Role of Immune Recognition of Stem Cells in the Aging Process

Kurt Whittemore

Stem cells are among the many different types of cells in the body which can become
cancerous through processes of mutation, chromosomal instability, and changes in gene
regulation. One of the requirements that must be met in order for a cell to become cancerous
is that it must be able to overcome the Hayflick limit presented by the end replication problem
of eukaryotic cells. In order to overcome this limit, a cell must either express telomerase or
develop an ALT (alternative lengthening of telomeres) mechanism. Since stem cells naturally
produce the enzyme telomerase, they already meet one of the requirements for becoming
cancerous and are just a few mutations away from becoming malignant. Over the course of a
lifetime, the immune system of an individual may have suppressed such rogue stem cells
many times. If the immune system incorrectly associates normal proliferating stem cells with
these cancerous stem cells, the ability of the organism to repair itself will degrade which may
eventually lead to death. With this research, we aim to test the unconventional hypothesis
that the aging process may be a type of acquired autoimmunity against stem cells.

Table of Contents

Research Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Specific Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Research Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Innovation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Bibliography & References Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Budget . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Biographical Sketch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Facilities & Other Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Research Plan

Significance
Aging is a significant problem which decreases quality of life and increases healthcare costs
for society. Advances in biomedical technology are allowing researchers to probe the causes
of aging better than ever before, and we may soon be able to use this knowledge to increase
the number of healthy years in a person's life. As we learn more about aging, scientists are
discovering that there is an intimate relationship between cancer development and the aging
process. This research will test a specific hypothesis about one possible relationship
between cancer and aging. Since stem cells normally involved in tissue repair also become
cancerous more frequently than other somatic cells, we propose that the aged immune
system may fail to distinguish between cancerous stem cells and normal stem cells needed
for regeneration. We propose that part of the aging process is due to an acquired
autoimmune disease against stem cells. If this hypothesis is true, steps may be taken to
mitigate the effects of a misguided immune system and extend lifespan.

Innovation
In order to test the hypothesis that the aged immune system responds to stem cells and that
this response affects lifespan, several well established and innovative techniques will be
applied. We will apply the well established techniques of ELISA and ELISPOT to measure the
immune response of mice and humans against specific selected stem cell antigens. Another
less established technique using peptide microarrays will be used to detect possible
variations in the immune responses between young and old mice and humans. The stem cell
marker which elicits the greatest immune response will then be used to modulate the immune
systems of mice. One group of mice will be vaccinated with the stem cell antigen, and
tolerance for the antigen will be induced in another group of mice. Two types of tolerance
induction will be attempted: oral tolerance, and a more novel genetically engineered dendritic
cell tolerance approach.

Specific Aims
Aim 1: Perform assays to measure the mouse and human immune responses against
several stem cell markers. ELISA will be used to measure the antibody titer against 10
stem cell markers. ELISPOT will be used to measure the T cell response against the same
10 stem cell markers.
Aim 2: Use a peptide microarray to observe differences in the antibodies produced by
old and young mice and humans. Mouse and human antibody serum will be applied to a
peptide microarray and differential binding events will be detected to determine if there is a
consistent pattern which can distinguish between young and old serum.
Aim 3: Modulate immune response for a specific stem cell marker in mice and observe
the effect on lifespan. Tolerance for a stem cell antigen will be induced in one group of
mice, and another group of mice will be vaccinated with a stem cell antigen.

Using these techniques, we will be able to answer the question about whether the immune
system responds to normal stem cells in old age. We will also be able to determine if this
response affects lifespan.

2
Research Strategy

Significance

The increase of diseases that occur with age places a great burden on aging
individuals as well as the society that must support them. Citizens in the U.S. over 65 spend
three to five times more on healthcare than younger Americans [1], and a vast infrastructure is
required to meet their needs. With the arrival of biotechnology, scientists have demonstrated
that single mutations and certain drugs can modulate the lifespan and healthy years of
animals. These new findings suggest that with greater understanding, we may be able to
apply these techniques as well as yet undiscovered techniques to humans to greatly enhance
quality of life and reduce healthcare costs for society.
There are currently many theories about why organisms age. Many of these theories
can be categorized into two groups: stochastic theories of aging and programmed theories of
aging. Stochastic theories of aging claim that aging is simply due to the accumulation of
damage over time. These theories are very intuitive since many of the real world objects we
deal with on a daily bases degrade and break down over time. Unlike inanimate objects such
as cars and bikes, however, living things possess the capability of repairing themselves.
Unfortunately, this repairing capability seems to diminish with age. Programmed theories of
aging claim that the aging process is somewhat hard-coded in our genes.
Several stochastic theories of aging have been proposed. In fact, one of the first
theories of aging was proposed by Raymond Pearl in 1928. He proposed that the aging
process was determined by the rate at which organisms lived since he noticed that organisms
with fast basal metabolic rates often had shorter lifespans than other animals [2]. Another
theory is the mutation accumulation theory proposed by Peter Medawar [3]. The theory
proposes that aging is caused by the DNA damage which accumulates with age due to
oxidation, radiation, and other types of environmental stressors. In addition to damage of the
DNA in the nucleus, mitochondrial DNA has demonstrated an accumulation of damage with
age [4]. This theory supports the “rate of living” theory in some ways since organisms with
fast metabolic rates may have more chances to accumulate oxidative damage. Perhaps the
most generalizable theory of aging is the reliability theory which predicts that even systems
composed of non-aging elements will deteriorate and break down if those elements are non-
replaceable and have a constant failure rate [5].
There are also many programmed theories of aging. The disposable soma theory
proposes that aging is the result of the genes of the organism investing resources in
reproduction rather than the maintenance of the body [6]. This theory seems quite
reasonable when one considers that natural selection will not exhibit any pressure on a
species to evolve to maintain health after the period during which individuals in that species
would normally already have reproduced and died from factors in their environment.
Therefore, evolution has influenced the genetic program of the organism so that it will only
maintain the organism up to a certain age. The antagonistic pleiotropy theory proposed by
Geoge C. Williams suggests that certain genes which increase fitness early in life may have
negative effects later on [7]. For example, calcium deposition may be good for juvenile
growth, but harden the arteries later on. Gene regulation theories of aging trace the aging
process back to specific genes or differences of aging in different species. For example,
scientists have demonstrated that reduction of the activity of the methuselah gene extends

3
the lifespan of drosophilia [8]. The existence of semelparous organsims such as salmon and
bamboo which die immediately after reproducing without any gradual decline in age also lend
strong support to gene regulation theories of aging [9]. Aging diseases such as Werner
syndrome and progeria result from genetic mutations often involved in DNA repair, and these
diseases provide further evidence for the roles of an individual's genetic make-up in the aging
process [10].
One programmed theory of aging which has received significant attention in recent
years involves cellular senescence. The study of cellular senescence has also demonstrated
that there is an intimate connection between cancer and the aging process. Eukaryotic cells
reach the Hayflick limit and become senescent after they divide a certain number of times.
The number of times a cell can divide is determined by the length of segments of DNA at the
ends of every chromosome. These segments of DNA are known as telomeres and they
consist of a repeating DNA sequence [11]. These telomeres shorten with each cell division.
Once the shortest telomere present in all of the chromosomes in a cell reaches a critically
short length, the cell will no longer divide or become cancerous [12]. A cancerous cell can
lengthen the telomeres by expressing an enzyme known as telomerase which is also
expressed in stem cells at low levels and germ line cells. Alternatively, the cell may develop
an ALT (alternative lengthening of telomeres) mechanism. However, more than 85% of
cancers overexpress the telomerase enzyme [13]. The telomerase enzyme not only
lengthens telomeres, but appears to be involved in the regulation of other genes involved in
cellular proliferation [14], which is another feature of cancer.
Many interesting findings have been found which link telomeres and telomerase to
cancer and aging processes. Long telomeres have been associated with good health and a
long healthspan in humans [15][16]. Long telomeres cannot account for the entire aging
process, however, since mice still age but appear to have much longer telomeres than
necessary for their lifespan. Telomerase knockout mice do not exhibit any noticeable aging
symptoms until the 4th or 6th generation [17][18]. On the other hand, telomerase
overexpressing mice develop more cancers on average during their first year of life, but live
longer than normal if they survive past the first year [19]. Supposedly this increase in lifespan
is due to telomerase activities not involved in lengethening telomeres. Interestingly, stem
cells which repair damaged tissue in the body actually already express the telomerase
enzyme so it would seem that senescence would not pose a problem for the aging body.
However, telomerase expression is not sufficient to maintain telomere length in many normal
stem cells [20]. Researchers have also discovered that the majority of cancers develop from
stem cells [21]. This result is somewhat intuitive since stem cells already meet one of the
requirements of tumor development: telomerase expression or an alternative method for
lengthening telomeres. All of these fascinating results, imply that there is an intimate
connection between cancer and aging, but they do not provide a clear means for addressing
either problem directly.
Regardless of which theory of aging is the most accurate, theoretically a system can be
maintained as long as one keeps investing energy into it. According to the reliability theory of
aging, the aging process could be averted if the damaged parts are simply replaced,
regardless of the cause of the damage. Stem cell therapies provide a means for replacing
damaged cells and tissues. However, stem cell therapies will not be possible if the aged
immune system attacks stem cells, which this research proposal intends to address. Why
would the immune system attack stem cells, and is there any evidence to support such
wayward behavior? One counterintuitive result that has come from stem cell research is that

4
stem cells from an aged organism are unable to repair damage in the aged organism
environment, but these same “old” stem cells are able to repair damage in a young
environment [22][23][24]. A number of explanations could account for this result. Perhaps the
aged environment is damaged beyond repair or perhaps epigenetic shifts in gene expression
may hinder proper cell to stem cell signaling. In reality, probably both the stem cells and the
environment become damaged with age. There may be another way that the old environment
becomes unsuitable for stem cells though. The immune system may have learned to
recognize and attack these stem cells.
Over time the immune system may incorrectly associate stem cells with harmful
behavior since the vast majority of cancers arise from stem cells. They only require a few
mutations before they cross a threshold and become cancerous. As stem cells become older,
their chances of experiencing such mutations due to radiation, oxidative, or other damage
increases. At this point the immune system may attack these cancerous stem cells as well as
stem cells involved in repair (Figure 1). Although the immune system's strength decreases
with age, the immune system also becomes less specific and autoimmune diseases are
known to increase [25]. Another recent finding which supports this stem cell autoimmune
theory is that the immunosuppresion drug rapamycin increases the lifespan of mice
maintained in pathogen free environments [26].

Figure 1: Contributions of Autoimmune Reactions to the Aging Process. In an old

mouse, we hypothesize that the immune system fails to discern between normal and
cancerous stem cells and attacks both cell types.

The implications of an acquired autoimmune stem cell disease hypothesis are great. If
it is true that part of the aging process is due to an acquired autoimmunity against the stem
cells normally involved in repair, then researchers may be able to develop methods to correct
the misguided immune system and slow the aging process. Longer life may be obtained by
inducing tolerance for stem cells using current techniques or developing new methods. One
possible life extension strategy could involve using stem cell treatments to repair damaged
tissues in old individuals after their immune systems have been reset to tolerate such stem
cells. Although some researchers have already demonstrated that old individuals can gain
some benefit from stem cell treatments without such modulations of the immune system, the
current stem cell treatments may not be optimal. Testing the hypothesis proposed here could
provide great insight into the mechanisms of the aging process.

5
Innovation

In order to test the hypothesis that the immune system begins to attack stem cells in
old age, three basic strategies will be used. The first strategy will be to measure the immune
response against several specifically selected stem cell antigens. There are hundreds of
stem cell antigens that have been identified, but testing all of these is not practical for our
research group. We will choose 10 different stem cell antigens from stem cell types which are
the progenitors of a variety of different kinds of tissues. One of the stem cell markers we
choose will be telomerase since this is one of the most well known antigens that is highly
expressed in cancers and is also present in normal stem cells at lower levels. Well
established protocols such as ELISA and ELISPOT will then be used to measure B and T cell
responses to these antigens. Both human and Balb/c mouse samples will be used.
Even if the immune system truly does respond to stem cells, we may not detect this
behavior from our ten selected antigens. Therefore a more general assay of the immune
system will also be employed. DNA microarrays have allowed researchers to measure the
expression of many different genes simultaneously by hybridizing cDNA or cRNA from a cell
with many different DNA transcripts laid out on a chip. Similar methods have been utilized to
create protein microarrays. These protein microarrays can be used to determine which
proteins the antibodies found in serum will bind to. Although protein microarrays are useful,
information can also be acquired from microarrays displaying peptides with as few as 20
amino acids as demonstrated by Stephen Johnston's lab which I have worked in [27]. Other
labs have also begun using protein and peptide arrays [28][29][30][31]. Antibodies can bind to
peptides which are similar to the original epitope they were produced against. We will take
advantage of this technology to determine if it is possible to obtain a consistent signature on
these arrays which will allow us to distinguish between young and old human serum. If we
can acquire such distinguishing signatures, this result will suggest that the immune system
does shift to recognize certain antigens in a repeatable way with age. If we cannot acquire
such a distinguishing signature, the result will suggest that each individuals' immune system
may deteriorate with age in a different way that is unique to that individual's environment and
genetic makeup.
The last technique we will use will be the modulation of the immune systems of mice.
The most immunogenic stem cell antigen will be used to both vaccinate some mice, and
induce tolerance in others. Tolerance will be induced using oral tolerance methods in one
group of mice, and we will also attempt to induce tolerance using genetically engineered
dendritic cells in another group of mice. The effect of these actions on lifespan will then be
measured. We expect that vaccination will shorten lifespan, and tolerance will lengthen
lifespan. If we cannot detect an immune response to any of our stem cell markers, then the
telomerase protein will still be used for these vaccinaion and tolerance experiments.
Researchers have actually vaccinated mice with telomerase before, and they determined that
the mice were able to fight tumors more effectively [32][13][33]. However, measuring
lifespan was not the focus of these studies, and no researchers have attempted to induce
tolerance for telomerase in mice. Therefore, the results of these new experiments will
provide further insight into cancer and the aging process.
Inducing tolerance with genetically engineered dendritic cells will be one of the more
novel methods used in this research. This concept has been demonstrated by several labs
[34][35][36][37]. Dendritic cells are collected from the mouse and re-engineered to code for
FasL (Fas ligand), and the antigen of interest is attached to MHC II (Major Histocompatiblity

6
Complex II). The FasL signals apoptosis in other cells which it comes into contact with. The
antigen of interest attached to MHC II will target the dendritic cell to T cells specific for the
antigen of interest. We will use our most immunogenic stem cell marker, or telomerase if
none of our stem cell markers are detectably immunogenic, to induce tolerance in a mouse
model.
Several other techniques could be used to address this hypothesis. For example, we
will try to induce tolerance orally and with genetically engineered dendritic cells. Instead of
inducing tolerance though, the immune system could also be completely replaced using a
bone marrow transplant procedure. The immune system could be irradiated, and then fresh
new B and T cells could be transferred into the mouse. Alternatively, a process of apheresis
could be used to selectively remove the old B and T cells, and the new cells could then be
transferred into the mouse. Yet another way to test whether the immune system attacks stem
cells would be to transfer young stem cells into an old mouse with SCID (Severe Combined
Immunodeficiency) and a wild type mouse, and then we could determine the effects of this
transfer on lifespan. If our hypothesis is correct, the SCID mouse would receive a much
greater benefit from the transferred stem cells than the wild type mouse. These types of
experiments could provide some interesting results, but we will test our innovative hypothesis
initially using more conventional approaches.
When our experiments are complete, we will have a a greater understanding about
whether or not the immune system attacks self stem cells in old age. We may also discover
some unexpected results along the way as well, particularly when attempting to vaccinate or
induce tolerance with a selected stem cell marker.

Approach

Biological Samples

Figure 2: Types and numbers of samples used in experiments.

These experiments will make use of the serum from 20 unrelated young humans and

7
20 old humans as illustrated in Figure 2. The cutoff point for the definition of young will be 65
and younger. These human samples will be obtained by collaborating with a clinic such as
the Mayo clinic.
These experiments will also make use of 60 Balb/cJ mice obtained from the Jackson
Laboratory company. 20 mice will be allowed to age normally without interference. The
serum from these mice will be used to asses stem cell marker immune reactions, and this
serum will also be applied to the peptide microarrays to produce unique signatures. Another
group of 20 mice will be used for vaccination protocols, and the last group of 20 mice will be
used for tolerance induction protocols. The tolerance induction group will be split into two
groups of 10. One group will undergo tolerance induction orally, and the other group will
receive genetically engineered dendritic cells designed to down-regulate T cells specific for
the stem cell marker of interest.

Aim 1: Perform assays to measure the mouse and human immune responses against
several stem cell markers.

Figure 3: Techniques used to detect immune responses to stem cell markers. ELISA
will be used to detect humoral B cell antibody reponses. ELISPOT will be used to detect
cellular T cell responses.

An immune response against specific stem cell markers will be determined using
ELISA and ELISPOT techniques. ELISA will be used to discover antibody responses, and
ELISPOT will be used to determine T cell responses as illustrated in Figure 3. The ten stem
cell markers selected are found in a variety of different progenitor cells for different tissue
types (Table 1). These stem cell markers were selected from a list published by the NIH [38].

8
These are human stem cell markers, but the mouse proteins which are homologous to these
proteins will also be tested using mouse B and T cells. For all of the following experiments,
the house-keeping protein beta actin will be used as a control since we do not expect a strong
immune response to this protein. If there is a detectable response to beta actin, then we will
use a completely foreign protein such as the novel tungsten-iron-sulfur protein found in a type
of archaebacteria as a control [39].

Table 1: Stem Cell Markers. EC: embryonal carcinoma. ES: embryonic stem. HSC:
hematopoietic stem cell. MSC: mesenchymal stem cell.

General Cell Marker name Specific Cell Type Significance

Type/Location
Blood Vessel Fetal liver kinase-1 Endothelial Cell-surface receptor
(Flk1) protein that identifies
endothelial cell
progenitor; marker of
cell-cell contacts
Bone Marrow and Stem cell antigen (Sca- HSC, MSC Cell-surface protein on
Blood 1) bone marrow (BM) cell,
Nervous System CD133 Neural stem cell, HSC Cell-surface protein that
identifies neural stem
cells, which give rise to
neurons and glial cells
Pancreas Cytokeratin 19 (CK19) Pancreatic epithelium CK19 identifies specific
pancreatic epithelial
cells that are progenitors
for islet cells and ductal
cells
Pluripotent Stem Cell Pax6 Ectoderm Transcription factor
expressed as ES cell
differentiates into
neuroepithelium
Pluripotent Stem Cell Stem cell factor (SCF or ES, EC, HSC, MSC Membrane protein that
c-Kit ligand) enhances proliferation
of ES and EC cells,
HSCs, and MSC; binds
the receptor c-Kit
Pluripotent Stem Cell Telomerase ES, EC An enzyme uniquely
associated with
immortal cell lines;
useful for identifying
undifferentiated PSCs
Skeletal MyoD and Pax7 Myoblast, myocyte Transcription factors

9
Muscle/Cardiac/Smooth that direct
Muscle differentiation of
myoblasts into mature
myocytes
Skeletal Myogenin and MR4 Skeletal myocyte Secondary transcription
Muscle/Cardiac/Smooth factors required for
Muscle differentiation of
myoblasts from muscle
stem cells
Bone Osteocalcin Osteoblast Mineral-binding protein
uniquely synthesized by
osteoblast; marker of
bone formation
These stem cell marker proteins will be produced using mammalian expression
systems to ensure that the proper post translational modifications are present since such
modifications can greatly affect structure and antibody binding [40]. We will use the pCDNA
3.3 TOPO TA cloning kit from Invitrogen to subclone the gene with a GST (Glutathione S-
transferase) tag for the protein into the pcDNA 3.3 plasmid. The GST tag will be used to aid
in protein purification. E. coli will then be transformed with this modified vector to replicate
and maintain the plasmid. The presence of the gene insert in the vector will be verified with
PCR. The plasmid will then be isolated and used to transfect mammalian FreeStyle 293-F
cells from Invitrogen. These cells will then be induced to express the protein of interest and
the cell lysate will be harvested. A Western blot procedure will then be performed in order to
determine if the protein has the expected size. The proteins will then be purified on a column
coated with glutathione which will bind to the GST tag of the fusion proteins.
An ELISA will be performed with these proteins to see if there are any antibodies in the
blood which bind them. Our ELISA protocol will be similar to the standard protocol outlined by
Loizou et al [41]. The ELISA plate will be coated with the stem cell marker. Then the plate
will be blocked with BSA (Bovine Serum Albumin) and serum from mouse or human will be
applied to the plate as appropriate. Secondary anti-IgG antibody conjugated to biotin followed
by streptavidin conjugated HRP (horse radish peroxidase) along with ABTS (2,2'-azino-bis(3-
ethylbenzthiazoline-6-sulphonic acid)) or TMB (3,3’,5,5’-Tetramethylbenzidine) substrate will
be used to detect antibodies which bound to protein.
For the ELISPOT technique, T cells must be collected. T cells will be collected from
serum using FACS (Fluorescence Activated Cell Sorting) [42] with an instrument such as the
Gallios from Beckman Coulter. FACS functions by separating electrically charged droplets
into separate containers. Cells which possess a certain marker will be bound by a fluorescent
antibody to that marker. The FACS instrument detects this fluorescence and imparts a charge
on a droplet of liquid containing this fluorescent cell antibody group and the cells of interest
are isolated. We will collect cytotoxic T cells with FACS using a commercially available
antibody from Abcam against the CD8 T cell specific marker.
These isolated T cells will then be used in an ELISPOT to see if they react against the
protein of interest [43]. T cells will be added to a plate coated with antibody which binds to the
cytokine IFN-γ. These T cells will then be stimulated with one of our stem cell marker
proteins and a solution containing other immune cells such as dendritic cells. If the T cells

10
respond to this antigen, they may produce the IFN-γ cytokine. The plate is then washed to
remove the cells. Biotinylated anti-IFN-γ antibody is then added to the well followed by
streptavidin AP (alkaline phosphatase). A precipitating substrate such as NBT/BCIP ((Nitro-
Blue Tetrazolium Chloride)/(5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt)) is added
to react with AP. The resulting spots obtained from the different samples will then be
compared.
If none of our cells produce any interferon gamma, then a T cell proliferation assay will
be used to determine if there is a T cell response against these stem cell markers. We will
follow the basic protocol outlined by Graziano et al. [44]. In brief, leukocytes will be collected
from blood after centrifugation. Standard tissue culture will be used to grow these cells, and
then the antigen of interest will be added. The cells will then be incubated for several hours
with tritiated thymidine. The cells will then be pelleted and redissolved in scintillation fluid,
and the radioactivity of the sample will be assayed in an instrument such as a Packard
Scintillation Counter.
We expect that serum and cells from old mice and humans will react to some of the
selected stem cell markers more than the serum and cells from young mice and humans. We
particularly expect to find these results for the telomerase stem cell marker. In other words,
the ELISA test will reveal that there is a high antibody titer to telomerase, and the ELISPOT
test will reveal that T cells produce IFN-γ when stimulated by telomerase. If the tritiated
thymidine cell proliferation assay becomes necessary in the event that no IFN-γ is produced,
then we expect that the T cells will proliferate more in the presence of the stem cell antigens
than in the presence of the control.

Aim 2: Use a peptide microarray to observe differences in the antibodies produced by

old and young mice and humans.

Figure 4: Concept of Immunosignature. The antibodies from the serum of an organism are used to probe a
10K array of 20 mer peptides, which produce a unique immunosignature. Binding events are detected with dye
conjugated secondary antibodies. This concept is similar to the concept used in a standard ELISA.

11
 Testing specific stem cell markers is useful, but also laborious and time consuming.
We may also not be lucky enough to choose a stem cell marker which the immune system
responds to. Therefore, we will also use a more general method to distinguish between the
immune responses of young and old humans and mice. We will react the antibody containing
serum of young and old humans and mice with a peptide microarray, which will consist of a
glass slide made up of 10,000 peptides with 20 amino acids each. The lab I currently work in,
the Center for Innovations in Medicine in the Biodesign Institute at Arizona State University,
produces these peptide microarrays. However, if we encounter any problems with the
manufacturing process of these arrays, we could collaborate with other labs producing similar
arrays. The numerous antibodies in the serum will separate on the array since different
antibodies will have varying affinities for different peptides. The ultimate result will be a
signature of unique peptide reactivities, an immunosignature (Figure 4). Using this method,
we will discover if the immunosignature from old mice and humans is consistent or varies
greatly. If the immunosignature is repeatable, this will support the hypothesis that the immune
system acquires immunity against antigens in the body in a specific manner. These antigens
may be stem cell antigens.
The procedure for using the peptide microarrays is actually very similar to a standard
ELISA protocol. The array is blocked with BSA to prevent non-specific binding interactions.
Then the serum is applied to the array followed by a biotinylated secondary anti-IgG antibody.
Then a streptavidin conjugated dye is reacted with the array. Fluorescence intensities are
then detected and a unique signature is acquired.
The massive amount of data from these experiments will be analyzed using standard
statistical techniques. First a t test will be used to determine which peptide signals have
statistically significant variable levels of fluorescence compared to peptide signals from young
human or mouse samples. Principal component analysis will then be used to determine
whether these peptides are capable of distinguishing old from young signatures which were
not used in the t test.
If we can obtain a specific signature capable of distinguishing old from young mice, this
will still not provide us with any information about the original antigens which these antibodies
were raised against. However, such positive results will encourage us to keep looking for
such antigens if the ten we choose do not yield any positive results.

Aim 3: Modulate immune response for a specific stem cell marker in mice and observe
the effect on lifespan.

The response of the immune system of mice, but not humans, against a certain stem
cell antigen will be modulated (Figure 5). This feat will be accomplished by vaccinating some
mice against a certain stem cell marker and attempting to induce tolerance in some mice for a
certain stem cell marker. The stem cell marker which elicited the greatest immune response
in Aim 1 as determined by our ELISA and ELISPOT assays will be used to perform
vaccination or tolerance studies. If none of the stem cell markers demonstrate that an
immune response exists, then we will still perform the immune modulation procedures using
telomerase. Telomerase is an interesting protein to test since it is found in >85% of all
cancers. Telomerase vaccination has also decreased the incidence of cancers [32], but the
effect of vaccination on lifespan has not been well documented.
Vaccination will be performed by injecting the protein of interest along with the adjuvant
KLH (keyhole limpet hemocyanin) into 10 mice. Another group of 10 mice will receive no

12
vaccine. This group will be the control group for both the vaccination and tolerance group.
The vaccination will be performed when the mouse has reached middle age. The overall
health of the mice will then be assessed qualitatively every week on a 1 to 10 scale, and the
ultimate lifespan will be determined and compared to normal mice.
Inducing oral tolerance will be performed with 10 mice by exposing the mouse to large
quantities of the stem cell antigen orally. Baking soda will be used to prevent digestion of the
protein so that the protein has a greater chance to be presented to the immune system. As
with the vaccinated mice, the overall health of the mice will then be assessed qualitatively
every week, and the ultimate lifespan will be determined and compared to normal mice.

Figure 5: Immune modulation of mice. Mice will be injected with our most immunogenic
stem cell antigen or telomerase. Tolerance will also be induced orally in some mice and with
the aid of engineered dendritic cells in other mice.

Another group of 10 mice will be subjected to a tolerance induction procedure using the
more innovative genetically engineered dendritic cell method. Dendritic cells will be collected
from mouse serum using a method outlined by Vremic et. al. [45]. These cells will then be
transfected with the mammalian plasmid pcDNA3.3 from Invitrogen. This plasmid will be
engineered to code for FasL (Fas ligand), our most immunogenic stem cell marker or
telomerase, the m-LAMP1 TmCy gene, and FADD. The FasL will signal apoptosis to T cells
specific for the tested stem cell marker. M-LAMP1 TmCy will direct the stem cell marker to be
presented by the cell by MHC II. FADD will prevent the dendritic cell from receiving apoptosis
signals from another cell. The overall health of the mice will then be assessed qualitatively
every week, and the ultimate lifespan will be determined and compared to normal mice.
If our hypothesis is true, the vaccinated mice will exhibit a decrease in health and
lifespan, while the opposite will be observed in the mice subjected to tolerance induction.
A timeline for all three aims is presented in Figure 6. Many of the experiments will be
stretched over a long period of time since they will need to be performed at certain time points
during the lifespan of the Balb/c mice. Balb/c mice live approximately 2 years and 2 months
[46]. The first serum will be collected for peptide microarray experiments when the mice are 1

13
month old. Serum will then be collected every 6 months in order to monitor how the peptide
binding profile changes with time. The mice that will have tolerance induced or exposed to a
vaccine will receive this treatment when they are 1 year old.

Figure 6: Timeline of Work

Summary

The ultimate goal of the proposed experiments is to address the hypothesis that part of
the aging process is due to an acquired autoimmune disease against stem cells. Specifically,
after these experiments are complete we will know whether or not the immune system reacts
to several selected stem cell antigens in old age. We will know if the immune system
changes in old age in a way that can be distinguished and used to separate old from young
samples. We will also know how vaccinating against or inducing tolerance for a specific stem
cell antigen affects the lifespan of mice. If the immune system does change as expected in
old age, we will have greater insight into the connection between aging and cancer. These
results will also guide the direction of future research aimed at developing therapies for the
aging process in order to improve quality of life and reduce healthcare costs for society.

14
Bibliography and References Cited

[1] U.E. Reinhardt, “Does the aging of the population really drive the demand for health care?,”
Health Affairs (Project Hope), vol. 22, Dec. 2003, pp. 27-39.
[2] T.I. Edwards, R. Pearl, and S.A. Gould, “THE GROWTH AND DURATION OF LIFE OF
CELOSIA CRISTATA SEEDLINGS AT DIFFERENT TEMPERATURES,” The Journal of
General Physiology, vol. 17, Jul. 1934, pp. 763-781.
[3] G.M. Martin, S.N. Austad, and T.E. Johnson, “Genetic analysis of ageing: role of oxidative
damage and environmental stresses,” Nature Genetics, vol. 13, May. 1996, pp. 25-34.
[4] T. Hamatani, G. Falco, M.G. Carter, H. Akutsu, C.A. Stagg, A.A. Sharov, D.B. Dudekula, V.
VanBuren, and M.S.H. Ko, “Age-associated alteration of gene expression patterns in mouse
oocytes,” Human Molecular Genetics, vol. 13, Oct. 2004, pp. 2263-2278.
[5] L.A. Gavrilov and N.S. Gavrilova, “The reliability theory of aging and longevity,” Journal of
Theoretical Biology, vol. 213, Dec. 2001, pp. 527-545.
[6] T.B.L. Kirkwood, “Evolution of ageing,” Mechanisms of Ageing and Development, vol. 123, Apr.
2002, pp. 737-745.
[7] P.A. Parsons, “Antagonistic pleiotropy and the stress theory of aging,” Biogerontology, vol. 8,
Oct. 2007, pp. 613-617.
[8] Y.J. Lin, L. Seroude, and S. Benzer, “Extended life-span and stress resistance in the Drosophila
mutant methuselah,” Science (New York, N.Y.), vol. 282, Oct. 1998, pp. 943-946.
[9] G.T. Crossin, S.G. Hinch, S.J. Cooke, M.S. Cooperman, D.A. Patterson, D.W. Welch, K.C.
Hanson, I. Olsson, K.K. English, and A.P. Farrell, “Mechanisms influencing the timing and
success of reproductive migration in a capital breeding semelparous fish species, the sockeye
salmon,” Physiological and Biochemical Zoology: PBZ, vol. 82, Dec. 2009, pp. 635-652.
[10] M. Aggarwal and R.M. Brosh, “WRN helicase defective in the premature aging disorder Werner
syndrome genetically interacts with topoisomerase 3 and restores the top3 slow growth phenotype
of sgs1 top3,” Aging, vol. 1, Feb. 2009, pp. 219-233.
[11] C. Harley, “Telomeres and telomerase in aging and cancer,” Current Opinion in Genetics &
Development, vol. 5, 1995, pp. 249-255.
[12] M.T. Hemann, M.A. Strong, L. Hao, and C.W. Greider, “The Shortest Telomere, Not Average
Telomere Length, Is Critical for Cell Viability and Chromosome Stability,” Cell, vol. 107, 2001,
pp. 67-77.
[13] X. Cortez-Gonzalez and M. Zanetti, “Telomerase immunity from bench to bedside: round one,”
Journal of Translational Medicine, vol. 5, 2007, p. 12.
[14] L.L. Smith, H.A. Coller, and J.M. Roberts, “Telomerase modulates expression of growth-
controlling genes and enhances cell proliferation,” Nature Cell Biology, vol. 5, May. 2003, pp.
474-479.
[15] E. Chang and C.B. Harley, “Telomere length and replicative aging in human vascular tissues,”
Proceedings of the National Academy of Sciences of the United States of America, vol. 92, Nov.
1995, pp. 11190-11194.
[1] D.F. Terry, V.G. Nolan, S.L. Andersen, T.T. Perls, and R. Cawthon, ?Association of longer
telomeres with better health in centenarians,? The Journals of Gerontology. Series A, Biological
Sciences and Medical Sciences, vol. 63, Aug. 2008, pp. 809-812.
[17] K.L. Rudolph, S. Chang, H.W. Lee, M. Blasco, G.J. Gottlieb, C. Greider, and R.A. DePinho,
“Longevity, stress response, and cancer in aging telomerase-deficient mice,” Cell, vol. 96, Mar.
1999, pp. 701-712.
[18] M.A. Blasco, H.W. Lee, M.P. Hande, E. Samper, P.M. Lansdorp, R.A. DePinho, and C.W.

15
 Greider, “Telomere shortening and tumor formation by mouse cells lacking telomerase RNA,”
Cell, vol. 91, Oct. 1997, pp. 25-34.
[1] E. González-Suárez, C. Geserick, J.M. Flores, and M.A. Blasco, ?Antagonistic effects of telomerase
on cancer and aging in K5-mTert transgenic mice,? Oncogene, vol. 24, 2005, pp. 2256-2270.
[20] H. Vaziri, W. Dragowska, R.C. Allsopp, T.E. Thomas, C.B. Harley, and P.M. Lansdorp, “Evidence
for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age,”
Proceedings of the National Academy of Sciences of the United States of America, vol. 91, Oct.
1994, pp. 9857-9860.
[21] M.S. Rao and M.P. Mattson, “Stem cells and aging: expanding the possibilities,” Mechanisms of
Ageing and Development, vol. 122, May. 2001, pp. 713-734.
[22] D.E. Harrison, C.M. Astle, and J.W. Doubleday, “Cell lines from old immunodeficient donors give
normal responses in young recipients,” Journal of Immunology (Baltimore, Md.: 1950), vol. 118,
Apr. 1977, pp. 1223-1227.
[23] B.M. Carlson and J.A. Faulkner, “Muscle transplantation between young and old rats: age of host
determines recovery,” The American Journal of Physiology, vol. 256, Jun. 1989, pp. C1262-1266.
[24] I.M. Conboy, M.J. Conboy, A.J. Wagers, E.R. Girma, I.L. Weissman, and T.A. Rando,
“Rejuvenation of aged progenitor cells by exposure to a young systemic environment,” Nature,
vol. 433, Feb. 2005, pp. 760-764.
[25] G. Wick and B. Grubeck-Loebenstein, “The aging immune system: primary and secondary
alterations of immune reactivity in the elderly,” Experimental Gerontology, vol. 32, Oct. 1997,
pp. 401-413.
[26] D.E. Harrison, R. Strong, Z.D. Sharp, J.F. Nelson, C.M. Astle, K. Flurkey, N.L. Nadon, J.E.
Wilkinson, K. Frenkel, C.S. Carter, M. Pahor, M.A. Javors, E. Fernandez, and R.A. Miller,
“Rapamycin fed late in life extends lifespan in genetically heterogeneous mice,” Nature, vol. 460,
Jul. 2009, pp. 392-395.
[27] C. Morales Betanzos, M.J. Gonzalez-Moa, K.W. Boltz, B.D. Vander Werf, S.A. Johnston, and
S.A. Svarovsky, “Bacterial glycoprofiling by using random sequence peptide microarrays,”
Chembiochem: A European Journal of Chemical Biology, vol. 10, Mar. 2009, pp. 877-888.
[28] J.R. Falsey, M. Renil, S. Park, S. Li, and K.S. Lam, “Peptide and small molecule microarray for
high throughput cell adhesion and functional assays,” Bioconjugate Chemistry, vol. 12, Jun.
2001, pp. 346-353.
[29] X. Duburcq, C. Olivier, F. Malingue, R. Desmet, A. Bouzidi, F. Zhou, C. Auriault, H. Gras-Masse,
and O. Melnyk, “Peptide-protein microarrays for the simultaneous detection of pathogen
infections,” Bioconjugate Chemistry, vol. 15, Apr. 2004, pp. 307-316.
[30] R.C. Panicker, X. Huang, and S.Q. Yao, “Recent advances in peptide-based microarray
technologies,” Combinatorial Chemistry & High Throughput Screening, vol. 7, Sep. 2004, pp.
547-556.
[31] S. Panse, L. Dong, A. Burian, R. Carus, M. Schutkowski, U. Reimer, and J. Schneider-Mergener,
“Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using
radioactive and fluorescence-based assays,” Molecular Diversity, vol. 8, 2004, pp. 291-299.
[32] J.A. Kyte, “Cancer vaccination with telomerase peptide GV1001,” Expert Opinion on
Investigational Drugs, vol. 18, May. 2009, pp. 687-694.
[33] C. Mennuni, S. Ugel, F. Mori, B. Cipriani, M. Iezzi, T. Pannellini, D. Lazzaro, G. Ciliberto, N. La
Monica, P. Zanovello, V. Bronte, and E. Scarselli, “Preventive vaccination with telomerase
controls tumor growth in genetically engineered and carcinogen-induced mouse models of
cancer,” Cancer Research, vol. 68, Dec. 2008, pp. 9865-9874.
[34] W.P. Min, R. Gorczynski, X.Y. Huang, M. Kushida, P. Kim, M. Obataki, J. Lei, R.M. Suri, and

16
 M.S. Cattral, “Dendritic cells genetically engineered to express Fas ligand induce donor-specific
hyporesponsiveness and prolong allograft survival,” Journal of Immunology (Baltimore, Md.:
1950), vol. 164, Jan. 2000, pp. 161-167.
[35] H. Hackstein, A.E. Morelli, and A.W. Thomson, “Designer dendritic cells for tolerance induction:
guided not misguided missiles,” Trends in Immunology, vol. 22, Aug. 2001, pp. 437-442.
[36] C. Siatskas, J. Chan, J. Field, K. Murphy, Z. Nasa, B. Toh, and F. Alderuccio, “Gene therapy
strategies towards immune tolerance to treat the autoimmune diseases,” Current Gene Therapy,
vol. 6, Feb. 2006, pp. 45-58.
[37] B. Wu, J.M. Wu, A. Miagkov, R.N. Adams, H.I. Levitsky, and D.B. Drachman, “Specific
immunotherapy by genetically engineered APCs: the "guided missile" strategy,” Journal of
Immunology (Baltimore, Md.: 1950), vol. 166, Apr. 2001, pp. 4773-4779.
[38] Appendix E: Stem Cell Markers . In Stem Cell Information [World Wide Web site]. Bethesda, MD:
National Institutes of Health, U.S. Department of Health and Human Services, 2009 [cited
Sunday, April 25, 2010] Available at <http://stemcells.nih.gov/info/scireport/appendixe>
[39] S. Mukund and M.W. Adams, “The novel tungsten-iron-sulfur protein of the hyperthermophilic
archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its
participation in a unique glycolytic pathway,” The Journal of Biological Chemistry, vol. 266,
Aug. 1991, pp. 14208-14216.
[40] D. Houde, Y. Peng, S.A. Berkowitz, and J.R. Engen, “Post-translational modifications
differentially affect IgG1 conformation and receptor binding,” Molecular & Cellular Proteomics:
MCP, Jan. 2010.
[41] S. Loizou, J.D. McCrea, A.C. Rudge, R. Reynolds, C.C. Boyle, and E.N. Harris, “Measurement of
anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization
and quantitation of results,” Clinical and Experimental Immunology, vol. 62, Dec. 1985, pp. 738-
745.
[42] W.A. Bonner, H.R. Hulett, R.G. Sweet, and L.A. Herzenberg, “Fluorescence activated cell
sorting,” The Review of Scientific Instruments, vol. 43, Mar. 1972, pp. 404-409.
[43] D.E. Hricik, V. Rodriguez, J. Riley, K. Bryan, M. Tary-Lehmann, N. Greenspan, C. Dejelo, J.A.
Schulak, and P.S. Heeger, “Enzyme linked immunosorbent spot (ELISPOT) assay for interferon-
gamma independently predicts renal function in kidney transplant recipients,” American Journal
of Transplantation: Official Journal of the American Society of Transplantation and the American
Society of Transplant Surgeons, vol. 3, Jul. 2003, pp. 878-884.
[44] K.D. Graziano, J.C. Ruckdeschel, and M.R. Mardiney, “Cell-associated immunity to measles
(rubeola). The demonstration of in vitro lymphocyte tritiated thymidine incorporation in response
to measels complement fixation antigen,” Cellular Immunology, vol. 15, Feb. 1975, pp. 347-359.
[45] D. Vremec, M. Zorbas, R. Scollay, D.J. Saunders, C.F. Ardavin, L. Wu, and K. Shortman, “The
surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the
CD8 expression by a subpopulation of dendritic cells,” The Journal of Experimental Medicine,
vol. 176, Jul. 1992, pp. 47-58.
[46] A.A. Morley and K.J. Trainor, “Lack of an effect of vitamin E on lifespan of mice,”
Biogerontology, vol. 2, 2001, pp. 109-112.

17
Budget

Year 1
Personnel
Name Position Months devoted to Project
Role Salary and fringe benefits
TBD Post doc 12 Animal work 55000
TBD Graduate Student 12 Work with post doc on animals 40000
TBD Graduate Student 12 Molecular Biology 40000
TBD Graduate Student 12 Molecular Biology 40000
Equipment
No major equipment needs to
be purchased
Supplies
Item Cost
Typical Lab Supplies 24000
Mice 45000
Year 1 Total 244000

Year 2
Personnel
Name Position Months devoted to Project
Role Salary and fringe benefits
TBD Post doc 12 Animal work 55000
TBD Graduate Student 12 Molecular Biology 40000
TBD Graduate Student 12 Molecular Biology 40000
Equipment
No major equipment needs to
be purchased
Supplies
Item Cost
Typical Lab Supplies 24000
Year 2 Total 159000

Year 3
Personnel
Name Position Months devoted to Project
Role Salary and fringe benefits
TBD Post doc 12 Animal work 55000
TBD Graduate Student 12 Molecular Biology 40000
TBD Graduate Student 12 Molecular Biology 40000
Equipment
No major equipment needs to
be purchased
Supplies
Item Cost
Typical Lab Supplies 24000
Year 3 Total 159000

18
 BIOGRAPHICAL SKETCH
Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2.

NAME POSITION TITLE

Kurt Whittemore Research Assistant
eRA COMMONS USER NAME (credential, e.g., agency login)

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency
training if applicable.)
DEGREE
INSTITUTION AND LOCATION MM/YY FIELD OF STUDY
(if applicable)
Southern Utah University Bachelors 2005-2008 Chemistry
Arizona State University Ph.D. 2008-2013 Biological Design

A. Personal Statement

The goal of the proposed project is to test the hypothesis that part of the aging process is due to
an acquired autoimmune disease against stem cells. Accomplishing this goal will require several
molecular biotechnology techniques as well as the use of some new innovative technologies. My
background in chemistry and molecular biology has made be particularly well suited for this research.
I received my undergraduate degree in Chemistry during which time I learned about a wide variety of
basic sciences, and performed well in the Chemistry department program, which was the most rigorous
physical science program at my institution. During this time, I also created and lead a team to compete
in the international Genetically Engineered Machines competition at MIT. This experience and the
corresponding classes gave me a foundation in molecular biology which I would not otherwise have
had as an undergraduate major in Chemistry.
As a graduate student, I have taken the Biological Design core courses which cover many
different biotechnology related topics. I have also taken an immunology course which will be directly
useful for this proposed aging research. Additionally, rotations in several research labs during my first
year as a graduate student have provided me with several varied experiences and allowed me to make
connections with many knowledgeable researchers who can assist me with this research. All of these
factors combined with the excellent environment of the Biodesign Institute which provides great
facilities and expertise, will allow me to accomplish the goal of exploring relationships between the
immune system and aging.

B. Positions and Honors

Positions and Employment

2005-2008 Southern Utah University Water Lab Analyst (2005-2008)
2008-present Research Assistant

Other Experience and Professional Memberships

2005-2008 Member, Honor Society
2005-2008 Member, Alpha Chi Member

19
2008 Member, Golden Key International Honor Society

Honors
2007 Listed on Dean's List
2007 Gold medal at iGEM (international Genetically Engineered Machines)
competition
2008 Graduated Summa Cum Laude with Bachelor of Science in
Chemistry with Honors from
Southern Utah University
2008 College of Science Outstanding Scholar at Southern Utah University

C. Selected Peer-reviewed Publications

2010 Work presented at the Graduate Student Recruitment Weekend

D. Research Support

ARCS (Academic Rewards for College Scientists) 08/01/10-05/31/13

The goal of this study is to identify uniquely tumor-expressing antigens that a cancer patient's
immune system can specifically recognize.

20
Facilities & Other Resources

CIM/Biodesign Institute:
The Center for Innovations in Medicine (CIM) in the Biodesign Institute is a unique center. It is housed
in ~10Ksf of unwalled space. It is one of 10 centers in the state of the art Biodesign Building (Lab of
the Year, RD Award 2006). All benches are mobile, desks and office are optimized for communication
and every detail is designed to maximize interaction and efficient science. All utilities are drop down to
the benches with 1GB Ethernet to bench sites. The Institute offers support for IT, IP, business and grant
submissions. The Center is well equipped with standard equipment for all aspects of this project. There
are 3 chemistry suites in the Center with 6, 6’ hoods. The robotics and spotters are in a clean room.
There is a large walk-in cold room and a tissue culture room with 4 hoods. CIM currently has ~45
members, including 4 postdocs, 11 graduate students, 2 tenure-track and 5 research faculty, 3 senior
researchers, technicians and undergraduates.

There are 9 other Centers in The Biodesign Institute with which we interact closely. These centers
represent research in photochemistry, single molecule physics, nanofabrication, biosensors,
computational-evolutionary biology, glycochemistry, bioengineering and vaccinology.

Animals:
The Biodesign Building has a full animal facility in the basement. This includes capacity to
handle ~5000 rodents and 20 primates. In addition there is a full ABSL3 suite with one
dedicated laboratory space and 3 animal holding rooms capable of handling 1500 rodents each at
once. Standard care is provided by the excellent ASU Animal Resources, including veterinary
primate care.

Computer:
In Biodesign CIM has access to a 40 CPU Linux system providing 300Gigflops of computing
power. It has alloted 10k cpu hours from the High Performance Computing Center. We can also
use the ASU supercomputer. Biodesign supplies excellent IT support for CIM. CIM itself has 4
servers, one of which is dedicated to the cancer vaccine projects. CIM has computer programmers for
assistance with the bioinformatic analyses and data management needed for this project .

Equipment

CIM/Biodesign:

The Center for Innovations in Medicine (CIM) has a complete microarray core. There are two
spotting systems for custom arrays and three array readers accompanied by current software.
Commercial Affymetrix equipment and software are also part of the core. CIM has a mass
spectrometry core with an Agilent LC MSD Ultratrap and SPR Flexichip and Symphony 12
channel Peptide Synthesizer. Other major supportive equipment in CIM includes Biomek FX
Robotic system, Nanodrop, Fluorescent plate readers, Luminex system, Typhoon, Animal and
Plate barcoding, HiGro Culture system, Gene guns and stations. We are the only, or one of 2
academic units with a BiaCore A100 High-throughput SPR system and a Corning EPIC
system. The equipment has skilled support including Bioinformatics, computer programmers
and technicians for the array core, peptide synthesis, robotics and animal care. In other

21
Centers in Biodesign there is available FACS, AFM, Nano-fabrication and Shop support. In
particular we can have custom, in-situ synthesized peptide arrays made to specifications.

22