U.S. patent application number 16/847334 was filed with the patent office on 2020-10-01 for use of glycans and glycosyltransferases for diagnosing/monitoring inflammatory bowel disease.
This patent application is currently assigned to Children's Hospital Medical Center. The applicant listed for this patent is Children's Hospital Medical Center. Invention is credited to Lee A. DENSON, Ardythe L. MORROW.
Application Number | 20200308644 16/847334 |
Document ID | / |
Family ID | 1000004896824 |
Filed Date | 2020-10-01 |
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United States Patent
Application |
20200308644 |
Kind Code |
A1 |
MORROW; Ardythe L. ; et
al. |
October 1, 2020 |
USE OF GLYCANS AND GLYCOSYLTRANSFERASES FOR DIAGNOSING/MONITORING
INFLAMMATORY BOWEL DISEASE
Abstract
Diagnostic methods for assessing risk of or presence of
inflammatory bowel disease in a patient based on
glycosyltransferase or histo-blood group antigen signatures or a
combination thereof. Also disclosed herein are prognostic methods
for monitoring inflammatory bowel disease progression in a
patient.
Inventors: |
MORROW; Ardythe L.;
(Cincinnati, OH) ; DENSON; Lee A.; (Wyoming,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Hospital Medical Center |
Cincinnati |
OH |
US |
|
|
Assignee: |
Children's Hospital Medical
Center
Cincinnati
OH
|
Family ID: |
1000004896824 |
Appl. No.: |
16/847334 |
Filed: |
April 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14767116 |
Dec 24, 2015 |
10626460 |
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PCT/US2014/017630 |
Feb 21, 2014 |
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16847334 |
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61767500 |
Feb 21, 2013 |
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Current U.S.
Class: |
1/1 ; 435/6.12;
435/7.9; 435/7.92 |
Current CPC
Class: |
G01N 2800/065 20130101;
C12Q 1/6883 20130101; G01N 33/5308 20130101; G01N 2800/56 20130101;
C12Q 2600/112 20130101; C12Q 1/48 20130101; C12Q 1/6876 20130101;
C12Q 2600/158 20130101; G01N 2333/91091 20130101; G01N 2800/52
20130101; G01N 33/80 20130101 |
International
Class: |
C12Q 1/6876 20060101
C12Q001/6876; C12Q 1/48 20060101 C12Q001/48; G01N 33/80 20060101
G01N033/80; C12Q 1/6883 20060101 C12Q001/6883; G01N 33/53 20060101
G01N033/53 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0003] This invention was made with government support under
DK078392 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for diagnosing and treating inflammatory bowel disease,
the method comprising: measuring a level of at least one
glycosyltransferase in a tissue sample of a subject suspected of
having an inflammatory bowel disease (IBD), determining an
expression profile of the least one glycosyltransferase in the
tissue sample, assessing whether the subject has or is at risk for
IBD based on the expression profile of the least one
glycosyltranferase, and treating the human subject who is at risk
for IBD with an anti-inflammatory agent, an immune suppressant
agent, an antibiotic agent, or a combination thereof; wherein the
at least one glycosyltransferase comprises one or more of
fucosyltransferase 3 (FUT3), fucosyltranferase 5 (FUT5), or
fucosyltransferase 7 (FUT7), ST3 beta-galactoside
alpha-2,3-sialyltransferase 3 (ST3Gal III), and ST3
beta-galactoside alpha-2,3-sialyltransferase 4 (ST3 Gal IV).
2. The method of claim 1, wherein the tissue sample is an
intestinal sample, a colon biopsy sample, a biofluid sample, a
saliva sample, or a stool sample.
3. The method of claim 1, wherein the at least one
glycosyltransferase includes ST3Gal IV, and wherein an expression
profile representing an elevated level of ST3Gal IV in the tissue
sample of the subject as compared with the expression profile of
ST3Gal IV in a control tissue sample indicates that the subject has
or is at risk for IBD.
4. The method of claim 1, wherein the at least one
glycosyltransferase includes one or more of FUT3, FUT5, FUT7, and
ST3Gal III; and wherein an expression profile representing a
reduced level of the one or more of FUT3, FUT5, FUT7, and ST3Gal
III in the tissue sample of the subject as compared with the
expression profile of the one or more glycosyltransferases of a
control sample indicates that the subject has or is at risk for
IBD.
5. The method of claim 3, wherein the at least one
glycosyltransferase further includes one or more of FUT3, FUT5,
FUT7, and ST3Gal III; and wherein an expression profile
representing an elevated level of ST3Gal IV and a reduced level of
the one or more FUT3, FUT5, FUT7, and ST3Gal III in the tissue
sample of the subject as compared with that in a control tissue
sample indicates that the subject has or is at risk for IBD.
6. The method of claim 1, wherein the at least one
glycosyltransferase further comprises fucosyltransferase 2 (FUT2)
and wherein an expression profile representing an elevated level of
ST3Gal IV and a reduced level of the one or more FUT2, FUT3, FUT5,
FUT7, and ST3Gal III in the tissue sample of the subject as
compared with that in a control tissue sample indicates that the
subject has or is at risk for IBD.
7. The method of claim 1, wherein the subject is suspected of
having Intestinal Colitis, Ulcerative Colitis, or Crohn's
disease.
8. The method of claim 1, wherein the expression level of the at
least one glycosyltransferase is determined by measuring the mRNA
level of the at least one glycosyltransferase in the tissue sample
or by measuring the level of a microRNA that regulates the
expression of the at least one glycosyltransferase.
9. The method of claim 8, wherein the mRNA and microRNA levels are
measured by PCR, in situ hybridization, RNA sequencing, or flow
cytometry.
10. A method for diagnosing and treating Ulcerative Colitis, the
method comprising: measuring the level of fucosyltransferase 2
(FUT2) in a tissue sample of a subject suspected of having
Ulcerative Colitis (UC), assessing whether the subject has or is at
risk for UC based on the expression level of FUT2, wherein an
expression level representing a reduced level of FUT2 in the tissue
sample of the subject as compared with that in a control tissue
sample indicates that the subject has or is at risk for UC, and
treating the human subject who is at risk for UC with an
anti-inflammatory agent, an immune suppressant agent, an antibiotic
agent, or a combination thereof.
11. The method of claim 10, wherein the tissue sample is an
intestinal sample, a colon biopsy sample, a biofluid sample, a
saliva sample, or a stool sample.
12. The method of claim 10, wherein the expression level of FUT2 is
the mRNA level of FUT2 or the level of a microRNA that regulates
FUT2 expression.
13. The method of claim 12, wherein the mRNA and microRNA levels
are measured by PCR, in situ hybridization, RNA sequencing, or flow
cytometry.
14-19. (canceled)
21. A method for monitoring and treating inflammatory bowel disease
progression, the method comprising: measuring a first expression
level of at least one glycosyltransferase in a first tissue sample
obtained from a subject having inflammatory bowel disease (IBD) at
a first time point and a second expression level of the at least
one glycosyltransferase in a second tissue sample obtained from the
subject at a second time point, which is later than the first time
point; determining a first expression profile of the at least one
glycosyltransferase in the first tissue sample and a second
expression profile of the at least one glycosyltransferase in the
second tissue sample; and assessing IBD progress in the subject
based on the second expression profile as compared with the first
expression profile, wherein a change between the first expression
profile and the second expression profile is indicative of IBD
progression or regression in the subject; and treating the subject
who shows IBD progression with an anti-inflammatory agent, an
immune suppressant agent, an antibiotic agent, or a combination
thereof; wherein the at least one glycosyltransferase comprises one
or more of fucosyltransferase 3 (FUT3), fucosyltranferase 5 (FUT5),
or fucosyltransferase 7 (FUT7), ST3 beta-galactoside
alpha-2,3-sialyltransferase 3 (ST3Gal III), and ST3
beta-galactoside alpha-2,3-sialyltransferase 4 (ST3 Gal IV).
22. The method of claim 21, wherein the first and second tissue
samples are both intestinal samples, colon biopsy samples, biofluid
samples, saliva samples, or stool samples.
23. The method of claim 21, wherein the at least one
glycosyltransferase includes ST3Gal IV, and wherein the second
expression profile representing an elevated level of ST3Gal IV as
compared with the first expression profile indicates disease
progression in the subject and the second expression profile
representing a reduced level of ST3Gal IV as compared with the
second expression profile indicates disease regression in the
subject.
24. The method of claim 21, wherein the at least one
glycosyltransferase includes one or more of FUT3, FUT5, FUT7, and
ST3Gal III; and wherein the second expression profile representing
a reduced level of the one or more of FUT3, FUT5, FUT7, and ST3Gal
III as compared with the first expression profile indicates IBD
progression in the subject and the second expression profile
representing an elevated level of the one or more of FUT3, FUT5,
FUT7, and ST3Gal III as compared with the first expression profile
indicates IBD regression in the subject.
25. The method of claim 21, wherein the at least one
glycosyltransferase further includes FUT2, wherein the second
expression profile representing an elevated level of ST3Gal IV and
a reduced level of the one or more FUT2, FUT3, FUT5, FUT7, and
ST3Gal III as compared with the first expression profile indicates
IBD progression in the subject, and wherein the second expression
profile representing a reduced level of ST3Gal IV and an elevated
level of the one or more FUT2, FUT3, FUT5, FUT7, and ST3Gal III as
compared with the first expression profile indicates IBD regression
in the subject.
26. The method of claim 21, wherein the subject is suspected of
having Intestinal Colitis, Ulcerative Colitis, or Crohn's
disease.
27. The method of claim 21, wherein the first and second expression
levels of the at least one glycosyltransferase are the mRNA level
of the at least one glycosyltransferase or the level of one or more
microRNAs that regulate expression of the at least one
glycosyltransferase.
28. The method of claim 27, wherein the mRNA and microRNA levels
are measured by PCR, in situ hybridization, RNA sequencing, or flow
cytometry.
29. The method of claim 21, wherein the subject has undergone a
treatment of IBD; wherein either the first and second tissue
samples are obtained before and after the treatment, respectively,
or at least one of the samples is obtained during the course of the
treatment; and wherein the method further comprises assessing the
efficacy of the treatment based on the second expression profile as
compared with the first expression profile.
30. The An in vitro method of claim 21, wherein the at least one
glycosyltransferase is fucosyltransferase 2 (FUT2), and wherein the
IBD is Ulcerative Colitis.
31-41. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/767,116, filed Aug. 11, 2015, which
is a U.S. national stage filing under 35 U.S.C. .sctn. 371 of.
[0002] International Application No. PCT/US2014/017630 entitled
"USE OF GLYCANS AND GLYCOSYLTRANSFERASES FOR DIAGNOSING/MONITORING
INFLAMMATORY BOWEL DISEASE", filed Feb. 21, 2014, which claims the
benefit under 35 USC .sctn. 119(e) of the filing date of U.S.
Provisional Application No. 61/767,500, filed Feb. 21, 2013. The
entire contents of each of the above-referenced applications are
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0004] Inflammatory bowel disease (IBD), including Intestinal
Colitis, Crohn's Disease (CD) and Ulcerative Colitis (UC), involves
chronic inflammation of all or part of the digestive tract.
Intestinal Colitis involves non-specific inflammation of the
intestine. CD involves inflammation anywhere along the lining of
the digestive tract, while UC involves chronic inflammation in a
subsection of the digestive tract and usually only affects the
innermost lining of the colon and rectum. Onset of IBD occurs from
early childhood to older adulthood and includes symptoms of bloody
stool, diarrhea, severe abdominal cramps and pain, and weight
loss.
[0005] IBD is a debilitating condition affecting an estimated 1.4
million Americans, and is a high public health priority. The
incidence of IBD has been increasing in the general population. It
is costly in health care utilization, lost productivity, and
quality of life, with estimated costs for privately insured IBD
patients ranging from $15,020 to $18,963 per year.
[0006] Currently available treatment for IBD includes a stepwise
application of antibiotics, corticosteroids, and immune modifying
treatments. However, not all patients respond to these regimes. The
loss of clinical response is a challenge that results in further
morbidity, reduced quality of life, and increased costs. To date,
there is no validated approach for monitoring patient health status
while under treatment. Considering the variability in patient
response and the frequent occurrence of flares or relapse in
disease, finding and validating novel approaches for patient
monitoring and self-monitoring holds great promise for improving
care as well as patient quality of life. It is therefore of great
interest to develop new approaches for monitoring IBD development
and progression.
SUMMARY OF THE INVENTION
[0007] The present disclosure is based on the unexpected discovery
that levels of certain glycosyltransferases and/or the glycan
antigens synthesized by these enzymes, e.g., the histo-blood group
antigens, are associated with IBD and therefore can be used in IBD
diagnosis and/or prognosis.
[0008] Accordingly, one aspect of the present disclosure provides a
method for diagnosing IBD in a subject, the method (e.g., an in
vitro method) comprising: (i) measuring a level of at least one
glycosyltransferase in a tissue sample (for example, an intestinal
sample, a colon biopsy sample, a biofluid sample, a saliva sample,
or a stool sample) of a subject (e.g., a human subject) suspected
of having inflammatory bowel disease (IBD); (ii) determining an
expression profile of the at least one glycosyltransferase in the
tissue sample; and (iii) assessing whether the subject has or is at
risk for IBD (e.g., Intestinal Colitis, Ulcerative Colitis or
Crohn's Disease) based on the expression profile of the least one
glycosyltransferase. The at least one glycosyltransferase can be
fucosyltransferase 3 (FUT3), fucosyltransferase 5 (FUT5),
fucosyltransferase 7 (FUT7), ST3 beta-galactoside
alpha-2,3-sialyltransferase 3 (ST3Gal III), ST3 beta-galactoside
alpha-2,3-sialyltransferase 4 (ST3Gal IV), or a combination
thereof.
[0009] In one example, the at least one glycosyltransferase
includes one or more of FUT3, FUT5, FUT7, and ST3Gal III. When the
tissue sample exhibits an expression profile representing a reduced
level of the one or more of FUT3, FUT5, FUT7, or ST3Gal III as
compared to a control sample, the subject is determined as having
or being at risk for IBD.
[0010] In another example, the at least one glycosyltransferase
includes ST3Gal IV. When the tissue sample exhibits an expression
profile representing an elevated level of ST3Gal IV as compared
with the expression profile of ST3Gal IV in a control tissue
sample, the subject is determined as having or being at risk for
IBD.
[0011] In yet another example, the at least one glycosyltransferase
includes ST3Gal IV and one or more of FUT3, FUT5, FUT7, and ST3Gal
III. If the tissue sample exhibits an expression profile
representing an elevated level of ST3Gal IV and a reduced level of
the one or more of FUT3, FUT5, FUT7, and ST3Gal III, the subject is
determined as having or being at risk for IBD.
[0012] In any of the examples described above, the at least one
glycosyltransferase can further include fucosyltransferase 2
(FUT2). For example, if the tissue sample exhibits an expression
profile representing an elevated level of ST3Gal IV and a reduced
level of the one or more of FUT2, FUT3, FUT5, FUT7, and ST3Gal III,
the subject is determined as having or being at risk for IBD.
[0013] In any of the methods described above, the expression level
of the at least one glycosyltransferase can be determined by
measuring the mRNA level of the at least one glycosyltransferase in
the tissue sample or by measuring the level of a microRNA that
regulates the expression of the at least one glycosyltransferase.
The mRNA and microRNA levels can be determined by PCR, in situ
hybridization, RNA sequencing, or flow cytometry.
[0014] In a further aspect, the present disclosure provides a
method for diagnosing Ulcerative Colitis in a subject (e.g., a
human subject), the method comprising: (i) measuring an expression
level of fucosyltransferase 2 (FUT2) in a tissue sample (for
example, an intestinal sample, a colon biopsy sample, a biofluid
sample, a saliva sample, or a stool sample) of a subject suspected
of having Ulcerative Colitis (UC), and (ii) assessing whether the
subject has or is at risk for UC based on the expression level of
FUT2 in the tissue sample. When the tissue sample exhibits a
reduced level of FUT2 as compared with that in a control tissue
sample, the subject is determined as having or being at risk for
UC. The expression profile of FUT2 can be determined by measuring
the mRNA level of FUT2 or the level of a microRNA that regulates
FUT2 expression. The mRNA and microRNA levels can be determined,
for example, by PCR, in situ hybridization, RNA sequencing, or flow
cytometry.
[0015] In another aspect, the present disclosure provides a method
for diagnosing inflammatory bowel disease (IBD) in a human subject,
the method comprising: (i) measuring a level of one or more
histo-blood group antigens in a tissue sample (for example, an
intestinal sample, a colon biopsy sample, a biofluid sample, a
saliva sample, or a stool sample) of a human subject suspected of
having IBD, (ii) determining an expression profile of the one or
more histo-blood group antigens in the tissue sample, and (iii)
assessing whether the human patient has or is at risk for IBD
(e.g., Intestinal Colitis, Ulcerative Colitis or Crohn's Disease)
based on the expression profile of the one or more histo-blood
group antigens, which can comprise one or more of sialyl Lewis x
(sLe.sup.x), sialyl Lewis a (sLe.sup.a), and Lewis b (Le.sup.b).
Optionally, the one or more histo-blood group antigen can further
comprise an H antigen.
[0016] In one example, the histo-blood group antigen(s) examined in
the methods described above is sLe.sup.x, sLe.sup.a, or both. In
another example, the histo-blood group antigen(s) examined in the
methods described above is sLe.sup.x. If the tissue sample exhibits
an expression profile representing an elevated level of the
histo-blood group antigen(s), the human subject is determined as
having or being at risk for IBD.
[0017] The above-noted histo-blood group antigen(s) can further
comprise Le.sup.b, and/or an H antigen. If the tissue sample
exhibits a reduced level of H antigen as compared to that of a
control sample, the human subject is further determined as having
or being at risk for IBD.
[0018] In any of the methods described above where applicable, the
level of the one or more histo-blood antigens can be measured by an
immune assay, agglutination inhibition assay, or flow
cytometry.
[0019] Any of the diagnosis methods described herein can further
comprise administering to the subject an effective amount of an
anti-IBD drug, if the subject is diagnosed as having or at risk for
IBD, such as Intestinal Colitis, Ulcerative Colitis, or Crohn's
Disease.
[0020] In yet another aspect, the present disclosure features a
method for monitoring inflammatory bowel disease (IBD) progression
in a subject (e.g., a human subject or a laboratory animal), the
method comprising: (i) measuring a first expression level of at
least one glycosyltransferase in a first tissue sample obtained
from a subject having inflammatory bowel disease (IBD) at a first
time point, and a second expression level of the at least one
glycosyltransferase in a second tissue sample of the subject at a
second time point, which is later than the first time point, (ii)
determining a first expression profile of the at least one
glycosyltransferase in the first tissue sample and a second
expression profile of the at least one glycosyltransferase in the
second tissue sample; and (iii) assessing IBD (e.g., Intestinal
Colitis, Ulcerative Colitis or Crohn's Disease) progress in the
subject based on the second expression profile as compared with the
first expression profile. If there is a change between the first
expression profile and the second expression profile, the subject
is determined as having IBD progression or regression. The first
and second tissue samples both can be an intestinal sample, a colon
biopsy sample, a biofluid sample, a saliva sample, or a stool
sample.
[0021] In some embodiments, the subject has undergone a treatment
of IBD. In that case, the first and second tissue samples can be
obtained before and after the treatment, respectively.
Alternatively, at least one of the samples can be obtained during
the course of the treatment. When necessary, the method described
above further comprises assessing the efficacy of the treatment
based on the second expression profile as compared with the first
expression profile. In one example, the at least one
glycosyltransferase includes one or more of FUT3, FUT5, FUT7, and
ST3Gal III. If the second expression profile represents a reduced
level of the one or more of FUT3, FUT5, FUT7, and ST3Gal III as
compared with the first expression profile, the subject is
determined as having IBD progression. On the other hand, if the
second expression profile represents an elevated level of the one
or more of FUT3, FUT5, FUT7, and ST3Gal III as compared with the
first expression profile, the subject is determined as having IBD
regression.
[0022] In another example, the at least one glycosyltransferase
includes ST3Gal IV. If the second expression profile represents an
elevated level of ST3Gal IV as compared with the first expression,
the subject is determined as having IBD progression. If the second
expression profile represents a reduced level of ST3Gal IV as
compared with the second expression profile, the subject is
determined as having IBD regression.
[0023] Alternatively, the just-noted at least one
glycosyltransferase further includes FUT2. When the at least one
glycosyltransferase includes ST3GalIV and one or more of FUT2,
FUT3, FUT5, FUT7, and ST3Gal III, if the second expression profile
represents an elevated level of ST3Gal IV and a reduced level of
the one or more FUT2, FUT3, FUT5, FUT7, and ST3Gal III as compared
with the first expression profile, the subject is determined as
having IBD progression. On the other hand, if the second expression
profile represents a reduced level of ST3Gal IV and an elevated
level of the one or more FUT2, FUT3, FUT5, FUT7, and ST3Gal III as
compared with the first expression profile, the subject is
determined as having IBD regression.
[0024] In any of the methods described above, the expression levels
of the at least one glycosyltransferase is determined by measuring
the mRNA levels of the at least one glycosyltransferase in the
tissue samples or by measuring the levels of a microRNA that
regulates the expression of the at least one glycosyltransferase.
The mRNA and microRNA levels can be determined, for example, by
PCR, in situ hybridization, RNA sequencing, or flow cytometry.
[0025] In yet another aspect, the present disclosure provides a
method for monitoring Ulcerative Colitis (UC) progression in a
subject, the method comprising: (i) determining a first expression
level of fucosyltransferase 2 (FUT2) in a first tissue sample of a
subject having UC at a first time point, and a second expression
level of FUT2 in a second tissue sample of the subject at a second
time point, which is later than the first time point, and (ii) and
(ii) assessing UC progression in the subject based on the second
expression level of FUT2 as compared with the first expression
level of FUT2. If the second expression level of FUT2 is reduced as
compared with the first expression level of FUT2, the subject is
determined as having UC progression. If the second expression level
of FUT2 is elevated as compared with the first expression level of
FUT2, the subject is determined as having UC regression.
[0026] The expression level of FUT2 can be determined by measuring
its mRNA level in the tissue sample or by measuring the level of a
microRNA that regulates FUT2 expression. The mRNA and microRNA
levels can be measured, for example, by PCR, in situ hybridization,
RNA sequencing, or flow cytometry.
[0027] In some embodiments, the just-described method can involve
taking samples from subjects having undergone a treatment of IBD.
Either the first and second intestinal samples can be obtained
before and after the treatment, respectively. Alternatively at
least one of the samples is obtained during the course of the
treatment. When desired, this method further comprises assessing
the efficacy of the treatment based on the second expression
profile as compared with the first expression profile.
[0028] In still another aspect, the present disclosure features a
method (e.g., an in vitro method) for monitoring inflammatory bowel
disease (IBD) progression in a human patient, the method
comprising: (i) measuring a first level of one or more histo-blood
group antigens in a first tissue sample of a human IBD patient at a
first time point and a second level of the one or more histo-blood
group antigens in a second tissue sample of the human patient at a
second time point, which is later than the first time point, (ii)
determining a first profile of the one or more histo-blood group
antigens in the first tissue sample and a second profile of the one
or more histo-blood group antigens in the second sample, and (iii)
assessing IBD (for example, Intestinal Colitis, Ulcerative Colitis
or Crohn's disease) progression or regression in the human patient
based on the second profile of the one or more histo-blood antigens
as compared with the first profile of the one or more histo-blood
antigens. The one or more histo-blood group antigens can comprise
one or more of sialyl Lewis x (sLe.sup.x), sialyl Lewis a
(sLe.sup.a), and Lewis b (Le.sup.b), and optionally an H antigen.
In one example, the one or more histo-blood group antigens is
sialyl Lewis x (sLe.sup.x). The first and second tissue samples
both can be an intestinal sample, a colon biopsy sample, a biofluid
sample, a saliva sample, or a stool sample. The level of the one or
more histo-blood antigens can be measured, for example, by an
immune assay, agglutination inhibition assay, or flow
cytometry.
[0029] In one example, the histo-blood group antigen(s) to be
measured in the above method is sLe.sup.x, sLe.sup.a, or both.
Optionally, the histo-blood group antigens further include
Le.sup.b, an H antigen, or both. The human subject is determined as
having IBD progression if the second expression profile indicates
elevated levels of the one or more histo-blood group antigens as
compared with the first expression profile. Conversely, the human
subject is determined as having IBD regression if the second
profile indicates reduced levels of the one or more histo-blood
group antigens as compared with the first profile.
[0030] When the histo-blood antigens include an H antigen, a human
subject is determined to have IBD progression if the second profile
indicates a reduced level of the H antigen and an elevated level of
other histo-blood group antigens as compared with the first
profile. Conversely, the human subject is determined to have IBD
regression if the second profile indicates an elevated level of the
H antigen and reduction of other histo-blood group antigens as
compared with the first profile.
[0031] In another example, the level of an H antigen, either alone
or in combination of any of the other glycan antigens described
herein, of a test subject is measured to assess whether that
subject has or is at risk for Ulcerative Colitis. If a reduced
level of the H antigen is observed, the test subject is determined
as having or being at risk for Ulcerative Colitis.
[0032] Also within the scope of this disclosure are kits for use in
diagnosing IBD or monitoring IBD progression/regression in a
subject, such as a human subject. Such a kit comprises reagents
(e.g., oligonucleotides) for determining the level(s) of one or
more of the glycosyltransferases described herein (e.g., FUT2,
FUT3, FUT5, FUT7, ST3Gal III, and/or ST3Gal IV), or reagents (e.g.,
antibodies) for determining the levels of one or more histo-blood
group antigens also described herein (e.g., sLe.sup.x, sLe.sup.a,
Le.sup.b, or an H antigen).
[0033] In addition, the present disclosure provides a method (e.g.,
an in vitro method) for monitoring Crohn's disease (CD)
progression, the method comprising: (i) measuring a first level of
one or more human blood group antigens in a first tissue sample of
a human CD patient at a first time point and a second level of the
one or more human blood group antigens in a second tissue sample of
the human CD patient at a second time point, which is later than
the first time point; (ii) determining a first profile of the one
or more human blood group antigens in the first tissue sample and a
second profile of the one or more human blood group antigens in the
second tissue sample; and (iii) assessing CD progression or
regression in the human patient based on the second profile as
compared with the first profile; wherein the one or more human
blood group antigens comprise sialyl Lewis x (sLe.sup.x), Lewis x
(Lex), or both. In one example, the one or more human blood group
antigens is sialyl Lewis x (sLe.sup.x). The first and second tissue
samples may both be intestinal samples, colon biopsy samples,
biofluid samples, saliva samples, or stool samples. The subject may
exhibit at least one symptom associated with CD. In one example,
the subject is undergoing a CD treatment and the disease
progression status as assessed by this method can be used to
evaluate the efficacy of the treatment on the subject.
[0034] In some embodiments, the second profile representing an
elevated level of the one or more human blood group antigens (e.g.,
an elevated level of sialyl Lewis x (sLe.sup.x), Lewis x (Lex), or
both) as compared with the first profile indicates CD progression
in the patient and the second profile representing a reduced level
of the one or more human blood group antigens as compared with the
first profile indicates CD regression in the patient.
[0035] In other embodiments, the levels of the one or more human
blood antigens can be measured by an immune assay, agglutination
inhibition assay, or flow cytometry.
[0036] Further, the present disclosure features a method (e.g., an
in vitro method) for diagnosing Crohn's disease (CD), the method
comprising: (i) measuring a level of one or more human blood group
antigens in a tissue sample of a human subject suspected of having
CD, (ii) determining a profile of the one or more human blood group
antigens in the tissue sample, and (iii) assessing whether the
human patient has or is at risk for CD based on the profile of the
one or more human blood group antigens; wherein the one or more
human blood group antigens comprise an H antigen, Lewis b
(Le.sup.b), Lewis y (Le.sup.y), or a combination thereof. In some
examples, the profile representing a decreased level of H antigen,
Lewis b (Le.sup.b), Lewis y (Le.sup.y), or a combination thereof,
indicates that the human subject has or is at risk for CD. In other
examples, the tissue sample can be an intestinal sample, a colon
biopsy sample, a biofluid sample, a saliva sample, or a stool
sample. The level of the one or more human blood antigens is
measured by an immune assay, agglutination inhibition assay, or
flow cytometry.
[0037] A subject identified as having or at risk for CD by the
methods described herein can be subjected to a treatment against
CD, such as those described herein.
[0038] The details of one or more embodiments of the invention are
set forth in the description below. Other features or advantages of
the present invention will be apparent from the following drawings
and detailed description of several examples, and also from the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The drawings are first described.
[0040] FIG. 1 is a scatterplot of sialyl Lewis and H antigen levels
in cases/patients and controls. Boxes represent patients with IBD
while diamonds represent controls. The solid line represents the
cut-off value for significant differentiation between patients and
controls.
[0041] FIG. 2 is a scatterplot of sialyl Lewis and Lewis b antigen
levels in cases/patients and controls. Boxes represent patients
with IBD while diamonds represent controls. The solid lines
represent the cut cut-off values for sialyl Lewis and Lewis b for
significant differentiation between patients and controls.
[0042] FIG. 3 is a boxplot showing optical density values for Lewis
a (lea) and Lewis x (lex) salivary antigens in several patient
populations. The y axis indicates the optical density (O.D.) value
of the salivary antigens. For each group (1-7), the left box is lea
and the right box is lex.
[0043] FIG. 4 is a boxplot showing optical density values for H
antigen (H), Lewis b (leb) and Lewis y (ley) salivary antigens in
several patient populations. The y axis indicates the O.D. value of
the salivary antigens. For each group (1-7), the left box is H, the
middle box is leb, and the right box is ley.
[0044] FIG. 5 is a boxplot showing optical density values for
sialyl Lewis a (slea) and sialyl Lewis x (slex) salivary antigens
in several patient populations. The y axis indicates the O.D. value
of the salivary antigens. For each group (1-7), the left box is
slea and the right box is slex.
[0045] FIG. 6 is a boxplot showing salivary antigens tested in
samples collected at the time of enrollment from Asymptomatic
Crohn's Disease (CD) patients (n=20) compared to Symptomatic CD
patients (n=9). slex=Sialyl Lewis x, slea=sialyl Lewis a, lex=Lewis
x, lea=Lewis a, uea=H antigen (identified by Ulex europaeus
antigen), leb=Lewis b, and ley=Lewis y. The y axis indicates the
O.D. value of the salivary antigens. For each group (asymptomatic
or symptomatic CD), the boxs from left to right are slex, slea,
lex, lea, uea, leb, and ley.
[0046] FIG. 7 is a boxplot showing salivary antigens tested in the
follow-up sample collected by the patient at home, following the
clinical/enrollment visit. The patient groups were Asymptomatic
Crohn's Disease (CD) patients (n=15) compared to Symptomatic CD
patients (n=8). The y axis indicates the O.D. value of the salivary
antigens. For each group (asymptomatic or symptomatic CD), the boxs
from left to right are slex, slea, lex, lea, uea, leb, and ley. The
_01 indicates that it is a follow-up sample.
[0047] FIG. 8 is two graphs showing Lexis x and sialyl Lewis X
values in asymptomatic and symptomatic CD patients measured by
ELISA. The values for the x and y-axes are optical density (O.D.)
values. The line through each graph marks the 1.8 O.D. value, which
may serve as a cut-point for a high sLe x value.
[0048] FIG. 9 is a dotplot of the values for each subject on the
short PCDAI scale (y-axis) in relation to the combination of
sLe.sup.x and Lewis x (high vs low).
[0049] FIG. 10 is a boxplot of sialyl Lewis x (sLe.sup.x) and Lewis
x (Lex) O.D. values by history of antibiotic use. The y axis
indicates the O.D. value. abx use=history of antibiotic use, no
abx=no history of antibiotic use. For each group in each plot, the
left box is sLe.sup.x and the right box is Lex.
[0050] FIG. 11 is a boxplot of sialyl Lewis x (sLe.sup.x) and Lewis
x (Lex) O.D. values by secretor status. The y axis indicates the
O.D. value. Non-sec=non-secretor, sec=secretor. For each group in
each plot, the left box is sLe.sup.x and the right box is Lex.
[0051] FIG. 12A shows two graphs of sialyl Lewis x (sLe.sup.x) O.D.
values from samples collected at the enrollment visit for
symptomatic and asymptomatic CD patients who either did or did not
have a history of antibiotic use.
[0052] FIG. 12B shows two graphs of sialyl Lewis x (sLe.sup.x) O.D.
values from samples collected at the followup visit for symptomatic
and asymptomatic CD patients who either did or did not have a
history of antibiotic use.
[0053] FIG. 13 shows two graphs of fecal calprotectin values in
relation to patient symptom group. The upper graph shows ELISA O.D.
values for calprotectin. The lower graph shows the amount of
calprotectin (ug/mL). P=0.074, comparing Calprotectin O.D. value
<0.5 between sx and asx Crohn's. 1=symptomatic CD,
0=asymptomatic CD at followup.
[0054] FIG. 14 shows two graphs of the IBD-SCAN.RTM. values in
relation to patient symptom group. The upper graph shows O.D.
value. The lower graph shows the amount (ug/mL). 1=symptomatic CD,
0=asymptomatic CD at followup.
[0055] FIG. 15 shows a scatterplot of the IBD-SCAN.RTM. and
salivary sialyl Lewis x (sLe x) O.D. values.
DETAILED DESCRIPTION OF THE INVENTION
[0056] Inflammatory bowel disease (IBD), including Intestinal
Colitis, Crohn's Disease (CD) and Ulcerative Colitis (UC), involves
chronic inflammation of all or part of the digestive tract. Onset
of IBD occurs from early childhood to older adulthood and includes
symptoms of bloody stool, diarrhea, severe abdominal cramps and
pain, and weight loss.
[0057] The present disclosure is based on the unexpected
discoveries that the levels of certain glycosyltransferases,
including sialyltransferases (e.g., ST3Gal III, ST3Gal IV, ST6Gal
1, ST6Gal 2, and ST6GalNAc 1) and fucosyltransferases (e.g., FUT2,
FUT3, FUT5, and FUT7), are associated with development of IBD.
Thus, these glycosyltransferases, either taken alone or in
combination, as well as their glycan products such as histo-blood
group antigens (e.g., H antigens, Lewis antigens, and/or sialyl
Lewis antigens) can be used as biomarkers for diagnosing IBD,
assessing the risk for IBD development, or monitoring IBD
progression/regression in a patient. Accordingly, described herein
are methods for diagnosing IBD in a subject based on the expression
profile of one or more glycosyltransferase, the expression profile
of one or more histo-blood group antigens, or both; and methods for
monitoring disease progression/regression of an IBD patient based
on the just-noted expression profiles.
IBD Diagnosis
[0058] One aspect of the present disclosure relates to methods
(e.g., in vitro methods) for diagnosing IBD in a subject (e.g., a
human subject), including determining presence/absence of IBD in
the subject and/or assessing the risk for developing IBD in the
subject, based on the expression profile (level) of one or more
glycosyltransferases.
[0059] Glycosyltransferases catalyze the transfer of a
monosaccharide unit from an activated nucleotide sugar to a
glycosyl acceptor molecule. Glycosyltransferases useful in the
methods described herein include both fucosyltransferases (FUT) and
sialyltransferases (ST), which are well-characterized enzymes
involved in biosynthesis of histo-blood group antigens.
[0060] Fucosyltransferases catalyze the addition of fucose to
precursor polysaccharides in the last step of histo-blood group
antigen biosynthesis via different linkages. For example, FUT2 adds
fucose residues to precursor polysaccharides in an alpha1,2-linkage
and FUT3 adds fucose residues to precursor polysaccharides in an
alpha1,3-linkage. Examples of fucosyltransferase genes to be used
as biomarkers in the methods described herein include, but are not
limited to, FUT2 (e.g., GenBank Accession Nos. NM_000511 and
NM_001097638), FUT3 (e.g., GenBank Accession Nos. NM_000149,
NM_001097639, NM_001097640, and NM_001097641), FUT5 (e.g., GenBank
Accession No. NM_002034), and FUT7 (e.g., GenBank Accession No.
NM_004479).
[0061] Sialyltransferases are involved in biosynthesis of sialyl
Lewis antigens, including sialyl Lewis x (sLe.sup.x), sialyl Lewis
y (sLe.sup.y), sialyl Lewis a (sLe.sup.a), and sialyl Lewis b
(sLe.sup.b), through addition of sialic acid. Examples of
sialyltransferase genes useful in the methods disclosed herein
include, but are not limited to, ST3Gal III (e.g., GenBank
Accession Nos. NM_174963, NM_174964, NM_174965, NM_174966,
NM_174967, NM_174968, NM_174969, and NM_174970) and ST3Gal IV
(e.g., GenBank Accession Nos. NM_006278, NM_001254757,
NM_001254758, and NM_001254759), ST6Gal 1 (NM_003032.2,
NM_173216.2, NM_173217.2), ST6Gal2 (NM_001142351.1, NM_001142352.1,
NM_032528.2) and ST6GalNAc1 (NM_018414.3).
[0062] To practice this method, the level(s) of one or more of the
glycosyltransferases noted above in a tissue sample of a candidate
subject can be determined by performing a method known in the art
or based on medical records of that candidate subject. In one
example, a tissue sample (e.g., saliva, intestinal biopsy, colon
biopsy, biofluid, or stool) can be obtained from a subject (e.g., a
human subject who can be suspected of having IBD) and the
expression level of one or more of the glycosyltransferases
described herein can be measured by conventional methods. In some
embodiments, the expression level of one glycosyltransferase, e.g.,
selected from those noted above, such as ST3Gal IV or FUT3, is
measured and used as a marker in the methods described herein. In
other embodiments, a combination of glycosyltransferases, e.g.,
selected from those noted above, are examined and their expression
pattern is used as a marker in the methods described herein. Any
combination of the above noted glycosyltransferases can be used
here. Specific examples include, but are not limited to, (a) ST3Gal
IV and one of the fucosyltransferases FUT2, FUT3, FUT5, and FUT7;
(b) ST3Gal III and ST3GalIV; or (c) ST3Gal III and one or more of
FUT2, FUT3, FUT5, and FUT7.
[0063] In one example, the expression levels of the
glycosyltransferases are determined by measuring the mRNA levels of
the enzymes via, e.g., quantitative PCR (real-time PCR) or
microarray hybridization. In another example, the levels of the
glycosyltransferases can be determined by measuring the level(s) of
one or more microRNAs that regulate the expression of these
glycosyltransferases. Levels of microRNAs can be determined via a
routine method, e.g., real-time PCR. Alternatively, the levels of
these glycosyltransferases can be determined by measuring their
protein levels via, e.g., immunoassays or by examining their
enzymatic activities. Methods for determining RNA levels and
protein levels are all well known in the art, including
hybridization, PCR, ELISA, sequencing, agglutination inhibition
assay, and flow cytometry. See, e.g., Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001,
Current Protocols in Molecular Biology, F. M. Ausubel, et al.,
eds., John Wiley & Sons, Inc., New York. Microarray technology
is described in Microarray Methods and Protocols, R. Matson, CRC
Press, 2009, or Current Protocols in Molecular Biology, F. M.
Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Methods for determining the activity of a glycosyltransferase as
described herein, e.g., FUT2, are also well known in the art. See,
e.g., U.S. Pat. No. 7,871,785, which is incorporated by reference
herein.
[0064] The data thus obtained can be normalized against the
expression level of an internal control RNA (e.g., a ribosomal RNA
or U6 RNA). The normalized expression level(s) of the
glycosyltransferase(s) can then be compared to the expression
level(s) of the same glycosyltransferase(s) of a control tissue
sample, which can be normalized against the same internal control
RNA, to determine whether the subject has or is at risk for
developing IBD (having a higher probability of IBD occurrence as
compared to a normal subject).
[0065] The control tissue sample can be obtained from a subject
free of IBD. Alternatively, the control tissue sample can be
obtained from a pool of IBD-free subjects. Optionally, these
IBD-free subjects match with the test subject in, e.g., age,
gender, and/or ethnic background. Preferably, the control tissue
sample and the tissue sample examined in the methods described here
are of the same type. When an intestinal biopsy or colon biopsy
sample is used, the control tissue sample can be obtained from the
same subject but from an area free of inflammation.
[0066] When necessary (e.g., when more than one glycosyltransferase
is investigated), the expression levels of the glycosyltransferases
(preferably normalized against an internal control RNA) can be
processed by, e.g., a computational program to generate an
expression profile (e.g., an mRNA signature), which can be
represented by a number or numbers, that characterize the
expression pattern of the glycosyltransferases. The expression
levels of these glycosyltransferases from the control tissue sample
can be processed by the same method to generate an expression
profile representing the expression pattern of these
glycosyltransferases of the control. The expression profile (mRNA
signature) of the test subject can be compared with the expression
profile of the control sample to determine whether the subject has
or is at risk for IBD development.
[0067] Various computational programs can be applied in the methods
of this disclosure to aid in analysis of expression data. Examples
include, but are not limited to, Prediction Analysis of Microarray
(PAM; see Tibshirani et al., PNAS 99(10):6567-6572, 2002);
Plausible Neural Network (PNN; see, e.g., U.S. Pat. No. 7,287,014),
PNNSulotion software and others provided by PNN Technologies Inc.,
Woodbridge, Va., USA, and Significance Analysis of Microarray
(SAM).
[0068] As described herein, elevated levels of ST3Gal IV and a
reduced level of FUT2, FUT3, FUT5, FUT7, or ST3Gal III were found
to be associated with IBD development, including the occurrence of
both Crohn's Disease and Ulcerative Colitis. Thus, if the
expression profile of one or more of the above-noted
glycosyltransferases of a test subject, relative to that of a
control subject, represents an elevated level of ST3Gal IV, and/or
a reduced level of one or more of FUT2, FUT3, FUT5, FUT7, and
ST3Gal III, the test subject is determined to have or be at risk
for IBD (e.g., Intestinal Colitis, Crohn's Disease or Ulcerative
Colitis).
[0069] In one example, the expression profile of FUT2, either alone
or in combination of any of the other glycosyltransferases
described herein, of a test subject is determined to assess whether
that subject has or is at risk for Ulcerative Colitis. If the
expression profile represents a reduced level of FUT2, the test
subject is determined as having or being at risk for Ulcerative
Colitis.
[0070] In another example, the expression profile of FUT2, either
alone or in combination of any of the other glycosyltransferases
described herein, in a saliva sample of a test subject is
determined to assess whether that subject has or is at risk for
IBD. If the expression profile represents a reduced level of FUT2,
the test subject is determined as having or being at risk for
Ulcerative Colitis.
[0071] In another example, the expression profile of FUT2, either
alone or in combination of any of the other glycosyltransferases
described herein, in a saliva sample of a test subject is
determined to assess whether that subject has or is at risk for
IBD. If the expression profile represents a reduced level of FUT2,
the test subject is determined as having or being at risk for
IBD.
[0072] In another aspect, the present disclosure relates to methods
for diagnosing IBD (e.g., diagnosing UC or CD) in a subject (e.g.,
a human subject), including determining presence/absence of IBD in
the subject and/or assessing the risk for developing IBD in the
subject, based on the expression profile of one or more glycans
that are products of one or more of the glycosyltransferases noted
above, including histo-blood group antigens. Human histo-blood
group antigens are a set of innate, fucosylated carbohydrates
expressed on red blood cells, in saliva, on epithelial surfaces,
and on intestinal mucosa. Examples include, but are not limited to,
H antigens (H1 and H2), Lewis antigens (Lewis a or Lea; Lewis b or
Le.sup.b; Lewis x or Lex; and Lewis y or Leg), and sialyl Lewis
antigens such as sialyl Lewis a (sLe.sup.a), sialyl Lewis b
(sLe.sup.b), sialyl Lewis x (sLe.sup.x), and sialyl Lewis y
(sLe.sup.y).
[0073] Histo-blood group antigens can act as cell binding sites for
both cells of the immune system and for microbial organisms, either
commensal or pathogenic (Henry 2001). Polymorphisms in certain
fucosyltransferase genes are known to determine expression of the
Lewis blood-group type, fucosylated oligosaccharide patterns in
human milk, and histo-blood group antigens on human epithelial cell
surfaces (Niverge et al 1990, Thurl et al 1997, Chaturvedi et al
2001). Thus, like the glycosyltransferases discussed herein, glycan
products of these glycosyltransferases such as histo-blood group
antigens can also be used as biomarkers in the methods described
herein.
[0074] In some embodiments, a single glycan antigen from those
described herein (e.g., sLe.sup.x, Le.sup.x, H, Le.sup.b, Le.sup.y,
and sLe.sup.a) is used as an IBD marker. In other embodiments, a
combination of the glycan antigens described herein is used as an
IBD marker, e.g., any combination of the histo-blood group antigens
described above. Examples include, but are not limited to, (a)
sLe.sup.x and Le.sup.b, (b) sLe.sup.a and Le.sup.b, (c) sLe.sup.x
and one or more of H, Le, Le.sup.b, Lex, and Le.sup.y, (d)
sLe.sup.a and one or more of H, Le, Le.sup.b, Lex, and Le.sup.y,
(e) sLe.sup.x and Lex, (f) H and one or more of sLe.sup.x,
Le.sup.b, and sLe.sup.a, and (g) at least two of H, Le.sup.b, and
Le.sup.y.
[0075] The levels of these glycan antigens can be measured by
routine practice, e.g., by an immunoassay such as ELISA or using
lectins such as UEA1, AIA, GSA II, WGA, sWGA, SNA, MAL-II, PWA,
SJA, LEA, and I-PHA. Other methods for determining the levels of
glycan antigens such as histo-blood group antigens include, but are
not limited to, flow cytometry, and agglutination inhibition
assay.
[0076] The level(s) of the glycan antigen(s) thus obtained can be
normalized and optionally processed following the procedures
described above to generate an expression profile, which is then
compared with that of a control sample to determine whether a test
subject has or is at risk for IBD. In one example, the expression
profile of sLe.sup.x, sLe.sup.a, or both is determined and if the
expression profile obtained from a tissue sample of the test
subject represents an elevated level of sLe.sup.x, sLe.sup.a, or
both, the test subject is determined as having or being at risk for
IBD. When desired, the level of Le.sup.b, an H antigen, or both can
also be determined. In that case, if an expression profile
represents a reduced level of the H antigen, and/or an elevated
level of Le.sup.b, the test subject is determined as having or
being at risk for IBD.
[0077] In one example, the level of an H antigen, either alone or
in combination of any of the other glycan antigens described
herein, of a test subject is determined to assess whether that
subject has or is at risk for Ulcerative Colitis. If the expression
profile represents a reduced level of the H antigen, the test
subject is determined as having or being at risk for Ulcerative
Colitis.
[0078] The above-described methods can be applied to a test
subject, which can be a human subject, e.g., a non-secretor human
subject (an individual who secretes a low level or no blood group
antigens into body fluids). In one example, the human subject is
suspected of having IBD. A subject suspected of having IBD may show
one or more symptoms associated with IBD, e.g., abdominal pain,
vomiting, diarrhea, rectal bleeding, severe internal cramps/muscle
spasms in the region of the pelvis, and/or weight loss, or may be
asymptomatic but exhibit one or more risk factors associated with
IBD. IBD-associated risk factors include genetic factors (specific
gene mutations), environmental factors (IBD is more common in
developed countries, urban areas, and colder climates, and among
people with high socioeconomic status), age (onset is usually
between the ages of 15 and 35), race (Caucasians have the highest
risk), family history, use of nonsteroidal anti-inflammatory drugs
(e.g., ibuprofen), smoking, and lack of breast-feeding.
[0079] In another example, the level of Le.sup.b, Le.sup.y, an H
antigen, or a combination thereof can be used to diagnose CD. A
reduced level of one or more of these antigens is indicative of
presence or risk of the disease.
IBD Prognosis
[0080] Any of the glycosyltransferases, combinations thereof, or
human histo-blood group antigens, and combinations thereof as
described herein can also be used as prognostic markers to monitor
the status of IBD (including Intestinal Colitis, Crohn's disease
and Ulcerative Colitis), including progression and regression, in a
subject, which can be a human subject or a laboratory animal.
Accordingly, also disclosed herein are prognostic methods for
monitoring the disease status of a subject based on changes in the
levels of one or more glycosyltransferases or changes of the levels
of one or more histo-blood group antigens of a subject. Optionally,
this method can be practiced during the course of an IBD treatment
to assess the efficacy of the treatment in a subject.
[0081] To practice this method, tissue samples (e.g., saliva,
intestinal biopsy, colon biopsy, biofluid, or stool) can be
collected via routine methods at various time points (e.g., along
the course of a treatment) from a subject, who has or is suspected
of having IBD. The expression level(s) of a glycosyltransferase
(e.g., ST3Gal IV or FUT3), a combination of glycosyltransferases
(e.g., ST3Gal IV and one or more of FUT2, FUT3, FUT5, FUT7, and
ST3Gal III), a histo-blood group antigen (e.g., sLe.sup.x or
sLe.sup.a) or a combination of the blood group antigens (e.g.,
sLe.sup.x or sLe.sup.a and one or more of H antigen and Le.sup.b)
can be measured as described above. The levels of the one or more
glycosyltransferases or the levels of the one or more histo-blood
group antigens thus obtained can be normalized and optionally
processed following the procedures described above to generate an
expression profile of the sample obtained at each time point.
[0082] The expression profile of a sample obtained from a later
time point is compared with that of a sample obtained from an
earlier time point. If there is a change between the two expression
profiles, the subject is determined to have IBD progression or
regression. For example, if a change of the expression profile
represents an increased level of a glycosyltransferase (e.g.,
ST3Gal IV) or a histo-blood group antigen that is correlated with
IBD (sLe.sup.x, sLe.sup.a, or Le.sup.b) develop along the course,
or if the change of the expression profile represents a decreased
level of a glycosyltransfearse (e.g., FUT2, FUT3, FUT5, FUT7, or
ST3Gal III) or a histo-blood group antigen (e.g., H1 or H2) that is
inversely correlated with IBD development, the subject is
determined as having IBD progression. On the other hand, if a
change of the expression profile represents a decreased level of a
glycosyltransferase (e.g., ST3Gal IV) or a histo-blood group
antigen that is correlated with IBD (sLe.sup.x, sLe.sup.a, or
Le.sup.b) develop along the course, or if the change of the
expression profile represents an increased level of a
glycosyltransfearse (e.g., FUT2, FUT3, FUT5, FUT7, or ST3Gal III)
or a histo-blood group antigen (e.g., H) that is inversely
correlated with IBD development, the subject is determined to have
IBD regression.
[0083] When the above method is performed on an IBD patient
undergoing a treatment, a first tissue sample or a first set of
tissue samples can be collected at a time point before the
treatment begins and a second sample or a set of samples is
collected from a subject at a later time point than the first time
point, e.g., during or after treatment. If necessary, multiple
samples/sample sets can be collected along the course of the
treatment. The expression profile(s) of the glycosyltransferase(s)
and/or histo-blood group antigen(s) can be determined as described
herein and the disease progression/regression can be assessed based
on changes of the expression profiles along the course of the
treatment (that is, changes of the expression profile in samples
taken at intervals later in the course of the treatment as compared
to those in samples taken at intervals earlier in the course of the
treatment). For example, if changes in the expression profile
represent an increased level of ST3Gal IV or sLe.sup.x and/or
sLe.sup.a, or a decreased level of FUT2, FUT3, FUT5, FUT7, or
ST3Gal III, the patient is determined to have IBD progression
during the course of the treatment. In that case, the treatment is
determined as have little or no effect on the patient. On the other
hand, if changes in the expression profile represent a decreased
level of ST3Gal IV or sLe.sup.x and/or sLe.sup.a, or an increased
level of FUT2, FUT3, FUT5, FUT7, or ST3Gal III, the patient is
determined to have IBD regression during the course of the
treatment. In that case, the treatment is determined as being
effective on the patient.
[0084] In some embodiments, the expression profile of FUT2, either
alone or in combination of any of the other glycosyltransferases
described herein, and/or the expression profile of an H antigen,
either alone or in combination with any of the other histo-blood
group antigens described herein, in a subject having is determined
to assess disease status in that subject over time. If the
expression profile of a later time point represents a reduced level
of FUT2 or the H antigen as compared to that of an earlier time
point, the subject is determined as having Ulcerative Colitis
progression. Conversely, if the expression profile of a later time
point represents an elevated level of FUT2 or an H antigen compared
to the expression profile of an earlier time point, the subject is
determined as having regression of Ulcerative Colitis.
[0085] In one example, the level of sLe.sup.x, Lex, or a
combination thereof is used as a marker for assessing progression
of Crohn's disease (CD). In another example, the level of sLe.sup.x
is used as a marker for assessing progression of Crohn's disease
(CD). An elevated level of sLe.sup.x, Lex, or both can be
indicative of disease progress. Alternatively, a reduced level of
one or both antigens can be indicative of disease regression. These
two markers, either alone or in combination, are particularly
useful in monitoring CD progression in human patient who exhibits
at least one symptom associated with CD, e.g., abdominal pain,
diarrhea (which may be bloody if inflammation is severe), vomiting,
or weight loss, as well as complications outside the
gastrointestinal tract such as anemia, skin rashes, arthritis,
inflammation of the eye, tiredness, and lack of concentration.
[0086] Any of the diagnostic and prognostic methods described
herein can further comprising subjecting the subject to an IBD
treatment or adjusting the current IBD treatment that was applied
to the subject.
[0087] Any of the above-described diagnosis or prognosis methods
can be applied to a test subject, which can be a human subject as
described above or a laboratory animal (e.g., a mouse, a rat, a
guinea pig, a rabbit, a goat, or a non-human primate). For example,
a subject may be a human known to have or suspected of having IBD.
Further, this human may be in the process of or planning to undergo
treatment of IBD. A subject may also be, e.g., an animal disease
model of IBD. Treatment of subjects with or suspected of having IBD
can include, but is not limited to, medications (e.g.
corticosteroids, aminosalicylates, immunomodulators, antibiotics,
and biologics), surgery (e.g. lleoanal anastomosis, proctocolectomy
with ileostomy, and strictureplasty), nutritional restrictions,
and/or any other IBD treatment described herein.
IBD Treatment
[0088] When a subject is diagnosed by any of the methods described
herein as having or at risk for developing IBD, this subject could
be subjected to a treatment for IBD, including any of the IBD
treatments known in the art and disclosed herein. For example,
medications such as sulfasalazine (Azulfadine), mesalamine (Asacol,
Pentasa), azathioprine (Imuran), 6-MP (Purinethol), cyclosporine,
methotrexate, infliximab (Remicade) and corticosteroids
(prednisone) can be administered to the subject in an amount
effective to treating IBD (e.g., UC or CD). In some embodiments,
the IBD treatment (e.g., UC or CD treatment) comprises an
anti-inflammatory agent, an immune suppressant agent, an antibiotic
agent, or a combination thereof. Non-limiting examples of
anti-inflammatory agents include sulfasalazine, mesalamine,
balsalazide, olsalazine, or corticosteroids (e.g., prednisone or
budesonide). Non-limiting examples of immune suppressant agents
include azathioprine, mercaptopurine, cyclosporine, infliximab,
adalimumab, certolizumab pegol, methotrexate, or natalizumab.
Non-limiting examples of antibiotics include metronidazole and
ciprofloxacin. In some embodiments, IBD treatment comprises an
anti-diarrheal (e.g., psyllium powder, methylcellulose or
loperamide), a laxative, acetaminophen, iron, vitamin B-12,
calcium, or vitamin D. In some embodiments, IBD treatment (e.g., UC
or CD treatment) comprises surgery or fecal bacteriotherapy (also
called a fecal microbiota transplantation or stool transplant).
Non-limiting examples of surgery include proctocolectomy,
ileostomy, or strictureplasty. In some embodiments, IBD treatment
(e.g., UC or CD treatment) comprises a therapeutic agent (e.g., an
anti-inflammatory agent, an immune suppressant agent, an antibiotic
agent, or a combination thereof) and surgery. It is to be
understood that any of the IBD treatments described herein may be
used in any combination. The term "treating" as used herein refers
to the application or administration of a composition including one
or more active agents to a subject, who has IBD, a symptom of IBD,
or a predisposition toward IBD, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disease, the symptoms of the disease, or the predisposition
toward the disease. An "effective amount" is that amount of an
anti-IBD agent that alone, or together with further doses, produces
the desired response, e.g. eliminate or alleviate symptoms, prevent
or reduce the risk of flare-ups (maintain long-term remission),
and/or restore quality of life. The desired response is to inhibit
the progression of the disease. This may involve only slowing the
progression of the disease temporarily, although more preferably,
it involves halting the progression of the disease permanently.
This can be monitored by routine methods or can be monitored
according to diagnostic and prognostic methods discussed herein.
The desired response to treatment of the disease or condition also
can be delaying the onset or even preventing the onset of the
disease or condition.
[0089] Such amounts will depend, of course, on the particular
condition being treated, the severity of the condition, the
individual patient parameters including age, physical condition,
size, gender and weight, the duration of the treatment, the nature
of concurrent therapy (if any), the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose of the individual components or combinations thereof be used,
that is, the highest safe dose according to sound medical judgment.
It will be understood by those of ordinary skill in the art,
however, that a patient may insist upon a lower dose or tolerable
dose for medical reasons, psychological reasons or for virtually
any other reasons.
[0090] Any of the methods described herein can further comprise
adjusting the IBD treatment performed to the subject based on the
results obtained from the diagnostic/prognostic method. Adjusting
treatment includes, but are not limited to, changing the dose
and/or administration of the anti-IBD agent used in the current
treatment, switching the current medication to a different anti-IBD
agent, or applying a new IBD therapy to the subject, which can be
either in combination with the current therapy or replacing the
current therapy.
[0091] In some embodiments, the present disclosure provides a
method for treating a subject (e.g., a human patient) having
inflammatory bowel disease (IBD), the method comprising
administering an effective amount of an IBD drug such as those
described herein (e.g., an anti-inflammatory agent, an immune
suppressant agent, an antibiotic agent, or a combination thereof)
to the subject, who exhibits an expression profile or expression
level of at least one glycosyltransferase that deviates from a
control. The at least one glycosyltransferase can comprises one or
more of fucosyltransferase 3 (FUT3), fucosyltranferase 5 (FUT5), or
fucosyltransferase 7 (FUT7), ST3 beta-galactoside
alpha-2,3-sialyltransferase 3 (ST3Gal III), and ST3
beta-galactoside alpha-2,3-sialyltransferase 4 (ST3Gal IV). In one
example, the subject has an elevated level of ST3Gal IV, a reduced
level of the one or more of FUT3, FUT5, FUT7, and ST3Gal III, or
both as compared to a control. Alternatively or in addition, the
subject can further exhibit a reduced level of fucosyltransferase 2
(FUT2) as compared to a control.
[0092] In other embodiments, the present disclosure features
methods for treating a human subject having inflammatory bowel
disease (IBD), the method comprising administering an effective
amount of an IBD drug (e.g., an anti-inflammatory agent, an immune
suppressant agent, an antibiotic agent, or a combination thereof)
to a subject having or at risk for IBD (e.g., a human patient). The
subject exhibits a level or profile of one or more human blood
group antigens that deviates from a control. The one or more human
blood group antigens comprise one or more of sialyl Lewis x
(sLe.sup.x), sialyl Lewis a (sLe.sup.a), and Lewis b (Le.sup.b). In
one example, the subject has an elevated level of sLe.sup.x,
sLe.sup.a, or both as compared to a control. Alternatively or in
addition, the subject exhibits a reduced level of H antigen
compared to a control.
[0093] In yet other embodiments, provided herein are methods for
treating a subject having Ulcerative Colitis (UC), the method
comprising administering an effective amount of an anti-UC agent
(e.g., an anti-inflammatory agent, an immune suppressant agent, an
antibiotic agent, or a combination thereof) to a subject having UC,
wherein the subject exhibits an expression level of
fucosyltransferase 2 (FUT2) that is reduced as compared to a
control. Such methods can further comprise monitoring the subject
for UC progression by a suitable method as those described
herein.
[0094] In addition, the present disclosure provides a method for
treating a subject having Crohn's disease (e.g., a human patient),
the method comprising administering an effective amount of an CD
drug (e.g., an anti-inflammatory agent, an immune suppressant
agent, an antibiotic agent, or a combination thereof) to the
subject having or at risk for CD, wherein the subject has an
expression profile of one or more human blood group antigens that
deviates from a control. In one example, the one or more human
blood group antigens comprise one or more of sialyl Lewis x
(sLe.sup.x), Lewis x (Lex), H antigen, Lewis b (Le.sup.b), and
Lewis y (Le.sup.y). In one example, the subject is a human patient
exhibiting an elevated level of sLe.sup.x, Lex, or both as compared
to a control. In another example, the subject is a human patient
having a decreased level of H antigen, Le.sup.b, Le.sup.y, or a
combination thereof, as compared to a control. Such methods can
further comprise monitoring the subject for CD progression by a
suitable method such as those described herein. In another example,
the one or more human blood group antigens comprise sialyl Lewis x
(sLe.sup.x). In one example, the subject is a human patient
exhibiting an elevated level of sLe.sup.x as compared to a
control.
[0095] The subject to be treated by any of the treatment methods
described herein can be identified as having or at risk for IBD by
any of the methods described herein. The treatment methods as just
described herein can further comprise monitoring the subject for
IBD progression by a method of any one of the methods described
herein. Based on the status of disease progression, a suitable IBD
treatment can be applied to the subject.
Kits for Use in IBD Diagnosis and/or Prognosis
[0096] Also within the scope of this disclosure are kits for use in
diagnosing IBD or monitoring IBD progression/regression in a
subject, such as a human subject. Such a kit can comprise reagents
for determining the level(s) of one or more of the biomarkers to be
used in the diagnostic or prognostic methods described herein.
[0097] The kit noted above can comprise one or more reagents for
determining the levels of one or more glycosyltransferases (e.g.,
FUT2, FUT3, FUT5, FUT7, ST3Gal III, and/or ST3Gal IV). The reagents
can be oligonucleotide probes/primers for determining the mRNA
levels of the one or more glycosyltransferases, or oligonucleotide
probes/primers for examining the levels of one or more microRNAs
that regulate the expression of these enzymes. Alternatively, the
kit can contain antibodies specific to one or more of these
enzymes. In one example, the kit comprises reagents for determining
the levels of ST3Gal IV and one of the fucosyltransferases FUT2,
FUT3, FUT5, and FUT7. In another example, the kit comprises
reagents for determining the levels of ST3Gal III and ST3GalIV. In
yet another example, the kit comprises reagents for determining the
levels of ST3Gal III and one or more of FUT2, FUT3, FUT5, and FUT7.
Alternatively, the kit comprises reagents for determining the
levels of ST3Gal IV and one or more of FUT2, FUT3, FUT5, and
FUT7.
[0098] When glycan markers are to be examined in the
diagnostic/prognostic methods described herein, the kit can
comprise one or more antibodies binding to the glycan markers
(e.g., antibodies binding to sLe.sup.x, Le.sup.x, sLe.sup.a,
Le.sup.b, Le.sup.y or H antigen) or one or more lectins binding to
these glycan markers. In one example, the kit comprises one or more
antibodies or lectins that bind to sLe.sup.x.
[0099] Any of the kits described herein can further comprise an
instruction manual providing guidance for using the kit to perform
the diagnostic/prognostic methods.
[0100] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference for the purposes or subject matter referenced herein.
Examples
Example 1: Identification of Glycosyltransferases Differentially
Expressed In Patients With IBD
[0101] RNA samples were prepared from colon biopsies of affected
segments of CD and UC patients and healthy controls, and the global
patterns of gene expression were examined using the HGU133 Plus V.2
Affymetrix GeneChip in the Cincinnati Children's Digestive Health
Center Microarray core.
[0102] As shown in Table 1, ST3Gal IV was found to be significantly
(p<0.01) overexpressed in IBD patients versus controls. ST3Gal
III and the fucosyltransferase genes, FUT2, FUT3, FUT5 and FUT7
were found to be significantly under-expressed (p<0.05) in IBD
patients versus controls. The above results indicate that the
expression level of any of these enzymes, or any combination
thereof, can be used as a marker for diagnosing IBD or monitoring
disease progress.
TABLE-US-00001 TABLE 1 Intestinal epithelial cell expression of
fucosyltransferase genes and sialyltransferase genes from colon
biopsy ST3Gal ST3Gal Gene expression FUT2 FUT3 FUT5 FUT7 III IV
Crohn's Disease 0.72 * 0.59 + 0.8 * 0.73 * 0.82.sup. 2.3 + N = 29
Ulcerative 0.58 * 0.45 + 0.8 * 0.82.sup. 0.73 + 2.9 + Colitis N = 8
Compared to Controls (N = 8) as reference (1.0). * p .ltoreq. 0.05,
+ p .ltoreq. 0.01
Example 2: Identification of Histo-Blood Group Antigens
Differentially Expressed in Patients with IBD
[0103] Saliva specimens were tested for secretor and Lewis types by
monoclonal antibody based phenotyping methods as previously
described. See Huang et al., J Infect Dis 2003; 188; 19-31.
Briefly, boiled saliva samples were coated on microtiter plates
(Dynex, Immulon; Dynatech, Franklin, Mass.) and then interacted
with monoclonal antibodies specific to individual human blood group
antigens. The bound antibodies were then detected by corresponding
secondary antibody-horseradish peroxidase (HRP) conjugates followed
by adding HRP substrate reagents (optEIA, BD Bioscience, San Diego,
Calif.).
[0104] As shown in FIG. 1 and Table 2, the level of sialyl Lewis
(sLewis) antigens, which refers to the average of sLe.sup.x and
sLe.sup.a levels, was found to be significantly higher in IBD
patients (i.e., cases) as relative to the controls.
TABLE-US-00002 TABLE 2 Optical density (OD) values for sialyl Lewis
in patients with Crohn's Disease (CD) or (UC) or controls. sLewis
O.D. values Case Control 0.24 CD 0.16 asx Crohn's 0.61 UC 0.16 0.62
UC 0.16 0.64 CD 0.18 0.75 UC 0.2 0.5 0.95 setting sLewis cutpoint
for O.D. value >0.5: Cases 4/5 (80%) vs Controls 1/7 (14%), p =
0.07 One control (asx Crohn's) was an asymptomatic Crohn's Disease
patient.
[0105] FIG. 2 shows that all five IBD patients showed elevated
sialyl Lewis levels (O.D.>0.5), elevated levels of Lewis b
(O.D.>1.5), or both, while only one out of seven controls showed
similar results (p=0.015, Fisher's exact). These data indicate that
Lewis b can be used as an adjunct marker with sLewis for IBD
diagnosis and/or prognosis.
Example 3. Salivary Glycan Biomarkers for Monitoring Crohn's
Patients
[0106] The goal of this study was to study salivary glycans
(histo-blood group antigens) as biomarkers of symptom flares in
Crohn's Disease by examining the levels of these antigens in the
saliva samples of symptomatic Crohn's disease patients, compared to
asymptomatic Crohn's Disease patients and healthy controls. For the
symptomatic and asymptomatic Crohn's Disease patients, a stool
sample was also collected after the clinic visit, which was tested
for fecal biomarkers. A second saliva sample was collected from the
patient concurrent with the stool sample collection. It was
determined that the primary candidate biomarker was sialyl Lewis x,
as it was found to be elevated in symptomatic patients.
[0107] It was found that salivary levels of sialyl Lewis x antigen
(and Lewis x antigen) were elevated in symptomatic Crohn's patients
compared to the asymptomatic Crohn's patients in the second saliva
collection. There was no association with these or other
histo-blood group antigens in the first sample, taken at the time
of a clinic visit for treatment. It is hypothesized that this
anomalous finding is a treatment effect: That when initially
enrolled and measured, the symptomatic Crohn's patients had already
initiated treatment with medications that worked to reduce their
inflammation. An additional piece of suggestive evidence was that
salivary Lewis x was significantly elevated in individuals with a
history of antibiotic use, including in controls.
Methods
Patient Enrollment
[0108] A total of 60 healthy control subjects were enrolled, of
whom 30 were pediatric (11 to 17 years) and 30 were adults (18 to
55 years). A total of 20 asymptomatic Crohn's subjects were
enrolled, ranging from 11 to 19 years of age. Subjects were
recruited at a semi-annual visit that was scheduled to administer
Infliximab (Remicade) in order to maintain remission in these
subjects. The frequency of their clinic visits was individualized
based on their history and scheduled to avoid symptom recurrence.
At the time of enrollment, some subjects were awaiting treatment,
but most had just initiated treatment. A total of 9 symptomatic
Crohn's subjects were enrolled, ranging from 12 to 18 years of age.
Three of these symptomatic patients were recruited as hospital
inpatients, while six were recruited as outpatients who were
experiencing a symptomatic flare. A detailed summary of the
demographic characteristics of these patients is provided as Table
3.
TABLE-US-00003 TABLE 3 Patient Demographic Data Symptomatic
Asymptomatic Crohn's Crohn's Pediatric Adult Disease Disease
Healthy Healthy Question on Patients Patients Controls Controls
Demographic Form (n = 9) (n = 20) (n = 30) (n = 30) Weight (pounds)
(mean + SD) 117.6 + 14.48 129.8 + 39.01 130.1 + 34.68 187.5 + 48.1
Height (inches) (mean + SD) 64.7 + 4.09 63.6 + 4.56 64.43 + 3.70
68.5 + 3.66 Health insurance? Yes 8 (88.8%) 20 (100%) 29 (96.6%) 27
(90%) No 1 (11.1%) 0 1 (3.3%) 3 (10%) Type of health insurance
Private 8 (88.8%) 14 (70%) 24 (80%) 24 (80%) Public 0 5 (25%) 5
(16.6%) 3 (10%) Both 0 1 (5%) 0 0 Unknown 0 0 0 0 Hispanic or
Latino/a? Yes 0 1 (5%) 0 4 (13.3%) No 8 (88.8%) 19 (95%) 30 (100%)
26 (86.6%) Race White 8 (88.8%) 17 (85%) 21 (70%) 20 (66.6%) Black,
African-American 1 (11.1%) 3 (15%) 10 (33.3%) 10 (33.3%) American
Indian/Alaskan Native 1 (11.1%) 2 (10%) 6 (20%) 1 (3.3%) Asian or
other 1 (11.1%) 0 0 0 Highest degree or diploma completed (if
<18 years old: mother, if adult controls - themselves) None 0 2
(10%) 0 1 (3.3%) GED 0 0 0 1 (3.3% High school diploma 3 (33.3%) 2
(10%) 10 (33.3%) 8 (26.6%) 2-year college degree 2 (22.2%) 4 (20%)
4 (13.3%) 5 (16.6%) (Associate's or Technical degree) 4-year
college degree 2 (22.2%) 4 (20%) 12 (40%) 9 (30%) (Bachelor's
degree) Graduate degree 1 (11.1%) 5 (25%) 4 (13.3%) 6 (20%)
(Master's, Doctorate, Medical, etc.) Seasonal allergies? Yes 7
(77.7%) 10 (50%) 17 (56.6%) 8 (26.6%) No 2 (22.2%) 10 (50%) 13
(43.3%) 22 (73.3%) Still have tonsils No, my tonsils have 2 (22.2%)
3 (15%) 3 (10%) 5 (16.6%) been removed Yes, I still have my 7
(77.7%) 17 (85%) 27 (90%) 25 (83.3%) tonsils I don't know 0 0 0 0
Family history of any of the following diseases Crohn's Disease 4
(44.4%) 4 (20%) 1 (3.3%) 1 (3.3%) Ulcerative Colitis 2 (22.2%) 2
(10%) 3 (10%) 3 (10%) Irritable Bowel 2 (22.2%) 2 (10%) 3 (10%) 3
(10%) Syndrome (IBS) Celiac Disease 0 1 (5%) 3 (10%) 0 Other 0 0 0
0 Taken antibiotics in the last year No 1 (11.1%) 6 (30%) 25
(83.3%) 13 (43.3%) Yes 8 (88.8%) 13 (65%) 5 (16.6%) 10 (33.3%) I
don't know 0 1 (5%) 0 1 (3.3%) Experienced any of the following
health problems? Heartburn in the last year 3 (33.3%) 5 (25%) 6
(20%) 5 (16.6%) Cavities in the last year 2 (22.2%) 3 (15%) 5
(16.6%) 7 (23.3%) Diarrhea in the last month 8 (88.8%) 4 (20%) 1
(3.3%) 1 (3.3%) Vomiting in the last month 3 (33.3%) 2 (10%) 2
(6.6%) 1 (3.3%) Estimated hours of sleep in the last night Less
than 4 hours 0 1 (5%) 0 3 (10%) 4-6 hours 3 (33.3%) 1 (5%) 1 (3.3%)
6 (20%) 6-7 hours 1 (11.1%) 5 (25%) 8 (26.6%) 6 (20%) 7-8 hours 2
(22.2%) 4 (20%) 7 (23.3%) 10 (33.3%) 8-9 hours 0 3 (15%) 7 (23.3%)
5 (16.6%) More than 9 hours 3 (33.3%) 6 (30%) 7 (23.3%) 0 Smoker in
the home? Yes 0 2 (10%) 5 (16.6%) 6 (20%) No 9 (100%) 18 (90%) 25
(83.3%) 24 (80%) Personal smoking history (for healthy controls 18
and older) Never smoked 1 (11.1%) 1 (5%) -- 19 (63.3%) Former
smoker -- -- -- 7 (23.3%) Current smoker -- 1 (5%) -- 4 (13.3%)
Weekly alcohol intake? (for healthy controls 18 and older) Less
than 1 drink/week 1 (11.1%) 2 (10%) -- 20 (66.6%) 1-7 drinks/week
-- -- -- 10 (33.3%) 7+ drinks/week -- -- -- 0
Sample Collection and Testing
[0109] Saliva sample collection and testing. For all subjects,
saliva samples were collected at the time of enrollment. For
Crohn's patients, saliva samples were also collected upon
follow-up, concurrent with stool sample collection. Sample
collection was performed by having patients spit into a conical
tube to provide 2 mL of saliva under the supervision of the study
staff. These tubes were then labeled, and transferred to the
laboratory, where sample was aliquoted into storage vials and
stored at -80 C until they were retrieved for analysis. One vial of
saliva sample was provided for testing using enzyme linked
immunoassay to quantitate the relative quantities of the
carbohydrate histo-blood group antigens: H, Lewis b, Lewis y, Lewis
a, Lewis x, sialyl Lewis a, and sialyl Lewis x.
[0110] Stool sample collection and testing. Stool sample collection
was limited to Crohn's patients, symptomatic and asymptomatic. At
the time of enrollment, patients were given a stool sample
collection kit, and requested to collect their sample at home and
send the sample via a courier service. The courier service
transported the samples on dry ice within 2 hours from the
patient's home to the lab. Samples were and immediately frozen at
-80 C, until shipment. Periodically, samples were removed from
storage and placed on dry ice for shipment for analysis. While
stool sample collection required significant additional work to
obtain samples from outpatients, an overall 79% collection rate was
achieved: 8 (89%) of 9 symptomatic patients and 15 (75%) of 20
asymptomatic patients.
Results
(i) Comparison of Salivary Glycans Across Patient Populations.
[0111] FIGS. 3-5 provide boxplots comparing the patient groups for
each salivary antigen. No significant differences were identified
among patient groups overall when analyzed univariately using
nonparametric tests. However, some important specific differences
in salivary glycan values were discovered after detailed analysis.
For example, H antigen, Lewis b and Lewis y values were
significantly higher (p<0.05) in healthy controls than CD
patients as a whole. Sialyl Lewis x and Lewis x, and the
combination thereof, were found to differ in symptomatic and
asymptomatic patients as described below.
(ii) Statistical Analysis of Samples Collected at the Time of
Patient Enrollment for Asymptomatic and Symptomatic CD
Patients.
[0112] For this analysis, only the 9 symptomatic patients, and 20
asymptomatic patients were included. By the non-parametric
Kruskal-Wallis test there were no significances identified in the
salivary glycan values comparing these patient groups. FIG. 6 shows
the optical density values of the glycans (histo-blood group
antigens) tested.
(iii) Statistical Analysis of Saliva Samples Collected after the
Enrollment Visit, at Home, by Crohn's Patients Concurrent with
Stool Sample Collection.
[0113] For this analysis, 8 symptomatic Crohn's patients and 14
asymptomatic Crohn's patients were assessed. There was a
significant difference between these patients in relation to sialyl
Lewis x (sLe x) and Lewis x (Le x). The best prediction was
obtained by the combination of these two antigens. FIG. 7 shows the
optical density values of all of the glycans (histo-blood group
antigens) tested. FIG. 8 shows the optical density values of Lewis
x and sialyl Lewis x.
[0114] The optical density cutpoints, including the predictive
value, sensitivity and specificity, are summarized below for sialyl
Lewis x, Lewis x, or the combination of both markers.
1. Single marker: sLe.sup.x>1.8
[0115] Symptomatic Crohn's 5/8 (62.8%)
[0116] Asymptomatic Crohn's 1/14 (7.1%)
[0117] Odds ratio=21. 7 (95% CI 1.4, 1100), exact p=0.011
[0118] Predictive value=77.7%, sensitivity=62.5%,
specificity=92.9%
2. Alternate single marker: Lewis x>1.0
[0119] Symptomatic Crohn's 6/8 (75.0%)
[0120] Asymptomatic Crohn's 3/14 (21.4%)
[0121] Odds ratio=11.0 (95% CI 1.05, 148), exact p=0.026
[0122] Predictive value=76.8%, sensitivity=75.0%,
specificity=78.6%
3. Combination of sLe.sup.x>1.8 and high Lewis x>1.0 if
sLe.sup.x is very low (<0.5)
[0123] Symptomatic Crohn's 6/8 (75.0%)
[0124] Asymptomatic Crohn's 1/14 (7.1%)
[0125] Odds ratio=39 (95% CI 2.2, 1924), exact p=0.002
[0126] Predictive value=83.9%, sensitivity=75.0%,
specificity=92.9%
[0127] The combination of sLe.sup.x and Lewis x was significantly
associated with the short Pediatric Crohn's Disease Activity Index
(PCDAI) scale (Kruskal-Wallis test, p=0.008, FIG. 9). The short
PCDAI scale is used clinically to determine Crohn's Disease symptom
severity.
(iv) Comparison of First and Second Samples: Change Over Time.
[0128] Of the 9 symptomatic Crohn's patients, a second sample at a
later time point was received from 8 subjects. Among these 8
subjects, three remained essentially the same over time and five
changed dramatically. There was no correlation between these two
samples. The sialyl Lewis X values for the first and second samples
are shown below in Table 4.
TABLE-US-00004 TABLE 4 Sample S Lex values for symptomatic Crohn's
patients. Patient 1.sup.st sample 2.sup.nd sample ID value value
Comments 402 1.949 2.18 The first & second samples of the
subject remained consistent. 406 2.248 1.852 The first & second
samples of the subject remained consistent. 409 .053 .045 The first
& second samples of the subject remained consistent. 401 .432
2.195 The first & second samples of the subject remained
consistent. 403 .35 2.186 The subject started with low values but
at followup had high values. 405 .136 2.178 The subject started
with low values but at followup had high values. The subject
started with low values but at followup had high values. 404 2.156
.172 The subject started with high values but at followup had low
values. 407 1.728 .062 The subject started with high values but at
followup had low values.
[0129] Of the 20 asymptomatic patients, a second sample at a later
time point was received from 15 subjects. The values of first and
second samples were highly correlated and are summarized in Table
5, Spearman's rho=0.64, p=0.011. Samples of symptomatic and
asymptomatic subjects indicate variation within individuals over
time, which may be highly relevant to using sLe.sup.x for
monitoring health status.
TABLE-US-00005 TABLE 5 Sample S Lex values for asymptomatic Crohn's
patients. Patient 1.sup.st sample 2.sup.nd sample ID value value
Comments 304 2.136 2.009 This patient had similar sLex values for
initial and follow-up samples. 330 2.088 1.724 This patient had
similar sLex values for initial and follow-up samples. 307 1.805
1.661 This patient had similar sLex values for initial and
follow-up samples. 317 1.683 1.716 This patient had similar sLex
values for initial and follow-up samples. 320 2.332 1.511 This
patient had similar sLex values for initial and follow-up samples.
308 .159 .062 This patient had similar sLex values for initial and
follow-up samples. 309 .392 .782 This patient had similar sLex
values for initial and follow-up samples. 313 .154 .048 This
patient had similar sLex values for initial and follow-up samples.
315 .257 .09 This patient had similar sLex values for initial and
follow-up samples. 301 2.097 1.197 This patient had high initial
sample values but intermediate follow-up sample values. 310 2.169
.975 This patient had high initial sample values but intermediate
follow-up sample values. 340 2.078 1.375 This patient had high
initial sample values but intermediate follow-up sample values. 312
1.238 .13 This patient had an intermediate initial value, with low
follow-up value. 303 2.026 .061 This patient had a high initial
value, with low follow-up value. 314 1.91 .785 This patient had a
high initial value, with low follow-up value.
(v) Factors Associated with Sialyl Lewis x and Lewis x.
[0130] Two factors were associated with salivary sialyl Lewis
x--history of antibiotic use in the past year (p=0.002), and
secretor status (p<0.001), in an ANOVA model that included study
group (which was not significant). No other factors were associated
with sLe.sup.x. Secretor status was significantly associated with
levels of Lewis x, but not history of antibiotic use. FIG. 10 shows
plots of sialyl Lewis X and Lewis X optical density values by
history of antibiotic use. FIG. 11 shows plots of sialyl Lewis X
and Lewis X optical density values by secretor status. FIG. 12
shows plots of sialyl Lewis X optical density values in symptomatic
and asymptomatic CD patients at two collection time points by
history of antibiotic use.
(vi) Comparison with Fecal Markers Measured.
[0131] Fecal Calprotectin values were measured and assessed in
relation to patient symptom group (FIG. 13). IBD-SCAN.RTM. optical
density values were also measured and assessed in relation to
patient symptom group (FIG. 14). The IBD-SCAN.RTM. test is a
commercially available quantitative ELISA for measuring
concentrations of fecal lactoferrin, a marker of fecal leukocytes.
An elevated level is an indicator of intestinal inflammation. The
IBD-SCAN.RTM. values were compared to the sialyl lewis X optical
density values. It was found that the sialyl Lewis x optical
density values were significantly correlated with the IBD-SCAN.RTM.
optical density value, Spearman's rho=0.49, p=0.016 (FIG. 15).
CONCLUSIONS
[0132] Sialyl Lewis x, alone or in combination with Lewis x,
appears promising to examine in larger, longitudinal studies of
patient self-monitoring, based on the samples collected by patients
after their initial enrollment visit. Single antigen predictive
value was 77% based on the identified cut-points. Use of
combinations of antigen values increased the predictive value to
84%. Salivary sialyl Lewis x was associated with history of
antibiotic use in the past year, whether in control patients or in
Crohn's patients. Sialyl Lewis x was associated with patient
symptom scores, and was correlated with the IBD-SCAN.RTM. O.D.
values. In summary, this pilot study provides intriguing evidence
of the potential role of salivary glycans, particularly sialyl
Lewis x, as a biomarker for patient self-monitoring of health
status and symptom flares.
REFERENCES
[0133] Chaturvedi P, Warren C D, Altaye M, et al (2001) Fucosylated
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Other Embodiments
[0144] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0145] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
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