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Steven
C. Castle, M.D.
Summary
of Research:
The
overall theme of our research effort is to identify specific
components of immune dysfunction, especially with regard to
cytokine balance and T cell subsets, and to determine how
these physiologic parameters relate to clinically relevant
issues, such as malnutrition, deconditioning, psychologic
stress and depression. A successful immune response to tumors
or infectious pathogens is dependent on the activation of
an appropriate set of effector functions, while a dysregulated
response contributes to chronic inflammation resulting in
disease. The type of immune cells at the site of infection
and the ability to recruit effector cells, together with the
initial and subsequent balance of regulatory cytokines dictate
the characteristics of an immune response. Interleukin-12
(IL-12) and IL-4 promote differentiation of naive T cells
towards either a Th1 phenotype (high production of IL-2 and
interferon-gamma [IFN-g]; promoting cell mediated immunity)
or a Th2 phenotype (high IL-4 and IL-10; promoting humoral
immunity), respectively. There is evidence that there are
age- and stress-related shifts toward a Th2 response. We are
investigating age-related changes in cell types and regulation
of cytokine balance.
Ongoing
projects:
- Age-related
change in differentiation of specific Th subsets in response
to a specific cytokine milieu. Specifically, we are testing
the hypothesis that there is an enhanced, age-related
dominance of IL-4 over IL-12 in the differentiation and
reversibility of immune phenotype differentiation. A sub-hypothesis
includes that enhanced IL-4 activity with age lessens
the impact of anti IL-10 in cross-regulation of Th differentiation.
- Effect
on Immunity of Stress and Depression of Caregiving. We
are studying the relative impact of shifts in T cell subsets
(loss of signal transduction receptor CD38, decline in
Natural T cells- CD56+8+, and increased suppressor cells-
CD57+8+) on immune function and the effect of acute stress
on chronic stress.
- Immune
dysregulation in Malnutrition and Chronic Deconditioning
in Community-Dwelling Elderly.
Mei-Ping
Chang, Ph.D.
Summary of Research:
Although
we and others have shown that T cells are most vulnerable
to ethanol, there is little information regarding the cellular
and molecular mechanisms of the immunosuppressive effect
of alcohol. Thus, we have investigated the cellular changes
associated with the suppressive effect of ethanol on T cell-dependent
B cell immune responses in mice. We hypothesize that the
diminished T cell-dependent antibody (Ab) response in chronic
ethanol (EtOH)-consuming mice is due in part to defects
in CD40-induced signals of T cells and/or a diminished potential
for clonal expansion of B cells. Our goal is to elucidate
the mechanism(s) of ethanol-induced immunosuppression of
T cell-dependent antibody response. A well-established C57BL/6
mouse model is used to examine whether the decreased sheep
red blood cell Ab response in EtOH-consuming mice is due
to (a) altered CD40-induced help signaling cascades; (b)
dysregulation in cytokine production by T helper (Th) cells;
(c) the inability of B cells to respond to these cytokine
signals; or (d) a limited potential for clonal expansion
of B cells. Isotype-specific ELISA, immunofluorescence labeling,
receptor binding, BrdU-Hoechst flow cytometric analysis
and propidium iodine dye method for cell kinetics, immunoblotting
technique and protein kinase assay are used for this study.
Ongoing
projects:
- Using
a mouse model, we are studying whether ethanol affects
CD40-induced signaling cascade pathways in chronic ethanol-consuming
mice.
- Using
the same mouse model, we are also elucidating the mechanism
of immunosenescence of T/B cell interactions by examining
cytokine-dependent Ab responses, cytokine-receptor interactions,
B cell cycle kinetics, and cyclin proteins and their
associated protein kinase activities.
- Using
select darked-skinned minority as a model, we study
immune responses to Coccidiodes immitis infection. We
hypothesize that an increase in infection with Coccidiodes
immitis in select dark-skinned minority populations
in the United States is due to an imbalance of Th1/Th2
cell functions.
Meika
A. Fang, M.D.
Summary
of Research:
Musculoskeletal
conditions such as osteoarthritis and osteoporosis often
lead to functional disability in the elderly, and result
in increased health care use and cost, increased caregiver
burden, and loss of independence. In order to minimize
the impact of musculoskeletal disorders on the quality
of life for older individuals, we need to improve our
knowledge about the pathophysiology and treatment of rheumatic
disorders common in the elderly. Current projects focus
on improving our understanding of bone biology, assessing
the impact of arthritis on the lives of the elderly, and
determining the efficacy of interventions in maintaining
independent functioning in the elderly.
Ongoing
Projects:
- Defining
the cellular and molecular mechanisms regulating the
effects of basic fibroblast growth factor on type
I collagen in osteoblasts.
- Determining
the functional consequences of upper extremity musculoskeletal
disorders in the elderly and studying the efficacy
of interventions to prevent or minimize factors leading
to upper extremity musculoskeletal disability.
- Describing
the biomechanical effects of knee osteoarthritis on
other joints and assessing the efficacy of pharmacological
and non-pharmacological interventions in preventing
these effects.
Theodore
J. Hahn, M.D.
Summary
of Research:
Osteoarthritis
(OA) is characterized by a progressive loss of articular
cartilage, in association with reduced differentiated
functioning and increased fibronectin (FN) production
by articular chondrocytes. Our studies utilize the recently
developed rat RCJ 3.1C5.18 (RCJ) chondrocyte line to
examine the hypothesis (I) that articular cartilage
loss in OA is accelerated by chondrocyte dedifferentiation
and reduced functioning due to the effects of abnormal
extracellular matrix (ECM) signals on chondrocyte gene
expression, (ii) that these signals are due to altered
ECM composition, particularly increased FN deposition,
and are transduced by the a5b1 integrin and other integrin
FN receptors, (iii) that the FN receptor second messenger
signals producing these effects include increased intracellular
free calcium concentration, increased intracellular
pH, tyrosine kinase activation, and effects on cytoskeletal
organization; and (iv) and that these second messengers
alter chondrocyte-specific gene expression via effects
on various gene response elements.
Ongoing
Projects:
- Defining
the effects of ECM-mediated and inflammatory cytokine-mediated
differentiation and dedifferentiation on the content
and activity of a5b1 integrin, a4b1 integrin and
other integrin fibronectin receptors in RCJ cells.
Examining the mechanisms underlying these effects.
- Examining
the nature and second messenger mechanisms of the
individual and combined effects of the a5b1 and
a4b1 integrin binding sites in the fibronectin molecule
on RCJ cell chondrocytic gene expression.
- Defining
the molecular basis of the effects of the a5b1 integrin
receptor on type II collagen gene regulation in
RCJ cells, using transient transfections of type
II collagen gene promoter constructs, deletional
analysis, and site-directed mutagenesis.
Takashi
Makinodan, Ph.D.
Summary
of Research:
T
Cell Immunosenescence. T cells, which modulate immune
response, are susceptible to aging. We propose that
defects in the transcription activation process and
the regulation of [Ca]i in memory T cells are contributory
factors.
Ongoing
Projects:
- Tuberculosis
Among Asian Americans. Tuberculosis (TB) morbidity
continues to occur in epidemic proportions in
Los Angeles, and Asian Americans bear disproportionate
burden of the TB problem. Demographic characterization
of Asian Americans with TB have been completed.
Models are now being developed to reduce the incidence
of Asian Americans with TB.
Skye
McDougall, Ph.D.
Summary
of Research:
Osteoarthritis
is a condition characterized by progressive degeneration
of articular cartilage leading to mechanical failure
of the joint. This disease is characterized by markedly
altered chondrocyte functioning, with increased
proliferation and loss of differentiated chondrocyte
phenotype leading to decreased production of normal
cartilage extracellular matrix (ECM). The molecular
mechanisms that underlie this profound change in
cell phenotype and function remain to be defined,
however altered ECM-derived cell regulatory signals
play an important role in this process. The focus
of our research is to develop an understanding of
the molecular mechanisms responsible for the development
of age-related bone and joint diseases such as osteoarthritis,
in order to design rational treatment approaches.
To
study the development of osteoarthritis, we are
currently using a chondrocyte cell line which is
capable of differentiating and dedifferentiating
in vitro, as a model system to investigate the role
of one family of regulatory molecules, the helix-loop-helix
family of transcription factors, in both chondrocyte
cell differentiation and osteoarthritis development.
Our preliminary data suggests that one member of
this family, Twist, may play an important role in
controlling cartilage cell development and function.
Ongoing
projects:
- Functional
role of Twist. To define the role of Twist in
chondrocyte dedifferentiation, the chondrocyte
cell-specific targets of Twist action are currently
being determined. Overexpression studies are
also in progress to analyze the effects of increased
Twist expression on chondrocyte-specific gene
expression and chondrocyte cell phenotype.
- Twist
gene regulation. The cis-acting regulatory elements
within the Twist gene that control changes in
Twist expression in chondrocytes are being identified,
using mutagenesis and functional assays. The
proteins that interact with these regulatory
elements are being identified and characterized
using a combination of in vivo footprinting
and in vitro protein-DNA interaction studies.
- The
rate of fracture repair in young and old bones
is being studied using in situ hybridization
and immunohistochemical techniques to monitor
changes in cell type growth factor gene excpression
during the earliest stages of fracture repair.
This will allow us to develop an understanding
of age-related differences which play a role
in fracture healing.
William
J. Peterson, Ph.D.
Summary
of Research:
Although
there is much descriptive information concerning
the proliferation and differentiation of bone
cells and how they may be affected by aging, very
little is known about the mechanism(s) of these
processes and their contribution to osteoporosis.
Osteoblasts, the bone-forming cells, have an intimate
relationship with the extra cellular matrix (ECM)
that they produce. Interaction between these cells
and their ECM is facilitated by cell surface receptors
called integrins. This project is based on the
hypothesis that interaction of integrins with
ECM can promote proliferation and differentiation
of osteoblasts and dysregulation of ECM-osteoblast
interaction via integrins may be a cause of some
forms of metabolic bone disease. Our research
effort is focused on the MC3T3-E1 cell line which
is a non-transformed pre-osteoblastic cell line
that is capable of bone formation in vitro. The
major phases of its developmental activity include:
proliferation, matrix maturation, and mineralization.
Our goal is to relate the function of integrins
to these processes and determine how they may
be affected by aging. This project was initiated
by assessing the in vitro culture variables involved
in adapting the MC3T3-E1 cell line to grow in
microculture.
Ongoing projects:
- Studying
the relationship between proliferating and
quiescent cells in post-confluent cultures.
- Examining
the role of transforming growth factor beta
in proliferation and differentiation.
- Developing
methods for studying the role of integrins
in proliferation and differentiation.
Dean
T. Yamaguchi, M.D., Ph.D.
Summary of Research:
There
are three major basic science projects ongoing
in the Bone Physiology and Optical Biology Laboratory:
- The
role of gap junctions in osteoblast development.
This project is a VA Merit Review- funded
program examining the role of gap junctions
and the proteins that comprise gap junctions
called connexins in the development of the
osteoblast. The project in general will
examine how gap junctions are important
in new bone formation and more specifically
how the expression of gap junctions differ
during the maturation of the bone-forming
cell, the osteoblast. Experiments are designed
to inhibit or overexpress connexin in osteoblastic
cell models and to then monitor osteoblastic
markers of proliferation, differentiation
and secretory products, and mineralization
of osteoblasts in culture.
- The
role of endothelial cell and osteoblast
interaction in the process of new bone formation.
This project is funded by the American Heart
Association, Southern California Affiliate,
and seeks to address the role of microvascular
endothelial cells in stimulating osteoblastic
differentiation and subsequent bone mineralization.
Endothelial cell interaction with osteoblasts
will be examined under conditions of cell
contact-dependency as well as in co-cultures
in the absence of direct cell-cell contact.
- The
effects of electromagnetic fields on functional
gap junctions in osteoblasts. This project
is funded by the Electric Power Research
Institute and involves the development of
extremely low frequency electromagnetic
field exposure systems for a tissue culture
incubator and the stage of an inverted fluorescence
microscope. These systems will be used to
examine how gap junctions may be affected
by these electromagnetic fields and the
potential mechanism(s) responsible for effects
on functional gap junction communication.
There
is also one clinical project underway:
- The
relationship between age-related renal
function decline in the elderly and the
potential etiology of hypoerythropoietin-induced
anemia in elderly geriatric outpatients
is being examined. Those patients with
non-nutritional or non-iron deficiency
anemia who have normochromic and normocytic
indices will be tested for renal function
by creatinine clearance. Determinations
of erythropoietin levels and various cytokines
which may antagonize the action of erythropoietin
will also be made to help profile those
patients whose etiology of anemia is unclear.
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