U.S. patent application number 10/427196 was filed with the patent office on 2007-02-01 for treatment of ulcerative colitis with tropomyosin isoforms and monoclonal antibodies to tropomyosin isoforms.
This patent application is currently assigned to University of Medicine & Dentistry of New Jersey. Invention is credited to Kiron M. Das.
Application Number | 20070025984 10/427196 |
Document ID | / |
Family ID | 21941306 |
Filed Date | 2007-02-01 |
United States Patent
Application |
20070025984 |
Kind Code |
A9 |
Das; Kiron M. |
February 1, 2007 |
Treatment of ulcerative colitis with tropomyosin isoforms and
monoclonal antibodies to tropomyosin isoforms
Abstract
This invention pertains to a method for treating ulcerative
colitis. Specifically, the method comprises orally or rectally
administering to a human having ulcerative colitis a
therapeutically effective amount of an antibody which binds to a
tropomyosin isoform associated with ulcerative colitis. In another
embodiment, the invention pertains to a method for treating
ulcerative colitis in a human which comprises the steps of (a)
obtaining from a human a colon epithelial cell extract containing a
tropomyosin isoform associated with ulcerative colitis; (b)
purifying the tropomyosin isoform until the tropomyosin isoform is
substantially homogeneous; (c) developing an antibody which binds
to the tropomyosin isoform; and (d) orally or rectally
administering to a human having ulcerative colitis a
therapeutically effective amount of the antibody to bind to the
tropomyosin isoform associated with ulcerative colitis. In yet
another embodiment, the invention pertains to a method for treating
ulcerative colitis in a human which comprises orally administering
to the human a therapeutically effective amount of a tropomyosin
isoform associated with ulcerative colitis.
Inventors: |
Das; Kiron M.; (Basking
Ridge, NJ) |
Correspondence
Address: |
LICATA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Assignee: |
University of Medicine &
Dentistry of New Jersey
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20030198635 A1 |
October 23, 2003 |
|
|
Family ID: |
21941306 |
Appl. No.: |
10/427196 |
Filed: |
May 1, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09046049 |
Mar 23, 1998 |
6605276 |
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10427196 |
May 1, 2003 |
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08630541 |
Apr 10, 1996 |
5869048 |
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09046049 |
Mar 23, 1998 |
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08437474 |
May 9, 1995 |
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08630541 |
Apr 10, 1996 |
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Current U.S.
Class: |
424/133.1 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 2039/505 20130101; C07K 16/18 20130101; C07K 14/4713 20130101;
A01K 2227/40 20130101; A61P 43/00 20180101; A61P 1/04 20180101 |
Class at
Publication: |
424/133.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Part of the work performed during development of this
invention utilized United States Government funds. The United
States Government has certain rights in this invention: NIADDK RO1
DK47673 from the National Institutes of Health (Bethesda, Md.).
Claims
1. A method for treating ulcerative colitis in a human which
comprises orally administering to the human a therapeutically
effective amount of a tropomyosin isoform, or an antigenically
active fragment thereof, associated with ulcerative colitis.
2. The method according to claim 1, wherein the tropomyosin isoform
is hTM1.
3. The method according to claim 1, wherein the tropomyosin isoform
is hTM5.
4. The method according to claim 1, wherein the tropomyosin isoform
is administered in an amount from about 50 .mu.g/day to about 1000
.mu.g/day.
5. A method for treating ulcerative colitis in a human which
comprises orally or rectally administering to the human a
therapeutically effective amount of an antibody which binds to a
tropomyosin isoform associated with ulcerative colitis.
6. The method according to claim 5, wherein the tropomyosin isoform
is hTM1.
7. The method according to claim 5, wherein the tropomyosin isoform
is hTM5.
8. The method according to claim 5, wherein the antibody is
monoclonal antibody CG1 (IgG1).
9. The method according to claim 5, wherein the antibody is
monoclonal antibody CG3 (IgM).
10. The method according to claim 5, wherein the antibody is
administered in an amount from about 50 .mu.g/day to about 500
.mu.g/day.
11. The method according to claim 5, wherein the antibody is
administered rectally.
12. A method for treating ulcerative colitis in a human which
comprises the steps of: (a) obtaining from a human a colon
epithelial cell extract containing a tropomyosin isoform associated
with ulcerative colitis; (b) purifying the tropomyosin isoform
until the tropomyosin isoform is substantially homogeneous; (c)
developing an antibody which binds to the tropomyosin isoform; and
(d) orally or rectally administering to a human having ulcerative
colitis a therapeutically effective amount of the antibody to bind
to the tropomyosin isoform associated with ulcerative colitis.
13. The method according to claim 12, wherein the tropomyosin
isoform is hTM1.
14. The method according to claim 12, wherein the tropomyosin
isoform is hTM5.
15. The method according to claim 12, wherein the antibody is
monoclonal antibody CG1 (IgG1).
16. The method according to claim 12, wherein the antibody is
monoclonal antibody CG3 (IgM).
17. The method according to claim 12, wherein the antibody is
administered in an amount from about 50 .mu.g/day to about 500
.mu.g/day.
18. The method according to claim 12, wherein the antibody is
administered rectally.
19. The method according to claim 12, wherein the antibody in step
(c) is a murine antibody.
20. The method according to claim 1, wherein the tropomyosin
isoform hTM5 is orally administered.
21. The method according to claim 5, wherein the tropomyosin
isoform hTM5 is orally administered.
22. The method according to claim 12, wherein the tropomyosin
isoform hTM5 is orally administered.
23. The method of claim 1, wherein the tropomyosin isoform is an
hTM.
24. The method of claim 23, wherein the tropomyosin isoform is
hTM1.
25. The method of claim 23, wherein the tropomyosin isoform is
hTM5.
26. The method of claim 1, wherein the tropomyosin isoform is
administered in an amount from about 50 .mu.g/day to about 1000
.mu.g/day.
27. The method of claim 26, wherein the tropomyosin isoform is
administered in an amount from about 100 .mu.g/day to about 750
.mu.g/day.
28. The method of claim 27, wherein the tropomyosin isoform is
administered in an amount from about 150 .mu.g/day to about 500
.mu.g/day.
29. The method of claim 1, wherein the tropomyosin isoform is
administered orally.
30. The method of claim 29, wherein the tropomyosin isoform is
administered in an amount of 100 .mu.g once or twice a day for
about 8 weeks.
Description
[0001] This Application is a Continuation of U.S. patent
application Ser. No. 09/046,049 filed Mar. 23, 1998, and now
pending and which is incorporated herein by reference. Priority to
this application is claimed under 35 U.S.C. .sctn. 120.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention pertains to a method for treating ulcerative
colitis. Specifically, the method comprises orally or rectally
administering to a human having ulcerative colitis a
therapeutically effective amount of an antibody which binds to a
tropomyosin isoform associated with ulcerative colitis. In another
embodiment, the invention pertains to a method for treating
ulcerative colitis in a human which comprises the steps of (a)
obtaining from a human a colon epithelial cell extract containing a
tropomyosin isoform associated with ulcerative colitis; (b)
purifying the tropomyosin isoform until the tropomyosin isoform is
substantially homogeneous; (c) developing an antibody which binds
to the tropomyosin isoform; and (d) orally or rectally
administering to a human having ulcerative colitis a
therapeutically effective amount of the antibody to bind to the
tropomyosin isoform associated with ulcerative colitis. In yet
another embodiment, the invention pertains to a method for treating
ulcerative colitis in a human which comprises orally administering
to the human a therapeutically effective amount of a tropomyosin
isoform associated with ulcerative colitis.
[0005] 2. Description of the Background
[0006] The disclosures referred to herein to illustrate the
background of the invention and to provide additional detail with
respect to its practice are incorporated herein by reference and,
for convenience, are numerically referenced in the following text
and respectively grouped in the appended bibliography.
[0007] Ulcerative colitis (UC) is a chronic inflammatory bowel
disease (IBD) of unknown etiology, although autoimmunity has been
emphasized in the pathogenesis of the disease (1). A marked
increase in the mucosal IgG immunocytes (2) and IgG antibodies
against colonic antigens (3) and neutrophil antigen (4) have been
reported in ulcerative colitis colon. However, the specific
antigen(s) involved in the IgG-immune recognition has not been
clarified. The mucosal IgG overproduction could play pathogenetic
role in ulcerative colitis as the subclass distribution of
IgG-producing cells in both active and inactive ulcerative colitis
lesions shows a disproportionate local overproduction of IgG1
(5,6). IgG1 antibodies are more effective in complement activation
than IgG2 (7) and because of this property, IgG1 antibodies can
contribute to the perpetuation of tissue damage in ulcerative
colitis.
[0008] The presence of tissue-bound IgG antibody in the ulcerative
colitis colon has been reported (8) and it was demonstrated that
this IgG antibody recognized an Mr 40K colonic protein, (p40) (9).
Tissue-bound IgG eluted from Crohn's disease of the colon (CD) and
controls did not recognize the p40, suggesting an autoantigenic
role of this protein in ulcerative colitis (9). By
immunocytochemical methods, other investigators reported the
deposition of IgG1 autoantibody and activated complement products
on the colonic epithelium from active ulcerative colitis (10,11),
but not in Crohn's colitis (12). Recently, p40 was purified from
the colon to homogeneity, partially sequenced, and the two peptides
derived therefrom showed 93-100% identity with the cytoskeletal
protein tropomyosins (TMs) (13). Tropomyosins are cytoskeletal
microtubular proteins present in all eukaryotic cells with organ
specific isoforms, and multiple isoforms may be present in the same
cell (14). At least 8 tropomyosin isoforms have been identified
from human fibroblast cells (15) and strong evidence for the
distinct functions performed by different tropomyosin isoforms has
recently been generated (14). Tropomyosins are capable of inducing
significant immune responses related to allergy (16) as well as
autoimmunity (17). In a computer-based physico-chemical analysis,
several sequences of tropomyosin-residues were considered to be
among the most potent autoantigens (17). It has been reported that
blood serum from patients with ulcerative colitis, but not from
Crohn's colitis and other controls, had IgG antibodies reactive to
tropomyosins (13). The specific tropomyosin isoform(s) present in
the human colon epithelium is unknown.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 illustrates an SDS-polyacrylamide gel electrophoresis
followed by Coomassie blue staining of purified recombinant human
tropomyosin isoforms (hTM1-5) and enriched hTMs from colon
epithelial cells and colon smooth muscle.
[0010] FIG. 2 illustrates an immunotransblot analysis of the
enriched TM extracts from colon and jejunal epithelial cells using
isoform specific monoclonal antibodies, CG1 (anti-hTM1), CGb6
(anti-hTM 2&3), LC24 (anti-hTM4), LC1 (anti-hTM5) and CG3
(anti-hTM5).
[0011] FIG. 3 illustrates an immunotransblot analysis of enriched
TM preparations from the smooth muscle of intestine probed with the
hTM isoform specific monoclonal antibodies against hTM1-5.
[0012] FIG. 4 illustrate a scatterogram showing the
immunoreactivity of spontaneously produced IgG by LPMCs against
hTM1,2,3 and 5.
SUMMARY OF THE INVENTION
[0013] The present invention pertains to a method for treating
ulcerative colitis in a human which comprises orally or rectally
administering to the human a therapeutically effective amount of an
antibody which binds to a tropomyosin isoform associated with
ulcerative colitis.
[0014] In another embodiment, the present invention pertains to a
method for treating ulcerative colitis in a human which comprises
the steps of: [0015] (a) obtaining from a human a colon epithelial
cell extract containing a tropomyosin isoform associated with
ulcerative colitis; [0016] (b) purifying the tropomyosin isoform
until the tropomyosin isoform is substantially homogeneous; [0017]
(c) developing an antibody which binds to the tropomyosin isoform;
and [0018] (d) orally or rectally administering to a human having
ulcerative colitis a therapeutically effective amount of the
antibody to bind to the tropomyosin isoform associated with
ulcerative colitis.
[0019] In one embodiment, the tropomyosin isoform is hTM1. In
another embodiment, the tropomyosin isoform is hTM5. Preferably,
the antibody is monoclonal antibody CG1 (IgG1) or monoclonal
antibody CG3 (IgM). The antibody may be administered in an amount
from about 50 .mu.g/day to about 500 .mu.g/day. Preferably, the
antibody is administered rectally. The antibody may be a murine
antibody.
[0020] In yet another embodiment, the present invention pertains to
a method for treating ulcerative colitis in a human which comprises
orally administering to the human a therapeutically effective
amount of a tropomyosin isoform associated with ulcerative colitis.
In one embodiment, the tropomyosin isoform is hTM1. In another
embodiment, the tropomyosin isoform is hTM5. The tropomyosin
isoform may be administered in an amount from about 50 .mu.g/day to
about 1000 .mu.g/day.
DETAILED DESCRIPTION OF THE INVENTION
[0021] In accord with the present invention, the hTM isoforms (hTM
1-5) present in intestinal epithelial cells and in smooth muscle,
and the immunoreactivity against hTMs by IgG produced in vitro by
colonic mucosal lymphocytes (LPMC) from patients with ulcerative
colitis, Crohn's colitis (CD), and controls, were examined.
Tropomyosins were extracted from colonic and jejunal epithelial
cells and smooth muscle and the hTM isoforms were identified using
isoform specific monoclonal antibodies by ELISA and transblot
analysis. Immunoreactivity of IgG produced by colonic LPMC was
analyzed against the recombinant hTM isoforms. This study showed
that the major hTM isoforms present in colonic and jejunal
epithelial cells are hTM5 and hTM4, whereas intestinal smooth
muscle contains hTM 1-3 isoforms. The IgG synthesized in vitro by
LPMCs from ulcerative colitis (n=19) had significantly
(p<0.04<0.001) higher reactivity against hTM5 and hTM1, and
not against hTM2 and hTM3, when compared to Crohn's colitis (n=12)
and controls (n=17). However, IgG produced by LPMC from Crohn's
colitis did not show such anti-hTM reactivity. Mucosal anti-hTM IgG
mainly belonged to IgG1 subclass. Accordingly, it was found that
intestinal epithelial cells and smooth muscle have distinct hTM
isoforms, and patients with ulcerative colitis, and not Crohn's
colitis, demonstrate mucosal autoantibody response against hTM
isoforms, particularly hTM5 and hTM1.
[0022] In one embodiment, the present invention pertains to a
method for treating ulcerative colitis in a human which comprises
orally or rectally administering to the human a therapeutically
effective amount of an antibody which binds to a tropomyosin
isoform associated with ulcerative colitis.
[0023] The tropomyosin isoform associated with ulcerative colitis
used in the present invention may be any tropomyosin isoform
associated with ulcerative colitis. Preferably, the tropomyosin
isoform is hTM1 or hTM5. More preferably, the tropomyosin isoform
is hTM5.
[0024] The antibody which binds to a tropomyosin isoform associated
with ulcerative colitis used in the present invention may be any
antibody. Preferably, the antibody is a murine antibody or a
humanized antibody. More preferably, the antibody is monoclonal
antibody CG1 (IgG1), also referred to as LC1, or monoclonal
antibody CG3 (IgM). Most preferably, the antibody is monoclonal
antibody CG3 (IgM). Monoclonal antibodies CG1 and CG3 are described
in more detail in Novy et al., Cell Motil Cytoskeleton
1993;26:248-261; Lin et al., J Cell Biol 1988;107:563-572; Warren
et al., J Cell Biol 1995;129:697-708; Lin et al., International
Review Cytology 1997;170:1-38; and Lin et al., Hybridoma
1985;4:223-242; which disclosures are incorporate herein by
reference.
[0025] The amount of antibody which binds to a tropomyosin isoform
associated with ulcerative colitis used in the present invention is
a therapeutically effective amount. A therapeutically effective
amount of antibody is that amount of antibody necessary to bind to
a tropomyosin isoform associated with ulcerative colitis blocking
the epithelial binding of circulating and/or local auto antibodies
causing ulcerative colitis. The exact amount of antibody is a
matter of preference subject to such factors as the type of
condition being treated as well as the dosage recommended or
permitted for the particular antibody. In general, the amount of
antibody agent employed is the dosage required to obtain the
desired result. In a preferred embodiment, the dosage of antibodies
in an enema for patients with ulcerative colitis will be in the
range from about 50 .mu.g/day to about 500 .mu.g/day, preferably
from about 100 .mu.g/day to about 400 .mu.g/day, and more
preferably from about 150 .mu.g/day to about 300 .mu.g/day. Most
preferably, the dosage of antibodies will be about 100 .mu.g in the
form of a retention enema given once or twice a day for up to about
8 weeks.
[0026] The antibody may be administered orally or rectally.
Preferably, the antibody is administered rectally.
[0027] The present invention extends to methods for preparing the
antibodies which bind to a tropomyosin isoform associated with
ulcerative colitis used in the present invention. The antibodies
may be developed using standard techniques and apparatus known to
those skilled in the art. In a preferred embodiment, the invention
is directed to a method for treating ulcerative colitis in a human
which comprises the steps of: [0028] (a) obtaining from a human a
colon epithelial cell extract containing a tropomyosin isoform
associated with ulcerative colitis; [0029] (b) purifying the
tropomyosin isoform until the tropomyosin isoform is substantially
homogeneous; [0030] (c) developing an antibody which binds to the
tropomyosin isoform; and [0031] (d) orally or rectally
administering to a human having ulcerative colitis a
therapeutically effective amount of the antibody to bind to the
tropomyosin isoform associated with ulcerative colitis.
[0032] In another embodiment, the present invention is directed at
a method for treating ulcerative colitis in a human through oral
tolerance. Oral tolerance is the state of hyporesponsiveness that
follows immunization with a previously fed protein. Animals fed
antigenic proteins have been shown not to respond as well to these
antigens when subsequently immunized but do respond normally to
other antigens. Immunological tolerance is a fundamental property
of the immune system that provides a mechanism for self/nonself
discrimination. Through immunological tolerance, the immune system
can protect the host from external pathogens (against nonself)
without eliciting autoimmune disease (against self). Oral tolerance
has been used to treat autoimmune diseases in animal models and is
now being applied to the treatment of human diseases. As with
immunological tolerance, oral tolerance has been found to involve
multiple mechanisms and is not a single immunological event. The
primary mechanisms by which oral tolerance is mediated include
deletion, anergy, and active cellular suppression. The determining
factor in this process is the dose of fed antigen. Low doses favor
active suppression, high doses favor deletion and anergy. Oral
tolerance is reviewed in detail in Weiner, H. L., Immunology Today,
19, 335-343 (1997), which disclosure is incorporated herein by
reference. In this embodiment, a patient having ulcerative colitis
is tolerized or desensitized by being treated with a tropomyosin
isoform associated with ulcerative colitis in multiple doses over a
period of time sufficient to develop tolerance in the patient.
Developing tolerance in the patient causes the immune system to
become non-reactive to the tissue-autoantigen.
[0033] In a specific embodiment, the present invention pertains to
a method for treating ulcerative colitis in a human which comprises
orally administering to the human a therapeutically effective
amount of a tropomyosin isoform associated with ulcerative
colitis.
[0034] The tropomyosin isoform associated with ulcerative colitis
used in this method may be any tropomyosin isoform, or an
antigenically active fragment thereof, associated with ulcerative
colitis. Preferably, the tropomyosin isoform is hTM1 or hTM5. More
preferably, the tropomyosin isoform is hTM5.
[0035] The amount of tropomyosin isoform associated with ulcerative
colitis used in this method is a therapeutically effective amount.
A therapeutically effective amount of tropomyosin isoform is that
amount of tropomyosin isoform necessary to develop tolerance in the
patient causing the immune system to become non-reactive to the
tropomyosin isoform associated with ulcerative colitis. The exact
amount of tropomyosin isoform is a matter of preference subject to
such factors as the type of condition being treated as well as the
dosage recommended or permitted for the particular tropomyosin
isoform. In general, the amount of tropomyosin isoform employed is
the dosage required to obtain the desired result. In a preferred
embodiment, the dosage of tropomyosin isoform for patients with
ulcerative colitis will be in the range from about 50 .mu.g/day to
about 1000 .mu.g/day, preferably from about 100 .mu.g/day to about
750 .mu.g/day, and more preferably from about 150 .mu.g/day to
about 500 .mu.g/day. Most preferably, the dosage of tropomyosin
isoform will be about 100 .mu.g in given orally once or twice a day
for up to about 8 weeks.
[0036] The present invention is further illustrated by the
following examples which are not intended to limit the effective
scope of the claims. All parts and percentages in the examples and
throughout the specification and claims are by weight of the final
composition unless otherwise specified.
EXAMPLES
Extraction of Tropomyosin from the Intestinal Tissue
Colonic and Small Intestinal Mucosal Extracts Enriched in
Tropomyosins
[0037] Colonic mucosa (including epithelium and stromal tissue and
muscularis mucosa) was stripped from operative specimens of colon
from patients with colon cancer (normal segments, n=4). Jejunal
mucosa was obtained from normal subjects (n=4) who underwent
gastric by-pass surgery for morbid obesity. Mucosa was minced,
washed with phosphate buffered saline (PBS), re-suspended in the
buffer containing 1 M NaCl, 50 mM Tris, 1 mM EDTA, 5 mM DTT, and 2
mM PMSF, and ultrasonicated for 3 minutes on ice. The sample was
centrifuged at 1,000 g for 5 minutes, the supernatant boiled for 10
minutes, centrifuged at 7,000 rpm for 30 minutes, and further
purified by ammonium sulfate precipitation (initially 40% and then
60%) (15) and stored until use.
Extraction of Tropomyosins from Colonic and Jejunal Epithelial
Cells
[0038] Stripped mucosa from the surgical specimens was washed with
Hank's balanced salt solution (HBSS) containing gentamycin 50
units/ml, 100 U of penicillin and 100 mg of streptomycin. To remove
the mucus, the tissue was treated with 1 mM DTT for 15 min at
37.degree. C. followed by 3 washings in HBSS. The tissue was
subjected to dispase treatment 3 mg/ml in RPMI 1640 for 30 min at
37.degree. C. Cells were collected from supernatant and epithelial
cells were further purified by Percoll (Pharmacia, NJ) gradient
(44% and 66%), and centrifugation at 1000 g for 30 min. The top
layer was transferred, washed with PBS and the cells were counted
and stored at -80.degree. C. until further extraction. The purity
of epithelial cells was found to be at least 90%. Tropomyosins were
extracted by homogenization of the cell pellet in the buffer
containing 0.35M NaCl, 50 mM Tris, 1 mM EDTA, 5 mM DTT and 2 mM
PMSF, centrifuged at 1,000 g for 5 minutes and the supernatant
boiled for 10 minutes, followed by centrifugation at 7000 rpm for
30 minutes. The supernatant was further purified by sequential
ammonium sulfate precipitation.
Extraction of Tropomyosins from Colonic and Small Intestinal Smooth
Muscle
[0039] Smooth muscle was dissected from the intestinal specimens
after stripping off the mucosa, and tropomyosins were extracted, as
described above.
Preparation of Recombinant Tropomyosin Isoforms
[0040] The full-length cDNAs encoding various tropomyosin isoforms
(hTM1, hTM2, hTM3 and hTM5) were prepared from human fibroblast, as
reported earlier (15). hTM4 clone was not available. We have
subcloned these 4 cDNA clones into prokaryotic expression vector
pET8c. The resulting plasmids were transformed into E coli BL21
(D3) LysS strain. Recombinant human tropomyosin isoforms were
purified from bacterial lysates by ammonium sulfate fractionation,
DE-52 ion-exchange chromatography, and hydroxyapatite column
chromatography. The purity of the hTM isoforms was examined by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) and the
immunoreactivity was analyzed by ELISA and transblot analysis using
isoform specific monoclonal antibodies.
Monoclonal Antibodies Against Various Tropomyosin Isoforms
[0041] The isoform specific murine monoclonal antibodies CG1 (IgG1)
against hTM1, CG.beta.6 (IgM) against hTM2/hTM3, and CG3 (IgM)
against hTM5 were generated and characterized earlier (14,15). CGb6
reacts with both hTM2 and hTM3 but not against hTM1, hTM4 or hTM5.
The isoform-specific monoclonal antibodies LC24 (IgG1) against hTM4
and LC1 (IgG1) against hTM5 were generated by immunizing mice with
the recombinant hTM isoform (18).
Determination of Specific Tropomyosin Isoform(s) in the Colon and
Small Intestine by Immunotransblot Analysis and by Elisa
[0042] Extracted tropomyosins from mucosa, isolated epithelial
cells and smooth muscle from the small and large intestine were
examined by SDS-PAGE followed by immunotransblot analysis (9). Five
ug of enriched tropomyosin extracts were separated by 12% SDS-PAGE,
stained with Coomassie brilliant blue. Separated proteins were also
transferred to nitro-cellulose sheet blocked with 1% bovine serum
albumin (BSA) and probed with isoform specific murine
anti-tropomyosin monoclonal antibodies (CG1--1:1,000, CG3--1:500,
CGb6--1:1,000, LC1--1:500, LC24--1:500), followed by horseradish
peroxidase conjugated appropriate anti-mouse IgM or IgG antibodies.
The reactivity was detected by chemiluminescence using Renaissance
Kit (Dupont, Boston, Mass.), and respective recombinant hTM isoform
(2 ug) was run in parallel as positive control. Unrelated isotype
specific murine monoclonal antibodies such as MOPC-IgM or MOPC-IgG
were also used to examine any non-specific reactivity.
[0043] Tropomyosin extracts from jejunal and colonic mucosa, smooth
muscle, and small intestinal enterocytes and colonocytes were also
analyzed by ELISA. The extracts were plated (0.3 ug/well) in
carbonate buffer (pH 9.6) on ELISA plate for overnight. The
tropomyosin isoform specific monoclonal antibodies, as described
above, were incubated at 37.degree. C. for 1 hour after blocking
the plate with 1% normal goat serum, followed by alkaline
phosphatase conjugated appropriate (anti-mouse IgM or anti-mouse
IgG raised in goat) second antibody. The O.D. values were measured
at 405 nm within 2 hours.
Examination for Antibodies Synthesized in vitro by LPMCs
[0044] Patients. Forty-eight patients undergoing colonic resection
(n=30) or colonoscopy (n=18) at the Division of Gastroenterology,
Universita La Spienza, Rome, Italy, were included for these
studies. There were 19 patients with ulcerative colitis (12 male
and 7 female; mean age.+-.SEM 51.+-.2 years; range 21-65), 10
symptomatic and 9 asymptomatic at the time of the study. Of these
19 ulcerative colitis patients, 13 underwent routine colonoscopy
while 6 underwent colon resection. Indication for surgery in these
6 patients was steroid-dependence in 4/6, diffuse polyposis in 1/6
and no response to medical treatment in 1/6. The colitis was
left-sided in 14 patients and extensive in 5 patients. At the time
of the study, 10 of the 19 ulcerative colitis patients were taking
oral steroids, 7 patients were on mesalamine medications (orally in
2 patients, rectally in 5), and 2 patients were not receiving any
treatment. Twelve of the 19 patients were clinically and
endoscopically active and 7 were in remission. The mean (.+-.SEM)
duration of the disease in patients with ulcerative colitis was
11.+-.2 years (range 1-31 years). Twelve patients had colonic
Crohn's colitis (7 males and 5 females; mean age.+-.SEM 42.+-.3
years; range 22-62 years). In the Crohn's colitis group, there were
6 symptomatic and 6 asymptomatic patients. Four of these 12 Crohn's
colitis patients underwent routine colonoscopy, while 8 underwent
surgery. Indication for surgery was steroid-dependence in 4, no
response to medical treatment in 2 and abdominal abscess in 2. Five
patients were taking steroids, and 7 were treated with oral
mesalamine. The mean duration of the disease (.+-.SEM) was 9.+-.2
years (range, 1-17 years). The non-inflammatory bowel disease group
included 1 patient with diverticular disease who underwent
colonoscopy and 16 patients with colonic carcinoma who underwent
colon resection. Among them, 11 were male and 6 females with mean
age(.+-.SEM) 58.+-.3 years; range 34-72 years. In all subjects, the
diagnosis was made according to the usual clinical, endoscopical,
radiological and histological criteria. Patients with indeterminate
colitis are not included in this study. Disease activity was
assessed in patients with ulcerative colitis by the clinical
history and endoscopy findings (19) and in patients with Crohn's
colitis by the CDAI (20).
[0045] Tissue samples. Colonic mucosal specimens for isolation of
LPMCs were taken from each disease group, immediately following
colonic resection (total n=30: ulcerative colitis n=6, Crohn's
colitis n=8, non-inflammatory bowel disease n=16). In case of
colonoscopy, 4-5 biopsy specimens were obtained during the
procedure from 18 subjects with ulcerative colitis (n=13), Crohn's
colitis (n=4), and non-inflammatory bowel disease (n=1). Surgical
and colonoscopic biopsy specimens were obtained from the distal 20
cm of the colon (ulcerative colitis: n=12; Crohn's colitis: n=7;
non-inflammatory bowel disease: n=10), and from the area proximal
to the sigmoid colon (ulcerative colitis: n=7; Crohn's colitis:
n=5; non-inflammatory bowel disease: n=9).
[0046] Blood samples. Venous blood samples were obtained from each
patient during the visit for colonoscopy or prior to surgery, sera
separated and stored at -70.degree. C.
[0047] Isolation of LPMCs from surgical specimens. Intestinal
mucosa was dissected from surgical specimens within 1 hour of
resection. LPMCs were isolated from surgical specimens using the
Bull and Bookman enzymatic methods (21) with minor modifications,
as described by Pallone et al (22). LPMCs were washed, counted, and
the viability assessed by 0.1% Trypan blue exclusion. Contamination
by epithelial cells of the LPMCs suspensions ranged between 10% and
16%, as shown by us and others (23-25). LPMC yield ranged from
2.5.times.10.sup.6 to 11.5.times.10.sup.6 LPMCs/g wet tissue. The
viability of LPMCs ranged from 85% to 90% before cultures and from
50% to 70% on the 10th day of culture.
[0048] Isolation of LPMCs from Biopsy Specimens. LPMCs were
isolated using the EDTA-collagenase as mentioned above with
modifications for small samples (23,26,27). LPMC preparations
obtained from biopsy specimens were comparable to surgical
specimens in terms of yield and epithelial cells contamination,
ranging from 0.9 to 4.times.10.sup.6 from 4-5 biopsy specimens. The
viability of LPMCs ranged from 85%-95% before culture to 50-70% on
the 10th day.
[0049] LPMC cultures. Cells were resuspended in a complete medium
(2.times.10.sup.6 per 2 ml) placed in 2.8 ml wells tissue culture
plates (Falcon Plastic, franklin Lakes, N.J.), and cultured
unstimulated at 37.degree. C. in 5% CO2. Previous studies by us
(23) and others (28,29) consistently demonstrated that "in vitro"
pokeweed mitogen (PWM) stimulation does not increase the total and
antigen-specific IgG antibodies release by LPMC, suggesting that
LPMCs are already maximally stimulated "in vivo" by luminal
antigens permeating the intestinal mucosa. In the present study,
we, therefore, used limited paired cultures of LPMC with and
without PWM (ulcerative colitis n=4; Crohn's colitis n=6;
non-inflammatory bowel disease n=8). PWM was added with 1 ug/well
(Gibco laboratories, Grand Island, N.Y.). On the 10th day, cells
were harvested, counted and checked for viability and supernatants
were collected, frozen within 1 hour, and stored at -20.degree. C.
until tested.
Estimation of IgG and IgG Subclass Specific Antibodies Against hTMs
in Culture Supernatants and Sera
[0050] Total IgG in culture supernatants were tested by ELISA as
previously described (22,23). For anti-hTMs IgG reactivities, ELISA
plates (Falcon Plastic) were separately coated with the four
recombinant human tropomyosin isoforms (hTM1, hTM2, hTM3, hTM5) at
150 ng/well diluted in the carbonate buffer (pH 9.6) (100 ul/well).
Purified recombinant hTM4 was not available for testing. As blank,
additional wells were coated with BSA (0.5 ug/100 ul). After an
overnight incubation at 4.degree. C., plates were washed as above
in PBS and 0.05% Tween 20 (200 ul/well). Non specific binding was
blocked with 0.25% BSA in PBS (100 ul/well) for 30 minutes at
37.degree. C. After three washings, diluted supernatants (1:1) or
sera (1:100) were added in triplicates (100 ul/well). Plates were
incubated for 2 hours at 25.degree. C. and then overnight at
4.degree. C. After three washings, the
alkaline-phosphatase-conjugated goat anti-human IgG (100 ul at
1:6000) (Sigma Chemical Co.) was added for the measurement of IgG
antibodies to tropomyosins. For the IgG subclass analysis, alkaline
phosphatase labeled anti-human IgG1, IgG2 and IgG3 were used
(Zymed, San Francisco, Calif.) at 1:6000 (100 ml). After 1 hour at
25.degree. C. and three washings, the substrate
p-nitrophenyl-phosphate-disodium was added (100 ul) (5 ug/ml).
After 1 hour at 25.degree. C., plates were read at 405 nm. Results
for each sample were expressed as the mean Optical Density
(OD).+-.SD of triplicate values with the blank value against BSA
subtracted from each value.
Statistical Analysis
[0051] ELISA results from both total and tropomyosin-specific IgG
antibodies were not normally distributed in all groups. Therefore,
the nonparametrical Mann-Whitney U Test was used for the
statistical analysis of the data among groups and results expressed
as mean.+-.standard error of mean (SE) both in the text and
tables.
Results
[0052] FIG. 1 demonstrates SDS-PAGE analysis followed by Coomassie
blue staining of the recombinant hTM isoforms (hTM1, 2, 3, 4 &
5 in lanes 2-6 respectively) and enriched tropomyosins from colon
smooth muscle (lane 7) (Mr 38K to Mr 40K) and colon epithelial
cells (lane 8) (Mr 30K to Mr 32K). Recombinant hTM isoforms were
synthesized by us from the respective cDNA clones (hTM1, 2, 3 &
5), expressed in the bacterial system and purified. A small amount
of recombinant hTM4 was available and this was used as a reference
sample to examine the specificity of the isoform specific anti-hTM
monoclonal antibodies. The homogeneity of each of the purified hTM
isoforms can be seen. The molecular weights of various isoforms
varied from Mr 30K to Mr 40K. As shown in FIG. 2, using isoform
specific anti-tropomyosin murine monoclonal antibodies, the
specificity of various hTM isoforms is demonstrated by transblot
analysis. The immunoreactivity was also tested by an ELISA. Each of
the hTM isoform was examined using all of the monoclonal antibodies
to determine the presence or absence of any cross reactivity.
Isoform specificity of the hTMs and monoclonal antibodies were
clearly evident. While CG1 mAb (IgG1 subclass) reacted exclusively
with hTM1, LC-24 mAb (IgG1 subclass) reacted with hTM4 only. LC1
(IgG1 subclass) and CG3 (IgM isotype) monoclonal antibodies both
reacted solely with hTM5. These 2 mAbs react with 2 different
epitopes of hTM5 at a.a. residues 1-18 and 29-44 respectively
(15,18). CGb6 mAb (IgM isotype) reacted with both hTM2 and hTM3. At
this time, we do not have a mAb to differentiate between hTM2 and
hTM3. As also shown in FIG. 1, colonic smooth muscle mainly
contained high Mr TM isoforms (lane 7), whereas colonic epithelial
cells contained low Mr TM isoforms (lane 8).
[0053] Western blot analysis of tropomyosins extracted from normal
colonic and jejunal epithelial cells and intestinal smooth muscle
are shown in FIGS. 2 and 3. The predominant hTM isoforms in normal
colonic and jejunal epithelium are hTM5 and hTM4 which are lower Mr
isoforms (Mr 30K-Mr 32K)compared to hTM1-3 (Mr 36K-Mr 40K) (FIGS. 1
and 2). Epithelial extracts did not contain any detectable amount
of hTM1,2&3 (FIG. 2). In contrast, intestinal smooth muscles
from both colon and jejunum contained predominantly higher hTM
isoforms i.e. hTM1, 2 & 3 isoforms (FIG. 3). In the smooth
muscle there was also a trace of hTM4 but no detectable amount of
hTM5. Colonic and jejunal mucosal extracts contributed by
epithelial and non-epithelial mucosal tissue, including muscularis
mucosa, contained all the 5 isoforms as can be expected (data not
shown).
Total IgG in LPMC Supernatants from Ulcerative Colitis in
Comparison with Crohn's Colitis and Non-Inflammatory Bowel Disease
Controls
[0054] Total IgG levels (ng/ml) were higher in LPMC supernatants
from patients with ulcerative colitis (452.+-.155), and Crohn's
colitis (404.+-.147) when compared with non-inflammatory bowel
disease (127.+-.29) (p=0.04). Total IgG released by LPMC from
ulcerative colitis and Crohn's colitis did not differ
significantly. LPMCs from patients with symptomatic ulcerative
colitis spontaneously released higher levels of total IgG
(870.+-.243) ng/ml than LPMCs from ulcerative colitis in remission
(93.+-.31) ng/ml (p=0.03), Crohn's colitis in remission (p=0.04)
and non-inflammatory bowel disease (p=0.003). Levels of total IgG
were also higher in LPMC supernatants from symptomatic Crohn's
colitis (670.+-.219) ng/ml than Crohn's colitis in remission
(86.+-.33) ng/ml (p=0.04), ulcerative colitis in remission (p=0.04)
and non-inflammatory bowel disease (p=0.002). There were no
significant differences in terms of total IgG release, between
LPMCs isolated from biopsy specimens and surgical specimens from
the inflammatory bowel disease patients and non-inflammatory bowel
disease controls.
[0055] In inflammatory bowel disease patients, total IgG levels in
LPMC supernatants did not significantly differ also in relation to
steroid treatment, disease duration or age. As expected,
PWM-stimulation did not significantly increase the release of total
IgG by LPMCs from all groups (data not shown).
IgG Antibodies Against Different hTM Isoforms Present in LPMC
Supernatants from Ulcerative Colitis in Comparison with Crohn's
Colitis and Non-Inflammatory Bowel Disease Controls
[0056] In the ELISA, using the supernatants from cultured LPMCs
against the four hTM isoforms (hTM 1, 2, 3 & 5), the OD values
are shown for each of the 48 patients in the scatterogram (FIG. 4).
The thick horizontal bar indicates the mean value for each
patients' group against the hTM isoforms. The immunoreactivity of
LPMC supernatants from ulcerative colitis against hTM1 and hTM5 was
significantly higher when compared to LPMC supernatants from both
Crohn's colitis and non-inflammatory bowel disease (hTM1: p=0.006
and p=0.001; hTM5: p=0.04 and p=0.01 respectively). However, the
reactivity against hTM1 and hTM5 did not differ between Crohn's
colitis and non-inflammatory bowel disease controls. Differently
from hTM1 and hTM5, the mean OD values for IgG antibodies against
hTM2 and hTM3 in LPMC supernatants. did not significantly differ
among total group of ulcerative colitis, Crohn's colitis and
non-inflammatory bowel disease controls.
[0057] The supernatants from cultured LPMC from 12 of 19 ulcerative
colitis patients (63%) had clearly higher anti-TM reactivity
against one or multiple hTM isoforms when compared to the mean plus
three standard deviation value for Crohn's colitis (shown in FIG. 4
by the long horizontal line for each of the four hTM isoforms).
These 12 patients included all 10 patients who were symptomatic
both clinically and endoscopically, and 2 patients who were in
clinical remission. Six of the 10 symptomatic patients had
reactivity against both hTM1 & hTM5 isoforms. Four additional
patients (two symptomatic and two in remission) showed high
immunoreactivity against hTM1 but not hTM5 and two other patients
(both symptomatic) showed reactivity against hTM5 and not against
hTM1. Five of these six patients' sera (3/4 in the hTM1 group and
2/2 in the hTM5 group) were also tested against hTM 1 and hTM5.
Four of the five sera (two in each group) showed parallel anti-hTM
reactivity, whereas one serum did not react with either of the two
hTM isoforms. The two sera in the hTM1 group reacted with both hTM1
and hTM5.
[0058] Although not statistically significant as a whole group of
ulcerative colitis, the LPMC supernatants from 7 of the 12
ulcerative colitis patients who were reactive against hTM1 and/or
hTM5 also showed higher reactivity against hTM2 and 5 of these 7
against hTM3 as well (FIG. 4), and all of them were symptomatic.
However, none of the 12 patients with Crohn's colitis (6 active and
6 in remission) had values beyond the cutoff line of mean.+-.3SD
for Crohn's colitis. Only one of the 17 patients in the
non-inflammatory bowel disease control group had high OD value
against hTM1, 2 and 3, but not against hTM5 (FIG. 4). In general,
LPMCs from the symptomatic ulcerative colitis group produced higher
IgG antibodies to hTM1 and hTM5 (mean OD.+-.SE: 0.386.+-.0.162
& 0.337.+-.0.140 respectively) than LPMCs from ulcerative
colitis in remission (mean OD.+-.SE: 0.049.+-.0.019, 0.051.+-.0.025
respectively) (p=0.04), symptomatic Crohn's colitis (mean OD.+-.SE:
0.007.+-.0.005 and 0.016.+-.0.009 respectively) (p=0.03) and
non-inflammatory bowel disease (mean OD.+-.SE: 0.006.+-.0.002 and
0.003.+-.0.002 respectively) (p=0.002). The OD values for the IgG
antibodies against any of the four hTM isoforms did not differ
between patients with Crohn's colitis symptomatic or in remission
and the non-inflammatory bowel disease controls.
[0059] The higher IgG immunoreactivity against hTM1 and hTM5 by
LPMC supernatants in ulcerative colitis was not related to higher
levels of total IgG levels in the same samples. Indeed, the total
IgG for ulcerative colitis+ve vs ulcerative colitis-ve for hTM1 was
351.+-.249 vs 538.+-.206 ng/ml respectively and for hTM5, the total
IgG was 466.+-.329 vs 445.+-.184 ng/ml; (p=not significant for
both). PWM-stimulation did not significantly increase the release
of hTM-specific IgG antibodies from LPMCs (data not shown). Within
each disease group, levels of total and hTM-specific IgG did not
significantly differ in supernatants from LPMC isolated from biopsy
or surgical specimens, and between LPMC isolated from the distal 20
cm of the colon or from the descending colon proximal to 20 cm.
Levels of total and hTM-specific IgG antibodies produced by LPMC
from ulcerative colitis did not also differ in relation to clinical
variables such as age, sex, duration of disease and therapy.
IgG Subclass Antibodies Against hTM1 and hTM5 Isoforms in Culture
Supernatants and Sera from Ulcerative Colitis in Comparison with
Crohn's Colitis and Non-Inflammatory Bowel Disease Controls
[0060] As shown in Table 1, the major IgG reactivities of LPMC
supernatants from patients with ulcerative colitis against hTM1 and
hTM5 belong to IgG1 subclass. Antibodies against hTM1 and hTM5
belonging to IgG2 and IgG3 subclasses were almost undetectable in
the LPMC supernatants. The IgG1 reactivities of the LPMC
supernatants from ulcerative colitis against hTM1 and hTM5 were
significantly (p=0.02 to 0.04) higher when compared to Crohn's
colitis and non-inflammatory bowel disease controls (Table 1). The
IgG1 reactivities against hTM1 and hTM5 for the ulcerative colitis
sera were also significantly (p=0.01 to 0.04) higher when compared
to sera from Crohn's colitis and controls. In addition, unlike
mucosal IgG subclass antibodies against hTM1 and hTM5, ulcerative
colitis sera also contained IgG2 subclass antibodies against hTM1
and hTM5. This reactivity was significantly (p=0.01 to 0.04) higher
in ulcerative colitis when compared to Crohn's colitis and
non-inflammatory bowel disease controls (Table 1). Such a
difference was not observed in ulcerative colitis sera for the IgG3
reactivity against hTM1 or hTM5. There was no difference in the
anti-hTM1 and anti-hTM5 reactivities in Crohn's colitis sera when
compared to non-inflammatory bowel disease controls (Table 1).
TABLE-US-00001 TABLE 1 Levels of IgG subclass antibodies (IgG1,
IgG2, and IgG3) against hTM1 and hTM5 isoforms in the supernatants
from cultured mucosal lymphocytes (LPMC) and sera from patients
with UC, CD and non-IBD Antibodies against hTM1 and hTM5 isoforms
(Mean OD .+-. SE) hTM1 hTM5 Patients LPMC Sera LPMC Sera UC IgG1
0.146 .+-. 0.248 .+-. 0.021.sup.b 0.208 .+-. 0.026.sup.c 0.691 .+-.
0.077.sup.d 0.100.sup.a IgG2 0.003 .+-. 0.108 .+-. 0.013.sup.e
0.014 .+-. 0.009 0.218 .+-. 0.042.sup.f 0.001 IgG3 0.010 .+-. 0.007
.+-. 0.004 0.012 .+-. 0.008 0.004 .+-. 0.002 0.005 CD IgG1 0.116
.+-. 0.152 .+-. 0.032 0.133 .+-. 0.010 0.420 .+-. 0.111 0.007 IgG2
0.001 .+-. 0.010 .+-. 0.002 0.003 .+-. 0.006 0.058 .+-. 0.028 0.002
IgG3 0.005 .+-. 0.002 .+-. 0.003 0.003 .+-. 0.003 0.002 .+-. 0.003
0.002 Non-IBD IgG1 0.113 .+-. 0.102 .+-. 0.008 0.135 .+-. 0.007
0.363 .+-. 0.080 0.007 IgG2 0.003 .+-. 0.003 .+-. 0.002 0.001 .+-.
0.003 0.063 .+-. 0.018 0.003 IgG3 0.003 .+-. 0.004 .+-. 0.002 0.002
.+-. 0.006 0.002 .+-. 0.003 0.002 LPMC: .sup.ap = 0.02 UC vs CD and
p = 0.03 UC vs non-IBD .sup.cp = 0.04 UC vs CD and non-IBD Sera: bp
= 0.01 UC vs CD and p = 0.03 UC vs non-IBD .sup.dp = 0.04 UC vs CD
and non-IBD ep = 0.03 UC vs CD and p = 0.01 UC vs Non-IBD fp = 0.04
UC vs CD and non-IBD
[0061] FIG. 1. SDS-Polyacrylamide gel electrophoresis followed by
Coomassie blue staining of purified recombinant human tropomyosin
isoforms (hTM1-5) and enriched hTMs from colon epithelial cells and
colon smooth muscle. Lanes 2 to 6 contain hTM1-5 respectively. The
molecular weights of various hTM isoforms vary from Mr 30K to Mr
40K. Lane 7 contains enriched TM preparation from smooth muscle of
colon and lane 8 contains enriched TM preparation from colon
epithelial cells. The colon epithelial cells contain low Mr hTMs
(Mr 30K to Mr 32K) whereas smooth muscle contain mostly high Mr
hTMs (Mr 38K to Mr 40K).
[0062] FIG. 2. Immunotransblot analysis of the enriched TM extracts
from colon and jejunal epithelial cells using isoform specific
monoclonal antibodies, CG1 (anti-hTM1), CGb6 (anti-hTM 2&3),
LC24 (anti-hTM4), LC1 (anti-hTM5) and CG3 (anti-hTM5). Lanes 1 in
each panel contain respective recombinant hTM isoforms. For CGb6
hTM2 was used. Lanes 2 and 3 in each panel contain enriched TM
preparations from 2 different specimens of colon epithelial cells
and lanes 4 contain enriched TM from a jejunal epithelial cells.
Both colonic and jejunal epithelial cells contain hTM5 and hTM4,
and there was no detectable hTM1,2 or 3. The two extreme right
lanes show recombinant hTM4 and hTM5 (Mr .sup..about.30K) separated
by SDS-polyacrylamide gel electrophoresis and stained with
Coomassie blue.
[0063] FIG. 3. Immunotransblot analysis of enriched TM preparations
from the smooth muscle of intestine probed with the hTM isoform
specific monoclonal antibodies against hTM1-5. Lanes 3 in each
panel contain respective recombinant hTM isoforms. Lanes 1 contain
enriched TM extract from colonic smooth muscle and lanes 2 contain
similar preparation from jejunal smooth muscle. Both colonic and
jejunal smooth muscle contain mainly hTM1, hTM2 and/or hTM3 and a
trace of hTM4. There was no detectable hTM5.
[0064] FIG. 4. Scatterogram showing the immunoreactivity of
spontaneously produced IgG by LPMCs against hTM1,2,3 and 5. The
individual OD value for each of the LPMC-supernatant from the 48
patients with UC (n=19), CD (n=12), and non-IBD controls (n=17) is
shown here. In the UC group, there were 12 symptomatic patients
(identified by a D) and 7 patients were in remission (closed
circle, f). Of the 12 CD patients with colonic disease, 6 were
active and 6 were in remission. The continuous horizontal line
shown for each hTM isoform represents the mean value plus three
standard deviations for the CD group against the respective hTM
isoforms. The short, thick horizontal bar indicates the mean value
for the group.
Discussion
[0065] The results from this study demonstrate that normal human
intestine contains several isoforms of tropomyosins and there are
clear differences between epithelium and smooth muscle. Epithelial
cells from both colon and jejunum contain mainly hTM5 followed by
hTM4, whereas smooth muscle contain hTM1, hTM2 and 3. Since the
monoclonal antibody CGb6 does not differentiate between hTM2 and
hTM3, at present we do not know whether intestinal smooth muscle
contains either or both of these 2 hTM isoforms. Since the mucosa
contains both epithelium and muscularis propria and other cells,
total mucosal tropomyosin preparation included all of the 5
isoforms. Indeed, these findings explain our earlier report
demonstrating multiple tropomyosin related bands in the colonic
mucosal extracts (23).
[0066] Tropomyosins can induce significant autoimmune responses
(17). Physicochemical and biochemical analyses of 109 human
autoantigens demonstrated that sequences longer than 27 residues
with coiled-coil alpha-helices are the forerunner autoantigens for
various human autoimmune diseases and several sequences of
tropomyosin residues were considered to be the most potent
autoantigens (17). Tropomyosin has also been found to be the major
allergen related to seafood and house dust mite (16,30), and about
80% of shrimp-allergic subjects contain IgE and IgG serum antibody
reactive with a 36KD tropomyosin extracted from shrimp (16).
[0067] Multicellular organisms exhibit multiplicity of tropomyosin
isoforms. For example, at least 12 isoforms have been identified in
rat on the basis of primary sequence differences with molecular
weights ranging from Mr 30K to 40K (14). At least 8 distinct
isoforms of tropomyosins are isolated from human fibroblast (15)
and organ specific isoforms have been well characterized (14). Four
genes are identified in human and various isoforms are synthesized
by alternate RNA splicing mechanism restricted to three exon
regions (14, 32-33). Each tropomyosin molecule is a rod-shaped
coiled-coil dimeric protein associated with the actin filaments,
caldesmone, and tropomodulin and involved in cell shape,
cytokinesis, intracellular granule movement (14,33), and other
functions such as maintenance of cellular polarity in developing
tissue (34). While by and large tropomyosins are intracellular
proteins, they have also been found in the cell membrane e.g. in
erythrocytes (35), including at the root of the brush border of the
small intestine (36). Independent protein with partial (35%)
homology to tropomyosin has also been isolated from the brush
border of the small intestine (37).
[0068] Several independent studies recently demonstrated IgG
autoantibody responses against tropomyosins in ulcerative colitis
but not in Crohn's colitis (13,38,39). Using a large number of
inflammatory bowel disease patients and their first degree blood
relatives, one study from Italy reported that sera from about
two-thirds of these Italian patients with ulcerative colitis
contained autoantibody against hTM-5 and about half of the patients
against hTM-1 (38). Twenty percent of these patients' healthy
first-degree relatives were found to have autoantibody against
hTM-1 only. In contrast, patients with Crohn's colitis and their
healthy relatives did not have such antibody response (p<0.001)
(38). Another study from Japan also reported the presence of
anti-tropomyosin antibody in the circulation of patients with
symptomatic ulcerative colitis (39), although hTM isotype was not
analyzed. Furthermore, these investigators reported that tryptic
peptide(s) of tropomyosin bind with specific class II molecule
(HLA-DPw9) and is expressed on the surface of L cells transfected
with class II genes, and the serum from ulcerative colitis contains
autoantibody that recognizes the tropomyosin-peptide on the surface
of transfected L cells (39). In one study from the United Kingdom,
anti-tropomyosin antibodies were not demonstrated in ulcerative
colitis (40). In this study, from colonic mucosa, 2 tropomyosin
related proteins of 37 kDa and 39 kDa, which co-migrated in
SDS-polyacrylamide gel electrophoresis with porcine tropomyosin,
were electroeluted and used in an ELISA as antigen. No reactivity
was seen with any of the human sera, including ulcerative colitis,
Crohn's colitis, non-inflammatory bowel disease controls. This
complete non-reactivity can be explained due to the loss of
antigenicity by SDS-treatment which is a common phenomenon with
autoantibodies (41). Using colonic mucosal extract enriched in
tropomyosins, we earlier reported non-reactivity of autoantibodies
in ulcerative colitis by immunotransblot analysis, although the
reactivity was evident in the ELISA using the same extract prepared
without SDS treatment (23).
[0069] Recently, IgG antibodies against tropomyosin have been
demonstrated in an animal model of TCR.sup.-/-mice who develop
spontaneous colitis. Interestingly, there was a positive
correlation of anti-tropomyosin antibody titer with the severity of
colitis (42). However, it is unknown whether the autoantibody
response in this mouse model of colitis is directed against any of
the known hTM isoforms. This group further demonstrated that
autoantibody producing B cells directed against tropomyosin was
increased in the appendix lymphoid follicle compared to Peyer's
Patches, suggesting selectivity of the mucosal immune response
against tropomyosin (43).
[0070] In this study, we have purified recombinant hTM isoforms and
using the four hTM isoforms (hTM1-3 and hTM5), we have demonstrated
that unstimulated LPMCs from the colon of 12 of 19 (63%) patients
with ulcerative colitis synthesize IgG autoantibody in vitro that
preferentially reacts against certain hTM isoforms, particularly
hTM5 and hTM1. Ten of these 12 patients were symptomatic. Following
antigenic stimulation by a tropomyosin-peptide(s), the autoreactive
B cells may be polyclonal and may produce antibodies to different
parts of the tropomyosin isoform which will show cross-reactivity
to other hTM isoforms because of the shared areas among the
isoforms. Indeed, five of the 12 symptomatic ulcerative colitis
patients did show anti-tropomyosin activities to all the 4 hTM
isoforms, while the others had more restricted antibody activity
against hTM5 and/or hTM1. However, none of the Crohn's colitis
patients showed any reactivity against any of the 4 hTM isoforms,
although each of the 12 Crohn's colitis patients had colonic
involvement, and 6 of these patients had active disease. This
suggests that the autoantibody response is not a secondary effect
of colonic inflammation. The absence of anti-tropomyosin
autoantibody response in the remaining 7/19 ulcerative colitis
patients who were mostly in remission may be due to insufficient in
vitro synthesis of IgG by LPMCs for detection by the ELISA. The
non-reactive group may also suggest a subgroup. Although we did not
examine the purified hTM5 extracted from patient's own colonocytes
and instead we used recombinant hTM5, it is expected that local
autoantibody should react with the autologous hTM5. This is
supported by our previous observation that colon-tissue bound IgG
from ulcerative colitis recognized the tropomyosin related Mr 40K
protein isolated from the autologous colon (9). In this study, we
did not examine the pANCA status of the patients. However, the
study from Italy (38) reported a significant (p<0.04)
correlation of positive pANCA status with the presence of anti-hTM5
antibody in sera from patients with ulcerative colitis. The
predominant hTM isoform present in the colon epithelium, as shown
here, is hTM5, and autoantibody response against hTM5 is evident.
hTM4 is also present in smaller amount in the epithelial cells.
Recombinant hTM4 was not available and antibody response against
hTM4 was not studied. It is interesting that a significant
autoantibody response in ulcerative colitis was demonstrated
against hTM1. This may suggest that the major autoantigenic
epitope(s) may be located in the common region(s) between hTM5 and
hTM1. The autoantibody response against hTM1 may also be due to an
ectopic expression of hTM1 related epitope in ulcerative colitis
colon epithelium which is currently unknown.
[0071] To understand a possible role of hTM5 or related protein in
the pathogenesis of ulcerative colitis, one needs to explain that
since hTM5 is present in both colonocytes and small intestinal
enterocytes, why isn't there small bowel involvement in patients
with ulcerative colitis? It is intriguing that we recently observed
a selective expression of hTM5 on the surface of colon epithelial
cells and not on small intestinal enterocytes (44). Of all the 5
hTM isoforms (hTM1-5), only hTM5 epitope was identified on the
surface of normal colonic epithelial cells by FACS analysis and
immunoelectronmicroscopy (44). Indeed, none of the monoclonal
antibodies against hTM1, 2, 3 & 4, reacted with the freshly
isolated normal colon epithelial cells; whereas CG3 monoclonal
antibody (anti-hTM5) consistently reacted on colonocytes in the
FACS analysis. In contrast to colonocytes, small intestinal (both
jejunum and ileum) epithelial cells, however, did not react with
any of the monoclonal antibodies, including CG3. This raises the
possibility that hTM5 related epitope expressed on the surface of
colon epithelial cells may be involved in stimulating the effector
immune system as well as a target for immune attack restricted to
the colon. Such an organ-specific antigenic display may play an
important role for a local trigger of immune response and
perpetuation of the disease in the colon and not in the small
intestine. Indeed, recently antigenic display of myosin has been
found to be involved in the pathogenesis of autoimmune myocarditis
(45), which may also be genetically determined (46). Molecular
mimicry, related to a specific peptide in streptococcal M protein
and tropomyosin, has been found to be an important pathogenetic
factor in autoimmune myocarditis (47). Such a molecular mimicry
against bacterial product (48) may influence the mucosal immune
functions and be responsible for perpetuation and flare-up of the
disease.
[0072] While IgG autoantibody response against tropomyosin in
ulcerative colitis has been demonstrated by the 3 groups of
investigators (13,38,39), T-cell response to tropomyosins in
ulcerative colitis is unknown. Physicochemically,
tropomyosin-peptide(s) could be strong potential autoantigen(s)
(17). The knowledge of normal colon epithelial specific hTM
isoforms and the autoantibody response against hTMs in ulcerative
colitis, will facilitate future studies to focus on the
identification of the hTM peptide(s) capable of cellular and
humoral immune responses in patients with ulcerative colitis. Such
investigation should provide important biochemical information to
explain autoimmune mechanism in ulcerative colitis and may help to
develop strategies for possible immune therapy.
[0073] The antibodies of the present invention may be used together
with pharmaceutically acceptable carriers to provide pharmaceutical
compositions which can be administered to a human orally or
rectally, or both, in amounts effective to provide a variety of
therapeutic activity. Of course, the type of carrier will vary
depending upon the mode of administration desired for the
pharmaceutical composition as is conventional in the art.
Preferably, the antibody is administered orally or rectally to the
human.
[0074] It is especially advantageous to formulate the
pharmaceutical compositions in dosage unit forms for ease of
administration and uniformity of dosage. The term dosage unit forms
as used herein refers to physically discrete units suitable for use
as a unitary dosage, each unit containing a predetermined quantity
of active ingredient calculated to produce the desired therapeutic
effect in association with the pharmaceutical carrier.
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[0123] Throughout this application, various publications have been
referenced. The disclosures in these publications are incorporated
herein by reference in order to more fully describe the state of
the art.
[0124] While the invention has been particularly described in terms
of specific embodiments, those skilled in the art will understand
in view of the present disclosure that numerous variations and
modifications upon the invention are now enabled, which variations
and modifications are not to be regarded as a departure from the
spirit and scope of the invention. Accordingly, the invention is to
be broadly construed and limited only by the scope and spirit of
the following claims.
* * * * *