Temple University Annual Progress Report: 2010 Formula Grant

Temple University
Annual Progress Report: 2010 Formula Grant
Reporting Period
July 1, 2012 – June 30, 2013
Formula Grant Overview
Temple University received $2,050,596 in formula funds for the grant award period January 1,
2011 through December 31, 2014. Accomplishments for the reporting period are described
below.
Research Project 1: Project Title and Purpose
The Effects of Music Therapy Entrainment on Pain, Vital Signs, and Bowel Function of Cancer
Patients - The purpose of this project is to gather preliminary data on the effectiveness of a
specialized music therapy approach to pain management with cancer outpatients who have
chronic pain. A cross-over design will be used to examine the effects of music therapy
entrainment, a live music intervention based on principles of physics, on reported pain levels,
vital signs, pain medication usage, and bowel function in 40 participants. Participants will
receive one music therapy entrainment session and one sham treatment consisting of listening to
pre-recorded music. In addition to measuring the above mentioned outcomes, three participants
will be chosen at random to undergo MRI scans. The purpose of this is to assess brain activity
relevant to entrainment music.
Duration of Project
7/1/2011 – 6/30/2012
Summary of Research Completed
This project ended during a prior state fiscal year. For additional information, please refer to the
Commonwealth Universal Research Enhancement C.U.R.E. Annual Reports on the Department's
Tobacco Settlement/Act 77 web page at http://www.health.state.pa.us/cure.
Research Project 2: Project Title and Purpose
Place-Based Interventions for Public Health: A Cross-Disciplinary Approach to the Study of
Policing - The purpose of this study is: 1) to determine if the benefits of focused, foot patrol
policing to reduce violent crime translates to a decrease in the incidence of drug-related illnesses
(such as overdose) and physical assaults; and 2) to identify community variables that are
associated with rates of crime-related illness or injury. Community variables include microplaces (e.g. alcohol outlets, parks) that serve as perpetual ‘crime generators’ as well as facilities
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(e.g. churches, health clinics) that contribute to the promotion of healthy behaviors. These data
will provide evidence to support ways in which the police, along with city agencies and services
can leverage their different tools and resources in a more targeted and collaborative manner to
prevent crime and achieve shared health and safety outcomes.
Duration of Project
1/1/2011 – 6/30/2011
Summary of Research Completed
This project ended during a prior state fiscal year. For additional information, please refer to the
Commonwealth Universal Research Enhancement Annual C.U.R.E. Reports on the Department's
Tobacco Settlement/Act 77 web page at http://www.health.state.pa.us/cure.
Research Project 3: Project Title and Purpose
Inflammation and Organ Tissue Damage - This project seeks to clarify the mechanisms by which
alteration in the inflammatory response cause tissue damage and organ failure. Specifically, we
will study critical steps of the inflammatory response to understand how: 1) adhesion receptors
expressed on endothelial cells and circulating leukocytes affect the immune response and
subsequent organ damage, 2) signal transduction processes initiated by such receptors regulate
the inflammatory response, and 3) cytokines produced by immune cells affect the overall
outcome of the inflammatory response toward resolution or chronic inflammation. The overall
goal is to uncover novel pharmacological strategies to prevent or treat the organ complications
associated with chronic inflammation (e.g., neurodegenerative disorders, cardiovascular disease,
obesity with insulin resistance, and physiological aging). These results will advance our
understanding of the mechanism by which systemic inflammation increases organ morbidity and
all-cause mortality in the population of the USA.
Duration of Project
9/1/2012 – 6/30/2013
Project Overview
The overall goal of this project is to uncover novel mechanisms by which acute and chronic
inflammation causes tissue damage and organ failure. Recently, basic and clinical research has
highlighted a role for local or systemic inflammation in the organ tissue injury associated with
acute (e.g., myocardial ischemia, viral and bacterial infections) and chronic (e.g., obesity,
atherosclerosis, and Alzheimer) diseases states. The ability to mount an inflammatory response is
essential for survival in the face of environmental pathogens and injury; however in some
situations and diseases, the inflammatory response may be exaggerated and sustained without
apparent benefit and even with severe adverse consequences. Understanding the mechanisms
responsible for aberrant inflammatory responses in disease states is a necessary preliminary step
to implement novel therapy. Accordingly, we seek to study three different, yet complimentary
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aspects of inflammation with the overall goal of understanding inflammation-mediated
mechanisms of tissue damage using in vitro and in vivo experimental models that are relevant to
human disease. Specifically, three specific aims have been developed to study: a) the endothelial
expressed adhesive receptors that regulate infiltration of bone marrow derived inflammatory
cells into organ tissues; b) the contribution to inflammation of Toll-like receptor (TLR) 2
receptor activation by curli amyloid fibrils; c) the modulation of the immune response by
interleukin-4 in response to virus-induced inflammation.
The results of these studies will provide important information on the intercellular mechanisms
of tissue inflammation in clinically relevant disease states. They will also provide new insight
into the integrative mechanisms by which endothelial cells, circulating leukocytes, activation of
Toll-like receptor, and cytokines synergize to deregulate the inflammatory response, thus
producing aberrant immune responses leading to tissue damage.
Principal Investigator
Rosario Scalia, MD, PhD
Professor of Physiology
Temple University, MERB 1049
3500 N. Broad Street
Philadelphia, PA 19140
Other Participating Researchers
Cagla Tukel, PhD; Stefania Gallucci, MD; AbdelKarim Sabri, PhD; Glenn J. Rapsinski, Jun Xu
Qi Zhao - employed by Temple University
Expected Research Outcomes and Benefits
We expect to uncover novel mechanisms responsible for inflammatory induced tissue damage, a
phenomenon that is common to several disease states (e.g., Alzheimer, obesity, cardiovascular
disease, bacterial and viral infections, cancer and arthritis). In particular we anticipate finding
that eCAMs, Toll-like receptor and selected cytokines play a key, mechanistic role in the fibrosis
and remodeling associated with chronic inflammation. More importantly we will test whether
that blockade of selected eCAMs, Toll-like receptor and changes in cytokine secretions prevents
or attenuates tissue damage in inflammation. We will also define the molecular mechanisms
responsible for deregulation and chronicity of the inflammatory response under our experimental
conditions. Overall, this project will uncover novel mechanisms of inflammation that will
provide a framework for developing new therapeutic strategies to avert vascular and organ
damage in humans.
Summary of Research Completed
Sabri
Myocardial ischemia due to vessel occlusion results in loss of cardiac myocytes in the ischemic
zone, which diminishes cardiac contractility and impairs cardiac repair. Because of the
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importance of inflammation and reparative mechanisms on cardiac remodeling, defects in
inflammatory pathways have been suggested to be responsible for adverse remodeling and heart
failure in a large number of patients surviving a MI.1 The mechanisms whereby inflammation
contributes to remodeling after cardiac injury has largely focused on action of reactive oxygen
species and cytokines/chemokines and their role on myocyte growth and extracellular matrix
(ECM) remodeling. However, little is known about the role of inflammatory serine proteases
(ISPs) (i.e. cathepsin G (CG), elastase, proteinase A) on cardiac remodeling and HF progression.
Because ISPs seem to have redundant functions, we propose to target dipeptidyl peptidase I
(DPPI), a lysosomal cysteine protease essential for the processing and activation of many ISPs,
to better understanding of the role played by this family of proteases during the development of
HF post MI.
DPPI KO mice did not differ from WT mice with regard to their baseline heart weight-to-body
weight ratio, heart rate, LV end systolic and diastolic pressures (data not shown). However, DPPI
KO mice showed a marked reduction in CG and elastase activity post-MI (-90% and -85% vs.
WT MI, respectively). Cardiac function as determined by echocardiography show a significant
improvement in cardiac function as assessed by an increase in LV fractional shortening
compared to WT mice subjected to MI (Figure 1A). Survival up to 4-weeks after MI was
comparable in WT and DPPI KO shams (97% versus 98%, respectively). Interestingly, DPPI
deficient mice displayed better post-infarction survival compared to WT (65% in DPPI KO mice
compared to 40% in WT mice, p<0.05) especially in the first week after the insult. Examination
of cell death in the border zone indicated less TUNEL positive cells in the DPPI KO mice
compared to WT animals subjected to MI for 2- and 7-days (Figure 1B). Infarct size was
significantly lower in DPPI than in WT mice at 7d (Figure 1C). Collectively, these data show
that the integrity of the infarcted area was well maintained after MI in DPPI KO mice, which
results in better cardiac function post-MI.
Another mechanism by which ISPs may improve cardiac function long term is through
regeneration of new myocardium. We found that the density of c-kit positive cell, stem cells
that have the capability to regenerate new myocardium, was significantly increased in DPPI KO
infarct at 7-days after MI compared to WT (Figures 1D and 1E). Using FACS analysis, we
found a slight increase in c- kit+/CD45- cells in WT mice subjected to MI. This increase was
further enhanced in DPPI KO infarcts (Figure 1E). These data suggest that DPPI deletion
improves CSC survival, thus leading to better cardiac function.
Tukel
Hela57A cells were transiently transfected with a combination of human expression vectors
containing the genes for human CD14, human TLR2, and human TLR1. Cells were then
stimulated with purified curli fibers, recombinant curli major subunit, GST-CsgA, synthetic
TLR2/TLR1 ligand, Pam 3 CSK 4 , as a positive control, or GST as a negative control. Cell lysates
were collected and assayed for luciferase activity 8 hours after stimulation. Consistent with the
previous findings, cells transfected with both TLR2 and TLR1 had increased levels of luciferase
activity, the indicator of NF-κB activation, upon stimulation with curli fibers, Pam 3 CSK 4 and
GST-CsgA. GST-CsgA polymerizes following purification process (data not shown). Hela57A
cells that were transfected with CD14 together with TLR2 and TLR1 showed significantly
higher levels of luciferase activity after treatment with curli, Pam 3 CSK 4 and GST-CsgA
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compared to cells transfected with only TLR2 and TLR1. As expected, cells stimulated with the
GST protein purified with a similar method to GST- CsgA did not induce NF-κB activation.
Similarly, cells transfected with CD14 alone lacked any significant NF-κB activation (Figure 2).
Overall, these data suggested that CD14 was not required for the activation of TLR2/TLR1
heterocomplex by curli fibers. Nonetheless, the presence of CD14 enhanced the TLR2/TLR1
heterocomplex activation by curli fibers as well as its major subunit, CsgA.
Both membrane bound and soluble CD14 increases the activity of TLR2/TLR1 complex in
response to curli fibers. Adaptor molecule CD14 is expressed by monocytes and epithelial cells
in its membrane-bound form. In addition, soluble CD14 is released into the serum by
hepatocytes. In the absence of membrane bound CD14, soluble CD14 has been shown to aid the
TLR4/MD2 complex to recognize LipidA. Although membrane bound CD14 was identified as
an adaptor molecule in the recognition of a synthetic ligand, tri-acylated lipopeptide,
Pam 3 CSK 4 , no studies have yet addressed whether soluble CD14 could replace the function of
membrane bound CD14 molecule in aiding TLR2/TLR1 complex. To determine the roles of
membrane bound and soluble CD14 in the immune recognition of curli fibers, Hela57A cells
were transfected with vectors for TLR2 and TLR1 alone or in combination with the vector for
CD14. Transfection of the cells with vector carrying the gene for CD14 results in the expression
of membrane bound CD14. All groups of Hela57A cells were then treated with curli fibers alone
or curli fibers that were pre-treated with 10 µg of soluble CD14. Both the presence of membrane
bound CD14 and the pre-treatment of curli fibers with soluble CD14 resulted in an increased NFκB activation through the TLR2/TLR1 heterocomplex. Additionally, when both membrane
bound and soluble CD14 were present during stimulation, no change in NF-κB was seen when
compared to cells with just one form of CD14 present during stimulation (Figure 3). This data
suggested that the presence of either membrane bound or soluble CD14 enhances the
TLR2/TLR1 activation by curli fibers.
CD14 Leads to Increased IL-6 and Nitric Oxide Production By Macrophages Stimulated with
Curli. Consistent with previous reports, BMDMs from C57BL/6 mice produced IL-6 in response
to the synthetic TLR2/TLR1 complex ligand, Pam 3 CSK 4 , whereas BMDMs deficient in CD14
or TLR2, showed significantly decreased IL-6 production (Figure 4A). Similarly, CD14 deficient
or TLR2 deficient BMDMs stimulated with curli fibers (Figure 4B) or GST-CsgA (Figure 4C)
produced significantly lower levels of IL-6 compared to wild type C57BL/6 macrophages. As
expected the GST protein purified with a similar method as the GST-CsgA did not induce IL-6
production in BMDMs (Figure 4C).
In addition, nitrite levels were significantly lower in the supernatants of TLR2 and CD14
deficient BMDMs compared to wild type C57BL/6 BMDM supernatants after stimulation with
curli fibers (Figure 3D). We published these findings in Journal of Biological Chemistry
(PMID:23548899).
Gallucci
Th2-conditioned immune responses, such as those induced to fight infestations with Th2inducing parasites increase susceptibility to viral infections through yet unclear mechanisms. IL4 is the major cytokine driving Th2 immune responses. We have reported before that IL-4
inhibits the response of bone-marrow-derived conventional dendritic cells (cDCs) and splenic
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DCs to Type I interferons (IFNs). During this period, we have analyzed cDC responses to R848
and CpGs, the nucleic acids that stimulate respectively TLR7 and TLR9. R848 and CpGs are
synthetic molecules that resemble the nuclei acids that are expressed by viruses, as well as by
endogenous cells dying by necrotic death, which are the main source of antigens in autoimmune
diseases such as lupus. We found that IL-4 significantly suppressed the response of cDC to these
stimuli. It decreased the production of pro-inflammatory cytokines and the full extent of cDC
maturation. The inhibition involved many signaling pathways, downstream of TLRs, including
those dependent on Type I Interferons. The inhibition of the expression of Type I Interferons was
specific of the response to nucleic acids, because IL-4 did not inhibit cDC response to
stimulation with the TLR4 ligand LPS, a bacterial molecule normally inducing a Th1 type of
response. To test the significance of these results for host defense, we also analyzed the effects of
IL-4 on the response of cDCs to a viral infection. We found that IL-4 also decreased IFN
response and increased permissiveness to infection with a DC-tropic virus. Our results indicate
that IL-4 modulates and counteracts the pro-inflammatory stimulation induced by TLR7 and
TLR9 and may affect responses against viruses and intracellular parasites.
The results of this project have been included in the below manuscript submitted for publication.
IL-4 suppresses the responses to TLR7 and TLR9 stimulation and increases the permissiveness
to retroviral infection of murine conventional dendritic cells. Uma Sriram, Jun Xu, Robert Chain,
Linda Varghese, Heather L. Bennett, Philip W. Zoltick , and Stefania Gallucci.
Figure 1
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Figure 2
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Figure 3
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Figure 4
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Research Project 4: Project Title and Purpose
Inhibition of Leptin Receptor Signaling in Cellular Models of Rheumatoid Arthritis and
Osteoarthritis - Osteoarthritis (OA) and rheumatoid arthritis (RA) are debilitating diseases
whose progression can be promoted and/or aggravated by obesity. Leptin is a satiety factor
regulating appetite and energy expenditure by acting on leptin receptors (ObR) in the
hypothalamus. Leptin represents a critical link among nutritional status, metabolism and
immunity. Recent evidence suggests a major role of the leptin / ObR system in the pathogenesis
of OA and, perhaps, RA. Consequently, pharmacological downregulation of leptin activity could
become a novel treatment for OA and RA. Our laboratories developed highly efficacious,
specific and safe leptin-based peptide antagonists of ObR. This collaborative project will
explore the applicability of our lead ObR antagonists in reversing characteristic OA and RA
processes in vitro.
Duration of Project
7/1/2011 – 12/31/2011
Summary of Research Completed
This project ended during a prior state fiscal year. For additional information, please refer to the
Commonwealth Universal Research Enhancement C.U.R.E. Annual Reports on the Department's
Tobacco Settlement/Act 77 web page at http://www.health.state.pa.us/cure.
Research Infrastructure Project 5: Project Title and Purpose
Research Infrastructure Project: Multiphoton Imaging Facility – This project will fund a
laboratory renovation to create a new Multiphoton Imaging Facility. Renovations will be made to
existing space in the Department of Biochemistry located on the 6th floor of Kresge Hall. The
updated facility will greatly improve capabilities for confocal microscopic imaging.
Duration of Project
7/1/2011 – 6/30/2012
Summary of Research Completed
This project ended during a prior state fiscal year. For additional information, please refer to the
Commonwealth Universal Research Enhancement C.U.R.E. Annual Reports on the Department's
Tobacco Settlement/Act 77 web page at http://www.health.state.pa.us/cure.
Research Project 6: Project Title and Purpose
Immunomodulatory Cannabinoids as Potential Therapeutics for Transplant Graft Rejection –
Compounds that bind to Cannabinoid Receptor 2 will be investigated for their potential to
increase survival of skin and organ grafts. Compounds of this type should suppress immune
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reactions but not have psychotropic activity. The efficacy of these compounds in combination
with standard anti-rejection therapy will also be investigated.
Anticipated Duration of Project
7/1/2011 – 12/31/2013
Project Overview
Current therapies for organ and skin transplantation have deficiencies, as long-term use results in
unwanted side effects including nephrotoxicity, neurotoxicity, diabetes, and liver dysfunction.
Synthetic cannabinoids that bind selectively to the cannabinoid receptor 2 (CB2) have been
shown to have anti-inflammatory and immunosuppressive activity. Compounds of this class do
not have the psychogenic effects of marijuana or its constituents because the CB2 receptor is
expressed almost exclusively on cells of the immune system. The psychogenic effects of abused
cannabinoids result from binding of ingredients in marijuana to the CB1 receptor, which is
expressed on neurons. This anatomical separation of expression of CB2 receptors permits design
of compounds that will affect the immune system but not the nervous system. This project seeks
to assess the potential of CB2 binding compounds, alone or in combination with standard antirejection therapies, to depress graft rejection. There are two Aims. One will use an in vitro assay
that is an accepted correlate of graft rejection, the Mixed Lymphocyte Reaction, to assess the
immunosuppressive capacity of various compounds, alone and in combination with standard
anti-rejection therapies. Aim 2 will test the potency of these compounds alone or in combination
with standard anti-rejection therapies in vivo using mice with grafted tissues. The in vitro assays
will be used as a screen for compounds and drug combinations. Those that are most effective in
vitro will be tried in vivo.
Principal Investigator
Toby K. Eisenstein, PhD
Professor
Temple University School of Medicine
3500 North Broad St.
Philadelphia, PA 19140
Other Participating Researchers
Rebecca Hartzell, BSc; Joseph J. Meissler, BSc; Senthil Jayarajan, MD – employed by Temple
University School of Medicine
Expected Research Outcomes and Benefits
This research has the potential to develop a new class of compounds that bind selectively to
Cannabinoid Receptor 2 (CB2), expressed on cells of the immune system, as effective therapies
in prolonging skin and organ grafts. Based on preliminary in vitro data, CB2 selective
compounds appear to have the ability to decrease reactivity of leukocytes for foreign tissue. CB2
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active compounds were shown by us to decrease lymphocyte proliferation in response to foreign
tissue and to decrease the secretion of proteins (cytokines) that activate cells of the immune
system. These results were obtained using the Mixed Lymphocyte Reaction, an assay that is
generally accepted as an in vitro correlate of graft rejection. This project will further evaluate the
potential of these compounds for preventing graft rejection by taking the experiments in vivo.
Mice receiving grafts will be treated with the CB2 active compounds and graft survival
compared with controls and with those of mice receiving standard anti-graft treatment. In
addition, combinations of reduced doses of standard therapies will be tested with addition of
CB2 compounds. Combination therapies may reduce toxicity of currently available anti-rejection
drugs. The new compounds may be effective by themselves, or may be useful in combination
with reduced doses of currently accepted anti-rejection regimens, which may reduce the longterm toxicity of the current standard regimens.
Summary of Research Completed
Aim 1: Test the effect of cannabinoids that are selective for the cannabinoid type 2 (CB2)
receptor, alone, and in combination with standard therapies for graft rejection, using the assay
that is an in vitro correlate of graft rejection, the Mixed Lymphocyte Reaction (MLR)
These experiments use two compounds, O-1966 and JWH-014, that are selective for the CB2
receptor. We have previously established using funds from this grant that these compounds
suppressed the immune cells (mouse splenic lymphocytes) in the MLR. During this year more
studies were carried out to further an understanding of the mechanisms of action of this CB2
agonist in the MLR. Selected figures are presented for the most important experiments. Data for
other findings are not shown but are available.
1.1. CB2 receptor expression increases in the MLR. We had shown in 2011-2012 that O-1966
caused suppression in the Mixed Lymphocyte Reaction (MLR) because of down-regulation of
the activity of T-cells as compared to macrophages. To answer the question of whether this
differential action was due to greater expression of the CB2 receptor on T-cells, qRT-PCR was
carried out to measure CB2 receptor (CB2R) RNA expression levels in purified CD3+ cells (Tcells) and CD11b+ cells (predominantly macrophages) immediately after spleens were removed
(T 0 ) and after 24 h in the MLR (T 24 ). CB2R expression was not significantly different between
these cell populations at T 0 . By T 24 , both populations had significantly increased CB2R
expression, with a 26.6-fold increase in CD11b+ cells and a 6.9-fold in CD3+ cells. These data
show 1) that mRNA for this receptor is markedly up-regulated by stimulation in an MLR culture,
and 2) the reason for the increased sensitivity of T-cells versus macrophages to the suppressive
effects of the cannabinoid was not due to a greater expression of CB2R message by the former.
1.2. O-1966 inhibits T-cell proliferation in response to activation by anti-CD3 and anti-CD28
antibodies. To further verify that the cannabinoids can directly suppress T-cells, splenocytes
were sorted into a CD3+ population. T-cells, highly purified by flow cytometery, were treated for
3 hours with O-1966 or DMSO vehicle and then activated with the anti-CD3 and anti-CD28
antibodies (antibodies to surface markers present uniquely on T-cells). In the presence of O-1966
there was a dose-dependent, marked decrease in cell proliferation. This experiment shows
conclusively that the cannabinoids can act directly on T-cells, as proliferation could be inhibited
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by the cannabinoid in the absence of accessory cells like macrophages.
1.3. O-1966 inhibits both CD4+ and CD8+ T-cells. The previous observations were extended to
determine which subset of T-cells was suppressed. These T-cell subsets are characterized by
their surface markers, designated CD4 (T-helper cells) and CD8 (T-cytotoxic cells). Splenocytes
were sorted by flow cytometry into CD4+ and CD8+ cells populations which were individually
treated with O-1966 or ethanol vehicle, before being added back to the remainder of the
untreated spleen cells, which were either CD4 or CD8 depleted, to restore the normal spleen
population for the MLR. Complete inhibition of the MLR was observed only in unsorted cultures
and cultures containing cannabinoid treated CD4+ plus CD8+ cells. In cultures that received
cannabinoid only treated CD4+ cells, the maximum suppression was 65% of the unsorted
maximum and treatment of only CD8+ cells resulted in only 50% of the maximum suppression
observed in unsorted, treated cultures. From these results it can be concluded that O-1966
suppressed both CD4+ and CD8+ cells. (Figure 1.)
1.4. Effect of O-1966 on CD4 and CD8 cell numbers in the MLR. An experiment was carried out
in which cells were treated with O-1966 in the MLR. At the end of the 48 hr incubation, cells
were harvested and analyzed for the percent of CD4 and CD8 cells. In the MLR cultures, the Tcells replicate. This experiment was to see if treatment with the cannabinoid differentially
affected replication of one of the subsets. The CD4:CD8 ratio was not altered during the course
of the MLR, showing that there was no differential effect on one cell subset versus the other.
1.5. O-1966 treatment in the MLR decreases CD4 expression in vitro. The expression of CD4 on
the cell surface of CD3+ cells was measured. MLR cultures were started using splenocytes from
wild-type or CB2R k/o mice. The cells were pretreated for 3 h with O-1966 or ethanol vehicle
and harvested 48 h into the assay, stained, and analyzed by flow cytometry. Figure 2A is a
representative comparative histogram of the fluorescent intensity of CD4 in cultures treated with
32 μM O-1966 or ethanol vehicle control. While the percentage of CD4+ cells in the CD3+
population did not change, O-1966 treatment caused a negative shift of fluorescence intensity of
these cells. Figure 2B shows that treatment with 8, 16, and 32 μM of O-1966 resulted in a dose
dependent decrease of the mean fluorescence intensity of CD4 expression on the cell surface.
Further, when splenocytes from CB2R k/o mice were used, there was no change in CD4
fluorescence intensity when treated with similar doses of O-1966, demonstrating that the
decreased expression in wild-type mice is CB2 mediated.
1.6. O-1966 suppresses the MLR when cells from IL-17 knock-out (k/o) mice are used. To test
whether the cannabinoid acts through a pathway involving the cytokine, IL-17, splenocytes from
animals lacking this molecule were used in the MLR. The degree of inhibition by O-1966 in the
MLR was the same using splenocytes from IL-17 k/o mice as with cells obtained from wild-type
mice, indicating that IL-17 modulation does not mediate suppression by CB2-agonists.
1.7. CB2 agonists do not induce apoptosis in the MLR. A possible mechanism that has been
proposed for cannabinoid mediated immunosuppression is through the induction of apoptosis of
activated immune cells (Lombard et al. 2007. J Immunol 122: 259-270). Experiments carried out
in the present studies show that membrane integrity of the cells in the MLR was unchanged by
cannabinoid treatment, as measured by LIVE/DEAD staining in each experimental condition. To
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verify our conclusion that the CB2 agonist did not induce apoptosis, more precise measurements
were used to detect and measure apoptotic cells. To detect cells in the early stages of apoptosis,
MLR cultures treated with JWH-015, O-1966, or vehicle were harvested at the start of the
culture, and 24 and 48 h into the assay. Levels of caspases 1, 3, 4, 5, 7, 8, and 9 were measured
by flow cytometry using a caspase assay kit (Vybrant® FAM Poly Caspases Assay Kit,
Molecular Probes, Inc., Eugene, OR). While the number of caspase positive cells increased as
time in culture increased, there were no differences between cells that received no treatment or
treatment with vehicle, as compared with treatment with a cannabinoid. Additionally, DNA
fragmentation was measured using a terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assay, to test cells from MLR cultures that were harvested at the start of the
assay (T 0 ), and 24 or 48 h after culture initiation. At all time points tested, there was no
difference between treatment groups, showing that apoptosis is not the mechanism by which
JWH-015 and O-1966 are suppressing the MLR.
1.8. O-1966 decreases levels of activated nuclear forms of NF-kB and NFAT in T-cells. To
examine a possible mechanism of suppression by O-1966, nuclear levels of the transcription
factors NF-κB and NFAT were measured in the presence or absence of O-1966. Splenocytes
from wild-type C57BL/6 mice or CB2 receptor knockout (CB2R k/o) mice were sorted by flow
cytometry and T-cells were negatively selected (CD11b-B220-). The T-cells were treated for 3
hours with O-1966 or ethanol vehicle and then activated with anti-CD3 and anti-CD28
antibodies and incubated for 18 h. The cells were then harvested and nuclear proteins extracted.
Levels of activated NF-κB and NFAT that were able to bind to their target promoters were
measured using the TransAM® transcription factor ELISA kits for NF-κB p50 and NFATc1.
Figure 3 shows that treatment of T-cells from wild-type mice with O-1966 significantly
decreased levels of both transcription factors in a dose-dependent manner, with suppression
observed at concentrations of the cannabinoid of 16 and 32 μM for NF-κB, and of 8 to 32 μM for
NFAT, as compared to ethanol vehicle controls. Suppression of neither transcription factor was
observed in cultures containing T-cells from CB2R k/o mice.
1.9. Anti-IL-10 antibody partially reverses suppression of proliferation by O-1966. We extended
studies carried out last year on whether adding antibody to neutralize IL-10 in MLR cultures
would block the immunosuppression induced by the cannabinoid. Last year we found only
partial neutralization of the suppression. This year we used higher doses of the antibody and
found that at 5 ug/ml the anti-IL-10 antibody gave approximately 50% reversal of suppression
induced by the 32 μM dose of O-1966.
1.10. Anti-Il-10 antibody blocks the increase of Tregs by O-1966. One mechanism of the
immunosuppression induced by these CB2 cannabinoid compounds that was uncovered last year
was the induction in the MLR of a class of T-cells called T-regulatory cells. These cells are
immunosuppressive. The current experiments tested the effect of blocking IL-10 on induction of
Tregs by O-1066, as IL-10 has been reported to up-regulate these cells (Groux et al., 1997,
Nature 389: 737-742). Tregs are detected by expression of a surface marker CD25 and another
antigen Foxp3. MLR cultures were treated with 2.5 or 5 μg/ml anti-IL-10 at T 0 and 24 h, and
cells harvested 48 h into the assay were stained and analyzed by flow cytometry for Tregs.
Figure 4 shows that the addition of both 2.5 and 5 μg/ml anti-IL-10 antibody completely blocked
the increase of CD25+Foxp3+ Tregs in the CD3+CD4+ population in cultures treated with 8 to 32
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μM O-1966. The addition of 5 μg/ml of an isotype control antibody did not affect the
suppression of proliferation or the increase of the percentage of Tregs at any dose of O-1966
tested. These results indicate that the increased levels of IL-10 seen in O-1966 treated cultures
are an important contributing mechanism for the suppression of cell proliferation and are
essential for the increase of Tregs in the MLR.
1.5. Combinations of a CB2 agonist and FK506, a standard anti-rejection therapy, in the MLR.
MLR assays were performed to continue the studies on combinations of O-1966 and FK506. The
combinations were calculated based on the effective dose of the compound resulting in 50% of
observed maximal suppression (ED 50 ). In the MLR, the ED 50 of O-1966 is approximately 10 μM
and the ED 50 of FK506 is approximately 5x10-9. The first assay retested two of the combinations
of O-1966 and FK506 ED 50 as reported previously, namely 1 O-1966: 1 FK506, and 50 O-1966:
1 FK506. In addition, another combination, 25 O-1966: 1 FK 506 was added. The second MLR
tested combinations using higher ratios of FK506 to O-1966 ED 50 . The combinations were 1 O1966: 20 FK506, and 1 O-1966: 5 FK506 (Figure 5). In both assays, the immunosuppression
produced by the combinations was different from that produced by either O-1966 or FK506
alone.
Aim 2: To test the effect of cannabinoids that are selective for the CB2 receptor, alone or in
combination, with standard therapies for graft rejection, on the course of graft rejection, using
mouse models of skin grafts and heart transplants.
2.1. A CB2 agonist prolongs skin grafts in mice. Last year we reported results on the effect of O1966 on skin grafts onto C57Bl/6 mice from C3HeB/FeJ mice. These two mouse strains are
histoincompatible. Starting one hour before grafting mice were treated by intraperitoneal
injection with either 5 mg/kg of the CB2 agonist, O-1966, or vehicle (volumes were based on
weight. but were usually approximately 0.2 ml) every other day for 14 days, and monitored for
graft rejection. Mice that received O-1966 treatment had viable grafts 2.7 days longer than
vehicle treated mice, which was statistically significant. Additional experiments were carried out
for dose response studies using 1 mg/kg and 10 mg/kg. In contrast to the 5 mg/kg dose, treatment
with 1 mg/kg or 10 mg/kg did not affect graft survival (Figure 6).
2.2. O-1966 treatment decreases CD4 expression in skin graft recipient mice. The expression of
CD4 on the cell surface of CD3+ cells was altered by O-1966 treatment in vivo. Figure 7A is a
representative comparative histogram of the fluorescent intensity of CD4 on T-cells from mice
treated with O-1966 or vehicle. O-1966 treatment caused a negative shift of fluorescence
intensity of these cells. Figure 7B presents the mean fluorescence intensity of CD4 from all
recipient mice (n=17 for each treatment group), and shows that the average intensity of CD4
expression on the cell surface is decreased.
Publications
Robinson RH, Meisser JJ, Breslow-Deckman JM, Gaughan J, Adler MW, Eisenstein TK (2013)
Cannabinoids inhibit T-cells via cannabinoid receptor 2 in an in vitro assay for graft rejection,
the mixed lymphocyte reaction. Journal of Neuroimmune Pharmacology: Online First. DOI
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10.1007/s11481-013-9485-1
Presentations
Robinson RH, Meissler JJ, Adler MW, Eisenstein TK. (2013) O-1966, a CB2-Selective
Cannabinoid Agonist, Blocks T-cell Activation in the Mixed Lymphocyte Reaction. Symposium
of the International Cannabinoid Research Society. Vancouver, Canada; oral presentation.
Robinson RH, Meissler JJ, Adler MW, Eisenstein TK. (2013) A CB2-Selective Cannabinoid
Increases Anti-inflammatory Response in the Mixed Lymphocyte Reaction. Philadelphia
Infection and Immunity Forum (American Society of Microbiology). Philadelphia, PA; poster
presentation.
Robinson RH, Meissler JJ, Adler MW, Eisenstein TK. (2012) CB2 Receptors and Graft
Rejection. Center for Substance Abuse Research Retreat. Philadelphia, PA; oral presentation.
Robinson RH, Meissler JJ, Adler MW, Eisenstein TK. (2012) A CB2-Selective Cannabinoid
Increases Anti-inflammatory Response in the Mixed Lymphocyte Reaction. Center for Substance
Abuse Research Retreat. Philadelphia, PA; poster presentation.
Figure 1. O-1966 inhibits both CD4+ and CD8+ T-cells. C57BL/6 splenocytes were sorted by flow
cytometry into CD3+CD4+, CD3+CD8+, or CD3-CD4-CD8- fractions. CD4+ fractions were treated with O1966 () or ethanol vehicle (). CD8+ fractions were treated with O-1966 () or vehicle (). Both
CD4+ and CD8+ fractions were treated with O-1966 () or vehicle (). Unseparated populations were
treated with O-1966 () or vehicle (), for 3 h. Treated CD4+ or CD8+ populations were combined with
untreated CD8+ or CD4+ , respectively, and then with the CD4-CD8- cell subset to reconstitute the normal
splenocyte population for carrying out the MLR. Data are the mean of 2 separate experiments, with
quadruplicate wells for each treatment. (ANOVA, CD4 treated versus CD8 treated)
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Figure 2. O-1966 treatment decreases CD4 expression in vitro. Wild-type (panels A and B) or CB2R k/o
(panels C and D) C57BL/6 responder splenocytes were pretreated for 3 h with O-1966 or ethanol vehicle.
MLR cultures were harvested at 48 hr and analyzed for CD4 expression on CD3+CD4+ populations by
flow cytometry. An equal number of CD3+CD4+ cells were analyzed for each treatment group.
Representative histograms of CD3+ cells from cultures treated with 32 μM O-1966 (gray filled) compared
to vehicle treated cells (white filled) with responder cells from wild-type (Panel A) and CB2R k/o (Panel
C). Mean Fluorescence Intensity (MFI) of CD4 in CD3+CD4+ populations from MLR cultures that
), ethanol vehicle (
), 8 μM O-1966 (
), 16 μM O-1966
received no treatment (
), or 32 μM O-1966 (
). Data are mean ± S.E.M. of 3 separate experiments. * p < 0.05, ** p <
(
0.01 (Student’s t-test, O-1966 versus vehicle). Values for vehicle are not significantly different from no
treatment.
Figure 3. O-1966 decreases levels of activated nuclear forms of NF-κB and NFAT in T-cells. Splenocytes from
CB2R k/o mice (open symbols) or wild-type mice (closed symbols) were treated for 3 h with O-1966 (WT: ,
k/o:) or ethanol vehicle (WT: , k/o: ) and then added to a plate coated with 25 μg anti-CD3 antibody/well
and soluble anti-CD28 antibody (0.4 μg/well) or left unstimulated ( WT: , k/o: ). The cultures were
incubated for 18 h and cultures were harvested and nuclear proteins extracted. Levels of activated nuclear
NFκB (Panel A) and NFAT ( Panel B) were measured using a TransAM ® Transcription Factor ELISA. Data
are the mean ± S.E.M. of 3 separate experiments with triplicate wells for each treatment. *p < 0.01, **p <
0.001. (ANOVA, WT versus k/o). Values for vehicle are not significantly different than no treatment.
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Figure 4. Anti-IL-10 antibody blocks the increase of Tregs by O-1966. Panels A and B: Representative
scatterplots of MLR pretreated with ethanol vehicle or 8, 16, or 32 μM O-1966. Panel B was treated with
5 μg/ml anti-IL-10 antibody. Cells were stained with antibodies for CD25 and Foxp3 and are gated on
live CD4+ cells. Quadrants are expressed as a percentage of total live CD4+ cells. Panel C: Average
number of CD25+Foxp3+ Tregs as a percentage of total live CD4+ cells from 3 experiments from cultures
pretreated as indicated along with with no antibody ( ), 5 μg/ml isotype control ( ), 2.5 μg/ml anti), or 5 μg/ml anti-IL-10 antibody (
), harvested 48 hr into culture and analyzed by
IL-10 antibody (
flow cytometry for percentage of Tregs. Data are mean percentage ± S.E.M. CD4+Foxp3+ Tregs of total
live CD4+ in the culture and the average of 3 separate experiments. *p < 0.01 **p < 0.001 (two-way
ANOVA, anti-IL-10 versus isotype antibody)
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Figure 5. Immunosuppression in the Mixed Lymphocyte Reaction (MLR) assay By O-1966, FK506
(tacrolimus) and combinations of both.
Figure 6. O-1966 treatment prolongs skin graft viability. Donor C3HeB/FeJ flank skin was transplanted to
the back of a recipient C57BL/6J mice, sutured, and bandaged. Doses of O-1966 or vehicle (0.03%
ethanol and 0.03% cremophor in saline) were injected i.p. every other day from 1 hour pre-op to 14 days
post-op. On Day 7 bandages were removed and grafts were monitored for rejection. Percent graft survival
of mice treated with A: 1 mg/kg O-1966 (), or vehicle (), B: 5 mg/kg O-1966 () or vehicle (), or
C: 10 mg/kg O-1966 () or vehicle (). Panels A and C are mean of 1 experiment (n=8 per group) and
Panel B is mean of 2 experiments (n=17 per group). ** p < 0.01 (Log-rank (Mantel-Cox) test (O-1966
versus vehicle)
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Figure 7. O-1966 treatment decreases CD4 expression in skin graft recipient mice. Donor C3HeB/FeJ
flank skin was transplanted to the back of a recipient C57BL/6J mice, sutured, and bandaged. Doses of O1966 (5 mg/kg) or vehicle (0.03% ethanol and 0.03% cremophor in saline) were injected i.p. every other
day from 1 hour pre-op to 14 days post-op. Splenocytes were harvested on day 14, stained for CD4, and
analyzed by flow cytometry. Panel A: Representative histograms of CD4 expression on CD3+ cells from
mice treated with O-1966 (gray filled) or vehicle (white filled). Panel B: Mean Fluorescence Intensity
(MFI) of CD4 in CD3+CD4+ populations from mice treated with O-1966 ( ) or vehicle ( ). Data are
mean of 2 experiments (n=17 for both groups). ** p < 0.01 (Student’s t-test, O-1966 versus vehicle).
Research Project 7: Project Title and Purpose
Differentiation Therapy for Leukemia – The purpose of the project is to evaluate the therapeutic
efficacy of the peptide, angiocidin, for the treatment of leukemia. We propose to determine if
angiocidin can block the engraftment of human leukemia cells in a mouse model of leukemia.
We will evaluate the effect of angiocidin on the engraftment of at least three patient leukemia
cells. These will be patients with acute myeloid leukemia.
Duration of Project
1/1/2011 – 12/31/2012
Project Overview
This research project will have a direct and positive impact on leukemia cancer research since it
proposes an entirely novel and unique strategy for improved treatment of leukemia cancer which
can readily be applied for clinical evaluation. Acute myelogenous leukemia (AML) is a
hematogenous malignancy in which the bone marrow makes abnormal myeloblasts that fail to
differentiate into mature leukocytes. These myeloblasts lack immune function, are invasive and
can quickly lead to mortality. Current treatment options involve chemotherapy and durable
remissions are low requiring stem cell transplants to achieve a cure. We propose that
recombinant angiocidin, a human protein that is easily expressed and purified from bacteria and
previously shown to possess potent anti-tumor activity can be used to treat AML based on our
novel discovery that angiocidin was able to differentiate a highly studied monocytic AML cell
line (THP-1) into normal-like macrophage cells. In this project, we plan to evaluate the antitumor activity of angiocidin against three human AML primary cells in a mouse model of
leukemia.
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Principal Investigator
George P. Tuszynski, PhD
Professor of Neuroscience
Temple University
3500 North Broad Street
Philadelphia, PA 19140
Other Participating Researchers
Vicki L. Rothman, BS – employed by Temple University
Expected Research Outcomes and Benefits
An estimated 245,225 people in the United States are living with, or are in remission from,
leukemia. Approximately 21,870 deaths in the United States were attributed to leukemia in
2009. The most common types of leukemia in adults are acute myelogenous leukemia (AML),
with approximately 12,810 new cases in 2009, and chronic lymphocytic leukemia (CLL), with
about 15,490 new cases in 2010. In 2009, in the United States, leukemia was the fifth most
common cause of cancer deaths in men and the seventh most common in women. AML
accounts for 80% of all acute leukemia in adults. The trend in overall incidence is slowly
increasing. In general, 60% to 80% of all treated AML patients achieve initial remission, but 5year survival rate is only 15% in the adult population of AML and has been stably or slowly
increasing. Standard AML therapy requires intensive combination chemotherapy which leads to
significant treatment-related toxicity, especially in elderly patients. Therefore, new therapeutic
options for the treatment of leukemia are urgently needed. Our therapeutic targets unique
proteins in the provisional tumor matrix and tumor surface such as α2β1 integrin, and previously
published data indicate that systemically administered angiocidin homes to both the tumor and
its vasculature and should be equally effective against the primary tumor as well as tumor
metastases. Our new data provide evidence that angiocidin may have the potential to treat
systemic circulating hematogenous tumors by differentiating them non-toxically into normal
cells. Therefore, these therapeutics could provide an alternative non-toxic treatment choice for
leukemia patients or could also be used in combination with currently approved cytostatic drugs
for the treatment of leukemia.
Summary of Research Completed
AML is a malignant proliferative disorder in which blast stage cells fail to terminally
differentiate and accumulate in the blood and bone marrow. AML represents a significant health
risk in the general population with rising incidence in aging adults. Standard AML therapy
requires intensive combination chemotherapy with a low rate of durable remission, often requires
stem cell transplantation, and is associated with significant treatment-related toxicity, especially
in elderly patients. Thus, many elderly patients with AML do not receive therapy with curative
intent. Therefore, new therapeutic options for the treatment of AML are urgently needed.
Four experimental AML cell lines (THP-1, Mono-mac-1, HL-60 and MV4-11) and 5 of 10
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primary human AML samples showed evidence of differentiation when cultured in vitro for 24
hours with 10 µg/mL angiocidin. Differentiation was assessed by flow cytometry measuring the
increase in expression of cell surface markers characteristic of normal macrophages. Three
macrophage markers were evaluated: CD14, a major receptor for the innate immune system,
CD15, a neutrophil phagocytosis receptor, and CD54 or ICAM-1, the major adhesion receptor
used for endothelial transmigration. We found that angiocidin up-regulated these macrophage
receptors by more than 10-fold in 7-70% of the respective AML cell populations, attesting to its
capacity to differentiate AML cell lines and primary human AML samples into macrophage-like
phenotypes. Additionally, we found that angiocidin promoted secretion of a cocktail of
cytokines from the cell lines as well as patient cells. In THP-1 cells, we previously showed that
these cytokines mediated the differentiation induced by angiocidin.
We next evaluated the effect of angiocidin on a xenotransplanted primary human AML sample
engrafted in NSG mice. We found angiocidin monotherapy reduced the human AML burden in
bone marrow by 63% relative to control, angiocidin + arabinofuranosyl cytidine (Ara-C)
combination therapy reduced human AML in bone marrow by 79%. We believe the combination
of in vitro data supporting the capacity of angiocidin to drive differentiation in multiple AML
cell lines and primary human AML samples and its activity in a xenotransplantation model that
reproduces the genetic diversity of human disease is significant. These observations support the
continued evaluation and development of angiocidin as a potential novel, non-toxic therapy for
AML.
Research Project 8: Project Title and Purpose
Social Influences on Alcohol Consumption in Adolescent Versus Adult Mice – The presence of
peers is associated with increased risk taking during adolescence, including alcohol and drug use,
but the underlying neural mechanisms for this “peer effect” are not understood. Importantly, a
similar effect is not seen among adults. Previous research by one of the present investigators
indicates that the effect may operate via the impact of peer presence on the adolescent brain’s
reward processing system, such that the presence of peers increases the salience of other rewards
and thereby biases individuals toward the potential benefits of a risky choice. This project will
examine the feasibility of modeling this process in rodents, and will examine the differential
impact of “peer” presence on alcohol consumption among juvenile and adult mice.
Duration of Project
7/1/2011 – 12/31/2012
Project Overview
The presence of peers increases risky behavior among adolescents, but not adults. This “peer
effect” is likely due to the impact of the presence of peers on reward processing and is mediated
neurally by hyperactivation of brain regions known to be associated with reward sensitivity.
Understanding the processes through which peers influence adolescent risk-taking has important
implications for public health, since a preponderance of adolescent recklessness, including binge
drinking, occurs in the presence of their friends. To date, experimental documentation of the peer
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effect has relied on simulated outcome variables that mimic real-world risky behavior. It is
important to show that the presence of peers affects actual risky behavior, such as drinking
alcohol, but legal and ethical issues preclude the conduct of experiments in which alcohol
consumption is compared among teenagers when they are alone versus with their peers.
However, this is a topic to which animal models can be readily applied, and lessons learned from
such work can inform our understanding of human behavior and development.
All mammals experience a stage of development comparable to the biological stage in humans
that is labeled adolescence. Importantly, the behavior of adolescent and adult rodents is quite
different, as are their responses to alcohol. For example, social interaction among adolescent,
but not adult, rats is facilitated in familiar environments by low doses of alcohol; whereas
inhibition of social interaction is seen at moderate doses of alcohol in adults and only at high
doses in adolescents. Adolescent and adult rodents also differ with respect to voluntary
consumption of alcohol, with adolescent rats and mice consuming more alcohol than adult rats
and mice. A key unanswered question is whether there is an interaction between social setting
and alcohol consumption among rodents. The aim of the proposed research is to test the
hypothesis that adolescent mice will consume more alcohol when the alcohol is available in a
social setting than they will consume when they are alone. Being able to evaluate social
behavior and alcohol consumption simultaneously in rodents will provide valuable novel
information that will aid in understanding the factors involved in alcohol consumption in
adolescent humans.
Principal Investigator
Laurence Steinberg, PhD
Distinguished University Professor
Temple University
Department of Psychology
1701 N. 13th Street, Weiss Hall
Temple University
Philadelphia, PA 19122
Other Participating Researchers
Thomas J. Gould, PhD; Sheree F. Logue, PhD – employed by Temple University
Expected Research Outcomes and Benefits
The chief contributors to morbidity and mortality among adolescents are self-inflicted, and one
of the most common threats to adolescent health and well-being is alcohol use. Adolescents
typically engage in risky behavior (including drinking) when they are with their peers. Previous
experimental research has demonstrated that effect of peers on adolescent risk taking is mediated
through the impact of peer presence on the brain’s reward processing system, but this “peer
effect” only has been demonstrated experimentally using laboratory tasks that merely simulate
real-world risk taking. It is important to know whether a comparable peer effect is seen when the
outcome in question is a genuine, health-compromising risk behavior, such as drinking. Legal
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and ethical issues, however, preclude the possibility of studying peer effects on adolescent
alcohol use experimentally. We believe that the peer effect on adolescent alcohol consumption
can be profitably studied using animal models, which permit a more detailed examination of the
underlying neural processes. In order to fully study this phenomenon, we must first demonstrate
that we can create it experimentally in a controlled laboratory context. The expected outcome of
this project therefore is the demonstration of the feasibility of studying peer influences on
adolescent alcohol consumption using mice. The long-term benefit of this program of research is
a fuller understanding of factors that influence alcohol use during adolescence and, ultimately,
more effective preventive interventions to reduce alcohol use and abuse.
Summary of Research Completed
During the current reporting period the project was completed. A sample of mice were raised in
same-sex triads and were tested for alcohol consumption either as juveniles or as adults, with
half in each age group tested alone and half tested with their cagemates. The presence of “peers”
increased alcohol consumption among adolescent mice, but not adults. The peer effect on human
adolescent reward-seeking may reflect a hard-wired, evolutionarily conserved process through
which the presence of agemates increases individuals’ sensitivity to potential rewards in their
immediate environment.
Procedure:
The mouse model we developed to assess the impact of peer presence on alcohol consumption
used juvenile and adult C57BL/6J mice. This strain was chosen because both male and female
C57BL/6J mice are known to self-administer ethanol (Yoneyama, Crabbe, Ford, Murillo, &
Finn, 2008; Belknap, Crabbe, & Young, 1993).
The sample included 86 animals, half male and half female. The mice were housed in same sex
triads from weaning at postnatal day 21 to testing, and were tested for alcohol consumption in a
novel environment, either between postnatal day 28 and 30 (the juvenile period in mice) or
between postnatal day 84 and 86 (adulthood). As in the human studies that motivated the current
experiment, exposure to peers was experimentally manipulated using random assignment, with
half of the mice in each age group tested alone (individual condition; Ns=11 male juveniles, 10
female juveniles, 9 male adults, and 9 female adults), and half tested in triads (peer condition;
Ns=9 male juveniles, 12 female juveniles, 11 male adults, and 12 female adults). The video from
one group of male juvenile mice tested in the social condition was unscorable.
Prior to testing, the fur on the back of each mouse was lightened with a peroxide solution to
allow each identification of each mouse in a triad. Mice were given free access to a 5% v/v
ethanol solution for 45 minutes. The ethanol solution was administered through 4 sipper tubes
arranged around the perimeter of a Plexiglas chamber (20x30x20cm). The chamber had two
identical compartments that were separated by an arched doorway with two sipper tubes
available in each compartment. To test the effect of peer presence on voluntary ethanol
consumption, mice were either placed in the chamber alone or with their two cagemates. Because
the mice tested in triads were in cages with four available sipper tubes, individuals did not have
to complete for access to ethanol.
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Each session was video recorded for 45 minutes. Video tapes were scored for each mouse’s time
spent drinking (in seconds), number of visits to any of the four drinking tubes, and, as a measure
of basic exploratory behavior, number of transitions through the arched doorway (crosses). For
the mice tested with peers, all of the measures were scored for each mouse independently.
Duration of drinking bouts was calculated by dividing each individual animal’s total time
drinking by the number of visits that animal made to a drinking tube.
Results:
Impact of Peers on Juveniles’ and Adults’ Alcohol Consumption
Time spent drinking. An omnibus analysis of variance (ANOVA) examining the impact of age,
sex, and test condition on individuals’ time spent drinking was significant [F(7, 85) = 4.05, p =
.001; η p = .27]. Time spent drinking differed across ages [F(1,85) = 13.62, p =.000; η p = .15],
with juvenile mice (M = 127.28, SD = 61.87) spending more time drinking than adult mice (M =
90.86, SD = 37.48). Neither the main effect for test condition nor the main effect of sex on time
spent drinking was significant (p > .05). Importantly, and as hypothesized, the interaction
between age and test condition was significant [F(1, 85) = 4.35,p =.040; η p = .05]. Juvenile mice
tested with their peers (M = 147.71, SD = 63.74) spent significantly more time drinking than
individually tested juvenile mice (M = 107.77, SD = 54.50), whereas adult mice in the peer (M =
90.22, SD = 33.38) and individual (M = 91.60, SD = 42.60) conditions did not differ. There was
also an interaction between test condition and sex on time spent drinking [F(1,85) = 3.91, p
=.052; η p = .05. ], with the peer manipulation having a stronger impact on males than females,
but the three-way interaction among age, sex, and test condition was not significant (p>.05).
Duration of drinking bouts. The omnibus ANOVA for duration of drinking bouts also was
significant, [F(7, 85) = 8.54, p =.000; η p = .43] (Fig. 2). There was a main effect of age, (F(1,85)
= 37.53, p =.000; η p = .33), wherein juvenile mice (M = 1.75, SD = .75) had longer drinking
bouts than adult mice (M = 1.05, SD = .29). Test condition also had a significant impact on
duration of drinking bouts (F(1,85) = 7.86, p =.006; η p = .09) with mice tested in the social
condition (M = 1.54, SD = .71) averaging longer drinking bouts than mice in the individual
condition (M = 1.24, SD = .57). There was a main effect for sex (F(1, 85) = 4.19, p =.044; η p =
.05), wherein females (M = 1.53, SD = .70) had longer drinking bouts than males (M = 1.25, SD
= .59). As with time spent drinking, the interaction between age and test condition was
significant, (F(1,85) = 6.40 p =.013; η p =.08). Juvenile mice tested in the social condition (M =
2.05, SD = .74) averaged longer duration drinking bouts than juvenile mice tested individually
(M = 1.43, SD = .63), whereas adult mice tested in the social (M = 1.08, SD = .15), and
individual (M = 1.03, SD = .40) conditions had the shortest drinking bouts. No other interactions
were significant (p > .05).
Number of drinks. Number of drinks was counted when the mouse began drinking from the
sipper tube and then moved away or ceased drinking. The omnibus ANOVA for number of
drinks was not significant (F(7,85) = .73, p =.647; η p = .06).
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Peer Influences on Juveniles’ and Adults’ Locomotor Activity
Because the marked difference in average time spent drinking and duration of drinking bouts
between juvenile mice tested with their peers versus those tested individually might be due to a
higher level of overall activity in the peer condition, we conducted an omnibus ANOVA for
locomotor activity level (i.e., crosses between compartments), which was significant [F(7, 85) =
3.65, p =.002; η p = .25], with main effects found for age [F(1,85) = 15.60, p =.000; η p = .17) and
sex [F(1,85) = 5.65, p =.020; η p = .07); juvenile mice (M = 138.81, SD = 33.71) were less active
than adult mice (M = 169.97, SD = 38.41) and male mice (M = 145.14, SD = 34.55) were less
active than female mice (M = 163.55, SD = 41.71). However, and importantly, there was no
effect of test condition on activity level (p > .05) and no significant interactions (p > .05).
Differences in overall activity could also have impacted the frequency of visits to a drinking
spout, but, as noted earlier, there were no differences across any of the groups on this variable.
Preliminary Evidence for Sex Differences in the Peer Effect
The significant interaction between sex and test condition on time spent drinking (but not on
duration of drinking bouts) raises the question of whether the presence of peers differentially
affects male and female juvenile mice; although the three-way interaction among age, sex, and
condition in the prediction of time spent drinking did not reach significance, tests of three-way
interactions are often underpowered, and inspection of the results when graphed (see Figure 1)
prompted us to examine this issue further. Accordingly, we conducted two-way (age by
condition) ANOVAs to analyze the data for each sex separately.
Analysis of the males’ total drinking time demonstrated the same pattern as for the sample as a
whole [omnibus ANOVA: F(3,42) = 6.29, p =.001; η p = .33]. Time spent drinking differed
between male juvenile and adult mice (F(1,42) = 8.97, p =.005; η p = .19), with juvenile males (M
= 123.86, SD = 72.46) spending more time drinking than adult males (M = 81.18, SD = 41.32).
In addition, there was a main effect of test condition (F(1,42) = 6.05, p =.018; η p = .13), with
male mice tested with peers (M = 120.80, SD = 71.42) spending significantly more time drinking
than male mice tested individually (M = 85.70, SD = 47.84). And, as in the sample as a whole,
the interaction between age and test condition was significant (F(1,42) = 6.16, p =.018; η p = .14).
Juvenile males tested in the peer condition (M = 169.44, SD = 77.36) spent significantly more
time drinking than individually tested juvenile males (M = 89.67, SD = 47.11), whereas adult
males tested in the peer condition (M = 81.00, SD = 32.12) and individually tested adult males
(M = 81.36, SD = 50.54) did not differ. In contrast, analysis of the females’ drinking time did
not yield a significant omnibus ANOVA [F(3,42) = 1.68, p =.188; η p = .11], and the only
significant contrast effect was in the difference between juvenile females (M = 130.55, SD =
51.29) and adult females (M = 101.00, SD = 30.77) [t-test: t(41) = 2.28, p =.017]. A similar
pattern of results was found for duration of drinking bouts (stronger evidence of peer effect on
juveniles but not adults among males than females) but not on number of drinks.
A paper reporting these findings has been accepted for publication in Developmental Science:
Logue, S., Chein, J., Gould, T., Holliday, E., & Steinberg, L. (in press). Adolescent mice, unlike
adults, consume more alcohol in the presence of peers than alone. Developmental
Science.
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Figure 1. Juvenile mice, but not adults, spend more time drinking alcohol in the presence of
peers than when alone.
Research Project 9: Project Title and Purpose
The Role of G Protein-Coupled Receptor Associated Sorting Protein 1 (GASP-1) in Breast
Cancer Progression – The purpose of the project will be to evaluate the prognostic significance
of GASP-1 in breast cancer detection and diagnosis.
Duration of Project
1/1/2011 – 12/31/2012
Project Overview
The “2-D High Performance Liquid Electrophoresis” (2-D HPLE) technology employing
polyvinylidene fluoride (PVDF) membranes instead of polyacrylamide gels for the high
resolution separation of protein complexes is a totally novel protein separation system. This new
system possesses many advantages over the currently used 2D-SDS-PAGE procedure. Among
the advantages are high resolution, reproducibility, sensitivity, speed and the capacity to detect
protein-protein interactions under non-denaturing conditions. 2-D HPLE has the capacity to
separate both hydrophobic and hydrophilic serum albumin complexes into distinct, well-resolved
spots which cannot be accomplished by conventional 2D-SDS-PAGE. The separation of
albumin complexes in 2-D HPLE is based on their net charge or isoelectric points (pI). The
association of a newly produced cancer protein (or its fragments) with a pre-existing albumin
complex changes its pI and this new complex will migrate to a different location on the PVDF
membrane, allowing its detection among hundreds of already present albumin complexes.
Therefore, 2-D HPLE technology can detect early “biochemical events” occurring in breast
tissue that lead to breast cancer. To illustrate the potential utility of 2-D HPLE for detecting
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breast cancer, we have compared the serum albumin complex profiles of sera from 5 matched
normal volunteers and 3 patients each with Early Stage 1, and Stages 1, 2, 3, 4 breast cancer (15
patients) and reproducibly found a series of new protein complexes appearing in the cancer sera
but absent in normal sera. The validity of our 2-D HPLE approach was confirmed by the
identification of one cancer protein (biomarker) from Stage 1 sera that is over-expressed in tumor
tissue with respect to adjacent normal tissue in all 7 cases of breast cancer patients. This
protein, G- protein coupled receptor-associated sorting protein 1 (GASP-1), has an important
function in mediating the trafficking of G-protein coupled receptors and targeting them for
degradation in lysosomes. In this project the expression of GASP-1 will be evaluated in a series
of breast cancer tumor sections and its expression will be correlated with the severity of the
disease.
Principal Investigator
George P. Tuszynski, PhD
Professor of Neuroscience
Temple University
3500 North Broad Street
Philadelphia, PA 19140
Other Participating Researchers
Vicki L. Rothman, BS – employed by Temple University
Expected Research Outcomes and Benefits
We anticipate that increased GASP-1 expression will correlate with worsening stage and
histological grade. We additionally anticipate that hyperplastic lesions, especially from those
patients that eventually develop carcinoma, will show increased GASP-1 expression. The health
benefit is that a new breast cancer biomarker will be described that can be used for the early
detection of cancer.
Summary of Research Completed
One paper has been published as a result of this research support:
Zheng, X. et al. Experimental and Molecular Pathology 93 (2012) 111–115
Using an innovative “2-D high performance liquid electrophoresis” (2-D HPLE) technology we
identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1
(GASP-1) was present in the sera of breast cancer patients and was over-expressed in early and
late stage breast tumors (Tuszynski, G.P. et al., 2011). The 2-D HPLE procedure separates
protein fragments complexed to serum albumin in sera from cancer patients according to the
charge of the complex. Using mass spectrometry we identified a fragment of G-protein receptor
associated sorting protein 1(GASP-1) in the albumin complex. An antibody was raised against
the GASP-1 fragment and used to evaluate the expression of GASP-1 in tumor tissue.
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In this study we further investigated the significance of GASP-1 as a tumor marker by
investigating the expression GASP-1 in different kinds of tumors as well as in the sera of patients
with various cancers. Over expression of GASP-1 was detected in brain, pancreatic, and breast
cancers as compared to their respective normal tissues as assessed by immunohistochemical
staining of tissue arrays using a peptide GASP-1 antibody. We found that across these cancers,
GASP-1 was expressed approximately 10 fold more in the cancer as compared to normal tissue.
The increase in GASP-1 expression was also seen in hyperplastic and inflammatory lesions of
breast and pancreatic cancers as compared to normal tissue. GASP-1 was primarily expressed in
the tumor epithelium of the epithelial-derived cancers and in the transformed glial cells of the
brain tumors. Using a sensitive “competitive ELISA” for GASP-1, we found that sera from
patients with brain, liver, breast and lung cancers expressed 4–7 fold more GASP-1 peptide than
sera from normal healthy individuals. These studies identify GASP-1 as a potential new serum
and tumor biomarker for several cancers and suggest that GASP-1 may be a novel target for
development of cancer therapeutics.
Research Project 10: Project Title and Purpose
p38SJ, a Novel DING Protein from St. John's Wort Inhibits Tumor Cell Growth via Induction of
Cell Cycle Arrest – p38SJ is a novel DING phosphatase isolated from St. John's Wort. Our
preliminary data demonstrate that p38SJ inhibits proliferation of human T98G glioblastoma
cells, as increased toxicity was observed upon treatment of cells with p38SJ. Furthermore, pretreatment of rapidly proliferating U87MG cells with p38SJ leads to cell growth delay and
induces cell cycle arrest in G0/G1 phase. Our observations provide evidence that p38SJ alters
signaling pathways that impact cell growth and proliferation. The purpose of this project is to
demonstrate that treatment of primary glioblastoma tumors from patients with p38SJ will result
in the suppression of tumor growth. Thus p38SJ can be developed and used as an anti-cancer
agent.
Duration of Project
1/1/2011 – 6/30/2012
Summary of Research Completed
This project ended during a prior state fiscal year. For additional information, please refer to the
Commonwealth Universal Research Enhancement C.U.R.E. Annual Reports on the Department's
Tobacco Settlement/Act 77 web page at http://www.health.state.pa.us/cure.
Research Project 11: Project Title and Purpose
Body Sensor Networks and Their Applications in Maternal Fetal Monitoring – Assessment of
fetal health during pregnancy constitutes a very important task of modern obstetrics. It is applied
in high risk patients in the third trimester and in almost all patients during labor and delivery.
Currently, the monitoring devices needed for fetal heart rate (FHR) and uterine contractions are
hardwired to a large monitor (about 15 lbs), and require the patient to remain relatively immobile
in order for the monitor to function optimally and continuously. Many women feel more
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comfortable if they are able to move during labor, and therefore feel constrained by the monitor.
The project seeks to design a body sensor network (BSN), a network consisting of one or more
on-body sensing units coupled with a smart local processing unit, to allow normal mobility
during the monitored period. The BSN system has to be both energy-efficient and secure.
Anticipated Duration of Project
7/18/2011 – 08/31/2014
Project Overview
A body sensor network (BSN) consists of one or more on-body sensing units coupled with a
smart local processing unit. The on-body sensors are placed on a person's body to collect
physiological information, such as blood pressure, body temperature, heart rate, oxygen
saturation, and so on. These data are transmitted periodically using a short-range wireless
channel, to the smart local processing unit, such as a microprocessor unit or a mobile device, like
smartphones, for temporary storage and impending long range data transmission. When
appropriate or necessary, the processing unit will relay the collected data to remote centralized
servers. Medical professionals can use the aggregated data for remote diagnosis and treatment.
The objective of this proposed project is to build a BSN platform using state-of-the-art wearable
wireless technology for the purpose of remotely monitoring the health and well-being of a person
in care. Our project will consist of both a working BSN prototype, as well as a comprehensive
suite of communication and security protocols to ensure reliable delivery and safe transmission
of the medical data according to HIPPA regulations.
The specific research aims are (1) Design a working BSN prototype with wearable on-body
sensors which provide unobtrusive support for daily life based on context and the situation of a
patient or an assisted elder. (2) Design algorithms to schedule the sleep/wake cycle of the sensors
together with data transmission so as to reduce power consumption while being responsive to the
application demands. (3) Design security framework to safeguard the data collected at various
states of a BSN application cycle.
Principal Investigator
Jie Wu, PhD
Chair and Professor
Department of Computer and Information Sciences
Temple University
1805 N. Broad St., Room 302
Philadelphia, PA 19122
Other Participating Researchers
Chiu C. Tan, PhD; Li Bai, PhD; Dimitrios S. Mastrogiannis, MD, PhD, MBA – employed by
Temple University
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Expected Research Outcomes and Benefits
We expect to develop one or more integrated on-body sensor units that combine various sensing
capabilities into a small hardware package. Custom designed software will be developed on
commercial smartphones, such as the iPhone, Windows Mobile phones, or Android phones, to
allow the on-body units to interface with the smartphone. Our prototype BSN system is intended
to have an extensible design to accommodate future developments, such as new wireless
frequencies.
We also expect to develop an algorithmic framework to better understand the tradeoffs between
the responsiveness and power consumption. This framework will be used to design scheduling
policies to regulate the different wireless communications and duty cycles of the on-body units
and the local processing unit. A BSN simulator will be developed to test the performance of
various algorithms.
A comprehensive study of the security requirements for BSNs to manage medical data in
accordance with the necessary regulations will be completed in anticipation of clinical
experiments on human volunteers. We also expect to adapt existing security protocols and design
new ones, as needed, to satisfy any additional requirements. Using our prototype design, we
expect to conduct isolated experiments designed to collect system microbenchmarks, such as
power consumption and data fidelity, to improve our system design.
This project has the potential for significant benefits to the research community. We intend to
make freely available the hardware schematics and software packages to help other researchers
develop their own BSN prototypes. A depository database on collected trace data (energy
consumption, physiological data, and so on) will also be made available to researchers.
Appropriate steps on concealing any personal identifiable information (PII) will be undertaken to
protect the privacy of the experimental volunteers.
Summary of Research Completed
In the second year of the project, we have completed (1) the hardware integration of the sensors
with the smartphone, and (2) the software necessary for storage, transmission and retrieval.
Details are given below.
Based on the evaluation of possible architecture designs completed in the first year of this
project, we have settled on a Bluetooth based design to integrate the sensors to the phone. The
reading of data from the sensors requires I/O capability that is not available on consumer devices
like smart phones and tablets. For this, an interface for the sensors must be created. Different
sensors will have a number of output protocols. Even though usually we just need to sense
analog voltages, the sensor interface must be able to support a number of methods like Universal
Asynchronous Receiver Transmitter (UART), Pulse Width Modulation (PWM), and Serial
Peripheral Interface (SPI), as well as some wireless means such as 802.11, 801.15.4, and
Bluetooth. There needs to be an A/D with a high resolution, and a general purpose I/O for
interaction with arbitrary digital input. We used a PIC24 microcontroller with IOIO firmware.
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The IOIO API allowed use to easily interface the microcontroller with an Android device. We
have successfully implemented hardware devices and sensors to measure and transmit patient’s
vital signs such as heart rate, blood pressure and uterine contractions, etc. We can wirelessly
transmit data to an Android smartphone using standard Bluetooth 2.0 communication. Also, this
data can be easily measured and collected without any intrusive devices. Figure 1 shows how the
IOIO is attached to a regular Bluetooth module. The tocodynamometer and ultrasound transducer
(labeled 3 and 4 in the Figure) is connected to the Bluetooth module. The data collected by the
sensors are transmitted to the smartphone via the Bluetooth module labeled 2 in Figure 1.
Based on our initial testing of the hardware, we found that there is a large variety of
commercially available wireless enabled medical sensors on the market. A large portion of these
operate with a standard Bluetooth protocol. However, depending on the manufacturer and the
age of the device, they may use different Bluetooth profiles. For example, older FDA approved
devices used Bluetooth’s Serial Port Profile (SPP) to emulate a serial (COM) port from device to
base station in order to transmit data. As a result, no standard data format was used, and
interoperability was not possible. We need to choose one of these profiles, while maintaining the
required throughput, latency, and power efficiency metrics to maximize the system’s lifetime.
Most importantly, the sensors, in collaboration with the interface device, need to adaptively
respond to changing conditions as they monitor events. Our next step will be to develop possible
solutions to implement this adaptive behavior. We are using the emerging, Bluetooth Low
Energy (BLE) as a starting point since it offers a star network topology, with master and slave
nodes, that makes it possible to have application control over some of the parameters associated
with the low energy operation of the protocol through the Host Controller Interface (HCI).
We have also completed the software design to collect, transmit, and display the collected
information. Our software allows the patient a limited number of options; control over when to
start and end the monitoring session, when to transmit the data to the medical facility, observe
the collected data in real time, and contact information for the physician. The software system
has notably limited configuration options. Specifically, the phone does not require the patient to
input where the data is to be sent, how the data should be transmitted, or any specific security
settings. This is done to simplify the monitoring task for the user. This simple GUI design has
the benefit of making it easier to train people to use the system, as well as reduce the chances of
human error in configuration. Figure 2 shows the different GUI available to the user. In addition,
we have also implemented several security measures to protect the data collected by the phone.
We used the Near Field Communication (NFC) wireless radio to regulate access to the phone.
Rather than relying on the user to remember their password, the user will use an RFID enabled
card to gain access to the phone. The data from the sensors are immediately encrypted when
received by the phone. Encryption is performed using public key cryptography, where the phone
only stores the public key to encrypt the data. The corresponding secret key is off-site. The
purpose is to use the hospital’s existing access control system to regulate who has access to the
decrypted data.
The next step is to develop the algorithms necessary for the phone to detect potentially medically
dangerous events independently. After the smartphone collects the necessary data, it will upload
to the hospital’s server. However, in conditions where the data indicate the patient may be in
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danger, the phone should immediately notify the patient to go to the hospital, instead of waiting
for the data to be transmitted to, and analyzed in, a hospital. This is difficult to do, since in order
to improve the safety, we will need the monitoring system to replicate some of the functions
currently performed by the physician. In a clinical setting, the physician has to make multiple
decisions based on the recorded data. However, many of these decisions are based on the
doctor’s experience and the patient’s condition, and can be rather complex. To address this
complexity, we are planning to explore ways to reduce the complexity. One promising avenue is
to use a formal and model-based analysis to determine the software design and development. A
state diagram model can be used to describe doctor’s diagnostic process based on test results.
The state diagram consists of a set of states (several partial diagnostic results and several final
diagnostic results with outputs). State transitions occur based on transition conditions (i.e., test
results). At the same time, we need to control the false positives and false negatives. Different
patients, depending on their underlying medical condition, will require modifications to a formal
model. Our system has two observing points (1) local and real-time observing point displayed
under the Android platform and (2) remote and asynchronous observing point at the hospital’s
end in the form of a PHR like Indivo X. All test results will be stored remotely at PHR, while
urgent abnormal test results will be displayed locally for immediate attentions. Both false
positive (non-urgent abnormal results shown locally) and false negative (urgent abnormal results
not shown locally) cases can occur. We are planning to start with a relatively informal end of the
spectrum, modeling language such as UML. UML is easy to learn and use among three teams
from three disciplines: medical, science, and engineering. It will be used to describe high-level
design. This approach facilitates the early discovery of design problems. Two sets of design
validation will be used. The first is the completeness check. This process validates that all
possible test results are represented as transition conditions and all states are reachable (i.e. live
states). The second is the minimization check. This process detects, and then, removes all
redundant states. This is done by finding a set of maximal compatible states (equivalent state
groups) that behave consistently for all inputs. By consistent behavior, we mean that all states in
the same equivalent class group will transit to the same next state group for a given input.
As of June 30, 2013, we have published 3 conference papers and 1 journal paper. Two of the
conference papers were accepted and published in the IEEE International Conference on EHealth Networking, Application and Services (HealthCom). The two papers were presented in
Beijing, China in October 2012. One of the conference papers was published in International
Conference on Body Area Networks (Bodynets), and we presented that paper in Oslo, Norway in
September 2012. One of the conference papers to HealthCom was selected for fast track to the
journal, International Journal of E-Health and Medical Communications.
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Figure 1. The IOIO, coupled with a COTS Bluetooth 2.0 module, connects to the TOCO sensor
and the ultrasonic transducer. Bluetooth communication with Android phone.
Figure 2. Different user interfaces to control the monitoring system.
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Research Project 12: Project Title and Purpose
Clinical Management of Anxiety and Access to Health Care – The purpose of this project is to
develop and evaluate a computer-administered dental anxiety management program that can
easily be implemented in dental and non-dental healthcare settings. Anxiety is a major reason
for avoidance of dental health care, especially among low-income individuals. The proposed
dental anxiety management program will have the following features: 1) A dental anxiety
screening questionnaire for patients; 2) a computer-administered algorithm to classify patients
according to their level of dental anxiety, 3) a menu of interventions for managing dental anxiety
endorsed by each patient and delivered using a tablet PC and 4) an evaluation component (prepost intervention) The program will also be designed so that it can be administered online and
accessed from a prospective patient’s home.
Anticipated Duration of Project
7/1/2012 – 6/30/2014
Project Overview
Dental and health anxiety are common and potentially distressing problems, for both patients and
healthcare providers. Anxiety has been identified as a barrier to regular dental visits and as an
important target for enhancement of oral health-related quality of life. The aims of this study are
1) to screen for dental anxiety among patients using a brief screening questionnaire; 2) to
evaluate a computer algorithm to determine patients’ level of dental anxiety; 3) to deliver a menu
of interventions for managing dental anxiety endorsed by each patient and delivered using a
tablet PC or via an online website, and 4) to evaluate the program in a small randomized
controlled trial in comparison to a delayed treatment control. The proposed intervention would
involve screening for dental anxiety in the reception area of the Temple University Kornberg
School of Dentistry (TUKSoD) dental clinic while the patient is waiting for his/her appointment
and employing a specifically adapted version of the motivational enhancement system (MES)
program which will use the concepts of cognitive-behavioral therapy (CBT) to assist the
participant in preparing a personal plan for managing his or her dental anxiety. The same
program would be available for online access. The respondent will have a range of CBT choices
presented to him/her as part of the intervention. Each option will be presented using audiovisual
clips of the dental experiences that he or she may encounter. Using the graduated exposure
approach typical of CBT, respondents will select the sequence of dental encounters they wish to
experience when they meet the dentist. When participants visit the dentist, information on their
anxiety score and graduated exposure plan will be made available to the dental provider. At
subsequent dental encounters, the dental anxiety module would be reviewed with the participant.
A CBT anxiety management component will also be delivered via video as part of the
intervention package to supplement the graduated exposure plan. All of the intervention
components will be primarily delivered via a video interface but all will be supported by
personnel (dental students, residents, faculty) providing treatment to their patients. Caregivers
participating in the project will be trained in cognitive-behavioral methods so that they can
answer questions and model procedures when appropriate. The range of potential CBT
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interventions will be presented to the patient in the waiting area via a tablet PC or via the internet
if anxious patients wish to prepare for the visit at home prior to their appointment. Primary
outcomes such as anxiety levels, attendance, and satisfaction will be assessed periodically during
the study. Secondary outcomes such as oral health related quality of life will also be evaluated.
Principal Investigator
Marisol Tellez, BDS, MPH, PhD
Associate Professor Pediatric Dentistry and Community Oral Health Sciences
Maurice H Kornberg School of Dentistry
Temple University
3223 N. Broad Street, Room 307
Philadelphia, PA 19140
Other Participating Researchers
Amid I Ismail, MPH, MBA, DrPH; Richard G. Heimberg, PhD – employed by Temple
University
Joseph Himle, PhD – employed by the University of Michigan, Ann Arbor, MI
Steve Ondersma, PhD – employed by Wayne State University, Detroit, MI
Expected Research Outcomes and Benefits
Expected Outcomes:
- Patients will find the program easy to use and practical.
- Caregivers (including dental students) will be able to assist patients with the program
without adding significant time to the dental visit.
- Caregivers (including dental students) and patients will experience high levels of
satisfaction with the program.
- Patients exposed to the program will experience reduced anxiety levels during, and after
post-intervention dental visits compared to those not receiving the intervention package.
- Patients exposed to the program will attend more dental appointments and will improve
their oral health outcomes compared to those receiving the intervention package.
Benefits:
- The intervention package could be commercialized and patented.
- The dental anxiety management training, if successful, will become a best practice model
to be replicated in other health care settings in the country.
- The information from the program will be used to predict dental attendance and referral
patterns at the school’s clinics, improving efficiency of clinic operations.
- The proposed study will provide an opportunity for fostering interdisciplinary research
between Psychology and Dentistry and will also allow one doctoral candidate in
Psychology to develop expertise in dental anxiety and dental health.
- The data generated from this pilot project, if successful, will provide the basis for the
design of a proposal for a randomized controlled trial to be submitted to NIH to test the
implementation of the program on a larger scale, consistent with the goals of the NIDCR
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Behavioral or Social Intervention Planning and Pilot Data Grant (R34) program which
supports the planning, design, documentation, and pilot data collection for investigatorinitiated studies of behavioral or social interventions relevant to oral, dental or
craniofacial health, and is intended to provide support for the development of a
comprehensive clinical trial protocol (i.e., behavioral or social intervention study
protocol) or R01 funding.
Summary of Research Completed
The computer-based dental anxiety intervention has been programmed, including elements of
cognitive behavioral therapy and motivational interviewing. Four modules were developed and
scripts were completed with duration of 1 hour for the main intervention:
Module 1: Psychological education
Module2: Motivational Enhancement
Module 3: Exposure
Module 4: Closing
Videos and Animations
The following videos were filmed at the school and document six of the most common feared
dental procedures for the population that attends the Kornberg School of Dentistry at Temple
University. In addition, we developed animations of all the specific steps in each dental
procedure that match the scripts developed. Both videos and animations were brought together in
the computer based tool programmed under the MES (Motivational Enhancement System
Software).
Having your teeth polished and cleaned
Having your teeth X-rayed
Drilling and getting a cavity filled
Receiving an injection in your mouth
Having a tooth pulled (tooth extraction)
Having a root canal done
MES Software and Pilot Testing
The main intervention has been programmed in MES and we will conduct pilot testing in July
2013. The objectives of this pilot are to test usability of the computer based dental anxiety
treatment program, elicit feedback from patients regarding anything confusing that requires
specialist support and gather preliminary data for NIDCR (National Institute of Dental and
Craniofacial Research) grant submission (and potentially for case series paper). Our goal is to
test 6 high anxiety patients who will not be randomized but who will receive anxiety treatment
immediately.
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Data Collected
Data for the low anxiety sample (n = 57), for use as a baseline comparison to the high anxiety
sample, has been collected. Data collection for the high anxiety sample (n=150) will begin in
July 2013. Demographically, the low anxiety sample is mainly composed of African Americans
with almost equal proportions by gender and a mean age of 37±13 years old. About 70% attend
regular dental treatment at Temple University while the remaining 30% are emergency patients.
The mean anxiety score (MDAS) is 8.3±2.1 (highest score possible is 25) and none of them met
criteria for the diagnosis of phobia of the dentist. Tables 1 and 2 describe basic demographic and
test statistics for this low anxiety benchmark group.
Publications
The following manuscript was published in the Journal of Anxiety Disorders.
Dina Gordon, Richard G. Heimberg, Marisol Tellez, Amid I. Ismail. A critical review of
approaches to the treatment of dental anxiety in adults. Journal of Anxiety Disorders 2013; 27:
365– 378.
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Table 1: Demographic Characteristics of the Control Group (N = 57)
Sex
Males
Frequency
28
Percent
49.1
Females
28
49.1
Transgender
1
1.8
Race
White
22
38.6
Black
26
45.6
Asian
4
7.0
Other
5
8.8
Education Completed
Some high school/GED
28
49.1
Some college/technical degree
29
50.8
Employment Status
Employed full-time
20
35.1
Employed part-time
18
31.6
Retired
2
3.5
Unemployed
16
28.1
Homemaker
1
1.8
Annual Family Income
Less than $9,999
13
22.8
$ 10,000- 19,999
12
21.1
$20,000- 29,999
7
12.3
$30,000- 39,999
7
12.3
≥40,000
18
31.5
Marital status
Married
10
17.5
In Relationship (not married)
14
24.6
Single (never married)
25
43.9
Divorced
6
10.5
Separated
1
1.8
Widowed
1
1.8
Ethnicity
Hispanic
6
10.5
Non-Hispanic
51
89.47
Patient Type
Regular
41
71.9
Emergency
16
28.1
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Table 2: Descriptive Statistics of Selected Psychological Constructs
MDAS Total Score
OHIP Total Score
AAQ -II Total Score
SAAS Total Score
FQ-BII Total Score
FFMQ-SF observing
FFMQ-SF describing
PCL_S Cluster B Total*
DTS Tolerance
DTS Appraisal
DTS Regulation
ADIS Phobia Clinical
Severity Rating
Min
5.0
0.00
27.0
16.0
0.00
8.0
12.0
5.0
1.0
1.67
1.0
Max
12.0
45.0
69.0
76.0
21.0
20.0
25.0
21.0
5.0
5.0
5.0
Mean
8.32
18.07
54.77
23.36
7.73
14.0
19.3
6.77
3.47
3.81
3.11
0.00
3.0
0.40
Standard
Deviation
2.14
13.02
10.30
10.04
6.01
3.10
3.36
3.28
1.02
0.80
1.09
0.79
Selected Images of screenshots from MES program
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Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Temple University - 2010 Formula Grant – Page 40
Research Project 13: Project Title and Purpose
Relationship between Neuropsychological Function and Monocyte Dysregulation in HIV
Infection – The purpose of this project is to investigate the expansion of a circulating monocyte
subset that may be important to development of neurocognitive impairment in HIV infection and
how it relates to neurocognitive function in a small cohort of HIV infected subjects.
Anticipated Duration of Project
3/20/2012 – 6/30/2014
Project Overview
The studies proposed here will expand on an R01-funded longitudinal study of HIV infected
subjects to include neuropsychological testing. The overall objective of this project is to test the
hypothesis that improved neurocognitive function in anti-retroviral therapy (ART) treated HIV
infected individuals is due, at least in part, to the ability of ART to restore monocyte/macrophage
homeostasis. To test this hypothesis, 20 ART-naïve patients with detectable plasma viremia will
be recruited into these studies and followed for 6 months. Whole blood will be obtained from
volunteers at the time of enrollment (pre-ART) and at weeks 2 and 4 post-ART initiation and 2,
3, 4 and 6 months thereafter. For each draw, peripheral blood monocytes from HIV-1 infected
donors will be assessed by flow cytometry for alterations in immune homeostasis using the
following antibodies: CD14, CD16, CD163, CD11b (activation epitope), CCR5, CD115, MCSF, IL-10 and IL-12. Patients will also be assessed for neurocognitive function prior to the
start of ART and at 3 and 6 months post-ART. Participants will be assessed across multiple
cognitive domains, including verbal learning and memory, motor speed and dexterity, auditory
attention/working memory, speed of information of processing and executive function. In
addition, several behavioral self-report measures will be included to assess psychological
functioning and independence of daily living activities. Collected data will be analyzed for
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Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Temple University - 2010 Formula Grant – Page 41
correlations between neurocognitive performance and restored or altered monocyte/macrophage
homeostasis.
Principal Investigator
Tracy Fischer-Smith, PhD
Assistant Professor
Temple University
3500 N. Broad Street
MERB, Rm. 748
Philadelphia, PA 19140
Other Participating Researchers
Christiane Geisler, RN; Ellen Tedaldi, MD; Nancy Minniti, DPsy – employed by Temple
University
Expected Research Outcomes and Benefits
These studies are designed to answer key questions in the area of neurocognitive impairment in
HIV infection:
1. Does ART restore monocyte/macrophage homeostasis comparable to that seen in
seronegative individuals?
2. Does restored monocyte/macrophage homeostasis relate to improved neurocognitive
performance in ART-treated HIV infected subjects?
We anticipate that this longitudinal study will provide information regarding the phenotype and
potential function of the expanded CD163+/CD16+ monocyte subset in HIV-1 infection, as well
as the kinetics of suppression of this expanded monocyte subset (restored monocyte homeostasis)
and how it relates to neurocognitive function in HIV infection. We further anticipate that the
information obtained from these studies may reveal valuable biomarkers and/or additional
insights into the pathogenesis of neurocognitive dysfunction in the setting of HIV infection.
Summary of Research Completed
To date we have recruited 7 HIV infected, anti-retroviral therapy (ART)-naïve subjects into this
longitudinal study. 20ml of blood was collected in K 3 EDTA containing Vacutainer collection
tubes by a qualified phlebotomist at the time of donor enrollment (pre-ART) and at weeks 2 and
4 and months 2, 3, 4 and 6 post-ART initiation. An additional 20ml was collected for CD4+ and
CD8+ T cell counts, complete blood count (CBC) and HIV-1 viral load. These data are collected
by Quest Diagnostics Nichols Institute, CA and reported to our lab. From the 20ml of blood
collected and brought to our lab, peripheral blood mononuclear cells (PBMC) are isolated using
a Ficoll density gradient and prepared for flow cytometric analysis. Using flow cytometry, we
are identifying monocyte subsets using different panels of antibodies to identify alterations in
CD4, CD11b, CD16, CD115, CD163, CCR5, IL-10, IL-12 and M-CSF expression. Antibodies
used in the flow cytometric analyses were titrated on normal PBMC prior to inclusion in tests
and purchased from the same lot.
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Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Temple University - 2010 Formula Grant – Page 42
The blood draws prior to beginning ART (pre-ART) and at 3 and 6 months post-ART are paired
with neuropsychological assessments performed by neuropsychologist, Dr. Nancy Minniti. The
tests include the Hopkins Verbal Learning Test-revised edition to measure verbal learning and
memory, the Wide Range Achievement Test-4th edition to estimate reading ability, the Grooved
Pegboard test for measuring fine motor speed and dexterity, the Mazes subtest from the
Neuropsychological Assessment Battery to measure spatial reasoning and psychomotor speed,
the Wisconsin Card Sorting Test-64 to assess novel problem solving and mental flexibility, the
Delis-Kaplan Executive Function System for use of the Color Word Interference subtest to assess
mental flexibility and inhibition, the Wechsler Adult Intelligence Scale-4th edition Digit Symbol
Coding subtest for estimating psychomotor speed, the Profile of Mood States-2nd edition for
assessing current mood. Additional neuropsychological tests include the Paced Auditory Serial
Addition Test, Trailmaking Test A & B, Animal Naming, Controlled Oral Word Fluency Test,
Action Fluency and self-report measures Activities of Daily Living and Patient’s Assessment of
Own Functioning Inventory.
At the time of this report, two of the seven recruited subjects have completed the 6-month study,
however, no conclusions can yet be drawn from the low “n”. Another two subjects had to be
excluded and were disqualified from future testing due to a history of head injury for one subject
and illicit drug use, revealed by the drug urine screen, by the other. Of the remaining subjects,
two have only to complete their 6-month blood collection and neuropsychological evaluation and
one has just begun the study 07/24/2013. We are actively recruiting additional ART-naïve
subjects, targeting a study group of approximately 10-15 at the time the study closes.
We anticipate that the knowledge that will be obtained from our research will help in
understanding the role of altered monocyte homeostasis in neurocognitive impairment, as well as
identify potential biomarkers for neurocognitive impairment in HIV-1 infection.
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Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Temple University - 2010 Formula Grant – Page 43
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