U.S. patent application number 10/852439 was filed with the patent office on 2005-03-17 for control samples for use as standards for evaluating apoptosis in a selected tissue.
Invention is credited to Hewitt, Charles W..
Application Number | 20050059043 10/852439 |
Document ID | / |
Family ID | 25417411 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059043 |
Kind Code |
A1 |
Hewitt, Charles W. |
March 17, 2005 |
Control samples for use as standards for evaluating apoptosis in a
selected tissue
Abstract
A method for producing standard control samples to be used to
evaluate disease states or trauma that involve apoptosis or
suppression of apoptosis is disclosed. Also disclosed are standard
control samples produced by the method. The control samples
comprise natural or artificial tissues treated in vitro to display
reproducible, predetermined indicators of apoptosis that are
equivalent to indicators of apoptotic status of corresponding
tissues and organs of a living subject.
Inventors: |
Hewitt, Charles W.;
(Blackwood, NJ) |
Correspondence
Address: |
Jane Massey Licata
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
25417411 |
Appl. No.: |
10/852439 |
Filed: |
May 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10852439 |
May 24, 2004 |
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09903381 |
Jul 11, 2001 |
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6756194 |
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Current U.S.
Class: |
435/6.18 ;
435/6.1; 435/7.23 |
Current CPC
Class: |
G01N 1/30 20130101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 001/68; G01N
033/567; G01N 033/574 |
Claims
1-25. (canceled).
26. A composition for use as a standardized measure of apoptosis in
a test sample of tissue from a subject, the composition comprising
at least one segment of a naturally occurring tissue that has been
subjected to a treatment that reproducibly results in a
predetermined, measurable amount of apoptosis in the segment.
27. A kit for evaluating apoptosis in a test sample of tissue, the
kit comprising a container containing at least one segment of a
naturally occurring tissue that has been subjected to a treatment
that reproducibly results in a predetermined, measurable amount of
apoptosis in the segment, and instructions for use of the segment
in evaluating the apoptosis in the test sample of tissue.
28. A method for producing an apoptosis tissue standard comprising:
a) obtaining a desired tissue; b) subjecting the tissue to
microgravity treatment to induce a predetermined, measurable amount
of apoptosis; c) collecting the treated tissue for use as an
apoptosis tissue standard.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the fields of
medicine and pharmacological research. More specifically, the
invention provides standard control samples to be used to evaluate
disease states or trauma that involve apoptosis or suppression of
apoptosis. The control samples comprise natural or artificial
tissues treated in vitro to display reproducible, predetermined
indicators of apoptosis that are equivalent to indicators of
apoptotic status of corresponding tissues and organs of a living
subject.
BACKGROUND OF THE INVENTION
[0002] Several scientific or patent publications are referenced in
this patent application to describe the state of the art to which
the invention pertains. Each of these publications is incorporated
by reference herein, in its entirety.
[0003] A coordination and balance between cell proliferation and
cell death is critical for normal development and homeostasis of
tissues and organs. Abnormalities in either of these processes may
cause tissue atrophy or hypertrophy, either of which can lead to
cancer, autoimmune disease or degenerative disorders.
[0004] It is becoming increasingly apparent that the processes of
cellular proliferation and programmed cell death, i.e., apoptosis,
are linked by many common signaling mechanisms. Thus, up-regulation
of one process may be accompanied by down-regulation of the other,
leading to the ultimate expression of a disease state or
pathological condition. For instance, increased cell cycling due to
overexpression of the c-myc oncogene and reduced apoptosis due to
bcl-2 oncogene deregulation are two factors observed in the
development of lymphomas and mammary tumorigenesis.
[0005] A variety of disease states and other pathological
conditions are linked to dysregulated apoptosis in a particular
tissue or organ. These include, for example, general conditions
related to tissue rejection, immune/inflammatory responses,
ischemia and injury, cardiovascular diseases such as dilated or
ischeric cardiomyopathy, myocarditis and atherosclerosis,
neurodegenerative disorders such as ALS, Alzheimer's disease,
Parkinson's disease and retinal degeneration, hepatic and
pancreatic disorders related to viral infection or alcohol
consumption, which can lead to development of insulin-dependent
diabetes mellitus or infection with certain viruses, such as
adenoviruses, influenzaviruses and human immunodeficiency
virus.
[0006] Additionally, a variety of cell proliferative diseases and
disorders are linked to dysregulated apoptosis. These include, for
example, psoriasis, lupus and other autoimmune conditions such as
Crohn's disease, Hashimoto's thyroiditis and arthritis, infection
with certain viruses, such as human papillomavirus, Epstein-Barr
virus and herpes simplex virus; as well as a variety of cancers,
including mammary carcinomas, lymphomas cervical and ovarian
cancers, and neuroblastomas. As mentioned above, such proliferative
diseases may be marked by a decrease in apoptosis in the affected
tissue or organ, and may also be identified by up-regulation of
enzymes and signaling molecules involved in cell growth or cell
cycling.
[0007] Clearly, the apoptotic status of a particular tissue is
relevant to the diagnosis and prognosis of type and severity of a
wide variety of diseases or physiological conditions. Moreover, the
efficacy of a selected therapeutic regime for treating such disease
may be evaluated by assessing the apoptotic status of the tissue.
For this reason, morphological and biochemical markers of apoptosis
and mitosis are of great interest to investigators in their
attempts to devise clinically relevant diagnostic and prognostic
indicators of disease status in a patient.
[0008] In the development of clinically relevant models of disease
states, current technology utilizes cultured cell lines, or tissues
from subjects having a particular disease, or animal models to
evaluate a particular pathological condition and/or to develop
agents to treat the condition. Each of these methodologies has
certain limitations.
[0009] For instance, cultured cell lines are often utilized for
screening therapeutic agents and for studying the cellular
physiology associated with diseases involving tissues comprising
the cell type. The information gathered from the use of cultured
cell lines is of limited value, however, because the physiological
profile of cells in culture often does not accurately reflect the
physiological profile of corresponding primary cells contained in a
tissue or organ.
[0010] As another example, particularly utilized in the study of
tumors, it is common practice to store excised tumor tissue of a
particular type for use as comparison in diagnosis of other
patients or evaluation of the efficacy of a patient's therapeutic
regimen. One disadvantage of this practice is the lack of
appropriate standardized controls. The excised tumor sample
represents the end result of a disease progression. As such, it is
of little value in diagnosis of early or intermediate stages of the
disease. Furthermore, biopsied tissues can be extremely variable in
their disease presentation and expression of biochemical markers of
disease. Biopsied tissue also is often not readily available when
needed, and moreover can present a health hazard in that it may
contain infectious agents.
[0011] As another example, animal models are often used for
studying a disease and developing new agents and methods of
treatment. Whereas an animal model may offer biologically relevant
information, their use has many disadvantages. From a practical
standpoint, it is expensive and time-consuming to use animals in
research. Moreover, results may not be reproducible from one animal
study to another. Furthermore, there are numerous diseases and
pathological conditions for which no relevant animal model exists.
Finally, the use of animals in certain types of research (e.g.,
wound research) is increasingly being called into question for
ethical reasons.
[0012] Thus, a need exists for a clinically relevant standardized
control for use in evaluating the apoptotic status of a selected
tissue or organ. It would be of great advantage to be able to
reproducibly produce such standardized controls for any tissue or
organ in which it is desirable to evaluate apoptosis. It would be
of further advantage for such controls to be of high biological
relevance, i.e., to display many, if not all, of the morphological
and biochemical features of apoptosis as would be observed in a
corresponding tissue in a living subject. Finally, such controls
preferably should be capable of being standardized for incremental
changes in apoptosis.
SUMMARY OF TIE INVENTION
[0013] The present invention satisfies the need in the art for a
clinically relevant standardized control for use in evaluating the
apoptotic status of a selected tissue or organ. The invention
provides compositions that can serve as standardized controls for
any tissue or organ in which it is desirable to evaluate apoptosis.
The controls are of high biological relevance, in that they display
a variety of morphological and biochemical features of apoptosis,
and can display incremental changes in apoptosis.
[0014] Thus, one aspect of the invention features a composition for
use as a standardized measure of apoptosis in a test sample of
tissue from a subject. The composition comprises at least one
segment of an equivalent tissue that has been subjected to a
treatment that reproducibly results in a predetermined, measurable
amount of apoptosis in the segment. These tissue segments are
sometimes referred to herein as "apoptosis tissue standards". In a
highly preferred embodiment, they are produced by culturing the
tissue segment in a microgravity bioreactor for a period of time
known to produce the predetermined amount of apoptosis in the
tissue segment. Other means of producing the apoptosis tissue
standards are also provided in accordance with the invention, as
described in detail below.
[0015] In preferred embodiments of the invention, at least two
tissue segments are featured, wherein one of the segments is a
negative control segment which has not been subjected to the
apoptosis-inducing treatment and the other segment is a positive
control segment which has been subjected to a level of the
treatment that reproducibly results in a maximum amount of
apoptosis obtainable in the segment as a result of the treatment.
Other preferred embodiments provide intermediate control segments
which have been subjected to a level of the treatment that
reproducibly results in a predetermined amount of apoptosis
intermediate between that of the negative control segment and that
of the positive control segment.
[0016] Another aspect of the invention features a kit for
evaluating apoptosis in a test sample of tissue. At minimum, the
kit comprises the apoptosis tissue standard(s) described above,
along with instructions for their use in evaluating apoptosis in a
test sample of tissue. The kits may comprise the tissue segments
themselves, or extracts of the tissues containing soluble proteins
or RNA, or, optionally, culture media in which the tissue was
cultured during induction of apoptosis. The kits may further
comprise reagents and instructions for performing various assays
relating to measuring the amount of apoptosis present in the
tissues.
[0017] In other embodiments of the inventions, the kit comprises
apoptosis tissue standards that have been processed for one or more
assays or methods to evaluate apoptosis in a tissue. Such
processing may include processing for histology,
immunohistochemistry, or TUNEL staining, among others.
[0018] Other features and advantages of the present invention will
be understood by reference to the detailed description of the
invention and examples that follow.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention arises from the inventors' insightful
appreciation that tissue samples induced to apoptose under
reproducible conditions to display a predetermined, measurable
amount of apoptosis would fill a need in the art related to the
evaluation of apoptosis, or suppression thereof, in the clinical
and research setting. The steps for making the apoptosis standard
control tissues of the present invention are as follows:
[0020] 1. Select and obtain the desired tissue;
[0021] 2. Place the tissues in the desired form-for subsequent
treatment (e.g., prepare slices or segments of the tissue);
[0022] 3. Subject the tissue to a treatment that reproducibly
induces a predetermined level of apoptosis; and
[0023] 4. Collect the treated tissue samples for use; or
[0024] 5. Optionally, process the samples for storage or for a
specific assay or method to measure apoptosis.
[0025] As a result of the inventors' discovery that apoptosis can
be induced in any tissue type by one or more of several means,
apoptosis standards for any desired tissue can be produced in
accordance with the present invention. These include, butare not
limited to, tissues from skin, brain, heart, lung, liver, spleen,
pancreas, thymus, thyroid, lymph node, stomach, kidney, bladder,
intestine, colon, testis, mammary, ovary, uterus, muscle and bone,
to name a few. These further include "normal" tissues; i.e.,
tissues that are not diseased, or abnormal tissues such as tumors.
Indeed, apoptosis standard tumor tissues are expected to be of
utility in clinical determinations of the efficacy of a particular
anti-cancer therapy, or prognosis for a patient having such a
tumor.
[0026] In a preferred embodiment, the tissue is obtained directly
from a living or newly-dead organism. In another embodiment, the
tissue comprises a bioartificially constructed tissue demonstrated
to be equivalent to naturally occurring tissue of the same type.
For instance, a bioartificial living skin equivalent (LSE), as
described by Strande et al. (Transplantation Proceedings 29:
2118-2119, 1997), can be utilized instead of skin taken directly
from an organism.
[0027] The guideline for choosing a tissue type to use as the
standardized control of the invention, whether naturally occurring
or artificial, is that the tissue is equivalent to the type of
tissue for which it is to serve as a standard for comparison. For
instance, if the apoptotic status of liver tissue in a patient is
to be evaluated, the standard control tissue also should be liver
tissue. Moreover, it should be obtained from an organism of
biological relevance to the patient. That is, if the patient is a
human, mouse liver tissue, for example, can serve as an appropriate
standard because it has been demonstrated that mammalian livers all
function in essentially the same manner.
[0028] Once obtained, the tissue is prepared for treatment. Such
preparation can comprise cleaning the tissue, then slicing or
otherwise segmenting the tissue into appropriately sized fragments.
Other similar preliminary preparations of tissues will be well
understood to those of skill in the art.
[0029] Next, the tissue is subjected to the treatment that results
in reproducible production of a predetermined level of apoptosis,
ranging from none (negative control) to the maximum amount
obtainable for the tissue type (positive control). Apoptosis can be
induced in a variety of ways, many of which are controllable and
reproducible. These methods generally comprise applying a
biological stress to the tissue, which induces an apoptosis
response.
[0030] One very useful means of applying an apoptosis-inducing
biological stress is simply to culture a natural or artificial
tissue in one of several culture systems. In a particularly
preferred embodiment of the invention, apoptosis is induced by
incubating the tissue segments in a microgravity culture system,
such as that provided by the rotating cylindrical cell culture
vessel (RCCS bioreactor) developed for the NASA space program, and
commercially available from Synthecon, Inc. (Houston Tex). The RCCS
bioreactor is described in detail in U.S. Pat. Nos. 5,763,279 and
5,437,998 to Schwartz et al. The rotating culture vessel system
creates the environment of simulated microgravity. By rotating the
culture system at a constant and appropriate speed, tissue
fragments are maintained in constant suspension. This achieves a
state of continuous "free-fall" so that the tissue is exposed to
random gravity vectors, thereby reducing the effect of gravity to
approximately 0.1 G and relieving the cells or tissues from the
deforming force of gravity. The rotating culture system also
increases contact between the tissue and the media and reduces
shear forces on the cells.
[0031] A surprising discovery made in accordance with the present
invention is that, even though the RCCS bioreactor can promote
growth of cultured cells and tissues, primary tissues of all types
are induced to undergo apoptosis simply by incubating the tissue in
the bioreactor for a period of time ranging from two to 96 hours,
in most cases (though the range can be shorter or longer in some
cases). Because standard incubation times that reproducibly lead to
a predetermined amount of apoptosis in a given tissue can be
readily determined with little effort, this method of apoptosis
induction is very convenient and highly preferred for use in making
the apoptotic tissue standards of the invention. The induction of
apoptosis in thymic tissue utilizing incubation in the RCCS
bioreactor is described in detail in Example 1. In this example,
the maximum level of apoptosis, as measured by TUNEL, was achieved
in 24 hours without the addition of dexamethasone, and in 10 hours
in the presence of dexamethasone.
[0032] The RCCS bioreactor is suitable for use with natural or
artificial tissues. Indeed, it has been shown that the
bioartificial LSE (Strande et al., 1997 supra) grows as well, if
not better, in the RCCS bioreactor (Doolin et al., Tissue
Engineering 5: 573-581, 1999) than in static culture systems. For
artificial tissues, such as the LSE, that do not naturally undergo
apoptosis upon incubation in the RCCS bioreactor, apoptosis is
induced by a different means (such as heat treatment as described
below), and the bioreactor is used to incubate the tissues for a
time sufficient to achieve the desired level of apoptosis resulting
from the stress treatment.
[0033] In further embodiments, other bioreactor systems may be
used, including, for example, standard impeller-type bioreactors,
roller bottles, shaker baths, or hollow-fiber bioreactors. Such
bioreactors are known to induce apoptosis in cell cultures; in
fact, their use for mass production of cells has been limited by
this problem. It should be noted that the variable induction of
apoptosis that can occur in the standard bioreactor can be a
disadvantage in the production of the apoptotic tissue standards of
the present invention. The microgravity system allows for low
shear-stress and high nutrient exchange, as tissues are cultured in
small organoid fragments and consistent conditions to induce
apoptosis. These parameters provide consistency and control in the
microgravity system, as described above.
[0034] In yet another embodiment, a static culture system can be
utilized. Static culture systems, i.e., those that do not employ
agitation of the media by impeller or rotation, can biologically
stress a tissue as a result of the effects of gravity or low
oxygenation. If a static culture is used to induce apoptosis, this
is enhanced by the application of additional stress conditions,
including physical or chemical stresses such as heat, mechanical
injury or other physical stresses, or induction by toxic
chemicals.
[0035] Many biological stresses are also known to induce apoptosis.
These include, but are not limited to, temperature stress (heat or
cold), UV irradiation, hypoxia, free-radical damage, serum
deprivation and exposure to certain chemicals or biological agents,
such as dexamethasone, TNFa (NB cells), hyperosmolality, Granzyme
B, FAS ligand, FAS (CD95), perforin, and Trail/DR4, to name a few.
Any of these biological stresses can be used, alone or in
combination with each other or with a selected culture regime as
described above.
[0036] In a preferred embodiment, heat is used to induce apoptosis
in the tissue segments. As one example, using natural skin or the
living skin equivalent (LSE) and a static culture system, apoptosis
can be reproducibly induced by scalding the tissue. For instance,
it has been demonstrated that scalding of natural skin or the LSE
induces apoptosis, measurable as DNA damage by TUNEL assays (Doolin
et al., J. Burn Care Rehabil. 20: 374-376, 1999) and by increases
in apoptosis-related molecules, such as FasL and Fas (Hewitt et
al., Burn Care & Rehabil. S169; Abstr. 66, 1999). In this
embodiment, the tissues are subjected to the heat stress (e.g.,
treatment with hot PBS, up to 70.degree. C.), then incubated in
static culture or in a bioreactor for a selected period of time and
under conditions known to produce a desired level of apoptosis. The
amount of apoptosis in the tissue is controlled by the temperature
and duration of the heat treatment and the duration of incubation
in static culture or in the bioreactor.
[0037] In another preferred embodiment, a chemical treatment is
used alone or in combination with another biological stress to
induce apoptosis in the tissue. One example of this is set forth in
Example 1, wherein 1 .mu.M dexamethasone in the culture medium
increases the rate of apoptosis of thymic tissue incubated in a
RCCS bioreactor. Dexamethasone can be added to culture media in a
concentration range of 0.01-100 .mu.M, preferably 1-50 .mu.M. Most
preferably, however, the concentration is 1 .mu.M (it is understood
that 0.01-0.05 .mu.M dexamethasone is a physiological range,
0.1-1.0 .mu.M is a pharmacological dose range, and a high dose
range is 25-100 .mu.M).
[0038] It also is advantageous in the study and evaluation of
apoptotic status in tumors to treat the control tissue samples with
a mitogen or regulator of cell growth or de-differentiation, to
stimulate cell proliferation. In this manner, apoptosis in a cell
proliferative environment is can be mimicked, and appropriate
standard control tissues generated for use in evaluating various
forms of cancer. Mitogens or stimulants of cell growth or
de-differentiation suitable for use in this embodiment of the
invention include, but are not limited to, concanavalin A,
phytohemaglutinin, endotoxins, LPS, various antibodies, various
lectins, growth factors (such as PDGF, BFGF, ECGS), II-2,
.gamma.-IFN, and many biologically or functionally similar
molecules, as would be appreciated by one of skill in the art.
Tissue can be pre-treated with the mitogen, or the mitogen can be
introduced directly into the culture medium. The amount of such
mitogenic or cell growth-promoting agents to be used depends on the
specific compound, and is easily determinable by persons of skill
in the art.
[0039] After the desired amount of apoptosis in a particular tissue
is achieved, the tissue segments are collected and prepared for use
as standard controls in the evaluation of apoptosis in test samples
of tissues in the clinic or research laboratory. Such preparation
may comprise simply rinsing and placing the segments in an
appropriate medium, then using the segments immediately, or
refrigerating them for later use. This sort of preparation enables
the investigator to process the control tissue segments right along
with test tissue segments for apoptosis assays in the clinical or
research laboratory.
[0040] In other embodiments of the invention, the apoptotic tissue
segments are prepared for one of several assays for measuring
apoptosis. For instance, the tissue segments may be fixed and
sectioned for histological examination, using light- or
electron-microscopy. It is well known,.that apoptotic cells display
distinctive morphological features, which are characteristic of the
stages of apoptosis ranging from early events to complete cell
death.
[0041] As another example, the apoptotic tissue segments may be
fixed and processed for assays that measure DNA damage, another
hallmark of apoptosis. Preferred for the present invention is the
processing of the tissue segments for TUNEL assays, as is well
known in the art and described in Example 1.
[0042] As another example, the apoptotic tissue segments are
processed for in situ hybridization or immunohistochemical
evaluation as is well known in the art. This embodiment is designed
for detecting mRNA and/or protein markers of apoptosis; i.e.,
molecules known to be increased or decreased in apoptotic cells.
Such markers are well known in the art. For instance, markers known
to be increased in apoptotic tissues include, but are not limited
to: caspases, annexin, DNAse I, DNAse II, NUC 18/cyclophilin,
transglutaminase, Fas, FasL, p53, Diva, Bak, Bcl-X.sub.s, Bik, Bim,
Bad, Bid, Egl-1, and Bax, to name a few. Markers known to be
decreased in apoptotic tissues include, but are not limited to,
Bcl2, Bcl-X.sub.L, Mcl-1 and CED-9.
[0043] As another example, extracts of the tissue segments having
various predetermined levels of apoptosis may be prepared for use
as controls for Western blots. Method of preparing tissue extracts
are well known in the art. Along these same lines, many of the
protein markers enumerated above are secreted into the culture
medium as the tissue segments undergo apoptosis in a bioreactor or
static medium. Accordingly, the culture medium also can be
concentrated and used as a control for Western blots in the
evaluation of apoptosis in a selected tissue.
[0044] The person of skill in the art will be able to appreciate
many uses for the apoptosis tissue standards of the present
invention. These include uses in basic research, as well as in the
clinic.
[0045] The apoptosis tissue standards of the invention have
clinical utility in diagnosis, prognosis, and evaluation of
therapeutic treatments of diseases in which apoptosis, or
suppression of apoptosis, is a measurable condition associated with
the disease state. One nonlimiting example is the evaluation of
mammary tumors, wherein research has already demonstrated that
dysregulation of normal programmed cell death mechanisms plays an
important role in the pathogenesis and progression of breast cancer
(Krajewski et al., Endocrine-Related Cancer 6: 29-40, 1999). In
accordance with the present invention, a set of apoptosis tissue
standards made from normal breast tissue and from breast tumor
tissue can be used to comparatively assess a patient's biopsied
breast tissue for apoptosis. Further, one or more of those same
standards may be used to evaluate regression of the patient's
breast tumors as a result of chemotherapy or radiotherapy, by
comparison.
[0046] As another nonlimiting example, control standards can be
utilized to ensure that any assays or tests being utilized in the
clinic or research laboratory are performing properly and
appropriately on test specimens. Gradations of positive standards
can be created and used along with negative controls. The
sensitivity and specificity of the test or assay can be evaluated
with these standards.
[0047] The use of standardized tissue samples for clinical
evaluation of diseases in which apoptosis is involved has not been
used, nor was it available, prior to the present invention. These
standardized controls thus provide the clinician with a level of
accuracy and sensitivity (to intermediate gradations of disease)
that represent an advance in the art of clinical diagnosis.
[0048] The apoptosis tissue standards are also of utility to basic
and applied research, wherein the same advances in accuracy and
sensitivity are achieved through the use of a standardized set of
tissue samples displaying various predetermined levels of
apoptosis. For instance, they can be used as positive, negative and
intermediate controls in experiments on animals in which a
particular therapeutic agent is under evaluation.
[0049] As another example, the methodology used to create the
apoptosis tissue standards can itself be used as a screening tool
for candidate drugs to treat a selected disease state. As an
example using the RCCS bioreactor, selected tissue segments are
incubated in the bioreactor under conditions known to produce a
specific amount of apoptosis. A candidate drug is added to the
culture medium, and its effect on the rate of apoptosis is
measured.
[0050] Also provided in accordance with the present invention are
kits to facilitate use of the apoptosis tissue standards of the
invention. These may take on many forms, an may include a wide
array of different reagents.
[0051] A simple kit comprises minimally processed apoptosis tissue
standards. For instance, a simple kit may comprise an untreated
segment of a selected tissue as a negative control, and a maximally
treated tissue segment as a positive control displaying maximum
apoptosis. The clinician or investigator utilizes the negative and
positive control tissues by processing them along with test tissue
samples from the clinic or laboratory, e.g., for histology,
immunohistochemistry, TUNEL, or some other evaluative measurement
of apoptosis. In a preferred embodiment of this type of kit, tissue
samples displaying amounts of apoptosis intermediate between those
displayed by the negative and positive controls, respectively, are
provided. This graded series of apoptotic tissue samples is used to
generate a standard curve for accurately estimating the amount of
apoptosis present in the test tissue.
[0052] The aforementioned kit may be supplemented with instructions
that describe different assays for measuring apoptosis, and
additionally with reagents for performing such assays. For example,
a set of apoptosis tissue standards may be supplied in a kit that
also supplies reagents and instructions for performing TUNEL
assays. Alternatively, the kit may comprise antibodies and reagents
for performing immunohistochemistry. Other biological molecules and
reagents that can be supplied in such kits will be apparent to one
skilled in the art, in accordance with customary usage of such kits
for the preparation of cells and tissues for these and similar
assays.
[0053] Another type of kit comprises positive and negative control
tissue samples, along with samples of intermediate apoptosis in a
preferred embodiment, already processed for a specific type of
assay or evaluation. For example, a kit may contain a series of
microscope slides containing fixed sections of variously-apoptotic
tissues to serve as standards of comparison for histological
evaluation of a test tissue. The sections may be further processed
by immunohistochemical staining for a selected marker of apoptosis
or suppression of apoptosis. These may include any of the markers
of apoptosis mentioned previously, or any signal transduction
protein involved in the apoptosis pathway. Alternatively, tissue
sections already subjected to TUNEL staining can be provided in a
kit. A variety of other kits containing the apoptosis tissue
standards in a fixed or pre-processed form will be apparent to
persons of skill in the art.
[0054] The following examples are provided to describe the
invention in greater detail. They are intended to illustrate, not
to limit, the invention.
EXAMPLE 1
Thymic Apoptosis in Microgravity Culture
[0055] Materials and Methods:
[0056] The following media and solutions were prepared:
[0057] 1. Phosphate buffered saline (PBS) for rinsing tissue.
[0058] 2. Control media, consisting of RPMi 1640 containing 15%
heat-inactivated fetal bovine serum, 100 .mu.g/mL penicillin, 100
.mu.g/mL streptomycin, 100 .mu.g/mL L-glutamine, and 2.5 .mu.g/mL
amphotericin B.
[0059] 3. Experimental media, consisting of control media
containing 1 .mu.M dexamethasone.
[0060] Two microgravity bioreactors (Synthecon, Inc., Houston Tex.)
were utilized; one containing control media and one containing
experimental media.
[0061] The thymus was removed from two Lewis rats, washed in cold
PBS to remove blood, and stripped of excess tissue. Thymus tissue
was cut into sections of about 3 mm.sup.3, while in cold media.
[0062] The bioreactors were filled with control (CON) or
experimental (DEX) media. Approximately 24 thymus pieces were
placed in each bioreactor, for incubation at 37.degree. C. at a
rotation speed appropriate for the size of tissue piece and
sedimentation rate, in order to provide constant free-fall. Tissue
pieces were removed from each bioreactor every half hour for the
first 3 hours, then every hour to the sixth hour, then at hour 10
and hour 24.
[0063] Tissue was fixed in 10% neutral buffered formalin or
Histochoice.TM. (Amresco, Inc., Solon Ohio), processed to paraffin
and sectioned for histochemical analysis. Formalin-fixed sections
were stained with hematoxylin and eosin. Immunohistochemical
staining for Lewis Y expression was performed with Histochoice TM
fixed samples on a DAKO autostainer, using a monoclonal mouse
anti-human Lewis Y antibody (DAKO, Carpinteria Calif.). A
peroxidase labeled streptavidin-biotin detection system (Zymed, So.
San Francisco Calif.) and diaminobenzidine (DAB) chromagen were
used.
[0064] In situ labeling of apoptosis was measured by terminal
deoxynucleotidyl transferase mediated dUTP nick end labeling
(TUNEL) method with a cell death detection kit (Boehringer Mannheim
Gmbh, Mannheim, Germany). Using digital image analysis,
immunostained sections were analyzed for intensity stain index
(ISI=[.SIGMA.P.sub.0.times.I.sub.- 0]/total tissue area in pixels),
where P0=number of pixels at each intensity and 10=the intensity
units (Doolin et al., J. Surg. Res. 59: 191-197, 1995). TUNEL was
quantified as total apoptotic cell number divided by total tissue
area.
[0065] Results:
[0066] Results are show in Table 1.
1TABLE 1 Percent Area of Stain Time (Hours) CON DEX Ratio DEX/CON 0
6.04 6.04 1.00 3 14.86 21.68 1.46 6 21.08 33.29 1.58 10 31.92 43.82
1.37 24 49.83 43.30 0.87
[0067] TUNEL assays showed that apoptosis was observable at three
hours following initiation of the cultures, and increased in a
linear fashion to 10 hours in the dexamethasone culture and to 24
hours in the control culture. Further, at six hours of incubation,
apoptosis was demonstrated in free thymocytes infiltrating the
media. In the tissue fragments, the inclusion of dexamethasone in
the culture media increased the rate of apoptosis up to 10 hours
post-initiation, where an apparent maximum was reached. In the
control culture (lacking dexamethasone) the rate of apoptosis was
comparatively less, but the maximum apoptosis finally reached at 24
hours post-initiation was greater than that observed for the
dexamethasone culture. Nonetheless, a time frame was observable for
both cultures in which the increase in percentage of apoptotic
cells was essentially linear.
EXAMPLE 2
Apoptosis of Several Tissue Types in Microgravity Culture
[0068] The experiment described in Example 1 was repeated with each
of the following tissue types: heart, kidney, liver, spleen, lymph
node and skin. Results paralleled those observed and set forth
above for thymic tissue.
EXAMPLE 3
Detection of Apoptosis Markers in Microgravity Cultured Tissues
[0069] The experiments described in Examples 1 and 2 were repeated
with one or more of the following tissue types: thymus, heart,
kidney, liver, spleen, lymph node and skin. Markers of apoptosis
were detected immunohistochemically in the treated tissue, and/or
by Western blot in culture fluid. Specific apoptosis marker
proteins assayed for included annexin, one or more caspases, and
Fas/FasL.
[0070] Results showed that markers of apoptosis increased in a
graded fashion, similar to the results observed from TUNEL assays
of the tissues. In addition, the marker proteins accumulated in the
culture fluid in a similar graded fashion over time.
[0071] This invention is not limited to the embodiments described
and exemplified above, but is capable of variation and modification
within the scope of the appended claims.
* * * * *