
identified that increased RAGE expression was noted in the vascular
and inflammatory cells of inflammatory lesions, such as in the
kidney tissue from patients with active lupus nephritis (FIG. 5).
We therefore hypothesized that RAGE might interact with alternative
ligand(s) in that setting in order to, perhaps, participate in the
inflammatory response.
[0232] Herein, the findings demonstrate that RAGE interacts with a
molecule with close homology to calgranulin C. We have termed this
molecule, EN-RAGE (Extracellular Novel RAGE binding protein) and
show that EN-RAGE:RAGE interaction activates cells such as
endothelial cells which are importantly involved in the
inflammatory response. In a model of murine delayed
hypersensitivity, administration of soluble RAGE (sRAGE), which
contains the ligand interaction domain, inhibits the development of
cellular activation and inflammation. These findings identify RAGE
as a new target for anti-inflammatory intervention.
Materials and Methods
Isolation and Purification of EN-RAGE.
[0233] Bovine lung acetone powder (SIGMA.RTM.) was subjected to
solubilization in buffer containing tris (0.02M, pH 7.4); NaCl
(0.15M); octyl-.beta.-glucoside (1%); and protease inhibitors (PMSF
and aprotinin). After serial chromatography onto SP sepharose
(Pharmacia LKB.RTM.), and affi-gel 10 resin (BIO-RAD.RTM.) to which
had been adsorbed purified soluble human RAGE (prepared from a
baculovirus expression system), RAGE-binding proteins were
identified based on a screening assay employing immobilized column
fraction (Nunc Maxisorp dishes) (NUNC.RTM.) and .sup.125-I-labelled
sRAGE as above. After elution with heparin-containing buffer (1
mg/ml), positive fractions were identified. RAGE-binding proteins
were subjected to sequence analysis.
[0234] Cloning of EN-RAGE. The cDNA for EN-RAGE was cloned from a
bovine lung library and placed into a baculovirus expression
system. In this system, EN-RAGE, which lacks a leader sequence, was
synthesized within Sf9 cells. EN-RAGE was then purified after
solubilization of the cells in detergent-containing buffer, and
sequential purification on hydroxylapatite and heparin-containing
resins. The final product displayed a single band on
Coomassie-stained SDS-PAGE gels and was devoid of endotoxin after
chromatography onto Detoxi-gel columns (PIERCE.RTM.). Absence of
detectable endotoxin was confirmed using limulus amebocyte assay
(SIGMA.RTM.).
[0235] Sequence analysis. After SDS-PAGE identified an .apprxeq.12
kDa polypeptide with RAGE-binding activity, the gel band was eluted
according to previously-published methods (17). The published
method was modified by addition of a final wash of two aliquots
(0.1 ml each) of guanidine (5.0M), urea (5.0M), trifluoroacetic
acid (0.2-0.2%), acetonitrile (10%), and Zwittergent 3-08 (1.0%)
(Calbiochem) to ensure that protein was completely washed from the
filter. Amino-terminal sequence analysis was performed. Automated
Edman degradation was carried out employing an HP-G1005A sequencer
(Hewlett Packard Analytical Instruments). In order to obtain
internal sequence, the gel bands were treated as above for elution,
except that the extraction buffer contained half the usual amount
of SDS (1). Endoproteinase Lys-C (1 .mu.g) (Boehringer Mannheim)
was added and the sample incubated overnight. The digest was then
fractionated by microbore HPLC (Michrom Bioresources) on a 1
mm.times.50 mm PLRP-S column (Polymer Laboratories, Ltd.). The
gradient utilized was 2% per minute from acetonitrile (5-75%) in
trifluoroacetic acid (0.1%) and fractions were collected at 30
second intervals. Absorbance was monitored at 214 nm and fractions
that corresponded to chromatographic peaks were then subjected to
sequence analysis.
[0236] Endothelial cell activation. Human umbilical vein
endothelial cells were isolated, characterized and maintained as
previously described (18). Cells were cultured in serum-free RPMI
1640 without endothelial cell growth factor for 24 hrs and then
stimulated with the indicated concentrations of EN-RAGE. Where
indicated, cells were pretreated with rabbit anti-human RAGE IgG,
nonimmune rabbit IgG; in certain cases, EN-RAGE was pretreated with
the indicated concentration of soluble RAGE (sRAGE) for 2 hrs prior
to stimulation with EN-RAGE. After eight hrs stimulation with
EN-RAGE, cells were fixed with paraformaldehyde (2%) for 30 mins,
washed twice with PBS, treated with PBS containing non-fat dry milk
(5%) and BSA (2.5%) to block nonspecific binding sites on the cell
surface. Cell surface ELISA employing anti-VCAM-1 IgG (Santa Cruz
Biotechnologies, Santa Cruz, Calif.) was performed. Assessment of
functional VCAM-1 activity was determined using .sup.51Cr-labelled
Molt-4 cells (ATCC) as previously described (10).
[0237] Delayed hypersensitivity model. A murine model of delayed
hypersensitivity was established based on previously-published
studies (19). Female CF-1 mice (Charles River laboratories), 6
weeks of age, were sensitized by subcutaneous injection over the
left inguinal lymph node of an emulsion (0.1 ml) containing
methylated BSA (mBSA; 25 mg/ml; SIGMA.RTM.), NaCl (0.9%), dextran
(5-40.times.10.sup.6 MW; 50 mg/ml; SIGMA.RTM.) and Freund's
incomplete adjuvant (50%; ICN Biomedical). Three weeks later, the
left plantar hind paw was injected subcutaneously with mBSA (0.4
mg/ml; 0.050 ml). Where indicated, mice were pretreated by
intraperitoneal injection with sRAGE (indicated dose), mouse serum
albumin (SIGMA.RTM.), immune or nonimmune F(ab').sub.2 fragments
(prepared using a kit from Pierce) 24 and 12 hrs prior to, and 6
and 12 hrs after local challenge with mBSA. 24 hrs after injection
of foot pad with mBSA, clinical score of foot pad was performed;
mice were then humanely sacrificed and feet fixed in formalin (10%)
or frozen for further analysis. Histologic score was performed on
sections of foot stained with hematoxylin and eosin (SIGMA.RTM.).
The clinical score was defined as follows (scale; 1-5): 1=no
inflammation and thus identical to untreated foot; 2=slight rubor
and edema; 3=severe rubor and edema with wrinkling of the skin of
the foot pad; 4=severe rubor and edema without wrinkling of the
skin of the foot pad; and 5=severe rubor and edema resulting in
spreading of the toes. The histologic score after hematoxylin and
eosin staining was defined as follows (scale; 1-5): 1=no leukocytic
infiltration with slight subcutaneous edema; 2=slight perivascular
leukocytic infiltration with slight subcutaneous edema; 3=severe
leukocytic infiltration without granulomata; and 4=severe
leukocytic infiltration with granulomata.
Results
[0238] Identification of EN-RAGE. After a serial series of
experiments designed to identify RAGE-binding proteins from bovine
lung extract (from where RAGE was originally purified), an
.apprxeq.12 kDa polypeptide was identified. Upon sequence analysis,
this polypeptide was found to bear significant homology to members
of the calgranulin C family of proteins (Table 1) (20-21). This
class of proteins exist intracellularly within inflammatory cells.
Upon release in inflamed loci, we postulated they might be able to,
in turn, engage and activate other cells already recruited into the
inflammatory response. Thus, this might represent an important
means by which the inflammatory response might be propagated and
sustained, thereby increasing the probability of cellular
injury.
[0239] EN-RAGE activates endothelial cells in a RAGE-dependent
manner. To test this hypothesis, EN-RAGE was purified as described
above and incubated with endothelial cells. Incubation of EN-RAGE
with HUVEC resulted in increased cell surface Vascular Cell
Adhesion Molecule-1 (VCAM-1) in a RAGE-dependent manner (FIG. 6).
These data suggested that in an inflammatory focus, interaction of
EN-RAGE with EC RAGE might represent a means by which to further
propagate an inflammatory response. Consistent with increased
VCAM-1 antigen on the surface of EN-RAGE-treated ECs, increased
binding for Molt-4 cells (which bear the ligand for VCAM-1, VLA-4),
ensued (FIG. 7). While incubation with either BSA or non-immune IgG
did not affect the ability of EN-RAGE to activate EC VCAM-1,
incubation with either sRAGE or anti-RAGE F(ab').sub.2
significantly attenuated the ability of EN-RAGE to increase Molt-4
binding to treated HUVEC.
[0240] We sought to test these hypotheses in in vivo models. We
demonstrated that in diabetic mice, in which the ligand for RAGE is
likely to be, at least in part, products of glycation/oxidation of
proteins/lipids, the Advanced Glycation Endproducts, or AGEs,
administration of the soluble, ligand-binding portion of RAGE
(soluble or sRAGE), suppressed accelerated atherosclerosis in
diabetic apolipoprotein E null mice (12) and improved wound healing
in genetically-diabetic db+/db+ mice (22). Thus, the biologic
effects of EN-RAGE in highly-inflammatory foci, such as those
characterized by models of granulomatous inflammatory lesions
(delayed hypersensitivity), could be suppressed in the presence of
sRAGE.
[0241] To test this, we studied a model of delayed hypersensitivity
(DH) in which mice were first sensitized by injection of methylated
BSA (mBSA; which does not bind RAGE) over the inguinal lymph nodes
of female CF-1 mice. Three weeks after sensitization, mice were
challenged with mBSA by injection into the hind foot pad. An
inflammation score was designed on a scale of 1-9 which included
both clinical score (1-4) and histologic score (1-5) as indicated
in FIG. 8.
[0242] Consistent with our hypothesis, administration of sRAGE
suppressed inflammation upon injection of mBSA into the foot pad of
mice previously-sensitized with mBSA over the lymph nodes, in a
dose-dependent manner (FIG. 8). At a dose of 100 .mu.g sRAGE,
inflammation was markedly suppressed (p<0.01). In contrast,
administration of mouse serum albumin, had no effect on the
appearance of the inflammatory lesion (FIG. 8). Consistent with an
important role for EN-RAGE and RAGE in the development of
inflammation in this model, treatment of the mice with either
anti-EN-RAGE F(ab').sub.2 or anti-RAGE F(ab').sub.2 considerably
suppressed inflammation (p<0.05 in each case compared with
treatment with nonimmune F(ab').sub.2. When mice were treated with
both anti-EN-RAGE and anti-RAGE F(ab').sub.2, even further
suppression of the inflammatory response eventuated (p<0.05
compared with treatment with nonimmune F(ab').sub.2 (FIG. 8).
Discussion
[0243] The inflammation phenotype observed in delayed-type
hypersensitivity reactions certainly represent the culmination of a
complex interplay and contribution of multiple cell types and their
cellular mediators. In the development of inflammation, an
important source of the stimuli may be from the inflammatory cells
themselves. Upon initial recruitment into an inflammatory locus,
cells such as neutrophils and macrophages may release mediators
such as those of the calgranulin family, including EN-RAGE, and
propagate and sustain the inflammatory response. Such mediators,
such as EN-RAGE, likely require cellular receptors to initiate
events that will culminate in altered gene expression.
[0244] Our data strongly suggest that EN-RAGE-RAGE interaction is
an important factor in these processes. Nearly complete suppression
of inflammation was noted in the presence of sRAGE, in a
dose-dependent manner. Based upon our studies, sRAGE may act as a
decoy in this setting to bind EN-RAGE prior to its ability to
engage RAGE-bearing cells implicated in the inflammatory response.
Furthermore, in the presence of anti-RAGE/anti-EN-RAGE or
anti-RAGE+anti-EN-RAGE F(ab').sub.2, substantial suppression of
inflammation was observed, further indicating a role of these
factors in the modulation of the inflammatory response.
[0245] It is important to note, of course, that alternate
mechanisms underlying the beneficial effects of sRAGE may be
operative in these settings. However, the studies noted above
employing the indicated F(ab').sub.2 fragments, strongly implicate
EN-RAGE and RAGE in the evolution of the inflammatory response in
this setting.
[0246] In conclusion, the studies presented herein implicate RAGE
centrally in the inflammatory response and identify soluble RAGE as
a prototypic structure for the development of novel,
anti-inflammatory agents.
[0247] Note: FIG. 9 shows the nucleic acid sequence (cDNA sequence)
of bovine EN-RAGE.
TABLE-US-00005 TABLE 1 Sequence analysis of EN-RAGE and comparison
with related proteins. 1 10 20 30 EN-RAGE
TKLEDHLEGIINIGHQYSVRVGHFDTLNKY N-TERM Endo Lys C B-COAg
TKLEDHLEGIINIFHQYSVRVGHFDTLNKR B-CAAFI
TKLEDHLEGIINIFHQYSVRVGHFDTLNKR 31 40 50 60 EN-RAGE
ELKQLGTKELPKTLQNXKDQ N-TERM Endo Lys C B-COAg
ELKQLITKELPKTLQNTKDQPTIDKIFQDL B-CAAFI
ELKQLITKELPKTLQNTKDQPTIDKIFQDL 61 70 80 90 EN-RAGE N-TERM Endo Lys
C DGAVSFEEFVVLVSRVLK B-COAg DADKDGAVSFEEFVVLVSRVLKTAHIDIHK B-CAAFI
DADKDGAVSFEEFVVLVSRVLKTAHIDIHK (SEQ ID NOS:9, 10, 11, 12,
respectively).
EN-RAGE (Extracellular Novel-RAGE Binding Protein) Activated
Endothelial Cells to Mediate Inflammatory Responses.
[0248] The expression of Receptor for AGE (RAGE) is enhanced in
inflammatory settings such as atherosclerosis and autoimmune
vasculitities. We hypothesized that Receptor for AGE (RAGE) might
interact with alternative ligands beyond Advanced Glycation
Endproducts (AGEs) in such settings. We isolated and purified an
.apprxeq.12 kDa polypeptide from extract of bovine lung which bore
homology to the calgranulin family of proinflammatory mediators.
This polypeptide, called EN-RAGE, binds immobilized RAGE and
endothelial (EC)/macrophage (MP) RAGE in culture wells with
Kd.apprxeq.75 nM, processes blocked in the presence of anti-RAGE
IgG or soluble (sRAGE; the extracellular two-thirds of RAGE). In
vitro, exposure of cultured ECs to EN-RAGE increased activation of
NF-kB, expression of cell-surface VCAM-1 (4.3-fold compared to
treatment with bovine serum albumin BSA), and adhesion of Molt-4
cells (which bear VLA-4, the counter-ligand for VCAM-1) (7-fold
compared with BSA), all in a manner inhibited in the presence of
anti-RAGE IgG or sRAGE. Exposure of macrophages to EN-RAGE resulted
in increased chemotaxis in a RAGE-dependent manner. To test these
concepts in vivo, we utilized a model of delayed hypersensitivity
in mice in which footpad injections of methylated BSA (mBSA) induce
localized inflammation. Pre-treatment (intraperitoneal; IP) with
sRAGE prevented mBSA-mediated inflammation in a dose-dependent
manner. At 100 .mu.g IP sRAGE, the mBSA-treated foot manifested no
inflammation and markedly diminished activation of NF-kB compared
with mice treated with vehicle, mouse serum albumin (MSA); further,
elaboration of TNF-alpha into the serum was completely prevented.
Partial anti-inflammatory responses were observed upon treatment of
the mice with either anti-RAGE or anti-EN-RAGE F(ab')2. Nonimmune
F(ab')2 was without effect. Taken together, these findings indicate
that ligands alternative to AGEs such as EN-RAGE activate ECs and
MPs, thereby linking RAGE to the generalized inflammatory
response.
sRAGE Results in Diminished Mortality After Endotoxemia: A
Potential Treatment for Septic Shock
[0249] The use of sRAGE or compounds which are capable of
inhibiting the interaction of EN-RAGE and RAGE could be useful
agents for the treatment of septic shock or sepsis in subjects. It
has been shown that a subject given lethal doses of LPS has reduced
mortality when the LPS is given in the presence of sRAGE.
sRAGE and Endotoxemia
[0250] Soluble Receptor for AGE (sRAGE) has been shown to prevent
inflammation in a model of delayed-type hypersensitivity. Unlike
certain anti-inflammatory-type agents, it was believed that sRAGE
might exert beneficial effects when administered in the setting of
endotoxemia, a prototypic result of, for example, profound gram
negative bacteremia.
[0251] When uniformly lethal doses of LPS were administered to
Balb/C mice (=750 .mu.g), administration of sRAGE (pre or post LPS
injection) prevented death in .apprxeq.50% of the mice in pilot
studies.
[0252] These data underscore the proposition that the potent
anti-inflammatory effects of sRAGE are not associated with an
untoward inclination toward morbidity/mortality due to the presence
of septicemia/endotoxemia. sRAGE, therefore, may be a selective
anti-inflammatory agent with selective protective effects against
maladaptive inflammatory responses.
REFERENCES FOR EXAMPLE 3
[0253] 1. Schmidt, A. M., Vianna, M., Gerlach, M., Brett, J., Ryan,
J., Kao, J., Esposito, C., Hegarty, H., Hurley, W., Clauss, M.,
Wang, F., Pan, Y. C., Tsang, T. C., and Stern, D. Isolation and
characterization of binding proteins for advanced glycosylation
endproducts from lung tissue which are present on the endothelial
cell surface. J. Biol. Chem. 267:14987-14997, 1992. [0254] 2.
Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D., Wang, F., Pan,
Y. C., Elliston, K., Stern, D., and Shaw, A. Cloning and expression
of RAGE: a cell surface receptor for advanced glycosylation end
products of proteins. J. Biol. Chem. 267: 14998-15004, 1992. [0255]
3. Schmidt, A-M, Hori, O, Brett, J, Yan, S-D, Wautier, J-L, and
Stern D. Cellular receptors for advanced glycation end products.
Arterioscler. Thromb. 14:1521-1528, 1994. [0256] 4. Schmidt, A. M.,
S D Yan, and D. Stern. The Dark Side of Glucose (News and Views).
Nature Medicine 1:1002-1004, 1995. [0257] 5. Yan, S-D, Schmidt,
A-M, Anderson, G, Zhang, J, Brett, J, Zou, Y-S, Pinsky, D, and
Stern, D. Enhanced cellular oxidant stress by the interaction of
advanced glycation endproducts with their receptors/binding
proteins. J. Biol. Chem. 269:9889-9897, 1994. [0258] 6. Schmidt,
A-M, Yan, S-D, Brett, J, Mora, R, Nowygrod, R, and Stern D.
Regulation of mononuclear phagocyte migration by cell surface
binding proteins for advanced glycosylation endproducts. J. Clin.
Invest. 92:2155-2168, 1993. [0259] 7. Wautier, J L, Chappey, O,
Wautier, M P, Hori, O, Stern, D, and Schmidt A M. Receptor-mediated
endothelial dysfunction in diabetic vasculopathy: sRAGE blocks
hyperpermeability. J. Clin. Invest. 97:238-243, 1996. [0260] 8.
Miyata, T., Hori, O, Zhang, J H, Yan, S D, Ferran, L, Iida, Y, and
Schmidt, A M. The Receptor for Advanced Glycation Endproducts
(RAGE) mediates the interaction of AGE-b.sup.2-Microglobulin with
human mononuclear phagocytes via an oxidant-sensitive pathway:
implications for the pathogenesis of dialysis-related amyloidosis.
J. Clin. Invest. 98:1088-1094, 1996. [0261] 9. Schmidt, A-M, Hasu,
M, Popov, D, Zhang, J-H, Chen, J, Yan, S-D, Brett, J, Cao, R,
Kuwabara, K, Gabriela, C, Simionescu, N, Simionescu, M, and Stern
D. Receptor for advanced glycation endproducts (AGEs) has a central
role in vessel wall interactions and gene activation in response to
circulating AGE proteins. PNAS(USA) 91:8807-8811, 1994. [0262] 10.
Schmidt, A M, Hori, O, Chen, J, Brett, J, and Stern, D. AGE
interaction with their endothelial receptor induce expression of
VCAM-1: a potential mechanism for the accelerated vasculopathy of
diabetes. J. Clin. Invest. 96:1395-1403, 1995. [0263] 11. Lander,
H. L., Tauras, J. M., Ogiste, J. S., Moss, R. A., and A. M.
Schmidt. Activation of the Receptor for Advanced Glycation
Endproducts triggers a MAP Kinase pathway regulated by oxidant
stress. J. Biol. Chem. 272:17810-17814, 1997. [0264] 12. Park, L.,
Raman, K. G., Lee, K. J., Yan, L., Ferran, L. J., Chow, W. S.,
Stern, D., and Schmidt, A. M. Suppression of accelerated diabetic
atherosclerosis by soluble Receptor for AGE (sRAGE). Nature
Medicine 4:1025-1031, 1998. [0265] 13. Wautier J L, Chappey O,
Wautier M P, Boval B, Stern D and A M Schmidt. Interaction of
diabetic erythrocytes bearing advanced glycation endproducts with
the endothelial receptor RAGE induces generation of reactive oxygen
intermediates and cellular dysfunction. Circ. 94 (8):#4139, 1996.
[0266] 14. Hori, O., J. Brett, T. Slattery, R. Cao, J. Zhang, J.
Chen, M. Nagashima, D. Nitecki, J. Morser, D. Stern, A. M. Schmidt.
The Receptor for Advanced Glycation Endproducts (RAGE) is a
cellular binding site for amphoterin: mediation of neurite
outgrowth and co-expression of RAGE and amphoterin in the
developing nervous system. J. Biol. Chem. 270:25752-25761, 1995.
[0267] 15. Yan, S D, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T.
Slattery, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, G.
Godman, D. Stern, and A. M. Schmidt. RAGE and amyloid-b peptide
neurotoxicity in Alzheimer's disease. Nature 382:685-691, 1996.
[0268] 16. Yan, S-D., Zhu, H., Fu, J., Yan, S-F., Roher, A.,
Tourtellotte, W., Rajavashisth, T., Chen, X., Stern, D. and
Schmidt, A-M. Amyloid-beta peptide-RAGE interaction elicits
neuronal expression of M-CSF: a proinflammatory pathway in
Alzheimer's disease. Proc. Natl. Acad. Sci. 94:5296-5301, 1997.
[0269] 17. Slattery, T. K. and Harkins, R. N. Techniques in protein
chemistry IV, ed. Angeletti, R. H., Academic Press, San Diego,
Calif., 1992. [0270] 18. Jaffe, E., Nachman, R., Becker, C., and
Minick, R. Culture of human endothelial cells derived from
umbilical veins. Identification by morphologic and immunologic
criteria. J. Clin. Invest. 52:2745-2756, 1973. [0271] 19. Dunn, C.
J., Galinet, L. A., Wu, H., Nugent, R. A., Schlachter, S. T.,
Staite, N. D., Aspar, D. G., Elliott, G. A., Essani, N. A.,
Rohloff, N. A., and Smith, R. J. Demonstration of novel
anti-arthritic and anti-inflammatory effects of diphosphonates. J.
Pharmacology and Experimental Therapeutics 266: 1691-1698, 1993.
[0272] 20. Wicki, R., Marenholz, I., Mischke, D., Schafer, B. W.,
and Heizmann, C. W. Characterization of the human S100A12
(calgranulin C, p6, CAAF1, CGRP) gene, a new member of the S100
gene cluster on chromosome 1q21. Cell Calcium 20:459-464, 1996.
[0273] 21. Dell'Angelica, E. C., Schleicher, C. H., and Santome, J.
A. Primary structure and binding properties of calgranulin C, a
novel S100-like calcium-binding protein from pig granulocytes. J.
Biol. Chem. 269:28929-28936, 1994. [0274] 22. Wu J, Rogers L, Stern
D, Schmidt A M and Chiu D T W. The soluble receptor for Advanced
Glycation Endproducts (sRAGE) ameliorates impaired wound healing in
diabetic mice. Plastic Surgery Research Council, Abstract #77, p.
43, 1997.
Example 4
Treatment of Collagen-Induced Arthritis as an Example of Treating
Inflammation
Rage (G82S) and Rheumatoid Arthritis: Increased Susceptibility and
Upregulation of the Inflammatory Response
[0275] Receptor for Advanced Glycation Endproducts (RAGE) and its
proinflammatory S100/calgranulin ligands (1) are enriched in joints
of patients with rheumatoid arthritis (RA) (2-4). Linkage
disequilibrium with an RA-associated HLA-DR4 haplotype (5-6), and a
polymorphism of the RAGE gene (G82S) (7-9), suggested a role for
RAGE in RA. We demonstrate here that the prevalence of RAGE (G82S)
is significantly increased in RA compared with controls, even in
DR4-negative subjects. Cells bearing mutant RAGE (82S), either
stably-transfected CHO cells or patient-derived mononuclear
phagocytes, display enhanced responses following engagement of a
prototypic S100/calgranulin, compared with cells expressing
wild-type RAGE. Blockade of RAGE in a collagen-induced arthritis
model (10-12) suppressed clinical and histologic evidence of
arthritis, in parallel with diminished levels of proinflammatory
mediators and markers of tissue degradation. These findings
associate RAGE (G82S) with increased susceptibility to RA, and
suggest that RAGE (G82S) primes joint tissue for enhanced
inflammation and destruction in evolving arthritis.
[0276] MHC-linked genes are widely accepted contributors to
susceptibility in autoimmune/inflammatory disorders, though the
identity of these genes has yet to be fully elucidated. Indeed, it
is highly likely that multiple genes within the MHC, and, perhaps,
outside this complex, are involved in human autoimmunity.
Specifically, in rheumatoid arthritis (RA), polymorphisms at the
HLA-DRB1 locus, particularly within the DRB1*04 and DRB1*01 groups
of alleles, have been most strongly associated with development of
disease (5-6, 13). However, despite intense investigation, a
definitive understanding of the link between these alleles and
their involvement in susceptibility and/or evolution of
proinflammatory phenomena in RA has not been achieved. The gene
encoding RAGE, a multi-ligand member of the immunoglobulin
superfamily of cell surface molecules, is located approximately 400
kb from HLA-DRB1 and 300 kb from HLA-DRA, near the junction of MHC
Class II and Class III (7). Furthermore, expression of RAGE and its
proinflammatory ligands of the S100/calgranulin family (termed
EN-RAGEs or Extracellular, Newly-identified RAGE binding proteins)
(1) is enhanced in affected rheumatoid synovial tissue (2-4). These
considerations led us to test whether the RAGE polymorphism (G82S),
predictably increasing hydrophilicity of a critical portion of the
receptor's extracellular domain involved in ligand binding, might
confer increased susceptibility to RA, as well as enhanced
generation of proinflammatory and tissue destructive mediators
important in the evolution of arthritis.
[0277] Genomic DNA from RA patients and controls was analyzed using
PCR amplification of RAGE exon 3 followed by digestion of the
product with Alu I in order to identify subjects bearing the GG, GS
and SS genotypes (FIG. 10). Among Caucasian subjects, 76/345 (22a)
of patients with RA carried the S allele, compared with 10/190
(5.3%) control subjects, thus yielding a highly-significant
association of RAGE (G82S) with RA, with an estimated relative risk
(RR) of 5.0 (95% confidence interval [CI] 2.5-10.0), and p<0.001
(Table 2). Since the (G82S) polymorphism has recently been found to
be in linkage disequilibrium with a common RA-associated haplotype
(9), DRB*0401--DQA1*0301--DQB1*0301, it is difficult to
definitively establish whether RAGE itself contributes to disease
risk in the context of this haplotype. Therefore, we focussed on
the subset of patients and controls who do not carry HLA-DR4
haplotypes. As shown in Table 2, when only DR4 negative subjects
are considered, the RAGE (G82S) allele continues to exhibit a
significant association with RA, which an estimated RR=5.9, (95% CI
1.3-28), and p=0.011. These observations indicate that presence of
the RAGE G82S allele confers enhanced susceptibility to RA, even in
DR4 negative subjects. Interestingly, no evidence of linkage
disequilibrium between RAGE (G82S) and DRB1*0101 was seen.
[0278] RA joints display high levels of S100/calgranulins, a family
of polypeptides associated with inflammatory processes, which
transduce their signal of cellular activation via RAGE. To move
from genetic associations to changes in cell function associated
with inflammatory pathways underlying RA, we investigated whether
RAGE (G82S) would display altered affinity and activation of signal
transduction pathways, compared with the wild-type receptor, when
exposed to a prototypic S100/calgranulin that we previously termed
EN-RAGE (1). Chinese hamster ovary (CHO) cells provide a convenient
model, as they are devoid of detectable RAGE prior to, or after
stable transfection with pcDNA3.1 vector alone (FIG. 11A).
Stably-transfected CHO cells were made with pcDNA3.1 containing
either wild-type RAGE or mutant RAGE (82S) (FIG. 11A). Radioligand
binding studies showed dose-dependent binding of .sup.125I-EN-RAGE
to CHO cells expressing wild-type (.apprxeq.122.+-.31 nM) and
mutant receptor (Kd.apprxeq.77.+-.21 nM), though the affinity of
binding was greater with the mutant receptor (FIG. 2b; p=0.008). In
contrast, CHO cells stably transfected with the empty vector (mock)
displayed no specific binding of .sup.125I-EN-RAGE (FIG. 11B). That
the interaction of RAGE-bearing CHO cells with .sup.125I-EN-RAGE
was specific for interaction with RAGE was shown by inhibition of
specific binding in the presence of an excess of soluble
extracellular domain of the receptor (sRAGE), or anti-RAGE IgG
(FIG. 1C). In contrast, addition of bovine serum albumin (BSA) or
nonimmune IgG was without effect.
[0279] These observations led us to test the concept that
engagement of RAGE (82S) on transfected CHO cells by EN-RAGE might
amplify cellular activation beyond that seen in cells bearing
wild-type RAGE. Incubation of mock-transfected CHO cells with
EN-RAGE (10 .mu.g/ml) did not increase intensity of the bands
corresponding to phosphorylated p44/42 MAP kinases (14-15) (FIG.
1D, lanes 1-5). However, exposure of wild-type RAGE CHO
transfectants to EN-RAGE increased by .apprxeq.2-fold
phosphorylated p44/42 MAP kinases, compared with cultures incubated
with BSA alone (FIG. 11D, lanes 7&6, respectively; p<0.01).
CHO transfectants bearing RAGE (82S) incubated with EN-RAGE
displayed .apprxeq.3.9-fold increase in phosphorylated p44/42
compared with BSA (FIG. 1D, lanes 12 & 11, respectively;
p<0.01). Compared with cells expressing wild-type RAGE, CHO
cells expressing mutant RAGE displayed significantly increased
phosphorylation of p44/p42 MAP kinases (FIG. 1D, lanes 12 & 7,
respectively; p<0.05). In both wild-type RAGE- and mutant
RAGE-transfected cells, cellular activation by EN-RAGE was due to
engagement of RAGE as demonstrated by suppression of
phosphorylation of p44/p42 in the presence of excess sRAGE (FIG.
11D, lanes 8 & 13, respectively), or anti-RAGE IgG (FIG. 11D,
lanes 9&14, respectively). Nonimmune IgG was without effect
(FIG. 11D, lanes 10 & 15, respectively). In all cases, levels
of nonphosphorylated p44/p42 MAP kinases were identical.
[0280] To further support the hypothesis that the presence of
mutant RAGE (82S) enhanced activation of key proinflammatory signal
transduction pathways, we assessed nuclear translocation of NF-kB
in CHO transfectants exposed to EN-RAGE. Electrophoretic mobility
shift assays (EMSA) using .sup.32P-labelled consensus NF-.kappa.B
probe and nuclear extracts from mock-transfected CHO cells showed
no increase in intensity of the gel shift band after cultures were
exposed to EN-RAGE (FIG. 11E, lane 2). When these experiments were
repeated with CHO transfectants expressing wild-type RAGE, there
was a prominent .apprxeq.5.4-fold increase in intensity in nuclear
binding activity following incubation of cultures with EN-RAGE
compared to BSA (FIG. 11E, lanes 7&6, respectively).
NF-.kappa.B activation was even more striking when RAGE (82S) was
substituted for wild-type RAGE; RAGE (82S) CHO transfectants
displayed .apprxeq.11.3-fold increased intensity of the gel shift
band consequent to the presence of EN-RAGE, compared to incubation
with BSA (FIG. 11E, lanes 12&11, respectively). Thus,
RAGE-mediated NF-.kappa.B activation due to EN-RAGE was enhanced by
.apprxeq.2.1-fold comparing mutant to wild-type receptor. That
activation of NF-.kappa.B in transfected CHO cells by EN-RAGE
resulted from ligation of wild-type or mutant RAGE was confirmed by
its inhibition in the presence of sRAGE (FIG. 11E, lanes 8&13,
respectively), or anti-RAGE IgG (FIG. 11E, lanes 9&14,
respectively). Incubation with nonimmune IgG had no effect (FIG.
11E, lanes 10&15, respectively).
[0281] A critical test of these concepts was whether mononuclear
phagocytes (MPs) retrieved from human subjects bearing a mutant
RAGE allele displayed enhanced activation and generation of
proinflammatory mediators in the presence of EN-RAGE.
Immunoblotting revealed that basal levels of RAGE did not differ
between MPs bearing wild-type (G82G), (G82S) or (S82S) alleles.
Signaling was compared in MPs from patients with RAGE (G82G) and
RAGE (G82S)/(S82S) by assessing activation of p44/p42 MAP kinases.
In the presence of EN-RAGE, MPs isolated from individuals bearing
mutant RAGE displayed an .apprxeq.4.8-fold increase in
phosphorylated p44 and p42 MAP kinases compared with unstimulated
cells (FIGS. 12A&B). However, MPs bearing wild-type RAGE
exposed to EN-RAGE revealed a significant, although smaller
(.apprxeq.2.2-fold) increase in activation of p44/p42 MAP kinases
(FIGS. 12A&B). The differences between EN-RAGE-mediated
activation of p44/p42 MAP kinases in mutant vs. wild-type
RAGE-expressing MPs were significant, p<0.05 (FIG. 12B).
[0282] In order to assess the functional consequences of enhanced
activation of signal transduction molecules stimulated upon
ligation of RAGE, we examined production of key inflammatory and
tissue-degradative mediators linked to RA (16-19) by MPs bearing
wild-type RAGE or the mutant allele. Exposure of wild-type
RAGE-bearing MPs to EN-RAGE caused an .apprxeq.4-fold increase in
generation of TNF-alpha detected in culture supernatant compared
with quiescent cultures (322.+-.51 vs 81.+-.8.3 ng/ml; p<0.001)
(FIG. 12C). However, upon incubation of human MPs bearing GS or SS
RAGE, an .apprxeq.17.8-fold increase in elaborated TNF-alpha was
observed in culture supernatants compared to basal levels
(1,623.+-.98 vs 91.+-.11.1 ng/ml; p<0.001) (FIG. 12C).
Importantly, although basal levels of TNF-alpha did not differ
between wild-type RAGE- and mutant RAGE-bearing MPs, levels of
TNF-alpha were .apprxeq.4.5-fold more in the presence of RAGE
(G82S)/(S82S) compared with wild-type RAGE, p<0.01 (FIG. 12C).
Similarly, wild-type RAGE-bearing MPs exposed to EN-RAGE displayed
a small, but significant .apprxeq.1.5-fold increase in generation
of IL-6 compared with basal expression (29.2.+-.4.1 vs 20.2.+-.3.2
ng/ml; p<0.05) (FIG. 12D). However, MPs bearing GS or SS
revealed an A10.9-fold augmented generation of IL-6 upon incubation
with EN-RAGE compared with unstimulated controls (229.3.+-.26.8 vs
21.+-.1.8 ng/ml; p<0.001) (FIG. 12D). Again, MPs bearing mutant
RAGE generated increased amounts of IL-6 compared with cells from
wild-type individuals (=7.3-fold; p<0.01) (FIG. 12D). In these
studies, no significant differences between cellular activation
induced by EN-RAGE in MPs bearing (G82S) or (S82S) RAGE were
observed.
[0283] A central means by which structural elements of joints, such
as cartilage and bone, are degraded in unchecked RA is by
generation of matrix metalloproteinases (MMP), such as MMP-9. We
hypothesized that RAGE-mediated MP activation would augment
generation of MMP-9 activity on cells bearing mutant receptor
(G82S, S82S) versus those expressing wild-type RAGE. MPs retrieved
from subjects bearing wild-type RAGE displayed an .apprxeq.2.3-fold
increase in MMP-9 activity in the presence of EN-RAGE compared with
basal expression (p<0.01; FIGS. 12E&F). However, MPs
isolated from individuals bearing RAGE (G82S)/(S82S) demonstrated
an .apprxeq.4.7-fold increase in EN-RAGE-mediated MMP-9 activity
compared with basal levels of expression, p<0.01. Although basal
levels of MMP-9 activity did not differ among GG- or GS/SS-bearing
MPs, the extent of EN-RAGE-mediated enhanced MMP-9 activity was
significantly enhanced, .apprxeq.2-fold, in MPs bearing mutant RAGE
allele vs. wild-type receptor (p<0.01; FIGS. 12E&F).
[0284] These findings strongly suggest that in human subjects, the
presence of the 82S allele contributes, at least in part, to
enhanced susceptibility to RA, as well as to EN-RAGE-mediated
increased expression of proinflammatory and tissue-destructive
mediators highly-prevalent in rheumatoid synovium. To determine if
the interaction of EN-RAGE with RAGE modulated joint inflammation
and destruction in vivo, we employed a murine model of
polyarticular inflammatory arthritis induced by sensitization and
challenge with bovine type II collagen (10-12), the predominant
protein of articular cartilage, in dba/1 mice. Bovine type II
collagen was emulsified in incomplete Freund's adjuvant and
injected intradermally at the base of the tail (time 0;
immunization). Three weeks later, mice were challenged with a
second intradermal injection of bovine collagen type II/incomplete
Freund's adjuvant (time 3 weeks; challenge). The contribution of
RAGE to the pathogenesis of arthritis was studied by treating
animals with sRAGE (20). The administered exogenous sRAGE functions
as a decoy by engaging RAGE ligands and preventing their access to
cell surface receptor. Treatment with sRAGE, 100 .mu.g/day, was
started at three weeks (time of challenge with bovine type II
collagen). In previous studies, blockade of RAGE at this dose
affected the greatest decrease in the proinflammatory phenotype in
a murine model of delayed-type hypersensitivity (1).
[0285] The relevance of RAGE-ligand interaction in collagen-induced
arthritis was underscored by the increased expression of RAGE and
EN-RAGE in joint tissues. At six weeks, compared with control mice,
joint tissue from the hindpaw of mice immunized/challenged with
type II collagen demonstrated hypertrophy and hyperplasia of
synovial cells (FIGS. 13A&B, respectively). RAGE and EN-RAGE
expression was increased in joint tissue from mice with arthritis
compared with controls (RAGE, FIGS. 13C&D; EN-RAGE, FIGS.
13E&F, respectively). Immunoblots of joint tissue from control
animals and those with arthritis to detect RAGE and EN-RAGEs showed
increased expression in each case. RAGE levels were enhanced
.apprxeq.2.2-fold (p<0.001) in arthritis versus control joints
(FIG. 13I). Although EN-RAGEs were not detectable in joint tissue
of control mice, expression of these proinflammatory mediators was
induced in vehicle (murine serum albumin [MSA])-treated mice
immunized/challenged with bovine type II collagen (FIG. 13J). In
mice treated with sRAGE, levels of RAGE and EN-RAGE antigen by
immunoblotting were significantly reduced compared to mice treated
with MSA (FIG. 13I&J, respectively).
[0286] Consistent with the observation that blockade of RAGE in
this model of inflammatory arthritis reduced expression of RAGE and
accumulation/expression of proinflammatory EN-RAGEs, mice treated
with sRAGE displayed little evidence of foot pad
swelling/thickening in contrast to prominent swelling observed in
MSA-treated mice evaluated at multiple time points between 3.5-8
weeks after immunization; p<0.001 (FIG. 14A). Similarly,
clinical scoring of inflammatory arthritis at the wrist joint of
immunized/challenged mice revealed a significant reduction in mice
treated with sRAGE versus MSA (p=0.0001; FIG. 14B).
[0287] In order to dissect the molecular mechanisms underlying the
apparent protection afforded by preventing ligands from engaging
cell surface RAGE by administration of sRAGE, we assessed plasma
and joint tissue markers of inflammation. Immunoblots revealed
undetectable TNF-alpha in joint tissue of control mice, whereas a
striking induction was observed in MSA-treated mice
immunized/challenged with bovine type II collagen (FIG. 14C).
Animals subjected to the arthritis protocol and treated with sRAGE
showed striking reduction in TNF-alpha (.apprxeq.25.7-fold;
p=0.001). Similarly, plasma TNF-alpha antigen, although
undetectable in control mice, was markedly induced in plasma
retrieved from mice immunized/challenged mice with type II collagen
and treated with MSA (FIG. 14D). Plasma TNF-alpha was suppressed
.apprxeq.2.4-fold in samples from mice treated with sRAGE
(46.+-.6.2 vs 19.+-.1.2 ng/ml, respectively; p=0.03). IL-6 antigen
in joint tissue increased in MSA-treated mice immunized/challenged
with type II collagen compared with those animals receiving sRAGE
(1,260.+-.465 vs 478.+-.153 ng/.mu.g tissue; p=0.04) (FIG. 14E). No
measurable levels of IL-6 were detected in tissue retrieved from
control mice. Similarly, levels of IL-2 were reduced in joint
tissue of mice treated with sRAGE (FIG. 14F).
[0288] As induction of TNF-alpha and other inflammatory cytokines
sets in motion events leading to activation of latent/proenzyme
MMPs (21), we assessed MMP antigen and activity in stifle joint
tissue retrieved from the mice employed in this model. Compared
with control joint tissue, that retrieved from MSA-treated mice
undergoing the collagen-induced arthritis protocol revealed
.apprxeq.2.4-fold increase in MMP-2 antigen by immunoblotting
(p=0.01; FIG. 15A). That activation of RAGE was critical in this
process was demonstrated by the significant reduction in MMP-2
expression in joint tissue of sRAGE-treated mice, to levels
observed in unaffected mice (p=0.02; FIG. 15A). In addition,
expression of MMP-9 antigen was increased .apprxeq.4.6-fold in
joint tissue retrieved from MSA-treated mice compared with animals
without arthritis (p=0.01; FIG. 15B). Levels of MMP-9 antigen were
significantly reduced in mice receiving sRAGE compared with those
mice receiving MSA; p=0.02 (FIG. 15B). In order to determine the
extent of activity of MMPs 2 and 9 in the joint tissue, we
performed zymography. Consistent with increased levels of MMP-2 and
MMP-9 antigen in mice with arthritis, an .apprxeq.11.6- and
.apprxeq.5.5-fold increase in activity of MMP-2 and MMP-9,
respectively, was observed in joint tissue from vehicle,
MSA-treated mice compared with unafffected mice; p=0.001 and
p=0.02, respectively (FIG. 15C-D). In the presence of sRAGE, levels
of MMP-2 and MMP-9 activity were reduced by .apprxeq.12.2- and
4.2-fold, respectively, compared with mice receiving MSA; p=0.004
and p=0.005, respectively (FIG. 15C-D).
[0289] Lastly, in order to determine if blockade of RAGE suppressed
immune/inflammatory responses to bovine type II collagen at
extra-articular sites, at 6 weeks after immunization, immediately
prior to sacrifice, mice receiving either MSA or sRAGE were
injected with bovine type II collagen (10 .mu.g) into ear tissue.
Although baseline ear thickness was essentially identical in both
groups of mice, 18 hrs after injection, mice receiving MSA revealed
an .apprxeq.2.1-fold increase in ear thickness compared with those
mice injected with sRAGE (p=0.03; FIG. 16A). Consistent with these
observations, splenocytes retrieved from MSA-treated mice at six
weeks revealed significantly increased proliferation, as measured
by incorporation of tritiated thymidine, upon stimulation with
bovine type II collagen compared with mice treated with sRAGE;
p=0.003 (FIG. 16B). However, no significant differences in basal
levels of proliferation, or proliferation in the presence of PMA,
were observed between mice treated MSA vs sRAGE (FIG. 16B).
[0290] The S1000/calgranulin family of proinflammatory molecules,
long-associated with classic immune/inflammatory disorders (22-23),
has been mechanistically linked to cellular activation resulting in
an inflammatory phenotype by the observation that these molecules
are signal-transducing ligands of RAGE. Their release by activated
inflammatory effector cells, and accumulation in synovial fluid and
plasma of patients with RA has been linked to indices of disease
severity (4), such as bony erosions. In this context, Czech
subjects with psoriasis vulgaris, an immune/inflammatory disease of
the skin, displayed enrichment for the RAGE (G82S) allele compared
with age-matched subjects without this skin disorder (24).
Furthermore, strongly increased expression of a member of the S100
family of proinflammatory molecules, "psoriasin", has been
demonstrated in psoriatic lesions compared with adjacent unaffected
skin (25). These observations further support the premise that
S100/calgranulin-RAGE interaction may provide a mechanism
contributing to immune/inflammatory disorders. Finally, the data
presented herein suggests the possibility that the RAGE (G82S)
allele might prime affected tissues for exaggerated inflammatory
processes.
[0291] Previous studies demonstrated that the ligands of RAGE
identified thus far, each effectively cross-compete in radioligand
binding assays (1). These ligands include EN-RAGE (S100A12) and
related members of the S100/calgranulin family of proinflammatory
cytokines (1); Advanced Glycation Endproducts (AGEs) and,
particularly, carboxy(methyl lysine) (CML) adducts of proteins and
lipids (26-27); amyloid-.beta. peptide (28); and amphoterin
(15,29). Our studies support the contention that the primary
binding site for each of these ligands is within the V-domain (27),
the same region in which the (G82S) substitution occurs. Indeed,
substitution of glycine with serine at this site is likely to alter
polarity within that region. Consistent with this concept, we
demonstrated enhanced affinity and cellular activation mediated by
one of the receptor's ligands, EN-RAGE, on interaction with mutant
RAGE (82S) compared with wild-type RAGE. Although studies to
identify and characterize the tertiary structure of RAGE are
underway, it is nevertheless certain that altered properties of
ligand engagement ensue in the face of this polymorphism.
[0292] The present studies have demonstrated an association between
the (G82S) RAGE polymorphism and susceptibility to RA, including
those without an HLA-DR4 allele, thus providing critical evidence
that our findings do not solely reflect the known linkage
disequilibrium between this polymorphism and HLA-DRB1*04
haplotypes. In the future, a detailed haplotypic analysis of the
MHC (30) may provide further evidence of genetic heterogeneity
underlying MHC-linked susceptibility to RA. Studies are ongoing to
define the precise molecular cues triggered by this polymorphism
that appear to augur enhanced susceptibility to, and, possibly,
accelerated evolution of proinflammatory and tissue-degradative
properties in rheumatoid synovium. Certainly, improved
understanding of these complex relationships may refine not only
diagnostic criteria, but, ultimately, optimal means of therapeutic
intervention in this perplexing class of human disorders.
Experimental Methods
[0293] Patient population. The rheumatoid arthritis patients used
for association studies meet the criteria of the American College
of Rheumatology (31) and were taken from patient populations (32)
collected by the North America Rheumatoid Arthritis Consortium and
the Arthritis Research Center in Wichita, Kans.
[0294] Detection of Gly82Ser polymorphism. The following primers
were synthesized for detection of the glycine82serine (G82S)
polymorphism of the RAGE gene (8): sense primer: 5'
GTAAGCGGGGCTCCTGTTGCA-3' (SEQ ID NO:13) and the antisense primer:
5' GGCCAAGGCTGGGGTTGAAGG-3' (SEQ ID NO:14). Whole blood (20 .mu.l)
was obtained from human volunteers in accordance with the standards
and policies of the Institutional Review Boards of the
participating institutions. Genomic DNA was prepared according to
the manufacturer's instructions using a kit from QIAGEN.TM.
(Valencia, Calif.); 10 ng was amplified using Taq I polymerase
(Life Technologies, Grand Island, N.Y.) in a final volume of 25
.mu.l. PCR conditions were as follows: 94.degree. C. for 30 secs,
62.degree. C. for 45 secs, and 72.degree. C. for 60 secs for a
total of 35 cycles. PCR product (25 .mu.l) was then digested with
Alu 1 (Life Technologies), 3U for 16 hrs at 37.degree. C., followed
by gel electrophoresis on agarose gels (2%).
[0295] Chinese Hamster Ovary (CHO) cell studies. Chinese hamster
ovary (CHO) cells were obtained from the American Type Culture
Collection (ATCC) (Manassas, Va.) and cultured in F12 medium
containing fetal bovine serum (10%) (Life Technologies). In order
to generate the mutant 82S allele, the cDNA encoding human RAGE33
was cloned using the TOPO TA.TM. cloning system into pCR2.1TOPO
vector for mutagenesis (Invitrogen, Carlsbad, Calif.).
Site-directed mutagenesis to insert the (Gly82) (wild-type) to
(Ser82) (mutant) change was performed using the GENEEDITOR.TM. In
Vitro SDM System (Promega, Madison, Wis.) according to the
manufacturer's instructions. Sequencing was performed using an
ABI310 automated DNA sequencer (Perkin Elmer Biosystems, Foster
City, Calif.) to confirm the inserted sequence changes and to
ensure that no other mutations were created during any of the
mutagenesis reactions. Both wild-type and mutant RAGE cDNA were
excised from pCR2.1TOPO using EcoR I and subcloned into the
pcDNA3.1 expression vector (Invitrogen). Cells were transfected
with plasmid DNA using lipofectamine (Life Technologies) encoding
the following: pcDNA3.1 containing full-length wild-type RAGE cDNA
(Gly82), pcDNA3.1 containing mutant RAGE cDNA (Ser82) or pcDNA3.1
containing no insert (mock-transfectant). 24 hrs after
transfection, selection was begun using G418 (1 mg/ml) (Life
Technologies). RAGE expression was assessed by immonoblotting in
stably-transfected cells after 6 weeks. Cells were incubated with
the indicated mediators (BSA or EN-RAGEL) and assessed for
activation of phosphorylated p44/p42 MAP kinase, or for nuclear
translocation of NF-kB.
[0296] Radioligand binding assays. Purified EN-RAGE was
radiolabelled using .sup.125-I and Iodobeads (Pierce, Arlington
Heights, Ill.) to a specific activity of approximately 5,000
cpm/ng. Radioligand binding assays were performed in 96-well tissue
culture dishes containing the indicated transfected CHO cells. A
radioligand binding assay was performed in the presence of the
indicated concentration of radiolabelled EN-RAGE.+-.an 50-fold
molar excess of unlabelled EN-RAGE in PBS containing
calcium/magnesium and BSA, 0.2%, for 3 hrs at 37.degree. C. Wells
were washed rapidly with washing buffer (PBS containing Tween 20
(0.05%)). Elution of bound material was performed in a solution
containing heparin, 1 mg/ml. Solution was aspirated from the wells
and counted in a gamma counter (LKB, Gaithersburg, Md.).
Equilibrium binding data were analyzed according to the equation of
Klotz and Hunston (34): B=nKA/1+KA, where B=specifically bound
ligand (total binding, wells incubated with tracer alone, minus
nonspecific binding, wells incubated with tracer in the presence of
excess unlabeled material), n=sites/cell, K=the dissociation
constant, and A=free ligand concentration) using nonlinear
least-squares analysis (Prism; San Diego, Calif.). Where indicated,
pretreatment with either antibodies, or human soluble RAGE, was
performed.
[0297] Activation of p44/p42 MAP kinases. CHO cells were incubated
with EN-RAGE, 10 .mu.g/ml, for one hr. Cells were lysed in lysis
buffer (New England Biolabs, Beverly, Mass.). Cell lysate was
subjected to centrifugation and protein concentration of the
supernatant determined using the Bio-Rad assay (Bio-Rad, Hercules,
Calif.). Equal amounts of protein were subjected to SDS-PAGE
(Novex/Invitrogen, Carlsbad, Calif.). Contents of the gels were
transferred to nitrocellulose and immunoblotting performed using
anti-phosphorylated p44/p42 MAP kinase (New England Biolabs). Bands
were scanned into a densitometer, and band density was quantified
using IMAGEQUANT.TM. (Molecular Dynamics, Foster City, Calif.).
[0298] Electrophoretic mobility shift assay. Nuclear extracts were
prepared and EMSA performed employing consensus .sup.32P-labeled
probe for NF-kB as described (1). Where indicated, cells were
treated with either nonimmune/anti-RAGE F(ab')2, or soluble RAGE,
as described (1,20).
Peripheral Blood-Derived Mononuclear Phagocyte (MPs) Studies.
[0299] Cellular isolation. Whole venous blood was obtained from
healthy volunteers (30 ml) bearing G82G, G82S, and S82S RAGE.
Mononuclear cells were isolated using Histopaque 1077 (Sigma, St.
Louis, Mo.) and cultured on plastic dishes for 3 hrs at 37.degree.
C. Nonadherent cells were removed by washing in phosphate buffered
saline (PBS). Adherent cells (MPs) were removed by incubation with
EDTA (2 mM) for 15 mins at 37.degree. C. Cells were seeded in
tissue-cultured coated wells for study.
[0300] Activation of p44/p42 MAP kinases. MPs were seeded into the
wells of 24-well tissue culture plates at a density of
5.times.10.sup.5 cells per well. Cells were stimulated with either
BSA or EN-RAGE, and immunoblotting for detection of phosphorylated
p44/p42 MAP kinases performed as above.
[0301] Detection of IL-6 and TNF-alpha. MPs were seeded into the
wells of 24-well tissue culture plates at a density of
5.times.10.sup.5 cells per well. Cells were stimulated with either
BSA or EN-RAGE (10 .mu.g/ml). Supernatant was assayed for IL-6 and
TNF-alpha using ELISA kits from R&D systems (Minneapolis,
Minn.) according to the manufacturer's instructions.
[0302] Murine studies: induction of bovine collagen type II-induced
arthritis. Male dba/1 mice were purchased from the Jackson
Laboratories (Bar Harbor, Me.). Mice weighing 20-30 gms were
injected intradermally at the base of the tail with bovine type II
collagen, 200 .mu.g (Sigma) dissolved in acetic acid (0.01M) and
emulsified in incomplete Freund's adjuvant (Sigma). Three weeks
after sensitization, mice were challenged by injection of bovine
collagen type II (200 .mu.g) as above in incomplete Freund's
adjuvant at the base of the tail. Beginning at three weeks after
immunization (at the time of challenge), mice were treated with
either murine soluble RAGE (20), or vehicle, murine serum albumin
(Sigma), both at 100 .mu.g per day by intraperitoneal injection.
Treatment was continued daily until sacrifice.
[0303] Assessment of arthritis. Evidence of arthritis was evaluated
at the indicated time points after initial immunization by an
observer blinded to the experimental conditions. Severity of
arthritis in the wrist joints was assessed according to the
following scale: 0=no redness or swelling; 1=slight/moderate
redness and swelling; and 2=severe redness and swelling. At the
same time points, extent of swelling in the distal footpads was
assessed by measurement of foot pad diameter using calipers. In
each case, the mean of score/footpad diameter was obtained and
reported.
[0304] Injection of bovine collagen II into the ear. Six weeks
after initial sensitization, bovine type II collagen (10 .mu.g) was
injected into the ear of each mouse. 18 hrs later, thickness of the
ear was assessed using calipers by an observer blinded to the
experimental conditions.
[0305] Retrieval of tissues at sacrifice. Mice were sacrificed 3 or
6 weeks after challenge. The stifle joint was removed and
homogenized in Tris-buffered saline containing protease inhibitors
(Complete Protease Inhibitor, Boehringer-Mannheim, Indianapolis,
Ind.). From each animal, joints from the wrist and foot paw were
fixed in formalin (10%) for 16 hrs followed by storage in PBS for
studies using hematoxylin and eosin (H&E) or the indicated
antibodies.
[0306] Assessment of splenocyte proliferation. Spleens were removed
at sacrifice and meshed in RPMI medium (Life Technologies) and
diluted in 10 ml of the same medium. The solution was subjected to
centrifugation at 1,200 rpm at 4.degree. C. for 10 mins. The pellet
was dissolved in RPMI medium (15 ml) and aliquoted. Bovine type II
collagen, or PMA, (0.1 .mu.g/ml in each case) was added for 24 hrs.
Tritiated thymidine (0.02 ml) was then added for an additional 18
hrs. Cells were retrieved and counted in a beta counter (LKB).
[0307] Assessment of plasma TNF-alpha. Upon sacrifice, plasma was
obtained and assessed by ELISA for levels of murine TNF-alpha using
a kit from R&D Systems according to the manufacturer's
instructions.
[0308] Immunoblotting and ELISA. SDS-PAGE and immunoblotting were
performed on extracts of stifle joint tissue using the following
antibodies: anti-RAGE IgG and anti-EN-RAGE IgG as previously
describedl (4.7 and 2.0 .mu.g/ml, respectively); anti-MMP 2 and
anti-MMP 9 IgG (1 .mu.g/ml; Chemicon (Temecula, Calif.); and
anti-TNF-alpha IgG (1 .mu.g/ml; R&D Systems). Bands were
scanned into a densitometer, and band density was quantified using
IMAGEQUANT.TM.. In other experiments, assessment of joint tissue
levels of IL-6 and IL-2 was performed by subjecting joint tissue
lysates to ELISA using kits from R&D systems.
[0309] Zymography. Zymography for detection of MMP-2 and MMP-9
activity were determined using gelatin-laden gels from
Novex/Invitrogen according to the manufacturer instructions.
[0310] Bands were scanned into a densitometer, and band density was
quantified as above.
[0311] Statistical analysis. Statistical comparisons among groups
were determined using one-way analysis of variance (ANOVA); where
indicated, individual comparisons were performed using students'
t-test.
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TABLE-US-00006 [0345] TABLE 2 Prevalence of the RAGE 82S allele in
patients with rheumatoid arthritis (RA) and controls. RA patients
Controls p value All subjects 76/345 (22%) 10/190 (5.3%) <0.001
DR4 negative 9/114 (7.9%) 2/141 (1.4%) 0.011 subjects
Example 5
Treatment of Autoimmune Diseases as an Example of Treating
Inflammation, EAE
Uses of Soluble RAGE Related to Models of Autoimmune: Experimental
Autoimmune Encephalitis and Adoptive Transfer Diabetes
[0346] The experiments below describe the use of soluble(s) RAGE
(Receptor for advanced glycation endproducts) to inhibit the
development of autoimmune (type I) diabetes in an adoptive transfer
model and the occurrence of experimental autoimmune encephalitis
(EAE) both in murine systems.
[0347] The adoptive transfer model of diabetes involves transfer of
splenocytes from diabetic NOD (non-obese diabetic) mice to NOD mice
with severe combined immunodeficiency (scid) (1-2). The latter mice
are termed NOD/scid animals, and they do not develop diabetes
spontaneously. Rather, they require the presence of immunocytes
capable of destroying islet cells for induction of diabetes. This
model was selected for study because: 1) the kinetics of disease in
this model allows for a rapid determination of efficacy; and, 2)
the model is relevant to human disease, especially in the clinical
settings in which future immune therapies and islet transplantation
is likely to occur (1-3). These settings include: arresting the
loss of .beta.-cell function in individuals with new onset of Type
1 diabetes, prevention of diabetes in individuals at high risk for
development of disease, and blockade of disease recurrence in
patients with Type 1 diabetes who received islet transplants. Many
immune and non-specific treatments have been found to prevent the
spontaneous development of diabetes in the NOD mouse, but with very
few exceptions, these approaches have not prevented disease at its
late stages or recurrent disease (3). A notable exception to this
general statement includes treatment with anti-CD3 monoclonal
antibody that can prevent recurrent autoimmune diabetes in
recipients of islet transplants. This drug is now in clinical
trials.
[0348] The myelin basic protein (MBP) model of EAE is a widely
accepted system for studying the pathogenesis of multiple sclerosis
(4-6). Our studies have employed the B10.PL mouse strain and two
means of inducing EAE: immunization with a peptide derived from MBP
and transfer of an encephalitogenic T-cell clone isolated from mice
previously immunized with MBP (1AE10 cells). Preparation of such
encephalitogenic T-cell clones is standard in the literature and
has been described (7-10). Whereas the MBP immunization model
provides a situation to study early disease, including the initial
phase of sensitization to MBP, the adoptive transfer model
simulates a later phase. Namely, the latter is a situation in which
CD4+ lymphocytes already sensitized to MBP and activated (i.e.,
fully capable of causing disease) are administered to an irradiated
recipient mouse which has very limited capacity to resist the
destructive properties of the transferred immunocytes.
Methods
[0349] RAGE-blocking reagents. Two methods were used to prevent
access of RAGE ligands to the receptor. According to the first
method, soluble RAGE (sRAGE) was prepared using recombinant DNA
technology (11). The murine form of sRAGE was expressed in the
baculovirus system and purified to homogeneity based on a single
band on SDS-PAGE. This material was required to have an
undetectable level of lipopolysaccharide using the Limulus
amebocyte assay (Sigma) at an sRAGE concentration of 2 mg/ml.
Polyclonal antibody to RAGE was prepared in rabbits, the IgG was
purified and characterized as described (11). Nonimmune IgG was
prepared from rabbits not sensitized to a particular antigen. This
material was similarly characterized for its content of
lipopolysaccharide.
[0350] Adoptive transfer model of Type 1 diabetes: NOD and NOD/scid
mice were purchased from Jackson Laboratories (Bar Harbor, Me.) and
housed in a pathogen-free facility in the Institute for Comparative
Medicine at Columbia University. Animals were monitored for
development of diabetes by screening for glycosuria. Plasma glucose
levels were measured in mice found to have glycosuria. Mice with
diabetes (two glucose values >250 mg/dl) were sacrificed by
humane euthanasia and a single cell suspension of red blood
cell-depleted splenocytes was prepared. These cells were given
intravenously (IV) to NOD/scid recipients (1.5.times.10.sup.7
cells/recipient). In addition, recipients were then treated with
either sRAGE (100 .mu.g/day, intraperitoneally [ip]) or mouse serum
albumin. Plasma glucose levels were measured in capillary blood
from tail veins. Mice with two values >250 mg/dl were considered
to have diabetes.
[0351] Once diabetes was documented, the mice were sacrificed by
humane euthanasia, and the pancreas was fixed in formalin and
embedded in paraffin. Immunohistologic studies were then performed
on histologic sections according to standard techniques (12).
Antibodies employed for immunohistologic studies were rabbit
anti-tumor necrosis factor-alpha (TNF-a) IgG and rabbit
anti-Interleukin (IL)-1.beta. IgG (Santa Cruz). The protocol for
these studies involved incubation of tissue sections with primary
antibodies (5 .mu.g/ml) overnight at 4.degree. C., followed by
addition of a secondary biotin-conjugated antibody (affinity
purified anti-rabbit IgG; ExtrAvidin kit from Sigma). The
incubation with secondary antibody was for 30 min a 37.degree. C.,
and then substrate (aminoethylcarbazole; AEC) was added (all
procedures were performed according to the manufacturer's
instructions; Sigma). Sections were counterstained with Mayer's
hematoxylin. In other cases, sections were stained with hematoxylin
and eosin (H&E) according to standard procedures (12).
EAE. Induction of EAE Involved Two Model Systems:
[0352] MBP immunization. A peptide comprising the N-terminal nine
amino acids of MBP (sequence:
Acetylated-Ala-Ser-Gln-Arg-Lys-Pro-Ser-Gln-Arg) (SEQ ID NO:15)
(13-14) was prepared in the Peptide Chemistry Core Laboratory of
Columbia University using standard techniques. The peptide (100
.mu.g/animal) was emulsified with complete Freund's adjuvant and
injected subcutaneously (the total volume was 0.1 ml). Animals
(B10.PL mice from Jackson Laboratory) then received two injections
of pertussis toxin (total of 1 .mu.g/mouse) intravenously (Liss
Laboratories) 24 and 72 hours after inoculation with MBP peptide.
Animals were then observed for about 8 weeks for the development of
symptoms of EAE. Following this period, animals were sacrificed by
humane euthanasia. Where indicated, mice were treated with sRAGE
starting at the time of MBP injection. Scoring of symptoms was
according to the following criteria (9): 0, no signs; 1, weakness
of tail; 2, mild paresis of hind limbs (paraparesis); 3, severe
paresis of hind limbs; 4, complete paralysis of him limbs
(paraplegia) or the limbs of one side (hemiplegia); 5, death. At
the time of symptoms, or as indicated, animals were sacrificed, and
the spinal cord was studied histologically. Spinal cord tissue was
fixed in formalin, embedded in paraffin and sections were cut for
H&E staining.
[0353] Transfer of an activated encephalitogenic T-cell clone. For
this model, B10.PL mice were sublethally irradiated (350 R) and
were then subject to adoptive transfer of an MBP-sensitized and in
vitro activated MBP-specific CD4.sup.+V.beta.8.sup.+, Th1 clone
termed 1AE10 (10-15.times.10.sup.6 cells/animal). The in vitro
activation protocol involved culturing cells with MBP peptide (10
.mu.g/ml) in the presence of antigen presenting cells (the adherent
population of splenocytes from B10.PL mice; 2:1 ratio of antigen
presenting cells to 1AE10 cells) for four days and addition of IL-2
(20 U/ml; Hoffmann-LaRoche) during the last 48 hrs to increase cell
number. Animals received intravenous pertussis toxin 24 and 72 hrs
(as above) after infusion of activated 1AE10 cells. Similar T-cell
clones and their use to induce EAE in mice have been described in
the literature (7-10). This T-cell clone has been termed 1AE10
cells. RAGE blockade was achieved using rabbit anti-RAGE IgG (50
Ag/animal/day) administered intraperitoneally) for fifteen days.
Control animals were treated identically except that nonimmune
rabbit IgG was used in place of anti-RAGE IgG. Mice were observed
for 4-6 weeks for the development of symptoms (as above).
Results
[0354] Adoptive transfer model of diabetes. Treatment of NOD/scid
recipients of splenocytes from diabetic NOD mice demonstrated a
strong protective effect of sRAGE against the development of
diabetes (FIG. 17). The islet-sparing effect of sRAGE was
reversible, as discontinuance of sRAGE in 4/4 mice resulted in
subsequent development of diabetes in two separate experiments. The
latter result suggests that diabetogenic splenocytes that had been
transferred to NOD/scid recipients retained their capacity to
induce .beta.-cell destruction, but, in the presence of sRAGE,
their pathogenic immune/inflammatory potential was held in
abeyance.
[0355] Histology analysis of islets demonstrated a striking
reduction in inflammatory infiltrates in animals treated with sRAGE
compared with controls. Immune/inflammatory cells were consistently
confined to the periphery of islets in sRAGE-treated animals (FIGS.
18A-B). Immunohistology showed strong expression of TNF-a and
IL-1.beta. in inflamed islets from control animals after the onset
of diabetes, whereas sRAGE-treated animals displayed only low
levels of these inflammatory markers constrained to the outermost
periphery of islets (i.e., peri-insulitis) (FIGS. 19A-B). Both
TNF-a and IL-1.beta. have been shown to have direct toxic effects
on .beta.-cells (15), hence the reduced expression of these
mediators may account, at least in part, for the protective effect
observed in sRAGE-treated animals.
[0356] EAE models. The data shown in FIG. 20 demonstrate strongly
symptomatic EAE in the vehicle-treated group, whereas sRAGE-treated
mice showed suppression of symptoms (all mice were immunized with
MBP (maltose binding protein) as described under Methods).
Histologic analysis of these mice displayed scant infiltrates in
the spinal cord of MBP-immunized mice treated with sRAGE (FIG. 21C;
this sample was obtained on day 35 postimmunization with MBP
peptide, and the mouse was asymptomatic), compared with greater
evidence of inflammatory infiltrates in the MBP-immunized group
receiving vehicle alone (FIG. 21B [this sample was obtained 35 days
after immunization with MBP and the mouse had symptoms of
full-blown EAE]; FIG. 21A shows a mouse not immunized with MBP as a
control). Semiquantitation of inflammatory infiltrates was
determined by counting nuclei per high power field (10 fields per
slide were counted) from representative spinal cord; sections from
vehicle-treated mice demonstrated a dramatic increase in nuclei
coinciding with inflammatory infiltrates, whereas administration of
sRAGE caused the number of nuclei/cells per high power field to
remain at the level present in normal spinal cord (FIG. 21D).
[0357] To provide a model of later-stage disease, B10.PL mice were
infused with 1AE10 cells. Animals developed symptoms of EAE during
weeks 3-4 following cell transfer whether receiving nonimmune IgG
(FIG. 22) or vehicle (saline) alone (not shown). In contrast, mice
treated with anti-RAGE IgG showed strong suppression of symptomatic
EAE.
Discussion
[0358] The results of these studies demonstrate that blockade of
RAGE, with sRAGE (which prevents access of ligands to the receptor
by acting as a soluble decoy) or anti-RAGE IgG prevents the
development of disease in murine models simulating type I diabetes
and multiple sclerosis (EAE). The advantage of this method is its
lack of toxicity and apparent effectiveness. An important caveat is
that is difficult to be certain that the results of our experiments
can be directly extrapolated to successful treatment of the human
conditions. A common feature of the pathologic features of each
model concerns the inability of immune/inflammatory cells to reach
the target tissue (pancreatic islets or spinal cord) in the
presence of RAGE blockade. In the autoimmune diabetes model, this
was demonstrated to be a reversible phenomenon, as stopping sRAGE
resulted in the occurrence of diabetes.
REFERENCES FOR EXAMPLE 5
[0359] 1. Castano, L. and G. S. Eisenbarth, Type-I diabetes: a
chronic autoimmune disease of human, mouse, and rat. Annu Rev
Immunol, 1990. 8: p. 647-79 [0360] 2. Pakala, S. V., M. O. Kurrer,
and J. D. Katz, T helper 2 (Th2) T cells induce acute pancreatitis
and diabetes in immune-compromised nonobese diabetic (NOD) mice. J
Exp Med, 1997. 186(2): p. 299-306. [0361] 3. Atkinson, M. A. and E.
H. Leiter, The NOD mouse model of type 1 diabetes: as good as it
gets? Nat Med, 1999. 5(6): p. 601-4. [0362] 4. Lafaille, J.,
Nagashima, K., Katsuki, M., and Tonegawa, S. High incidence of
spontaneous EAE in immunodeficient anti-MBP T-cell receptor
transgenic mice. Cell 78:399-408, 1994. [0363] 5. Chen, Y.,
Hancock., Marks, R., Gonnella, P., and Weiner H. Mechanisms of
recovery from EAE: T cell deletion and immune deviation in MBP T
cell receptor transgenic mice. [0364] 6. Graesser, D., Mahooti, S.,
and Madri, J. Distinct roles for matrix metalloproteinase-2 and
alpha-4 integrin in autoimmune T-cell extravasation and residency
in brain parenchyma during EAE. J. Neuroimmunol. 109:121-131, 200.
[0365] 7. Chem. D, Mosmann T: Two types of murine helper T cell
clones. II. Delayed type hypersensitivity is mediated by Th1
clones. J Immunol 1987; 138:3688-3694. [0366] 8. Mosmann T,
Cherwinski H, Bond M, Giedlin M, Coffman R: Two types of murine
helper T cell clones. I. Definition according to profiles of
lymphokine activities and secreted proteins. J Immunol 1986;
136:2348-2357 [0367] 9. Zamvil S, Nelson P, Trotter J, MItchell D,
Knobler R, Fritz R, Steinman L: T cell clones specific for myelin
basic protein induce chronic relapsing paralysis and demyelination.
Nature 1985; 317:355-358 [0368] 10. Raine C, Mokhtarian F, McFarlin
D: Adoptively transferred chronic relapsing EAE in the mouse:
neuropathologic analysis. Lab Invest 1984; 51:534-536 [0369] 11.
Hofmann M, Drury S, Caifeng F, Qu W, Lu Y, Avila C, Kambhan N, RAGE
mediates a novel proinflammatory axis: the cell surface receptor
for S100/calgranulin polypeptides. Cell 1999; 97:889-901 [0370] 12.
Brett, J., Schmidt, A-M., Zou, Y-S., Yan, S-D., Weidman, E.,
Pinsky, D., Neeper, M., Przysiecki, M., Shaw, A., Migheli, A., and
Stern, D. Tissue distribution of the receptor for advanced
glycation endproducts (RAGE): expression in smooth muscle, cardiac
myocytes, and neural tissue in addition to the vasculature. Am. J.
Pathol. 143:1699-1712, 1993. [0371] 13. Tabira T: Cellular and
molecular aspects of the pathomechanism and therapy of murine EAE.
Crit. Rev Neurobiol 1989; 5:113-142 [0372] 14. Raine C:
Experimental allergic encephalomyelitis, in Koetsier J (ed):
Handbook of Clinical Neurology. Amsterdam, Elsevier, 1985, pp
429-466 [0373] 15. Rabinovitch, A., An update on cytokines in the
pathogenesis of insulin-dependent diabetes mellitus. Diabetes Metab
Rev, 1998. 14(2): p. 129-51.
Sequence CWU 1
1
151112PRTHomo sapiens 1Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro
Leu Val Leu Lys Cys1 5 10 15Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg
Leu Glu Trp Lys Leu Asn 20 25 30Thr Gly Arg Thr Glu Ala Trp Lys Val
Leu Ser Pro Gln Gly Gly Gly 35 40 45Pro Trp Asp Ser Val Ala Arg Val
Leu Pro Asn Gly Ser Leu Phe Leu 50 55 60Pro Ala Val Gly Ile Gln Asp
Glu Gly Ile Phe Arg Cys Gln Ala Met65 70 75 80Asn Arg Asn Gly Lys
Glu Thr Lys Ser Asn Tyr Arg Val Arg Val Tyr 85 90 95Gln Ile Pro Gly
Lys Pro Glu Ile Val Asp Ser Ala Ser Glu Leu Thr 100 105
1102332PRTHomo sapiens 2Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro
Leu Val Leu Lys Cys1 5 10 15Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg
Leu Glu Trp Lys Leu Asn 20 25 30Thr Gly Arg Thr Glu Ala Trp Lys Val
Leu Ser Pro Gln Gly Gly Gly 35 40 45Pro Trp Asp Ser Val Ala Arg Val
Leu Pro Asn Gly Ser Leu Phe Leu 50 55 60Pro Ala Val Gly Ile Gln Asp
Glu Gly Ile Phe Arg Cys Gln Ala Met65 70 75 80Asn Arg Asn Gly Lys
Glu Thr Lys Ser Asn Tyr Arg Val Arg Val Tyr 85 90 95Gln Ile Pro Gly
Lys Pro Glu Ile Val Asp Ser Ala Ser Glu Leu Thr 100 105 110Ala Gly
Val Pro Asn Lys Val Gly Thr Cys Val Ser Glu Gly Ser Tyr 115 120
125Pro Ala Gly Thr Leu Ser Trp His Leu Asp Gly Lys Pro Leu Val Pro
130 135 140Asn Glu Lys Gly Val Ser Val Lys Glu Gln Thr Arg Arg His
Pro Glu145 150 155 160Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu Met
Val Thr Pro Ala Arg 165 170 175Gly Gly Asp Pro Arg Pro Thr Phe Ser
Cys Ser Phe Ser Pro Gly Leu 180 185 190Pro Arg His Arg Ala Leu Arg
Thr Ala Pro Ile Gln Pro Arg Val Trp 195 200 205Glu Pro Val Pro Leu
Glu Glu Val Gln Leu Val Val Glu Pro Glu Gly 210 215 220Gly Ala Val
Ala Pro Gly Gly Thr Val Thr Leu Thr Cys Glu Val Pro225 230 235
240Ala Gln Pro Ser Pro Gln Ile His Trp Met Lys Asp Gly Val Pro Leu
245 250 255Pro Leu Pro Pro Ser Pro Val Leu Ile Leu Pro Glu Ile Gly
Pro Gln 260 265 270Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr His Ser
Ser His Gly Pro 275 280 285Gln Glu Ser Arg Ala Val Ser Ile Ser Ile
Ile Glu Pro Gly Glu Glu 290 295 300Gly Pro Thr Ala Gly Ser Val Gly
Gly Ser Gly Leu Gly Thr Leu Ala305 310 315 320Leu Ala Leu Gly Ile
Leu Gly Gly Leu Gly Thr Ala 325 330330PRTartificialportion of
V-domain of RAGE 3Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu
Val Leu Lys Cys1 5 10 15Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu
Glu Trp Lys 20 25 30430PRTartificialportion of V-domain of RAGE
4Gly Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val Leu Ser Cys1 5
10 15Lys Gly Ala Pro Lys Lys Pro Pro Gln Gln Leu Glu Trp Lys 20 25
30530PRTartificialportion of V-domain of RAGE 5Gly Gln Asn Ile Thr
Ala Arg Ile Gly Glu Pro Leu Met Leu Ser Cys1 5 10 15Lys Ala Ala Pro
Lys Lys Pro Thr Gln Lys Leu Glu Trp Lys 20 25
30630PRTartificialportion of V-domain of RAGE 6Asp Gln Asn Ile Thr
Ala Arg Ile Gly Lys Pro Leu Val Leu Asn Cys1 5 10 15Lys Gly Ala Pro
Lys Lys Pro Pro Gln Gln Leu Glu Trp Lys 20 25
30710PRTartificialfirst 10 residues of V-domain of RAGE 7Ala Gln
Asn Ile Thr Ala Arg Ile Gly Glu1 5
10850PRTbovinemisc_feature(47)..(47)Xaa can be any naturally
occurring amino acid 8Thr Lys Leu Glu Asp His Leu Glu Gly Ile Ile
Asn Ile Gly His Gln1 5 10 15Tyr Ser Val Arg Val Gly His Phe Asp Thr
Leu Asn Lys Tyr Glu Leu 20 25 30Lys Gln Leu Gly Thr Lys Glu Leu Pro
Lys Thr Leu Gln Asn Xaa Lys 35 40 45Asp Gln 50918PRTbovine 9Asp Gly
Ala Val Ser Phe Glu Glu Phe Val Val Leu Val Ser Arg Val1 5 10 15Leu
Lys1090PRTbovine 10Thr Lys Leu Glu Asp His Leu Glu Gly Ile Ile Asn
Ile Phe His Gln1 5 10 15Tyr Ser Val Arg Val Gly His Phe Asp Thr Leu
Asn Lys Arg Glu Leu 20 25 30Lys Gln Leu Ile Thr Lys Glu Leu Pro Lys
Thr Leu Gln Asn Thr Lys 35 40 45Asp Gln Pro Thr Ile Asp Lys Ile Phe
Gln Asp Leu Asp Ala Asp Lys 50 55 60Asp Gly Ala Val Ser Phe Glu Glu
Phe Val Val Leu Val Ser Arg Val65 70 75 80Leu Lys Thr Ala His Ile
Asp Ile His Lys 85 901190PRTbovine 11Thr Lys Leu Glu Asp His Leu
Glu Gly Ile Ile Asn Ile Phe His Gln1 5 10 15Tyr Ser Val Arg Val Gly
His Phe Asp Thr Leu Asn Lys Arg Glu Leu 20 25 30Lys Gln Leu Ile Thr
Lys Glu Leu Pro Lys Thr Leu Gln Asn Thr Lys 35 40 45Asp Gln Pro Thr
Ile Asp Lys Ile Phe Gln Asp Leu Asp Ala Asp Lys 50 55 60Asp Gly Ala
Val Ser Phe Glu Glu Phe Val Val Leu Val Ser Arg Val65 70 75 80Leu
Lys Thr Ala His Ile Asp Ile His Lys 85 901221DNAartificialsense
primer 12gtaagcgggg ctcctgttgc a 211321DNAartificialantisense
primer 13ggccaaggct ggggttgaag g 21149PRTartificialn-terminal
sequence of MBP 14Ala Ser Gln Arg Lys Pro Ser Gln Arg1
515395DNAbovine 15atgactaagc tggaggacca cctggaggga atcatcaaca
tcttccacca gtactccgtt 60cgggtggggc atttcgacac cctcaacaag cgtgagctga
agcagctgat cacaaaggga 120acttcccaaa accctccaga acaccaaaga
ccaacctacc attgacaaaa tattccaaga 180cctggatgcc gataaagacg
gagccgtcag ctttgaggaa ttcgtagtcc tggtgtccag 240ggtgctgaaa
acagcccaca tagatatcca caaagagtag gtttccagca atgttcccaa
300gaagacttac ccttctcctc cctgaggctg ctccccgagg gagagagaat
tataaacgta 360ctttggcaaa ttcttagcaa aaaaaaaaaa aaaaa 395
* * * * *