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PI
Name:
HARBRECHT, BRIAN G.
Title:
Hepatocyte Nitric Oxide Synthase
Regulation by Glucagon and Insulin
Grant Number:
2R01DK055664-06A2
Fiscal year search: 2008
DESCRIPTION: The immune response
to sepsis involves a series of
complex, highly integrated
homeostatic responses that, if
prolonged and excessive, can lead to
organ dysfunction and death. Nitric
oxide (NO) synthesis is upregulated
by sepsis in many tissues and is an
essential component of the host
immune response. Nitric oxide
synthesis can be beneficial and
improve immune and organ function,
but if synthesis is excessive and
prolonged, NO can promote organ
injury, tissue inflammation, and
death. NO is produced in hepatocytes
by the inducible nitric oxide
synthase (iNOS) that is stimulated
by cytokines and proinflammatory
stimuli. Excessive NO from iNOS
produces cellular dysfunction and
hepatic injury. Glucagon and cyclic
adenosine monophosphate (cAMP)
regulate hepatic iNOS expression in
vitro and in vivo, and by doing so,
decrease NO-mediated hepatic injury.
Our preliminary data demonstrate
that insulin also down-regulates
cytokine-induced iNOS expression.
Both glucagon and insulin alter
specific intracellular signaling
pathways in hepatocytes, but the
mechanisms involved in the
regulation of hepatocyte function in
sepsis by glucagon and insulin, and
specifically the regulation of
hepatocyte iNOS expression, have not
been identified. In this proposal,
we will determine the mechanisms
responsible for the regulation of
hepatocyte iNOS expression by
glucagon and insulin. In Aim I, we
will continue our work in
determining the mechanism for the
glucagon and cAMP-induced inhibition
of hepatocyte iNOS expression. We
will focus on protein kinase A (PKA)-independent
pathways induced by cAMP and
evaluate the role of the guanine
nucleotide exchange factor Epac and
the role of calcium. In Aim II, we
will determine the mechanisms
responsible for the inhibition of
iNOS by insulin. By defining how
these hormones regulate hepatocyte
iNOS expression, we will provide a
framework for understanding the
basic pathophysiologic cellular
events in shock and sepsis that may
lead to novel cellular-based
therapies for critically ill
patients. Project Narrative: Nitric
oxide is synthesized in critically
ill patients during septic shock,
and when overproduced, can increase
cellular dysfunction, tissue injury,
and death. Glucagon and insulin
primarily regulate blood glucose,
which has become an important facet
of the care of critically ill
patients, but we have found that
they also regulate hepatic nitric
oxide production. We will determine
the mechanisms responsible for the
regulation of hepatocyte inducible
nitric oxide synthase (iNOS)
expression by glucagon and insulin.
By defining these mechanisms, we
will provide a framework for
understanding the basic cellular
events in shock and sepsis, which
may lead to novel cellular-based
therapies for critically ill
patients.
PI Name: GARRISON, RICHARD N.
Title: Direct Peritoneal
Resuscitation from Hypovolemic Shock
Grant Number: 5R01HL076160-04
Fiscal year search: 2008
DESCRIPTION:
The pathophysiology of hemorrhagic
shock consists of disruption of the
cellular metabolic functions
endothelial barrier properties,
redistribution of body fluids and
electrolytes, a systemic
inflammatory response, and a
deleterious intestinal
vasoconstriction and a
disproportionate splanchnic
hypoperfusion, which persists even
after adequate fluid replacement
that restores and maintains
hemodynamics. These pathophysiologic
events cause local and remote tissue
injury that culminates in multiple
organ failure (MOF) by mechanisms,
which are poorly understood. It is
hypothesize that initiation of
direct intraperitoneal resuscitation
(DPR) with a balanced physiologic
salt solution supplemented with
glucose (2.5%), at the time of
conventional resuscitation (CR) from
hemorrhagic shock can reverse the
pathophysiology of this syndrome,
improving resuscitation outcome,
potentially preventing (MOF) and
improving survival: a) by
suppressing the hemorrhage-induced
systemic inflammatory response, b)
by direct intestinal resuscitation
to enhance visceral perfusion and
cardiac output, c) by improving
endothelial cell responsiveness, to
vasodilators, and d) by preventing
the hemorrhage-induced water and
electrolytes imbalance. To address
this hypothesis, we will utilize
intravital microscopy, and
quantitative autoradiography (QAR)
to perform in vivo studies in a
rodent model of hemorrhagic shock,
which will be resuscitated with
either CR or CR+DPR and determine
the following: 1) Serum cytokines
profile and level of prostanoid
metabolites; 2) Endothelial cell
function with dose response curves
to endothelial-dependent,
receptor-mediated and non-receptor
mediated as well as
endothelial-independent agonists; 3)
The pattern of distribution of
tissue water (intravascular thetaiv,
interstitial thetaif, intracellular
thetaic) in the gut and abdominal
wall with QAR; 4) The role of
neutrophils in the derangement of
intestinal microvascular endothelium
by measuring microvascular responses
in the presence of anti-PMN serum
and specific antibodies; and 5) The
mechanisms involved in the ability
of DPR to reverse the
pathophysiology of the shock
syndrome. Long-term objectives are
to develop a preclinical protocol
that utilizes DPR as a prelude to
translation of the results to trauma
patients and finally a clinical
protocol that utilize DPR to prevent
multiple organ failure in hemorrhage
shock with resuscitation.
PI Name:
CHAGPAR, ANEES B.
Title: Predicting Non-Sentinel Node
Metastasis in Breast Cancer Patients
Grant Number: 1R21CA131688-01A1
Fiscal year search: 2008
DESCRIPTION:(provided by
applicant): Sentinel lymph node (SLN)
biopsy is a minimally invasive means
of accurately staging the axilla in
breast cancer patients. While
finding negative SLNs avoids
axillary lymph node dissection (ALND),
the finding of micrometastasis in
the SLN mandates a completion ALND.
Up to 80% of all SLN-positive
patients will have no further
disease in the axilla. There is a
significant gap in our current
knowledge in predicting which SLN-
positive patients have a low
likelihood of non-SLN metastasis and
can therefore avoid ALND. A novel
intra- operative RT-PCR based assay
(GeneSearch(r)) for detecting SLN
micrometastases has been shown to
correlate with SLN tumor burden in
breast cancer patients. The
potential utility of this new
technology in predicting which
patients will not have further
disease in their non-SLNs has not
been investigated. Developing a
robust clinical prediction rule for
non-SLN status using these data will
fill this gap in our knowledge and
provide timely conclusions, which
will have a direct impact on patient
care. In order to achieve this
over-arching objective, we propose
the following specific aims: (1) To
determine the impact of quantitative
cycle time values of the
GeneSearch(r) assay on non-SLN
status using both bivariate and
multivariable logistic regression
analyses, and (2) to create and
validate (using bootstrap
techniques) a clinical prediction
rule to predict non-SLN status in
SLN-positive patients using
preoperatively- and intraoperatively-available
clinicopathologic factors, including
data obtained from the GeneSearch(r)
assay. We hypothesize that the
quantitative RT-PCR cycle time for
both mammoglobin (MMG) and
cytokeratin 19 (CK19), as evaluated
as continuous values using the
GeneSearch(r) assay, will be
significantly associated with non-SLN
metastasis on bivariate analyses. In
addition, the cycle time for these
markers will predict non-SLN
metastasis independent of other
factors. We hypothesize that a valid
clinical prediction rule can be
created using preoperatively- and
intraoperatively available
clinicopathologic factors, including
data obtained from the GeneSearch(r)
assay that will identify a subset of
patients in whom the likelihood of
non-SLN metastasis is = 5%. It is
anticipated that the addition of
data from the GeneSearch(r) assay
will significantly improve the
ability to predict this group of
patients over the use of
clinicopathologic factors alone. The
creation and validation of a robust
clinical prediction rule that
incorporates novel molecular data to
identify a subgroup of patients at
low risk of having non-SLN
metastasis will fill a significant
gap in our current knowledge and
will reduce unnecessary morbidity of
ALND, helping to meet the NCI's
Challenge of eliminating suffering
due to cancer by 2015. RELEVANCE: Up
to 80% of breast cancer patients who
have minimal disease in their first
draining (sentinel) lymph nodes will
have no further disease in their
axilla. However, currently there is
no accurate means of predicting
which patients will not have
residual disease, and therefore all
patients who have a tumor deposit =
0.2 mm in their sentinel lymph nodes
will have the remaining lymph nodes
removed -- a procedure that is
associated with considerable
morbidity. We seek to create and
validate a clinical prediction rule,
incorporating novel molecular data,
to identify a subgroup of patients
at low risk of having non-SLN
metastasis, thereby filling a
significant gap in our current
knowledge and reducing unnecessary
morbidity for breast cancer
patients.
PI Name: Sufan Chien, MD
Title: INTRACELLULAR ENERGY DELIVERY
AND DIABETIC WOUNDS
Grant Number:
5R01DK074566-02
Fiscal year
search: 2008
DESCRIPTION: The long-term goal
of our program is to develop a safe
and effective technique to combat
various tissue ischemic damages. The
specific aim of this proposal is to
use our newly developed proprietary
intracellular energy delivery
technique to promote healing of
diabetic wounds. Of the 17 million
Americans with diabetes,
approximately 2.5 to 4.5 million
will develop a chronic wound in
their lifetime. The overall cost of
diabetic foot problems, including
loss of productivity, could be as
high as $20 billion per year.
Despite thousands of dressing
products developed to treat wounds,
none have shown consistent effect.
We propose a new approach for
chronic wounds. Our central hypoth-
esis is that wound tissue hypoxia
results in depletion of adenosine
triphosphate (ATP), which is the
fundamental cause of non-healing
chronic wounds, and a direct
intracellular ATP delivery will
improve microenvironment of wound
tissue and facilitate healing
process. Direct energy supply for
wound treatment has never been
attempted before, and the
relationship between increased
energy supply and wound healing
process is entirely unknown. During
the tenure of the Pi's NIH grant
entitled "Enhanced glycolysis for
hypothermic heart preservation", a
new technique for direct
intracellular delivery of ATP has
been developed in which a special
carrier is used to encapsulate ATP.
The composition of this carrier is
similar to the cell membrane. When
the carrier meets with the cell
membrane, it fuses with it and
delivers the contents into the
cytosol. Preliminary results
indicate that this new energy
delivery technique can provide
significant protection to ischemic
cells and tissues. The technique has
shown very promising effects on
normal and ischemic wounds. Three US
patents and more than 12
international patents have been
filed and the innovation has also
been reported to the NIH. Our
preliminary results also indicated
that high-energy phosphate contents
were severely depleted in human
chronic wounds, and treatment with
ATP vesicles in animal wounds
increased tissue high-energy
contents. Five hypotheses will be
tested: (1) high-energy phosphate
contents are decreased in chronic
diabetic wounds; (2) an ischemic
wound model created using a
minimally invasive surgical
technique can be tolerable to
diabetic animals; (3) intracellular
ATP delivery will increase wound
tissue energy levels to facilitate
healing; (4) by providing energy to
wound tissue, improved healing is
achieved through coordinated
upregulation of growth factors and
other healing mechanisms; and (5)
direct intracellular energy delivery
will enhance wound healing by
improved tissue perfusion. These
issues have not been explored in the
past, but our preliminary results
have established the basis for the
success of this project. The
expansion of usage of the direct
intracellular energy delivery is
likely to have a major impact on
medicine. It will not only improve
chronic wound care, but also help
our treatment to various ischemic
conditions, such as severe trauma,
shock, stroke, heart attack, spinal
cord injury, cardiopulmonary bypass,
organ transplant, and many other
acute and chronic ischemic diseases.
PI Name: Suzanne T. Ildstad
Project title: Tolerance Induction to Islet
Transplants
Grant Number: 5R01DK069766-03
Fiscal year search: 2008
Abstract: The focus of
this proposal is to develop a novel "conditioning" approach that will
replace myelotoxic agents to establish chimerism in NOD mice. We will induce
immune deviation to promote host-versus-graft hyporesponsiveness, thereby
giving the hematopoietic stem cell (HSC) an opportunity to engraft and
establish subsequent self-perpetuating deletional tolerance to islet
allografts. Our recent studies in a mouse model suggest that the primary role
for conditioning for HSC transplantation is to suppress host-versus-graft
alloreactivity, rather than to prepare vacant niches in the recipient's bone
marrow compartment. This observation suggests that one could replace myelotoxic
agents with antigen-specific approaches to induce host-versus-graft
hyporeactivity or anergy at the time of HSC transplantation. As the mechanisms
underlying T cell activation are defined, highly specific approaches to
suppress this alloreactivity have emerged. In
AIM
I.
we will
ESTABLISH CHIMERISM THROUGH IMMUNE DEVIATION OF THE RECIPIENT. We will
immunomodulate the recipient: (a) targeting alloreactive cells in the host
microenvironment; (b) inducing anergy and/or antigen-specific apoptosis of
alloreactive host cells; and (c) through generation of regulatory T cells
(Treg), and develop a novel nonmyeloablative conditioning regimen to induce
antigen-specific hyporesponsiveness to the HSC and islet allografts. Cell-based
therapies have great potential for inducing transplantation tolerance. Of
greatest interest are the new subpopulations of bone marrow-derived dendritic
cells (DC) that have recently been shown to be potently tolerogenic in vitro
under certain circumstances. We are the first to demonstrate an in vivo
engraftment-enhancing effect for precursor plasmacytoid DC (p-preDC). The
exploitation of this discovery in vivo and its potential to reduce the need for
myelotoxic conditioning has not yet been tested. Hematopoietic growth factors
have also been used to drive the immune response to a tolerogenic T helper 2
(Th2) phenotype through production of p-preDC or other tolerance-promoting
cells (graft facilitating cells {FC}) that in turn generate Treg. In AIM II, we
will USE PRE-TRANSPLANT IMMUNOMODULATION OF THE DONOR WITH HEMATOPOIETIC GROWTH
FACTORS TO GENERATE TOLEROGENIC CELLS IN THE HSC ALLOGRAFT. We will use these
factors and the cells they generate to modulate the tolerogenicity of the donor
marrow inoculum in vivo to tip the immune milieu in favor of graft acceptance,
enhancing bone marrow chimerism without myelotoxic conditioning. We will
examine the mechanism by which this occurs and identify which cell types in the
graft are critical to tolerance induction. P-preDC exposed to apoptotic donor
antigens are potently tolerizing in vitro through generation of Treg. The
therapeutic application of this approach has not been tested in vivo. In AIM
III, we will USE EX VIVO IMMUNOMODULATION OF THE MARROW to expand p-preDC and
FC and induce a tolerogenic inoculum for HSC transplantation.
PI Name:
WILLIAMS, STUART K.
Title: A prevascularized islet
immunoisolation device
Grant
Number: 1R01DK078175-01A1
Fiscal
year search: 2008
DESCRIPTION: Beta-cell replacement therapy via islet transplantation remains a
promising technology for the
reversal of type 1 diabetes. A
significant barrier to the clinical
utilization of beta cell transplants
has been the lack of a host-derived
blood supply to maintain the
viability and thus the function of
transplanted cells. We have
developed a new cell-based therapy
for the generation of pre-vascularized
tissue engineered constructs. We
have also developed a new generation
of biomaterials that support
extensive neovascularization. The
combined cell and material construct
to be evaluated is termed a
Prevascularized Immuno-Isolation
Device or PVID. We propose to use
these materials in the development
of a new beta-cell immuno-isolation
device to prolong beta cell
viability and function. These
constructs represent a pre-formed
microcirculation that can be
constructed from a patient's own
fat-derived microvascular
endothelial cells, avoiding the use
of immuno- suppressive drugs.
Specific aim 1 will evaluate the
maturation of the microcirculation
within a prevascularized construct
following implantation in an animal
model. Specific aim 2 will evaluate
novel porous biomaterials and
material surface modification to
support the neovascularization of
the porous material to assure
perfusion of encapsulated islets.
The biomaterial developed is a two
component hybrid system that also
provides immunoisolation for the
encapsulated islets. Specific aim 3
will evaluate the viability and
function of islets encapsulated in
the prevascularized immunoisolation
devices in an animal model of
diabetes.
PI Name:
Hoying, James B.
Title: Fabricated Microvascular
Networks
Grant Number:
5R01EB007556-03
Fiscal year
search: 2008
DESCRIPTION:
Fabricated
Microvascular Networks. The
importance of an effective vascular
supply for tissue health is
universally accepted. In developing
strategies to build vasculatures for
tissue engineering and other
therapeutic applications, it is
important to recognize that,
foremost, the new vasculature must
quickly provide sufficient blood
flow to the target tissue to
preserve cell viability. We have
found that new microvessels formed
in vitro can begin to carry blood
within the first days following
implantation. However, flow patterns
are atypical and likely ineffective
at establishing normoxia until many
days later. The delay is primarily
due to a lack of organization within
the network at the time of
implantation and the time needed to
develop new mature inflow and
outflow pathways. We hypothesize
that pre-determining an appropriate
network organization prior to
implantation would reduce the amount
of time needed for the new
microvasculature to effectively
perfuse a tissue. We have
established generic technologies
utilizing a direct-write tissue
printing tool for patterning and
organizing tissue components for
tissue engineering applications. We
propose to implement this technology
to design and fabricate
pre-patterned, 3-dimensional
microvascular networks with
pre-existing inflow and outflow
pathways. Also, we will use an in
vitro, intravascular-perfusion
bioreactor system to establish flow
through the networks to further
organize and mature the
microvascular networks prior to
implantation. Computational modeling
and physiological analyses serve to
direct design strategies and
characterize the architectures and
functionality of the fabricated
vasculatures both in vitro and in
vivo. In addition to providing an
enabling technology platform for
assembling pre-determined
microvascular networks, this work
will provide a foundation from which
to explore the importance of network
architectures in vascular function.
PI Name: CHAGPAR, ANEES B.
Title:
Louisville Breast Cancer Updateam
Grant Number: 5R13CA124224-02
Fiscal year
search: 2008
DESCRIPTION: (provided by applicant): The purpose of the Louisville Breast Cancer
Update is to provide a high quality
forum for the dissemination of
advances in the state-of-the-art
multidisciplinary management of
breast cancer, and to allow for an
exchange of ideas regarding
controversial issues regarding care
of breast cancer patients. Lectures
from some of the most prominent
breast cancer researchers and
clinicians will be presented
covering all aspects of breast
cancer care. This year, topics to be
covered will include: Indications,
utility, and challenges of MRI;
minimally invasive mastectomy;
Innovations in reconstruction;
Advances in systemic chemotherapy;
Hormones in breast cancer: to
replace or inhibit?; Targeted
therapy for breast cancer; Genomics
in the management of breast cancer;
Advances in radiation therapy:
should we be doing more or less?;
Prevention, genetics and high risk
breast cancer and Supportive Care
Issues. In addition, this meeting
will be expanded to facilitate the
interaction between basic scientists
and clinicians with a scientific
poster session that will promote
fruitful collaborations and a common
understanding of progress in the
breast cancer arena, from bench to
bedside. The meeting will therefore
further the NCI's mission for the
alleviation of suffering and death
due to cancer by providing a forum
for the dissemination of advances in
the multidisciplinary care of
patients with breast cancer, and the
building of collaborations that will
promote further research in this
area. This annual event is the only
one of its kind in Kentucky, and
provides an important resource for
education of clinicians, scientists
and advocates.
PI Name:
WIGHTMAN, FREDERIC L.
Title:
Listening Skills in children with
and without cochlear implants
Grant
Number:
5R01HD023333-16
Fiscal Year
Search 2008
DESCRIPTION:
The long term goal of this program
is a complete understanding of the
capabilities and limitations of
auditory information processing in
children, the development of
auditory processing skills, and how
that developmental course may be
affected by hearing impairment,
deafness, and the use of a cochlear
implant. The current focus of the
project is on the strategies used by
children to segregate desired
auditory signals from interfering
sounds. Results to date suggest that
most young children display a
remarkable an ability to perform
this segregation in simple detection
tasks and in tasks requiring
attention to a target speech message
in the presence of other speech
distracters. The data also reveal
large individual differences in
source segregation ability, with
some teenagers demonstrating
child-like performance, and some
young school-aged children
demonstrating adult-like
performance. The adult-child
differences are largely consistent
with what results. The new work also
addresses the special selective
attention problems faced by children
and adults with cochlear implants
and evaluates strategies by which
selective attention can be enhanced.
Auditory selective attention is
quantified in four kinds of tasks:
1) simple signal detection, in which
the target signal is masked by
random multicomponent tonal
complexes; 2) a speech fundamental
frequency discrimination task; 3)
speech recognition tasks, both with
and without a visual representation
of the speaker; 4) spatial
segregation tasks in which the
target message and the distracter
are spatially separated. The aim of
the first task is to quantify
auditory selective attention within
the constraints of a simple
one-parameter model. The second task
will produce a scale of talker
similarity which will then be used
to predict the impact of talker
similarity in the selective
attention tasks. The third and
fourth tasks explore means by which
selective attention can be enhanced.
The overall importance of this
project lies in its study of
communication situations common in
everyday life. Speech communication
in noisy environments is an
especially important issue for
children who are developing speech
and language, and the results of
this project will add important new
knowledge about how children and
adults are able to address this
issue.
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