U.S. patent application number 14/132187 was filed with the patent office on 2014-06-26 for methods and compositions for diagnosing osteoarthritis in a feline.
This patent application is currently assigned to COLGATE-PALMOLIVE COMPANY. The applicant listed for this patent is COLGATE-PALMOLIVE COMPANY. Invention is credited to Samer Waleed Khedheyer Al-Murrani, William David Schoenherr.
Application Number | 20140179552 14/132187 |
Document ID | / |
Family ID | 39944201 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140179552 |
Kind Code |
A1 |
Al-Murrani; Samer Waleed Khedheyer
; et al. |
June 26, 2014 |
METHODS AND COMPOSITIONS FOR DIAGNOSING OSTEOARTHRITIS IN A
FELINE
Abstract
Methods, compositions and kits for diagnosing osteoarthritis in
a feline are disclosed. The methods of the invention comprise
detecting differential expression of at least one biomarker in a
body sample, preferably a blood sample, where the biomarker is
differentially expressed in osteoarthritis.
Inventors: |
Al-Murrani; Samer Waleed
Khedheyer; (Topeka, KS) ; Schoenherr; William
David; (Hoyt, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COLGATE-PALMOLIVE COMPANY |
New York |
NY |
US |
|
|
Assignee: |
COLGATE-PALMOLIVE COMPANY
New York
NY
|
Family ID: |
39944201 |
Appl. No.: |
14/132187 |
Filed: |
December 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12600064 |
Sep 10, 2010 |
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PCT/US08/62225 |
May 1, 2008 |
|
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14132187 |
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60927167 |
May 1, 2007 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/6.12; 435/6.13; 435/7.92; 506/16; 506/18; 530/388.2;
530/389.1 |
Current CPC
Class: |
G01N 2800/105 20130101;
Y10T 436/143333 20150115; A61P 19/02 20180101; C12Q 2600/136
20130101; C12Q 2600/112 20130101; C12Q 1/6883 20130101; G01N
33/6893 20130101 |
Class at
Publication: |
506/9 ; 435/6.12;
435/6.11; 506/16; 506/18; 435/6.13; 435/7.92; 530/389.1;
530/388.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/68 20060101 G01N033/68 |
Claims
1. A method of diagnosing OA in a feline comprising: (a) isolating
nucleic acid from a body sample of a feline; (b) determining the
level of a nucleic acid encoding one or more biomarkers set out in
FIGS. 1-7 in a body sample from said feline; and (c) comparing the
level of nucleic acid in the body sample from said feline with
levels in a control feline without OA, wherein differential
expression of the nucleic acid between the individual and the
control is indicative of OA in the feline.
2. The method of claim 1, wherein the level of nucleic acid is
determined using methods comprising the use of quantitative
RT-PCR.
3. The method according to claim 1, wherein the level of nucleic
acid is determined using methods comprising the use of
microarrays.
4. The method according to claim 3, wherein said microarray
comprises a plurality of isolated polynucleotides selected from the
group consisting of RNA, DNA, cDNA, PCR products, or ESTs
corresponding to one or more of the biomarkers of FIGS. 1-7.
5. The method according to claim 1, wherein the body sample is a
blood sample.
6. The method according to claim 1, wherein the nucleic acid is
RNA.
7. A composition comprising at least one or more isolated
polynucleotides, wherein each isolated polynucleotide selectively
hybridizes to a nucleic acid encoding a biomarker set out in FIGS.
1-7 and wherein the composition permits measurement of the level of
expression of at least one or more biomarkers in a body sample from
a feline.
8. The composition of claim 7 wherein the isolated polynucleotide
is RNA.
9. A composition comprising at least one or more antibodies,
wherein each antibody binds selectively to a protein product of a
biomarker selected from the biomarkers set out in FIGS. 1-7 and
wherein the composition permits the measurement of the level of
expression of at least one or more biomarkers in a body sample from
a feline.
10. The composition according to claim 9, wherein the antibodies
are monoclonal antibodies.
11. A kit for diagnosing OA in a feline, said kit comprising: (a) a
microarray comprising a plurality of isolated polynucleotides
selected from the group consisting of RNA, DNA, cDNA, PCR products,
or ESTs corresponding to one or more of the biomarkers of FIGS.
1-7; and/or (b) a composition comprising at least one or more
antibodies, wherein each antibody binds selectively to a protein
product of a biomarker set out in FIGS. 1-7 wherein components (a)
or (b) may comprise a majority of the components of said kit.
12. A method for identifying a component to be tested for an
ability to treat or ameliorate osteoarthritis in a feline
comprising: (a) contacting a cell capable of expressing an RNA or
protein product of one or more OA biomarkers disclosed in FIGS. 1-7
with a test component; (b) determining the amount of said RNA
and/or product produced in the cells contacted with the test
component, and (c) comparing the amount of said RNA and/or protein
product in the cells contacted with the test component to the
amount of the same said RNA or protein product present in a
corresponding control cell that has not been contacted with the
test component wherein if amount of the RNA or protein product is
altered relative to the amount in the control, the component is
identified as one to be tested for an ability to treat or
ameliorate osteoarthritis in a feline.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of application
Ser. No. 12/600,064, with a 371 entry date of 10 Sep. 2010, which
is a national stage entry under 35 U.S.C. .sctn.371 of
International Patent Application No. PCT/US2008/062225, filed 1 May
2008, which claims priority to U.S. Provisional Patent Application
No. 60/927,167, filed on 1 May 2007, which is incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the identification of novel
osteoarthritic biomarkers in felines and diagnostic methods,
compositions, and kits related thereto.
BACKGROUND OF THE INVENTION
[0003] Arthritis, more particularly osteoarthritis (OA), is a
degenerative joint disease commonly occurring in humans and in
companion animals. OA involves progressive deterioration of
articular cartilage, with loss of proteoglycan and collagen and
proliferation of new bone, accompanied by a variable inflammatory
response within the synovial membrane. It is the most common form
of joint and musculoskeletal disease affecting dogs, but cats may
also suffer from this condition.
[0004] Feline OA is disease primarily affecting aged felines 10
years of age or older. Animals suffering from this disease
characteristically jump less, reduce the height of their jumps or
stop jumping entirely, avoid going up or down stairs, and tend to
use their litter box less. Cats with OA also appear to be less
friendly, exhibit changes in their sleep-wake patterns, and may
have grooming problems. The management of OA in cats is similar to
treatment regimens in other species which include environmental
modification, treatment of obesity, controlled moderate exercise,
pain control, and surgery.
[0005] Environmental modification begins with the placement of food
bowls and litter pans in locations that do not require leaping or
stair climbing. Small ramps can be built to a feeding station or
into the litter box. The attempt is made by the pet owner to reduce
large leaps up or down, to encourage moderate exercise, and to
create an environment where the cat is not faced with an obstacle
course to maintain a daily routine. Environmental modification does
not slow the progression of the disease.
[0006] Overweight cats with OA can benefit limn weight control. A
reduction in body weight will alleviate the pressure and pain on
the affected joint(s). Though weight loss in overweight or obese
cats can help alleviate the pain caused by OA, it does not stop the
progression of the disease.
[0007] Pain control in cats is a problem because drug regimens that
are safe in other species are not necessarily safe in cats. Though
many pharmaceutical companies are evaluating non-steroidal
anti-inflammatory drugs (NSAIDs) in cats for treatment of pain,
aspirin is the only NSAID for which a safe, chronic dose has been
established in the cat. Corticosteroids have been used to alleviate
pain and inflammation, but their use may cause progression of OA.
NSAIDs may help alleviate pain but will not alter the progression
of OA.
[0008] Nutraceuticals have been used to alleviate pain associated
with OA in cats. Chondroitin sulfate and glucosamine HCl used in
combination or separately have been used as a treatment regimen in
cats. There are no published clinical studies that indicate these
nutraceuticals alter the progression of OA. Recent data in humans
indicates that chondroitin and glucosamine may help alleviate pain
in humans with severe OA.
[0009] Although helpful in some respect to provide symptomatic
relief, the approaches described above are not entirely successful
in disease management, as they clearly do not treat the underlying
pathology. Indeed, not only are improved treatment methods needed,
but also improved methods to monitor the clinical progress of an
animal with OA and even to diagnose an animal that has OA, and
those that may be genetically predisposed to developing OA but do
not as yet display any clinical signs of the disease. Currently,
extensive radiographic tests must be carried out to confirm a
diagnosis of OA in an animal and these tests are useful only to
identify animals that have manifest joint and tissue damage. Thus,
there is a need for a simple diagnostic test for detecting OA in
felines as well as improved methods for monitoring the clinical
progress of an animal with OA.
BRIEF SUMMARY OF THE INVENTION
[0010] The current invention relates to the identification of novel
biomarkers for OA in felines and methods for the detection of
arthritic animals based on a characteristic pattern of gene
expression for these OA biomarkers in vivo. Specifically, the
methods of the invention comprise detecting differential
expression, compared to a control expression level, of at least one
biomarker, in a body sample, preferably a blood sample, wherein the
detection of differential expression of said biomarker specifically
identifies animals that have OA. Thus, the method relies on the
detection of at least one biomarker that is differentially
expressed in OA in comparison to cells from normal, or control,
animals.
[0011] The biomarkers of the invention are proteins and/or nucleic
acids that are differentially expressed in OA in felines. In one
aspect, the biomarkers of particular interest include the
biomarkers listed herein on FIGS. 1-7.
[0012] Biomarker expression can be assessed at the protein or
nucleic acid level using various methods. Thus, in another aspect,
the invention relates to methods that utilize antibodies to detect
the expression of OA biomarker proteins in feline samples. In this
aspect of the invention, at least one antibody directed to a
specific OA biomarker of interest is used. In a further aspect,
expression levels can also be detected by nucleic acid-based
techniques, including, for example, hybridization, microarray
technologies and RT-PCR, including quantitative RT-PCR. Mass
specnometry, fluorescence activated cell sorting (FACS) or Luminex
Xmap.RTM. bead technology may also be used to detect expression
levels at both the protein or nucleic acid level.
[0013] It is further contemplated herein that the methods of the
present invention may be used in combination with traditional
diagnostic techniques that are able to detect the physical and
morphological characteristics of degenerative joint disease. Thus,
for example, the characterization of differential expression in
genes for OA biomarkers in cells obtained from a blood sample of an
animal may be combined with conventional diagnostic (e.g.,
radiological) techniques in order to corroborate a diagnosis of
OA.
[0014] In a further aspect, the invention relates to compositions
comprising one or more nucleic acid probes that specifically
hybridize to a nucleic acid, or fragment thereof, encoding an OA
biomarker of the present invention.
[0015] In an additional aspect, the invention relates to
compositions comprising antibodies that specifically bind to a
polypeptide encoded by a gene of an OA biomarker of the present
invention.
[0016] The invention also relates to kits to diagnose OA in a
feline comprising components that can be used to detect expression
of the OA biomarkers of the present invention, including, but not
limited to, the compositions and microarrays described herein.
[0017] It is also contemplated herein that the present invention
relates to the use of the OA biomarkers and compositions disclosed
herein in methods to diagnose OA in a feline.
[0018] In another aspect, it is also contemplated herein that the
invention relates to methods for identifying. bioactive dietary
components or other natural compounds (referred to hereafter as
"components") that may be tested for an ability to treat or
ameliorate osteoarthritis in a feline comprising: (a) contacting a
cell capable of expressing an RNA or protein product of one or more
OA biomarkers disclosed in FIGS. 1-7 with a test component; (b)
determining the amount of said RNA and/or product produced in the
cells contacted with the test component; and (c) comparing the
amount of said RNA and/or protein product in the cells contacted
with the test component to the amount of the same said RNA or
protein product present in a corresponding control cell that has
not been contacted with the test component; wherein if amount of
the RNA or protein product is altered relative to the amount in the
control, the component is identified as one to be tested for an
ability to treat or ameliorate osteoarthritis in a feline.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 discloses feline OA biomarkers identified based on a
selection criteria wherein the p value <0.001 and expression,
levels display a fold change of >2.0. With regard to FIGS. 1 and
FIGS. 2-6 as well, where mean-ratio >1, the gene is up regulated
in OA and equates to a positive fold change. In contrast, where
mean-ratio <1, the gene is down regulated in OA animals and
equates to a negative fold change.
[0020] FIG. 2 discloses feline OA biomarkers identified based on a
selection criteria wherein the p value <0.001 and expression
levels display a fold change of >1.5.
[0021] FIG. 3 discloses feline OA biomarkers identified based on a
selection criteria wherein the p value <0.000 and expression
levels display a fold change of >1.3.
[0022] FIG. 4 discloses feline OA biomarkers identified based on a
selection criteria wherein the p value <0.01 and expression
levels display a fold change of >2.0.
[0023] FIG. 5 discloses feline OA biomarkers identified based on as
selection criteria wherein the p value <0.01 and expression
levels display a fold change of >1.5.
[0024] FIG. 6 discloses feline OA biomarkers identified based on a
selection criteria wherein the p value <0.01 and expression
levels display a fold change of >1.3.
[0025] FIG. 7 discloses feline OA biomarkers identified based on an
analysis of feline data using qRT-PCR.
[0026] FIG. 8 discloses relative quantity of TGF-beta in normal and
arthritic cats fed different diets. With regard to FIG. 8 and FIGS.
9-11, as indicated, "j/d" and "senior" refer to diets containing
different amounts of omega-3 fatty acids as disclosed in WO
2007/002837 A2 and WO 2006/074089 A2, respectively.
[0027] FIG. 9 depicts the relative quantity of IL-1 alpha in normal
and arthritic cats on j/d and senior diets.
[0028] FIG. 10 depicts levels of Nix in cats fed diets high in EPA
and DHA. Levels are detected using conventional ELISA methods.
[0029] FIG. 11 depicts levels of CTX-II in cats fed diets high in
EPA and DHA. Levels are detected using conventional ELISA
methods.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides compositions and methods for
identifying or diagnosing osteoarthritis in felines. The methods
comprise the detection of the differential expression of specific
biomarkers that are either selectively over expressed or under
expressed in osteoarthritis. In this way, the biomarkers of the
invention are capable of distinguishing between animals that have
OA and those that do not. It is also contemplated herein that it
may be possible to identify those animals that may be predisposed
to developing OA or those that have OA but have not yet manifested
morphological or physical changes. Said methods for diagnosing
osteoarthritis involve detecting the differential expression of at
least one biomarker that is indicative of osteoarthritis in a
tissue or body fluid sample from a feline. In particular
embodiments, antibodies and immunocytochemistry techniques or
nucleic acid probes and hybridization techniques are used to detect
expression of the biomarker of interest. Kits for practicing the
methods of the invention are further provided.
[0031] "Diagnosing osteoarthritis" is intended to include, for
example, diagnosing or detecting the presence of OA or a genetic
predisposition thereto, monitoring the progression of the disease,
and effectiveness of therapeutic intervention, as well as
identifying or detecting cells or samples that are indicative of
osteoarthritis. The terms diagnosing, detecting, and identifying
osteoarthritis are used interchangeably herein. By "osteoarthritis"
is intended those conditions characterized by degeneration of
articular cartilage on the ends of bones that forms the surface of
the joints and which may include, later stages, accompanying
changes in surrounding tissue in and around joints, e.g., bone,
muscle, ligaments, menisci and synovium. Such physical changes are
manifested by pain, swelling, weakness and loss of joint
function.
[0032] OA has been classified into various grades or stages. Stage
1 is characterized by changes in chondrocyte metabolism such that
there is an increase in cartilage matrix destroying enzymes such as
metalloproteases. Protease inhibitors are synthesized in
insufficient levels to combat the breakdown of the cartilage
matrix. Stage 2 is characterized by erosion of the cartilage
surface which causes a detectable increase in levels of
proteoglycan and collagen fragments in the synovial fluid. During
stage 3, the synovium is chronically inflamed, as a result of the
breakdown products of cartilage. Macrophages in the synovium
produce cytokines which can damage cartilage by directly destroying
tissue or stimulating the chondrocytes to produce more
metalloproteinases. Pro-inflammatory molecules may also cause
damage at this stage. The resulting damage to the joint triggers an
increase in bone growth as the body attempts to stabilize the
joint, thus changing the normal mechanical and architectural
features of the joint.
[0033] As discussed above, it is contemplated that the methods of
the present invention may permit the identification of felines that
may be predisposed to developing OA in future. In these animals it
is contemplated that the methods of the present invention may be
particularly useful, since conventional methods that rely on
morphological changes to joint tissue would not typically identify
these patients as being in need of therapeutic intervention. Use of
haplotype markers and single nucleotide polymorphisms (SNPs) of the
GA biomarkers disclosed herein may he particularly useful in this
regard. In these cases, prophylactic measures or other therapies
may be started as a means to ward off the more debilitating
symptoms or physical damage associated with the later stages of
OA.
[0034] In addition, it is contemplated herein while the OA
biomarkers disclosed herein may he useful to diagnose a feline with
OA, the OA biomarkers may also be useful targets for therapeutic
intervention. For example, it is contemplated herein that a
therapeutic benefit may be achieved prior to the manifestation of
pathological physical changes in a feline (or even after physical
changes have occurred), by altering the expression of any one or
more of the biomarkers described in FIGS. 1-7 provided herein,
e.g., by decreasing expression levels of genes overexpressed in OA,
and/or by increasing expression in genes that are underexpressed in
OA. In the case of some biomarkers as described in detail in the
Examples below, this may he achieved by administration of a
formulation high in EPA and DHA to a feline in need thereof.
[0035] It is further contemplated herein that components that may
be of use to treat or ameliorate OA in felines may be identified by
exposing cells to test components in vitro and assaying, for
changes in gene expression of one or more of the OA biomarkers.
Such in vitro assays are familiar to one of skill in the art and
may be performed according to conventional methods. Primary
cultures of feline cells may be used as well as cell lines isolated
from different feline tissues such as, e.g., blood, kidney, brain,
tongue or lung, Feline cells for in vitro analysis may he obtained
commercially, e.g, from American Type Culture Collection (ATCC,
Manassas, Va.). Candidate components which show potential to
influence the expression of OA biomarkers can then be slated for
further experimentation, including as components of pet food
formulations such as described in the Examples below.
[0036] While not intending to be limited to a particular mechanism,
it is contemplated herein that the genotypic fingerprint of
osteoarthritis in felines may be characterized by the differential
expression of discrete genes, or biomarkers. The use of these
molecular biomarkers of OA in molecular diagnostic assay formats
can improve the detection of OA compared to current methods. Thus,
in particular embodiments, a method for diagnosing osteoarthritis
comprises detecting differential expression of a biomarker, wherein
differential expression of the biomarker may be indicative of a
disruption in a biochemical pathway related to or associated with
OA, e.g., either as cause or effect. In still other embodiments,
the methods comprise detecting differential expression of one or
more DA biomarkers, e.g., a subset of biomarkers provided, herein
on FIGS. 1-7. In this way, the methods of the present invention may
not only permit the identification of an animal with
osteoarthritis, but may also allow identification of an animal who
may be genetically predisposed to developing OA.
[0037] The methods disclosed herein provide superior detection of
osteoarthritis in comparison to conventional diagnostic testing.
"Conventional methods to diagnose OA" are familiar to one of skill
in the art and include X-ray, magnetic resonance imaging or the use
of ultrasound. In particular aspects of the invention, the accuracy
of the present methods are equal to or greater than that of
conventional radiological or magnetic resonance testing used to
detect the presence of OA.
[0038] The biomarkers of the present invention include genes and
proteins, and variants and fragments thereof Such biomarkers
include polynucleotides, e.g, DNA comprising the entire or partial
sequence of the nucleic acid sequence encoding the biomarker, or
the complement of such a sequence. Biomarker polynucleotides may
also include RNA comprising the entire or partial sequence of any
of the nucleic acid sequences of interest. A biomarker protein is a
protein encoded by or corresponding to a DNA biomarker of the
invention. A biomarker protein comprises the entire or partial
amino acid sequence of any of the biomarker proteins or
polypeptides disclosed herein.
[0039] A "biomarker" is any gene or protein whose level of
expression in a tissue or cell is altered compared to that of a
normal or healthy cell or tissue. Biomarkers of the present
invention are differentially expressed in felines with
osteoarthritis. By "differentially expressed in osteoarthritis" is
intended that the gene expression levels of the biomarkers of
interest are either up or down regulated in a subject having, or
predisposed to having, osteoarthritis compared to levels in a
control subject, i.e., a subject not having or predisposed to
having the condition. It is contemplated herein that detection of
the biomarkers of the invention not only permits the identification
of a subject having OA, but may also provide a means to identify an
animal predisposed to developing this condition, ideally before
physical symptoms manifest. If possible, such early detection would
allow for improved patient care and could possibly prevent disease
associated irreversible joint damage.
[0040] In some embodiments, the methods for diagnosing
osteoarthritis disclosed herein may be performed as a primary means
to screen for osteoarthritis in a feline. Said feline may be
screened as part of a routine physical evaluation. A feline may
also be screened according to the methods of the present invention
because information indicates that the feline may be genetically
predisposed to OA based on the medical history of its dam and/or
sire, or because the feline is suspected to have the condition
based on observed changes in behavior patterns. The methods of the
present invention may also be used as part of a clinical
examination in conjunction with conventional methods for diagnosing
OA in a feline, e.g. X-ray or MRI, when said conventional methods
are inconclusive or when confirmation of a diagnosis based on
conventional techniques is desired.
[0041] The biomarkers of the invention include any polynucleotide
or protein that is selectively differentially expressed in
osteoarthritis, as defined herein above. Although any biomarker
indicative of osteoarthritis may be used in the present invention,
in certain embodiments the biomarkers include any one or more, or
subsets of biomarkers set forth herein on FIGS. 1-7.
[0042] Although the methods of the invention require the detection
of at least one biomarker in a patient sample for the detection of
osteoarthritis, it is contemplated herein that several or more
biomarkers may be used to practice the present invention.
Therefore, in some embodiments, one or more biomarkers are used,
more preferably, two or more complementary biomarkers. By
"complementary" is intended that detection of the combination of
biomarkers in a body sample results in the successful
identification of osteoarthritis in a greater percentage of cases
than would be identified if only one of the biomarkers was used.
Thus, in some cases, a more accurate determination of
osteoarthritis can be made by using at least two biomarkers.
Accordingly, where at least two biomarkers are used, at least two
antibodies directed to distinct biomarker proteins can be used to
practice the diagnostic methods disclosed herein. For example,
antibodies or nucleic acid probes may be contacted with a body
sample simultaneously or concurrently.
[0043] In particular embodiments, the diagnostic methods of the
invention comprise collecting a blood sample from a feline patient,
contacting the sample with at least one antibody specific for a
biomarker of interest, and detecting antibody binding. Samples that
exhibit differential expression of a biomarker of the invention, as
determined by detection of antibody binding, are deemed positive
for osteoarthritis. In particular embodiments, the body sample is a
blood sample obtained from a feline by conventional methods such as
density gradient separation methods, e.g., Ficoll-hypaque
technique, or using cell preparation tubes (CPT.TM. tubes) from
Becton Dickinson or other methods familiar to one of skill in the
art.
[0044] By "body sample" is intended any sampling of cells, tissues,
or bodily fluids in which expression of a biomarker can be
detected. Examples of such body samples include, but are not
limited to, blood, lymph, urine, biopsies, and smears. Body samples
may be obtained from a feline by a variety of techniques including,
for example, by scraping or swabbing an area or by using a needle
to aspirate bodily fluids. Methods for collecting various body
samples are well known in the art. In particular embodiments, the
body sample comprises blood cells.
[0045] Any methods available in the art for identification or
detection of the OA biomarkers of the present invention are
encompassed herein. The differential expression of a biomarker of
the invention can be detected on a nucleic acid level or a protein
level. As described above, in order to determine differential
expression, the body sample to be examined may be compared with a
corresponding body sample that originates from a healthy subject.
That is, the "normal" level of expression is the level of
expression of the biomarker in the cells of a subject feline not
afflicted with or predisposed to osteoarthritis. In some cases
where the ratio of expression of a biomarker in an arthritic animal
compared to a control is known, differential expression of said
biomarker may be characterized in an animal without direct
comparison to a normal.
[0046] Methods for detecting biomarkers of the invention comprise
any methods that determine the quantity or the presence of the
biomarkers either at the nucleic, acid or protein level. Such
methods are well known in the art and include but are not limited
to Western blots, Northern blots, Southern blots, ELISA,
immunoprecipitation, immunofluorescence, flow cytometry,
immunocytochemistry, nucleic acid hybridization techniques, nucleic
acid reverse transcription methods, and nucleic acid amplification
methods. In particular embodiments, differential expression of a
biomarker is detected on a protein level using, for example,
antibodies that are directed against specific biomarker proteins.
These antibodies can be used in various methods such as Western
blot, ELISA, immunoprecipitation, or immunocytochemistry
techniques. In addition, data from conventional diagnostic imaging
using X-ray, or magnetic resonance may be obtained and compared to
the immunocytochemical or nucleic acid probe hybridization
information. In this manner, the detection of the biomarkers can
confirm results from conventional diagnostic methods or provide
clarity when data from conventional methods are inconclusive.
[0047] In one embodiment, antibodies specific for biomarker
proteins are utilized to detect the differential expression of a
biomarker protein in a body sample. The method comprises obtaining
a body sample from a subject, contacting the body sample with at
least one antibody directed to a biomarker that is selectively
differentially expressed in osteoarthritis, and detecting antibody
binding to determine if the biomarker is similarly differentially
expressed in the sample. A preferred aspect of the present
invention provides an immunocytochemistry technique for diagnosing
osteoarthritis using a blood sample from a subject.
[0048] In a preferred immunocytochemical method, a blood sample is
collected from a subject using methods familiar to one of skill in
the art. For example, as described in the examples provided herein,
PAXgene blood RNA tubes (for use in PAX gene blood RNA isolation)
may also be employed where isolation of nucleic acid is desired.
The blood sample may be assayed immediately or stored under
appropriate conditions familiar to one of skill in the art for
later analysis.
[0049] Alternatively, an antibody, particularly a monoclonal
antibody, directed to a biomarker of interest may be incubated with
a blood sample from a subject. As noted above, one of skill in the
art will appreciate that a more accurate diagnosis of
osteoarthritis may be obtained in some cases by detecting more than
one biomarker in a patient sample. Therefore, in particular
embodiments, at least two antibodies directed to two distinct
biomarkers are used to detect osteoarthritis. Where more than one
antibody is used, these antibodies may be added to a single sample
sequentially as individual antibody reagents or simultaneously as
an antibody cocktail. Alternatively, each individual antibody may
be added to a separate sample from the same patient, and the
resulting data pooled. In particular embodiments, an antibody
cocktail may comprises several antibodies, wherein said antibodies
bind to, e.g., a subset of the OA biomarkers disclosed in FIGS.
1-7.
[0050] The terms "antibody" and "antibodies" broadly encompass
naturally occurring forms of antibodies and recombinant antibodies
such as single-chain antibodies, chimeric and humanized antibodies
and multi-specific antibodies as well as fragments and derivatives
of all of the foregoing which fragments and derivatives have at
least an antigenic binding site. Fully assembled antibodies and
antibody fragments that can bind antigen are included in this
definition. Antibody derivatives may comprise a protein or chemical
moiety conjugated to the antibody.
[0051] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen-binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al, (1995) Protein Eng. 8(10):1057 1062); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments. Papain digestion of antibodies produces two
identical antigen-binding fragments, called "Fab" fragments, each
with a single antigen-binding site, and a residual "Fc" fragment.
Pepsin treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen. Described herein are methods for the production of
antibodies capable of specifically recognizing one or more
differentially expressed gene epitopes. Such antibodies may
include, but are not limited to, polyclonal antibodies, monoclonal
antibodies (mAbs), humanized or chimeric antibodies, single chain
antibodies, Fab fragments, F(ab').sub.2 fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies, and epitope-binding fragments of any of the above.
[0052] For the production of antibodies to a differentially
expressed gene, various host animals may be immunized by injection
with a differentially expressed gene protein, or a portion thereof
Such host animals may include but are not limited to rabbits, mice,
rats, and chickens to name but a few. Various adjuvants may be used
to increase the immunological response, depending on the host
species, including but not limited to Freund's (complete and
incomplete). mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
[0053] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as target gene product, or an antigenic functional
derivative thereof. For the production of polyclonal antibodies,
host animals such as those described above may be immunized by
injection with differentially expressed gene product, supplemented
with adjuvants as also described above.
[0054] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. These include, but are not
limited to the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983. Immunology Today 4:72:
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2010), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
[0055] In addition, techniques developed for t le production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452454) by splicing the genes from
a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity, can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable or hypervariable region
derived from a murine mAb and a human immunoglobulin constant
region.
[0056] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 334;544-540 can be
adapted to produce differentially expressed gene-single chain
antibodies. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0057] Most preferably, techniques useful for the production of
"humanized antibodies" can be adapted to produce antibodies to the
polypeptides, fragments, derivatives, and functional equivalents
disclosed herein. Such techniques are well known to those of skill
in the art and are disclosed in, e.g., U.S. Pat. Nos. 5,932,448;
5,693,762; 5,693.761; 5,585,089; 5,530,101; 5,910,771; 5,569,825;
5,625,126; 5,633,425; 5,789,650; 5,545,580; 5,661,016; and
5,770,429, the disclosures of all of which are incorporated by
reference herein in their entirety.
[0058] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, such fragments include
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity. Particularly preferred,
for ease of detection, is the sandwich assay, of which a number of
variations exist, all of which may be employed in the methods of
the present invention. Specifically, Elisa methods, including
standard, sandwich and microformat Elisa methodologies familiar to
one of skill in the art, may be used.
[0059] In some cases, an unlabeled antibody is immobilized on a
solid substrate and the sample to be tested brought into contact
with the bound molecule. After a suitable period of incubation, for
a period of time sufficient to allow formation of an
antibody-antigen binary complex, a second antibody, labeled with a
reporter molecule capable of inducing a detectable signal, is then
added and incubated, allowing time sufficient for the formation of
a ternary complex of antibody-antigen-labeled antibody. Any
unreacted material is washed away, and the presence of the antigen
is determined by observation of a signal, or may be quantitated by
comparing with a control sample containing known amounts of
antigen. Variations include a simultaneous assay, in which both
sample and antibody are added simultaneously to the bound antibody,
or an assay in which the labeled antibody and sample to he tested
are first combined, incubated and added to the unlabeled surface
bound antibody. These techniques are well known to those skilled in
the art, and the possibility of minor variations will be readily
apparent.
[0060] The most commonly used reporter molecules in this type of
assay are either enzymes, fluorophore- or radionuclide-containing
molecules. In the case of an enzyme immunoassay, an enzyme is
conjugated to the second antibody, usually by means of
glutaraldehyde or periodate. As will be readily recognized,
however, a wide variety of different ligation techniques exist,
which ae well-known to the skilled artisan. Commonly used enzymes
include horseradish peroxidase, glucose oxidase, beta-galactosidase
and alkaline phosphatase, among others. The substrates to be used
with the specific enzymes are generally chosen for the production,
upon hydrolysis by the corresponding enzyme, of a detectable color
change. For example, p-nitrophenyl phosphate is suitable for use
with alkaline phosphatase conjugates; for peroxidase conjugates,
1,2-phenylenediamine or toluidine are commonly used. It is also
possible to employ fluorogenic. substrates, which yield a
fluorescent product rather than the chromogenic substrates noted
above. Spectophotometric methods may also be used to evaluate the
presence of antigen in the serum sample.
[0061] Alternately, fluorescent compounds, such as fluorescein and
rhodamine, may be chemically coupled to antibodies without altering
their binding capacity. When activated by illumination with light
of a particular wavelength, the fluorochrome-labeled antibody
absorbs the light energy, inducing a state of excitability in the
molecule, followed by emission of the light at a characteristic
longer wavelength. The emission appears as a characteristic color
visually detectable with a light microscope. Immunofluorescence and
ER techniques are both very well established in the art and are
particularly preferred for the present method. However, other
reporter molecules, such as radioisotopes, chemiluminescent or
bioluminescent molecules may also be employed. It will be readily
apparent to the skilled artisan how to vary the procedure to suit
the required use.
[0062] With regard to detection of antibody staining, in the
immunocytochemistry methods of the invention, there also exist in
the art video-microscopy and software methods for the quantitative
determination of an amount of multiple molecular species (e.g.,
biomarker proteins) in a biological sample wherein each molecular
species present is indicated by a representative dye marker having
a specific color. Such methods are also known in the art as
calorimetric analysis methods. In these methods, video-microscopy
is used to provide an image of the biological sample after it has
been stained to visually indicate the presence of a particular
biomarker of interest. Some of these methods, such as those
disclosed in U.S. Pat. No. 7,065,236, incorporated herein by
reference, disclose the use of an imaging system and associated
software to determine the relative amounts of each molecular
species present based on the presence of representative color dye
markers as indicated by those color dye markers optical density or
transmittance value, respectively, as determined by an imaging
system and associated software. These techniques provide
quantitative determinations of the relative amounts of each
molecular species in a stained biological sample using a single
video image that is "deconstructed" into its component color
parts.
[0063] The antibodies used to practice the invention are selected
to have high specificity for the biomarker proteins of interest.
While methods for making, antibodies and for selecting appropriate
antibodies are known in the art and described above, it is also
contemplated herein that in some embodiments, commercial antibodies
directed to specific biomarker proteins may be used to practice the
invention.
[0064] One of skill in the art will recognize that the
concentration of a particular antibody used to practice the methods
of the invention may vary depending on such factors as time for
binding, level of specificity of the antibody for the biomarker
protein, and method of body sample preparation. Moreover, when
multiple antibodies are used, the required concentration may be
affected by the order in which the antibodies are applied to the
sample, i.e., simultaneously as a cocktail, or sequentially as
individual antibody reagents. Furthermore, the detection chemistry
used to visualize antibody binding to a biomarker of interest must
also be optimized to produce the desired signal to noise ratio.
[0065] In other embodiments, the expression of a biomarker of
interest is detected at the nucleic acid level. Nucleic acid-based
techniques for assessing expression are well known in the art and
include, for example, determining the level of biomarker mRNA in a
body sample. Many expression detection methods use isolated RNA. As
used herein, "RNA" includes total RNA as well as mRNA. Any RNA
isolation technique that does not select against the isolation of
mRNA can he utilized for the purification of RNA from blood cells
(see, e.g., Ausubel et al., ed., (1987 1999) Current Protocols in
Molecular Biology (John Wiley Sons, New York). Additionally, large
numbers of tissue samples can readily be processed using techniques
well known to those of skill in the art, such as, described in U.S.
Pat. No. 4,843,155.
[0066] The term "probe" refers to any molecule that is capable of
selectively binding to a specifically intended target biomolecule,
for example, a nucleotide transcript or a protein encoded by or
corresponding to an OA biomarker. Probes can be synthesized by one
of skill in the art, or derived from appropriate biological
preparations. Probes may he specifically designed to be labeled.
Examples of molecules that can he utilized as probes include, but
are not limited to, RNA, DNA, proteins, antibodies, and organic
molecules.
[0067] Isolated mRNA can he used in hybridization or amplification
assays that include, but are not limited to, Southern or Northern
analyses, polymerase chain reaction analyses and probe arrays. One
method for the detection of mRNA levels involves contacting the
isolated mRNA with a nucleic acid molecule (probe) that can
hybridize to the mRNA encoded by the gene being detected. The
nucleic acid probe can be, for example, a full-length cDNA, or a
portion thereof, such as an oligonucleotide of at least 7, 15, 30,
50, 100, 250 or 500 nucleotides in length and sufficient to
specifically hybridize under stringent conditions to an mRNA or
genomic. DNA encoding a biomarker of the present invention.
Hybridization of an mRNA with the probe indicates that the
biomarker in question is being expressed.
[0068] In one embodiment, the mRNA is immobilized on a solid,
surface and contacted with a probe, for example by running the
isolated mRNA on an agarose gel and transferring the mRNA from the
gel to a membrane, such as nitrocellulose. A skilled artisan can
readily adapt known mRNA detection methods for use in detecting the
level of mRNA encoded by the biomarkers of the present
invention.
[0069] In an alternative embodiment, the probe(s) for an OA
biomarker are immobilized on a solid surface and the mRNA is
contacted with the probe(s). As contemplated herein, gene chips,
(e.g., high density oligonucleotide arrays), microarrays (e.g.,
cDNA arrays or oligonucleotide arrays printed on glass slides),
microarrays, (e.g., PVDF membranes on which genes are printed) and
bead-based arrays (e.g. Illumina bead based microarrays) are among
the possible assay platforms that may be used in the methods of the
present invention. Thus, in one embodiment of the invention,
microarrays are used to detect biomarker expression, and such
methods are useful to detect expression levels of a number of
different genes. Microarray technologies, e.g., such as
commercially available from Affymetrix, are familiar to one of
skill in the art. It is contemplated herein that microarrays or
"chips" designed for the detection of expression of the OA
biomarkers disclosed herein may be created for use in the methods
of the present invention. As used herein, "microarray" is meant to
include all array platform technologies, which may include, e.g.,
gene chips, or bead arrays, and may include peptides or nucleic.
acids, c.a., RNA, DNA, cDNA, PCR products or ESTs, on beads, gels,
polymeric surfaces, fibers such as fiber optics, glass or any other
appropriate substrate.
[0070] An alternative method for determining, the level of
biomarker mRNA in a sample involves the process of nucleic acid or
oligonucleotide amplification, e.g., by RT-PCR, including
quantitative or qRT-PCR, ligase chain reaction (Barmy (1991) Proc,
Natl. Acad. Sci. USA 88:189 193), self sustained sequence
replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA
87:1874 1878), transcriptional amplification system (Kwoh et al.
(1989) Proc. Natl. Acad. Sci. USA 86:1173 1177), Q-Beta Replicase
(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle
replication or any other nucleic acid amplification method,
followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers, in
particular aspects of the invention, biomarker expression is
assessed by quantitative RT-PCR.
[0071] Biomarker expression levels of RNA may be monitored using
conventional methods, e.g, a membrane blot (such as used in
hybridization analysis such as Northern, Southern, dot, and the
like), or microwells, sample tubes, gels, beads or fibers (or any
solid support comprising bound nucleic acids). The detection of
biomarker expression may also comprise using nucleic acid probes in
solution.
[0072] Kits for practicing the methods of the invention are also
contemplated herein. By "kit" is intended any manufacture (e.g., a
package or a container) comprising the components necessary to
detect differential expression of an OA biomarker in a feline. Said
kits may comprise, for example, at least one reagent, e.g., an
antibody, a nucleic, acid probe, etc. for specifically detecting
the expression of a biomarker of the present invention. As
contemplated herein, the kits of the present invention may focus on
the diagnostic use of a single type of component (for example,
reagents such as antibodies or nucleic acid probes) or may comprise
different types of components and the relative amounts of each may
vary, such that the majority of the components may be of one type
or another, or the reagents may be of equal amounts. A kit of the
present invention may also comprise a microarray comprising one or
more nucleic acids specific. for the OA biomarkers of the present
invention or subsets of said biomarkers. The kit may be promoted,
distributed, or sold as a unit for performing the methods of the
present invention. Additionally, the kits may contain a package
insert describing the kit and methods for its use.
[0073] In a particular embodiment, kits for practicing the
immunocytochemistry methods of the invention are provided. Such
kits are compatible with both manual and automated
immunocytochemistry techniques (e.g., cell staining). These kits
comprise at least one antibody directed to a biomarker of interest,
chemicals for the detection of antibody binding to the biomarker,
and a counterstain. Any chemicals that detect antigen-antibody
binding may be used in the practice of the invention. In some
embodiments, the detection chemicals comprise a labeled polymer
conjugated to a secondary antibody. For example, a secondary
antibody that is conjugated to an enzyme that catalyzes the
deposition of a chromogen at the antigen-antibody binding site may
be provided. Such enzymes and techniques for using them in the
detection of antibody binding are well known in the art.
[0074] In another embodiment, the kits of the invention
additionally comprise at least two or more reagents, antibodies,
for specifically detecting the expression of at least two or more
distinct biomarkers. Each antibody may be provided in the kit as an
individual reagent or, alternatively, as an antibody cocktail
comprising all of the antibodies directed to the different
biomarkers of interest. Furthermore, any or all of the kit reagents
may be provided within containers that protect them from the
external environment, such as in sealed containers.
[0075] Positive and/or negative controls may be included in the
kits to validate the activity and correct usage of reagents
employed in accordance with the invention. Controls may include
samples, such as tissue sections, cells fixed on glass slides,
etc., known to be either positive or negative for the presence of
the biomarker of interest. The design and use of controls is
standard and well within the routine capabilities of those of
ordinary skill in the art.
[0076] In other embodiments, kits for identifying OA in a feline
comprising detecting differential expression of a bioniarker at the
nucleic acid level are provided. Such kits comprise, for example,
at least one nucleic acid probe that specifically binds to a
biomarker nucleic acid or fragment thereof. In particular
embodiments, the kits comprise at least two or more nucleic acid
probes that hybridize with distinct biomarker nucleic acids and may
additionally comprise a microarray comprising nucleic acid encoding
the OA biomarkers.
[0077] It is contemplated that the invention described herein is
not limited to the particular methodology, protocols, and reagents
described as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention in any way.
[0078] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices and materials are now
described. All publications mentioned herein are incorporated by
reference for the purpose of describing and disclosing the
materials and methodologies that are reported in the publication
which might be used in connection with the invention.
[0079] In practicing the present invention, many conventional
techniques in molecular biology may be used. These techniques are
well known and are explained in, for example, Current Protocols in
Molecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel
ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
[0080] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
the "antibody" is a reference to one or more antibodies and
equivalents thereof known to those skilled in the art, and so
forth.
[0081] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Gene Expression in Cats with Osteoarthritis Compared to Control
Cats
[0082] Studies are conducted using non-arthritic cats and cats with
osteoarthritis (OA) to determine the underlying gene expression
differences between non-arthritic, cats and cats with OA. In one
study, a baseline comparison is performed between the two groups to
determine the underlying gene expression differences between
non-arthritic cats and cats with OA. In a second study, another
group of non-arthritic cats and cats with OA are used, however, in
addition to the baseline comparison between all normal animals and
all OA animals, a diet is tested over time for the ability to
dampen the progression of the disease. Quantitative real-time PCR
studies are also performed using the samples obtained from the
second study.
[0083] With regard to the studies provided herein, cats with OA are
graded according, to a previously published method, i.e., all
non-arthritic cats are "grade 0" indicating that the joint appears
to be normal, cats with OA have grades that are either 1 (small
enthesophytes or small osteophytes present) or 2 (more prominent
enthesophytes and osteophytes). Cats with severe OA (grade 3) are
not included in this study.
[0084] Whole blood is obtained from the cats in the studies
provided herein using PAXgene.TM. RNA tubes and total RNA is
isolated from whole blood samples using PAXgene.TM. RNA isolation
kit according to the methods detailed below.
PAXgene.TM. Blood RNA Isolation
[0085] PAXgene.TM. Blood RNA tubes and the PAXgene.TM. Blood RNA
Kit (Qiagen) are used together to isolate and purify intracellular
RNA from whole blood obtained from felines as provided below (see
also PAXgene.TM. Blood RNA Kit Handbook, PreAnalytix, June 2005).
Briefly, blood is collected using a Vacutainer.RTM. needle,
directly into the PAXgene.TM. Blood RNA tube and then subjected to
several rounds of centrifugation, wash and purification steps which
ultimately result in high-quality RNA. The RNA then undergoes a
quality control step and is then used in future quantitative
real-time PCR and/or microarray analyses using a custom
manufactured proprietary feline gene chip produced on the
Affymetrix platform.
Assay Preparations
[0086] Incubate PAXgene.TM. tubes (containing blood) for at minimum
of 2 hours at room temperature before beginning the assay. If the
tubes are frozen, and are not allowed to incubate for 2 hours prior
to freezing, they will need to sit at room temperature to thaw an
additional 2 hours. Invert each PAXgene.TM. tube 8-10 times before
the first centrifugation. If using Buffer BR4 (buffers are included
with the PAXgene.TM. Blood RNA Kit) for the first time, add 4
volumes of 96-100% ethanol to the concentrated buffer to obtain a
working solution. Preheat two heating blocks prior to beginning the
assay--65.degree. C. and 55.degree. C. Prepare the DNase I stock
solution (the RNase-Free DNase Set is included with the PAXgene.TM.
Blood RNA Kit). Dissolve the solid DNase I enzyme in 550 .mu.L of
RNase-free water provided with the kit. Be sure not to lose any
DNase I when removing the lid. Mix gently by inverting the tube. Do
not vortex or centrifuge. Make a mixture of DNase I enzyme and
Buffer RDD (kit component) (enough volume for the number of samples
being processed per batch). Each sample needs 70 .mu.L of Buffer
RDD and 10 .mu.L of DNase I (i.e. 20 samples would require a
cocktail of 1.4 mL Buffer RDD and 200 .mu.L DNase I). The cocktail
should be stored at 2-8.degree. C. until needed. The reconstituted
enzyme is good for up to 6 weeks at 2-8.degree. C.
Sample storage
[0087] PAXgene.TM. tubes (which contain blood) can be stored at
room temperature for up to 3 days before processing. According to
the product insert provided with the PAXgene.TM. Blood RNA tubes,
the cellular RNA profile is stable under these conditions for up to
3 days. This, however. may vary between species. PAXgene.TM. tubes
can also be stored at 4.degree. C. for up to 5 days. If long term
storage is required, PAXgene.TM. tubes can be stored at -20.degree.
C. or -70.degree. C. for up to 6 months. Tubes should be frozen in
a loose wire rack in an upright position. It is recommended to
freeze first at -20.degree. C. and then transfer to -70.degree. C.
if tubes will be stored at -70.degree. C. Upon removing the tubes
from the freezer they should be thawed at room temperature
(temperature not to exceed 22.degree. C.). Each tube is to be
inverted 10 times before proceeding with the assay.
RNA Isolation from Whole Blood
[0088] Centrifuge the PAXgene.TM. Blood RNA tubes at 4000 .times. g
for 10 minutes. Remove the supernatant by decanting and discard.
Blot excess supernatant remaining on rim of PAXgene.TM. tube. Add 4
mL of RNase-free water to the pellet and cap with a new Hemogard
closure. Resuspend the pellet by vortexing and then centrifuge at
4000 .times. g for 10 minutes. Remove the supernatant by decanting
and discard. Blot excess supernatant remaining on rim of
PAXgene.TM.. Add 360 .mu.L, of Buffer BR1 (kit component) to the
pellet and gently pipette until pellet is completely resuspended.
Transfer the sample to a sterile 1.5 mL microcentifuge tube and add
300 .mu.L Buffer BR2 (kit component) and 40 .mu.L Proteinase K (do
not mix Buffer BR2 and Proteinase K prior to adding to the sample).
Mix each tube thoroughly by vortexing and place into a thermomixer
preheated to 55.degree. C. Incubate/shake the tubes for 10 minutes
at 1400 rpm. Pipet the lysate into a QIAshredder spin column placed
into a 2 mL collection tube. Centrifuge at 14,000 rpm for 3
minutes. Transfer the supernatant of the flow-through fraction to a
sterile 1.5 mL microcentrifuge tube. Add 350 .mu.L of 96-100%
ethanol and gently mix by pipetting. Add 700 .mu.L of the sample to
the PAXgene.TM. spin column placed in a 2 mL collection tube and
centrifuge at 14,000 rpm for 1 minute. Transfer the PAXgene.TM.
spin column into a new 2 mL collection rube and discard the
flow-through and old collection tube. Add the remaining volume of
the sample to the PAXgene.TM. spin column. Centrifuge at 14,000 rpm
for 1 minute.
[0089] Discard the old collection tube and the flow-through from
the centrifugation of the spin column described immediately above.
Place the PAXgene.TM. spin column into a new 2 mL collection tube.
Add 350 .mu.L of Buffer BR3 (kit component) to the PAXgene.TM. spin
column and centrifuge at 14,000 rpm for 1 minute. Discard the
flow-through and collection tube. Place the column into a new 2 mL
collection tube and add 80 .mu.L of the DNase I/Buffer RDD cocktail
(see "Assay Preparations") directly to the column membrane and
incubate for 15 minutes at room temperature. Add another 350 .mu.L
Buffer BR3 to the PAXgene.TM. spin column. Centrifuge at 14,000 rpm
for 1 minute. Transfer the PAXgene.TM. spin column to a new 2 mL
collection tube and discard the old collection tube and
flow-through.
[0090] Add 500 .mu.L of Buffer BR4 (kit component) to the
PAXgene.TM. spin column. Centrifuge at 14,000 rpm for 1 minute.
Place the PAXgene.TM. spin column into a new 2 mL collection tube
and discard the old collection tube and flow-through. Add another
500 .mu.L Buffer BR4 to the PAXgene.TM. spin column. Centrifuge at
14,000 rpm for 3 minutes to dry the spin column membrane. Discard
the collection tube and flow-through and place the columns in
another 2 mL collection tube. Spin the samples again at 14,000 rpm
for an additional minute to further dry the column membrane.
Discard the flow-through and the collection tube. Transfer the
PAXgene.TM. spin column to a 1.5 mL elution tube. Add 40 .mu.L
Buffer BR5 (kit component) directly to the PAXgene.TM. spin column
membrane. Centrifuge at 14,000 rpm for 1 minute. Remove the
PAXgene.TM. spin column and pipette the eluate in the 1.5 mL tube
onto the same PAXgene.TM. spin column. Return the PAXgene.TM. spin
column to the same 1.5 mL elution tube and centrifuge at 14,000 rpm
for 1 minute. Incubate the final eluate at 65.degree. for 5 minutes
and immediately chill on ice. Store final RNA sample at -80.degree.
C. for future use.
Example 2
Gene Chip Analyses
[0091] A proprietary, custom made feline gene chip (Affymetrix) is
used to evaluate base line gene expression in cats with and without
OA (10 normals. 10 arthritic animals). As provided above, gene chip
analyses are performed using conventional methods and according to
the manufacturer's instructions in order to obtain a baseline
comparison between the two groups to determine the underlying, gene
expression differences between non-arthritic cats and cats with
OA.
[0092] The raw gene chip data is normalized using the Robust
Multiarray Average (RMA) normalization algorithm (Irizarry, et al,.
Biostatistics 2003 Vol 4, Page 249-264) and is then subjected to
statistical analysis using Support Vector Machine (SVM) algorithm
(Partek Genomic Suite, Version 6) to determine the gene expression
differences that can differentiate between arthritic and
non-arthritic animals. Genes identifying OA biomarkers are selected
based on p value cut off and fold change (FC) according to the
following: genes with p value <0.001 and displaying, a fold
change of either >2.0, >1.5 or >1.3; and genes with a p
value <0.01 and displaying a fold change of either >2,
>1.5 or >1.3. The lists of feline OA biomarkers thus
identified are provided herein in FIGS. 1-6.
[0093] The results from these studies indicate that gene expression
can be used to differentiate between normal cats and cats with OA.
The lists of differentially expressed genes include sequences that
act as cell surface markers, receptors and other signaling
molecules.
Example 3
Effect of Diet on Gene Expression in Feline Arthritis
[0094] In this study, quantitative real-time PCR assays are
performed using RNA isolated from normal and arthritic cats, hi
addition to a baseline comparison between arthritic and normal
animals, the effect of diet is also measured. Specifically,
following standard animal nutrition testing procedures familiar to
one of skill in the art, arthritic and normal cats are fed test
diets comprising, components reported to be of use to combat
inflammatory disease, including polyunsaturated fatty acids such as
omega-3 fatty acids, such as provided in WO 2007/002837 A2 ("j/d")
and WO 2006/074089 A2 ("senior") and then changes in gene
expression in the animals is analyzed using qRT-PCR. OA serum
markers are also assayed using conventional methods (ELISA).
[0095] With regard to q RT-PCR, Taqman probe technology is used and
all analyses are carried out using an Applied Biosystems 7500
real-time PCR machine. The data is analyzed using the sequence
detection software package version 1.2.2. provided by the
manufacturer.
[0096] A baseline comparison of normal and arthritic animals using
qRT-PCR detect OA biomarker genes associated with proteases and
cartilage degradation: Caspase 1, Caspase 3, MMP 2, MMP16,
Inhibitor of MMP1, Inhibitor of MMP2, Inhibitor of MMP3, Cysteine
protease, PUMP-1 and Progesterone-dependent protein and genes
associated with inflammation: IFN-gamma; TGF-beta; MIP-1 alpha;
IL-1 alpha; IL-1 beta; IL-2; IL-6 and IL-10. Data also indicate
that IL-1 and TGF-beta are significantly different between the
arthritic and nonarthritic animals (see FIGS. 8 and 9). IL-1 is
known to induce arthritic lesions in experimental animals. This
result is corroborated by the gene chip analysis (data not
shown).
[0097] Also, using conventional ELISA methods, blood levels of a
peptide of type I collagen (NTx, see FIG. 10) and Collagen II
(CTX-II, see FIG. 11) are measured in the animals and data indicate
that cats fed a diet containing high levels of EPA and DHA show a
marked reduction in circulating levels of these OA markers.
[0098] Clinical data obtained from nutritional studies involving
the arthritic and non-arthritic animals described above indicate
that dietary intervention can affect the expression of sonic OA
biomarkers. Specifically diets containing high levels of n-3 fatty
acids DHA (0.3%) or DI-IA and EPA (0.3% and 0.46% respectively),
can cause a decrease in the OA serum markers, collagen I (Nix) and
a fragment of collagen II (CTX-II) but do not have an effect on
levels of TGF-beta. With regard to TGF-beta, a lack of change which
would be desired as this protein may play a protective role in
arthritis. Furthermore, quantitative real-time PCR analysis reveals
that the expression of IL-1 is also dramatically decreased in
animals administered a diet containing DHA and EPA and this is
desired as this protein is a known arthrogenic molecule. Thus, it
is shown that feeding a diet rich in n-3 fatty acids EPA and DHA
(based on TG oil) can cause a reduction in expression of IL-1, NTx
and CTX-II.
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