ESAT-6 Augments Inflammatory Responses to Multiwall Carbon Nanotubes (MWCNT) in a
Murine Chronic Granuloma Model.
Anagha Malur1, Barbara P. Barna1, Janki Patel1, Matthew McPeek, Larry Dobbs2, Isham Huizar1 and
Mary Jane Thomassen1.
Departments of: Internal Medicine, Division of Pulmonary and Critical Care Medicine 1; and
Pathology2, East Carolina University, Greenville, NC.
Background. Pulmonary granuloma formation represents a complex and poorly defined response
involving environmental and host factors that can culminate in persistent and chronic inflammatory
disease. We have described a murine granuloma model (AJRCMB 2011, 45: 858) in which multiwall
carbon nanotubes (MWCNT) elicit a granulomatous disease markedly similar to that found in
sarcoidosis, a prototypical human granulomatous disease. MWCNT-elicited granulomatous disease
is chronic (granulomas persist up to 90 days), and characterized by elevated pro-inflammatory
cytokines together with T cell and macrophage recruitment – all traits found in sarcoidosis. ESAT-6 is
an M. tuberculosis secreted protein and T cells from patients with Sarcoidosis have also been
reported to react to ESAT-6 peptides. Hypothesis. We hypothesized that ESAT-6 might exacerbate
granulomatous inflammation induced by MWCNT. Methodology. MWCNT (100 µg) +/- ESAT-6
peptide 14 [NNALQNLARTISEAG] (20 µg) were instilled into wild-type C57Bl/6 mice. Controls
consisted of (a) sham-instilled and (b) ESAT-6 alone. Animals were sacrificed after 60 days for
analyses of granuloma incidence and bronchoalveolar lavage cell (BALC) expression of CCL5,
MMP12, osteopontin (OPN), and IFN- mRNA. Results. Semi-quantitative morphologic analysis
indicated more abundant and larger granulomas (2-fold increase, p=0.03, n=6/group) in mice
receiving MWCNT+ESAT-6 than in mice receiving only MWCNT. Immunostaining revealed
prominent CD3+ cell infiltrates in granulomatous tissue of ESAT + MWCNT instilled mice compared
to MWCNT alone. Mice receiving ESAT-6 alone had no granulomas and BALC cytokine expression
did not differ from sham controls. BALC expression of OPN and IFN- was comparable in MWCNT vs
MWCNT + ESAT-6 groups but higher than sham control (p<0.05). In MWCNT+ESAT-6 mice,
however, BALC expression of CCL5 and MMP12 exceeded that of mice receiving MWCNT alone
(p<0.05). Summary and Conclusions. Instillation of MWCNT plus ESAT-6 exacerbated granuloma
formation and augmented BALC expression of the pro-inflammatory chemokine, CCL5 as well as
MMP12. Data suggest that simultaneous exposure to mycobacterial antigen and MWCNT may
worsen chronic granulomatous disease.
AEROALLERGEN-INDUCED HYPOXIC BRADYCARDIA IN HOUSE DUST MITE SENSITIVE NON-HUMAN PRIMATES
Robert L Wardle1, Stuart R Ellis1, J Leslie Gaddis1, Stephen G Olmstead, III1, Benjamin D Putnam1,
Shaun P Reece1, Robert B Fick, Jr.2, Michael R Van Scott1. 1Physiology, Brody School of Medicine
at East Carolina University, Greenville, NC, 2Merck Research Labs, Palo Alto, CA
RATIONALE: House dust mite-sensitive nonhuman primates (NHP) exhibit hallmark features of
allergic asthma including bronchoconstriction, nonspecific bronchial hyperresponsiveness, airway
inflammation, and nocturnal apnea and hypopnea. A small number of these animals exhibit lifethreatening bradycardia after aeroallergen challenge. This study was performed to elucidate the
mechanisms underlying the adverse cardiac response.
METHODS: House dust mite (HDM)-sensitive NHP were anesthetized, intubated, and instrumented
to record ETCO2, SpO2, HR (pulse oximetry & ECG), and chest & abdominal wall movements. The
animals were paralyzed and ventilated; and then subjected to hypoxia, hypercapnia, apnea and
hypopnea, before and after challenge with aerosolized HDM.
RESULTS: Before HDM challenge, apnea was associated with tachycardia in all animals. With apnea
during the early response to HDM, SpO2 fell to below 70% and ETCO2 rose to 45 to 50 mm Hg.
Bradycardic responders exhibited an initial tachycardia (peak HR at SpO2 of 80% ± 3%) followed by
sinus bradycardia that reversed immediately upon resuming ventilation (Figure).
With repeated periods of apnea, the onset of bradycardia occurred at progressively higher levels of
SpO2. Bradycardia during apnea was prevented by 3 cm H 2O PEEP or pretreatment with atropine.
Control animals exhibited only tachycardia. Complete cessation of ventilation was not required to
elicit hypoxic bradycardia. Decreasing minute ventilation by 55% led to hypoxia and bradycardia as
SpO2 fell below 70%. Similarly, bradycardia was observed during weaning from the ventilator when
tidal volumes were decreased (Figure).
When minute ventilation was held constant and O2 content of inspired air was decreased to 9%, SpO2
fell below 70%, but bradycardia was only observed if mechanical ventilation was interrupted. Hypoxic
bradycardia was also observed 24 hours after HDM exposure. At that time, if minute ventilation and
O2 content of inspired air were both maintained but CO2 content of inspired air was gradually
increased up to 5%, variability in HR increased, but pronounced bradycardia was not observed unless
ventilation was interrupted.
CONCLUSIONS: In susceptible NHP, repetitive hypoxic challenges following an allergic response
lead to life-threatening bradycardia. Hypoxic bradycardia during respiratory stasis has been described
previously in humans and animals. Our data indicate that HDM allergy can lower the O2 and tidal
volume thresholds for the reflex. The results have implications in allergic asthmatic and sleep apnea
patients at risk for sudden cardiorespiratory arrest.
Mechanism of Central Atypical Cannabinoid Receptor GPR18-Mediated Hypotension in
Conscious Rats
Anusha Penumarti, Abdel-Rahman AA
Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University,
Greenville, NC-27834
Background and objectives: Vascular GPR18 activation causes vasodilation, and our preliminary
findings are the first to show: (i) GPR18 expression in tyrosine hydroxylase (TH) immunoreactive
neurons in the rostral ventrolateral medulla (RVLM); (ii) RVLM GPR18 activation (Abn CBD) and
blockade (O-1918) dose-dependently reduced and increased blood pressure (BP), respectively, in
conscious male Sprague Dawley rats. Here we tested the hypothesis that NO generation, via ERK1/2
activation, coupled with reduced reactive oxygen species (ROS) generation in the RVLM underlies
GPR18-mediated hypotension. We complemented the integrative studies with signaling studies in
differentiated PC12 cells (catecholamine expressing cells with neuronal phenotype) to elucidate the
molecular mechanisms implicated in GPR18 signaling.
Key results: Ex vivo studies on RVLM neuronal tissues, collected during the hypotensive response
elicited by intra-RVLM GPR18 activation in conscious rats, revealed increases in ERK
phosphorylation and NO level and reduced ROS while RVLM GPR18 blockade, which increased BP,
elicited opposite molecular responses. In PC12 cells, GPR18 is associated with lipid rafts, and its
activation replicated the molecular responses obtained in the RVLM.
Conclusion: RVLM GPR18 activation lowers BP by reducing oxidative stress and increasing NO (via
ERK1/2 phosphorylation). Similar molecular findings in PC12 cells support the validity of this model
system in ongoing pharmacologic/genetic studies that aim at elucidating the role of the lipid rafts and
their major protein, caveolin, in GPR18 signaling. Our findings yielded new insight into a novel role for
RVLM GPR18 in the neurobiology of BP control.
Incidence and Impact of De Novo Donor-Specific Alloantibody in Primary Renal Allografts
Lorita M. Rebellato, PhD1, Matthew J. Everly, PharmD2; Kimberly P Briley, BS1; Miyuki Ozawa, PhD4;
Carl E. Haisch, MD3; Karen Parker, RN, BSN5; Paul Bolin, MD5; William T. Kendrick, MD6; Scott A.
Kendrick, MD6; Robert C. Harland, MD3,; and Paul I. Terasaki, PhD2
Author Institutions:
1
Department of Pathology, Brody School of Medicine at ECU, Greenville, NC, USA.
2
Terasaki Foundation Laboratory, Los Angeles, CA, USA.
3
Department of Surgery, Brody School of Medicine at ECU, Greenville, NC, USA.
4
One Lambda, Inc., Los Angeles, CA, USA.
5
Department of Medicine, Brody School of Medicine at ECU, Greenville, NC, USA.
6
Eastern Nephrology Associates, Greenville, North Carolina, USA.
Abstract
Background: To date, limited information is available describing the incidence and impact of de novo
donor specific anti-HLA antibodies (dnDSA) in the primary renal transplant patient. This report details
the dnDSA incidence and actual 3-year post-dnDSA graft outcomes.
Methods: The study includes 189 consecutive non-sensitized, non-HLA-identical patients who
received a primary kidney transplant between 3/1999 and 3/2006. Protocol testing for DSA via
LABScreen® single antigen beads (One Lambda, Inc) was done pre-transplant, and at 1, 3, 6, 9, and
12 months post-transplant, then annually and when clinically indicated.
Results: Of 189 patients, 47 (25%) developed dnDSA within 10 years. The 5-year post-transplant
cumulative incidence was 20% with the largest proportion of patients developing dnDSA in the first
post-transplant year (11%). Young age (18-35 years old at transplant), deceased donor transplants,
pre-transplant HLA (non-DSA) positive patients, and patients with a DQ mismatch were the most
likely to develop dnDSA. From DSA appearance, 9% of patients lost their graft at 1 year. Actual 3year death-censored post-dnDSA graft loss was 24%.
Conclusion: We conclude that 11% of patients without detectable DSA at transplant will have
detectable DSA at 1 year and over the next 4 years the incidence of dnDSA will increase to 20%.
After dnDSA development 24% of patients will fail within 3 years. Given these findings, future trials
are warranted determine if treatment of dnDSA positive patients can prevent allograft failure.
Patient Pages to Improve Cardiovascular Care
Katherine Cutitta, MA1, Lindsey Rosman, MA1, Jessica Ford, MA1, Kevin Woodrow, MA1, Kari
Kirian, PhD2 , Garret Hazelton, PhD3, & Samuel F. Sears, PhD 1, 4
1
Department of Psychology, East Carolina University
Department of Family Medicine, East Carolina University
3
Department of Psychiatry, East Carolina University
4
Department of Cardiovascular Sciences, East Carolina University
2
Background: Cardiac psychology is a specialized branch of psychology that focuses on the care of
cardiology patients after diagnoses of heart disease. Patients often have a difficult time adjusting to
their life and the physical changes that accompany heart disease. Cardiac psychologists have the
potential to facilitate patient acceptance and improve self-care and disease management behaviors.
Results: The Cardiac Psychology lab at ECU, under the direction of Dr. Sam Sears, has developed
a series of patient pages designed for use in cardiology clinics around the country and are
ppublished in Circulation. These articles are written in patient-centered terms and designed as an
educational tool to improve patient knowledge and understanding of their cardiovascular condition.
The cardiac psychology lab has particular interest in electrophysiology and patient reaction to
implantable cardioverter defibrillator (ICD) shock. The ICD is a medical device that is implanted in a
patient to detect and eliminate abnormal heart rates and rhythms via electrical leads attached to
cardiac walls. Patient pages have been developed specifically to address important quality of life
concerns for patients with ICDs related to sexual health concerns, pediatric issues, trauma, and
coping with a partner’s cardiac illness.
Conclusion: The Cardiac Psychology lab at ECU has a vision to enhance patient engagement in
cardiology clinics across the country by providing patients and providers with access to free, peerreviewed, patient-friendly educational tools in the form of patient pages. These pages can also
facilitate appropriate referrals to cardiac psychology specialists or mental health professionals, if
indicated. Patient pages are an important tool for cardiology patients and providers.
Deletion of claudin-7 in renal collecting duct cells impairs paracellular chloride permeability
Junming Fan, Rodney Tatum, John Hoggard, Beverly G. Jeansonne, and Yan-Hua Chen,
Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University,
Greenville, NC 27834, USA
We have reported previously that claudin-7 knockout mice (Cldn7-/-) display severe salt wasting
and chronic dehydration phenotypes. To investigate the role of claudin-7 in ion permeability in renal
epithelial cells, we generated stable collecting duct (CD) cell lines isolated from Cldn7+/+ and
Cldn7-/- mouse kidneys. Deletion of claudin-7 significantly increased the transepithelial electrical
resistance (TER) and decreased the absolute permeability for Cl- (PCl) and Na+ (PNa). The ratio of
PCl/PNa was <1, suggesting an impairment of Cl- permeability along with a suppression of Na+
permeability in Cldn7-/- CD cells. TER and dilution potential values were unchanged after inhibition
of Cl- and Na+ channels and the current-voltage curves were linear in Cldn7+/+ and Cldn7-/- CD cells,
indicating that the resulting ionic flux changes were through the paracellular pathway. The
impairment of paracellular Cl- permeability can be rescued by transfecting Cldn7-/- CD cells with
claudin-7 cDNA. In addition, we also found that the expression level of WNK4 was dramatically
reduced in Cldn7-/- CD cells, which was consistent with our previous finding that claudin-7 is a
substrate of WNK4 and can be phosphorylated by WNK4. Collectively, our findings indicate that
deletion of claudin-7 in CD cells impairs Cl- permeability, and that claudin-7 could be one of the key
factors in WNK4 mutations-induced hypertension. This research is supported by NIH grant
HL085752.
Integrative Biology of Vascular Disease
Joseph M. McClung. Department of Physiology, Diabetes and Obesity Institute, Brody School
of Medicine, East Carolina University.
The McClung lab is focused on understanding the complex signals regulating vascular biology
during disease, including cancer, cardiovascular disease, and muscle myopathy. Our laboratory
is involved in numerous projects to achieve our goals. 1) Genetic Variation Regulates
Susceptibility to Ischemic Necrosis: Using congenic chromosome substitution mouse strains
and viral re-expression we found that a single variant at residue 81 (snp81) in BCL2-associated
athanogene 3 (Bag3) differentiates the necrotic responses of C57BL/6 (BL6, Ile81) and BALB/c
(Met81) parental mouse strains to hindlimb ischemia (HLI). 2) Muscle Tissue Pathology in
Refined Clinically Relevant Models of PAD: Using ameroid constrictors, we have refined
traditional laboratory models of PAD to more accurately reflect the etiology of the human
disease and established a muscle tissue pathologic response to ischemia that is disconnected
from both the chronic level of limb perfusion and the outward level of peripheral tissue necrosis.
3) Paracrine/Autocrine Regulation of Endogenous Progenitor Cell Determination in the
Ischemic Limb: Using lineage tracing (ROSAmTmG) and genetic cellular specific ablation
(ROSA26DTA) mice bred to peri-endothelial (NG2) and muscle satellite cell (PAX7) Cre
drivers, we verified marked muscle degeneration in the ischemic limb of mice with the loss of
Pax7+ satellite cells.
Intriguingly, we also observed that the loss of efficient muscle
regeneration
resulted in a failure of NG2+ cells to co-localize to either the nascent muscle or vascular
compartments and a substantial deficit in limb tissue capillary density, an effect potentially
linked to a deficit in the local production/paracrine secretion of growth factors such as
angiopoietin-1 (Ang-1). 4) Heterogeneity in Cellular Expression and Function of Traditional
Vascular Growth Factors:
The primary target in anti-angiogenic strategies is vascular
endothelial growth factor (VEGF), and the anti-VEGF monoclonal antibody bevacizumab
(Avastin) was shown to prolong survival in patients with metastatic colorectal cancer. It is
increasingly clear that patients treated with anti-angiogenic therapy suffer from impaired
cardiorespiratory fitness and intolerable fatigue, which often results in the forced cessation of
drug therapy and, as a result, inadequate treatment. We have determined that both VEGF and
angiopoietins have direct effects on skeletal muscle cells and that targeting VEGF or Tie
receptors inhibits skeletal muscle function and physiological adaptation. These collective lines of
experimentation represent a novel integrative approach to traditional vascular disease and
function, integrating the biology of multiple tissue compartments for a unified response to
pathology.
FISH OIL OMEGA-3 FATTY ACIDS ACTIVATE PPARγ AND AUGMENT REDOX BALANCE AND
FATTY ACID METABOLIC CAPACITY IN HUMAN ATRIAL MYOCARDIUM
Ethan J. Anderson PhD1, 2†, Kathleen A. Thayne MS1, Mitchel Harris MS3, Saame Raza Shaikh PhD3,
Timothy Darden BS1, Daniel S. Lark MS4, J. Mark Williams MD2, W. Randolph Chitwood MD,2 Alan P.
Kypson MD2, Evelio Rodriguez MD2,5
Departments of 1Pharmacology & Toxicology, 2Cardiovascular Sciences and East Carolina Heart
Institute, 3Biochemistry and Molecular Biology, 4Kinesiology, East Carolina University, Greenville, NC;
5
St. Thomas Heart Cardiovascular Surgery, Nashville, TN
Background – Therapeutic benefit of fish oil omega-3 polyunsaturated fatty acids (n-3 PUFAs) in
cardiovascular disease remains controversial despite decades of study and millions of patients
treated. Consensus is lacking among investigators regarding formulation, dose, and mechanism of
action, and cellular and molecular data in human myocardium following n-3 PUFA treatment is
nonexistent.
Methods and Results - Patients ingested 4 g/day of n-3 PUFA ethyl esters (EPA + DHA) for a period
of 15-21 days prior to having cardiac surgery. Blood was obtained before treatment and at time of
surgery, and myocardial tissue from right atrium was also dissected during surgery. Eicosapentanoic
acid (EPA) increased 3.5-fold in blood with treatment, and n-3 PUFA-treated patients had ~3-fold
greater EPA in their atrial tissue compared to untreated, control patients (Ctl).
Docosahexaneoic
acid (DHA) levels were modestly increased with treatment. Interestingly, n-3 PUFA treatment led to
increased nuclear transactivation of peroxisome proliferator-activated receptor-γ (PPARγ) and
expression of genes involved in fatty acid metabolism, and greater mitochondrial respiration
supported by palmitoyl-carnitine, despite no differences in mitochondrial content. Atrial tissue from n3 PUFA patients also displayed greater levels of glutathione, thioredoxin reductase-2, and expression
of key antioxidant/anti-inflammatory enzymes.
Conclusions – This is the first translational evidence showing that fish oil n-3 PUFA therapy
enhances redox balance and capacity for fatty acid metabolism in human myocardium, and that 2-3
weeks is sufficient for this effect to occur. Further investigation is needed to explore whether postoperative clinical outcomes can be affected by this therapeutic regimen.
Glutathione Peroxidase-4 plays a critical role in protection from metabolic syndrome and
cardiac remodeling caused by diet-induced obesity
Lalage A. Katunga1, Taylor A Mattox1, Scott Abernathy1, Timothy M. Darden1, Jitka Virag2, Ethan J
Anderson1.
1
Pharmacology & Toxicology, East Carolina University, Greenville, NC
2
Physiology, East Carolina University, Greenville, NC
Peroxidation of polyunsaturated fatty acids (PUFAs) generates a number of highly reactive lipid
peroxidation products (LPPs), and the seleno-enzyme glutathione peroxidase-4 (GPx4) is an
antioxidant enzyme that selectively neutralizes LPPs. To determine the contribution of LPPs to the
pathogenesis of cardiomyopathy precipitated by diet-induced obesity, male WT and litter-mate GPx4deficient (GPx4+/-) mice were placed on a n-6 PUFA enriched high fat high sucrose (HFHS) diet for 24
weeks, along with a control group of WT mice (CTL) fed low-fat standard chow. At termination of diet,
adipose tissue mass and glucose intolerance were markedly higher in the GPx4+/- HFHS group
compared to WT-HFHS group, although both HFHS groups were significantly more than CTL. Blood
pressure and cardiac function were not significantly different between groups, although left ventricular
size and cardiomyocyte diameter was increased in WT-HFHS group compared to CTL. Cardiac
fibrosis was also markedly higher in both HFHS groups, but substantially more so in GPx4+/compared to WT. Mitochondrial O2 consumption was higher in WT-HFHS but not GPx4+/- HFHS,
compared to CTL. Surprisingly, the HFHS diet reduced mitochondrial H2O2 by ~3fold in WT vs. CTL,
but stayed similar between CTL and GPx4+/-. These findings implicate GPx4 as a critical enzyme
necessary for the heart to positively adapt to the stress imposed by obesity.
MONOAMINE OXIDASE IS A MAJOR DETERMINANT OF REDOX BALANCE IN HUMAN ATRIAL
MYOCARDIUM AND IS ASSOCIATED WITH POSTOPERATIVE ATRIAL FIBRILLATION
Ethan J. Anderson PhD,1,2,3† Jimmy T. Efird PhD,2,4 Stephen W. Davies MD, MPH,2,4 Wesley T.
O’Neal MD, MPH,5 Timothy M. Darden BS,1 Kathleen A. Thayne MS,1 Linda C. Kindell BSN, RN,2,3 T.
Bruce Ferguson MD,2,3 Curtis A. Anderson MD,2,3 W. Randolph Chitwood MD,2,3 Theodore C. Koutlas
MD,2,3 J. Mark Williams MD,2,3 Evelio Rodriguez MD,6 Alan P. Kypson MD2,3
Departments of 1Pharmacology & Toxicology, 2Cardiovascular Sciences, East Carolina Heart
Institute, and 3Center for Health Disparities,4 Brody School of Medicine, East Carolina University,
Greenville, NC; 5Department of Internal Medicine, Wake Forest University School of Medicine,
Winston-Salem, NC, and 6Saint Thomas Heart Cardiovascular Surgery, Nashville, TN
Background - Onset of post-operative atrial fibrillation (POAF) is a common and costly complication
of heart surgery despite major improvements in surgical technique and quality of patient care. The
etiology of POAF, and the ability of clinicians to identify and specifically treat high-risk patients,
remains elusive.
Methods and Results - Myocardial tissue dissected from right atrial appendage (RAA) was obtained
from 244 patients undergoing cardiac surgery. Reactive oxygen species (ROS) generation from
multiple sources was assessed in this tissue, along with total glutathione (GSHt) and its related
enzymes GSH-peroxidase (GPx) and GSH-reductase (GR). Monoamine oxidase (MAO) and NADPH
oxidase were observed to generate ROS at rates ten-fold greater than intact, coupled mitochondria.
POAF risk was significantly associated with MAO activity (adjusted P trend<0.0001). In contrast,
myocardial GSHt was inversely associated with POAF (adjusted Ptrend=0.016). GPx also was
significantly associated with POAF, however, a linear trend for risk was not observed across
increasing levels of the enzyme. GR was not associated with POAF risk.
Conclusions - Our results are the first to show that MAO is an important determinant of redox
balance in human atrial myocardium, and its activity is associated with an increased risk for POAF.
Further investigation is needed to validate MAO as a predictive biomarker for POAF, and to explore
this enzyme’s potential role in arrhythmogenesis.
Altering hyaluronan metabolism in cartilage and chondrocytes through the introduction of select transgenes
Shinya Ishizuka, Emily B Askew, Yohei Ono, Daisuke Hida, Cheryl B Knudson and Warren Knudson
Department of Anatomy and Cell Biology, BSOM, East Carolina University, Greenville, NC
During osteoarthritis (OA), hyaluronan (HA) is lost from articular cartilage in close coordination with the loss of the
proteoglycan, aggrecan. Thus, optimal cartilage repair requires the same tight coordination in reverse. We have
accumulated evidence that the level of HA within the extracellular matrix is controlled by the activity of hyaluronan
synthases (HAS2, [1]) and via CD44-mediated endocytosis [2]. This study focuses on modulating these two proteins as
means to regulate matrix levels of HA in cartilage. We hypothesize that overactive CD44-mediated endocytosis occurs in
OA, reducing matrix HA levels and thus, limiting repair by chondrocytes. However, the most direct approach to modify
HA levels is by expression of HAS2. Several studies have documented the use of adenoviral and rAAV constructs for
gene therapy in humans and successful gene transfer into cartilage explants. For our preliminary studies, viral
transduction of human CD44 isoforms and HAS2 into monolayers, neocartilage disks and human OA cartilage explants
were examined for their ability to modulate levels of matrix HA.
Adenovirus particles containing a CD44-dominant negative construct (Ad-CD44-DN) were applied to bovine articular
cartilage explant cultures and allowed to incubate ex vivo for 1 week. Western blot analysis confirmed expression of the
human transgene products in the bovine cartilage explants. When applied to bovine chondrocytes, an increase in HA
released into the medium was observed in proportion to increase in Ad-CD44-DN. HA also displayed a pronounced
accumulation in the media of intact bovine cartilage explants transduced with Ad-CD44-DN. No such increase was
observed with comparable vp/c transduction with Ad-CD44wt. This suggests blocking CD44 function (including receptormediated endocytotic turnover of HA) shunts higher levels of HA into the medium. Next, chondrocytes were incubated
with Ad-ZsGreen-HAS2 viral particles. At 24 hour post-transduction, 2-3 fold increases in cell-associated HA were
observed and 5x increases of HA detected in the cell culture medium. To test our hypothesis that local enhancement of
HA could be used to promote homeostasis in chondrocytes, human OA chondrocytes wee transduced with Ad-ZsGreenHAS2 or Ad-LacZ, followed by treatment with IL-1β. HAS2 transduction reduced IL-1β-induced stimulation of MMP13
mRNA expression as compared to Ad-LacZ transduced control cultures. When Ad-ZsGreen-HAS2 viral particles were
applied to human OA cartilage explants, ZsGreen-positive cells were readily observed in situ, primarily in superficial zone
chondrocytes. A second approach being explored is to establish bioengineered neocartilages that contain chondrocytes
expressing human transgenes. Ad-ZsGreen-HAS2 transduced neocartilage disks were observed to be Safranin O
positive (indicative of hyaline cartilage) and populated with ZsGreen-positive cells. Binding of a probe for HA (red
fluorescence) verified that HA levels were elevated in the Ad-ZsGreen-HAS2 transgene-expressing neocartilage as
compared to control, Ad-LacZ transduced neocartilages.
Our work has moved toward viral transduction because obtain >93% transduction efficiencies when applied to human or
bovine chondrocytes in vitro—a value never before obtained with plasmid transfection [3]. Moreover, since chondrocytes
(especially human) exhibit little proliferative capacity, adenoviral and rAAV expression of transgenes for long periods is
possible without genome insertion. Hyaluronan and aggrecan are both lost from articular cartilage during the development
-/and progression of OA. Furthermore, cartilage of mice lacking the lysosomal enzyme hyaluronidase-1 (Hyal1 ) exhibit
intense pericellular staining of HA as well as increased intracellular accumulation of HA [4], indicating that HA endocytosis
and degradation is an essential component of HA turnover in cartilage. In the current study, we explored the hypothesis
that overactive CD44-mediated endocytosis occurs in OA, resulting in decreased matrix HA and limited repair by
chondrocytes. Ad-CD44-DN transduction of bovine chondrocytes and explants resulted in an increased accumulation of
HA within respective culture media, suggesting that CD44-mediated endocytosis of HA was blocked. We also examined
the effect of directly enhancing HA production by the introduction of a human HAS2 transgene. Using the new AdZsGreen1-HAS2 virus, we can observe green fluorescence due to ZdGreen1. Since expression of both ZsGreen1 and
HAS2 are driven by dual CMV1E promoters on the same viral vector backbone, both inserts are co-expressed. We can
detect by RT-PCR, high mRNA levels of both ZdGreen1 and HAS2 mRNA. Likewise, we have observed increases in HA
in both cell layers and culture media of chondrocytes as well as within neocartilage tissues in Ad-ZsGreen1-HAS2
transduced samples. In this study, we also observed in chondrocyte monolayers, a diminution in IL-1β-induced MMP13,
suggesting that enhancing HA might diminish expression of catabolic markers such as MMP13.
Significance: During OA, chondrocytes attempt repair but this response ultimately fails and cartilage undergoes
progressive degeneration. We hypothesize that this is due in part to a deficiency in HA. Increasing HA helps to retain
aggrecan and, via binding to CD44, signal chondrocytes to become quiescent and return to homeostasis.
[1] J Biol Chem 274: 21893-99, 1999; [2] J Biol Chem 285: 36216-24, 2010; [3] Ortho Trans, 38: 1313, 2013; [4] Hum Mol
Genet 17: 1904-15, 2008. Supported in part by NIH grants R01-AR043384 (WK), and R01 AR039507 (CBK).
Isolation and expansion of murine mesenchymal progenitor cells to investigate the role of hyaluronanCD44 interactions during differentiation
Roberta Veluci, Warren Knudson and Cheryl B Knudson, Department of Anatomy and Cell Biology,
BSOM, East Carolina University, Greenville, NC
Hyaluronan (aka hyaluronic acid) is a ubiquitous component of the extracellular matrix. Its characteristics
enable hyaluronan to influence many physiological processes, including cell migration, proliferation,
differentiation and cell signaling. Hyaluronan is also important for tissue and organ development and
architecture. Evidence from several laboratories points to hyaluronan as critical for many facets of stem cell
biology both at the cell surface and as an element of the stem cell niche. Mesenchymal stem cells (MSC)
represent a subset of stem cells that have the potential to differentiate to all mesenchymal lineage cells
including chondrocytes, osteoblasts, and adipocytes and may contribute to the repair of damaged connective
tissues. The potential use of MSCs for tissue regeneration and treatment of osteoarthritis involves many
factors that regulate their differentiation, including members of the TGFβ superfamily of growth factors. In
articular cartilage hyaluronan serves as the central filament of the extracellular cartilage proteoglycan
aggregate. Within the pericellular matrix hyaluronan binds to the transmembrane receptor CD44 that serves as
a mechanism for chondrocytes to sense changes in the matrix. The presence of hyaluronan in the stem cell
niche and the expression of CD44 by stem cells have led us to explore whether hyaluronan or a receptor for
hyaluronan is involved or required for the differentiation of MSCs.
Previously, we found that removal of pericellular hyaluronan diminished SMAD1/4-signaling via bone
morphogenetic protein-7 (BMP7) but not SMAD2/4-signaling through TGFβ in adult bovine articular
chondrocytes [1]. Our hypothesis is that clustered CD44 may function as a SARA for SMAD1 and removal of
CD44-hyaluronan interactions reduces the cellular response to BMP7.
Our current studies aim to determine whether potential differences exist between mesenchymal progenitor
cells (MPC) isolated from wildtype (WT; BALB/c) and Cd44-/- mice induced by TGβ3 and/or BMP7 to
differentiate into chondrogenic and osteogenic lineages. MPCs were selected through media and plastic
adherence from bone marrow and endosteum-associated cells of 1 and 2 month old mice. Femurs and tibiae
were harvested, crushed and digested with 0.2% collagenase in serum free DMEM to release cells. Adherent
cells were expanded for 5 days in DMEM + 20% FBS, then lifted with 0.025% trypsin-EDTA, plated, expanded,
lifted and plated as passage-2 (P2) cells. These P2 cells exhibited a CFU = 20 ± 1.94 and at confluence were
used for experiments. The chondrogenic potential of P2-MPCs was tested using direct pellet cultures,
micromass cultures or a strategy that combined pre-culture in micromass prior to the establishment of pellet
cultures with chondrogenic media supplemented with TGβ3 and/or BMP7. WT direct-pellet cultures treated
with TGFβ for 5 days and then with TGFβ3+BMP7 for 14 days exhibited cartilage morphology and enhanced
Safranin O staining as compared with parallel Cd44-/- pellet cultures. Alcian Blue staining was observed across
all treatments in micromass cultures, with subtle differences between WT and Cd44-/- murine MPCs treated
with BMP7. Conditioning P2-MPCs in micromass cultures prior to pellet cultures significantly enhanced MPCs
chondrogenic potential. This approach may by-pass the need for hyaluronan-CD44 signaling in Cd44-/- pellet
cultures treated with BMP7. Surprisingly, Cd44-/- MPCs in monolayer cultures treated under optimal osteogenic
conditions demonstrated higher Alizarin Red staining and Runx2 mRNA expression than WT MPCs. Therefore,
hyaluronan-CD44 interactions may influence signaling pathways to modulate chondrogenic versus osteogenic
differentiation potentials of MPCs.
Future studies will include CD44-gain-of-function by using transduction with human full length-CD44 to provide
a rescue in Cd44-/- murine MPCs as well as CD44-loss-of-function by using human dominant negative DNCD44 [2] to reduce bioactivity of endogenous CD44 in WT murine MPCs. To further explore the role of
hyaluronan, synthesis will be enhanced by expression of the synthase, HAS2 [3], to verify if increasing
hyaluronan promotes BMP7 responses by MPCs.
[1] RA Andhare et al., 2009, Osteoarthritis Cartilage 17:892-902. [2] H Jiang et al., 2002, J Biol Chem
277:10531-38. [3] Y Nishida et al., 1999, J Biol Chem 274: 21893-99. Supported in part by NIH grants R01AR043384 (WK) and R01-AR039507 (CBK).
The Department of Comparative Medicine (DCM)
The Department of Comparative Medicine (DCM) is a basic science department in the Brody
School of Medicine at East Carolina University that supports high quality science through
excellent and humane animal care. It was established over 35 years ago and has been fully
accredited by AAALAC International since 1987. The department provides husbandry; veterinary
care and maintenance of animal health for all animals used in biomedical research, teaching,
testing, and field studies at ECU; technical and diagnostic laboratory services; surgical services
and surgery support; research protocol-design assistance; and collaborative biomedical and
veterinary research aimed at improving the health and welfare of humans and animals. DCM has
25 employees and cares for multiple species. There are two primary facilities operated by DCM,
located on ECU’s Health Sciences Campus. Additional housing space is located on the Main
Campus and West Research Campus. Total square footage of facilities exceeds 75,000 square
feet. DCM facilities are capable of housing a wide variety of species, including but not limited to
birds, fish, amphibians, reptiles, rodents, rabbits, and larger animal species. Quarantine, barrier,
and containment housing are also available. A fully staffed and equipped surgical suite and
diagnostic laboratory are available for research support. The surgical-support area includes a
state-of-the-art postoperative recovery chamber for large and small animals. The diagnostic
laboratory has hematology, clinical chemistry, parasitology, and microbiology capabilities. The
laboratory services are expanded through the use of outside testing services. The DCM support
staff consists of a PhD in laboratory animal sciences, an LATg-certified facility manager, an LATcertified surgery supervisor, a medical laboratory technologist, two LAT-certified veterinary
technicians, a special projects coordinator, a staff of 12 animal-care technicians, and 3
departmental faculty members that hold Doctor of Veterinary Medicine (DVM) degrees. The
veterinarians have a combined total of approximately 50 years of laboratory animal care and use
experience. The department chair is a diplomate of the American College of Laboratory Animal
Medicine (ACLAM). AT DCM, our overarching goal is to ensure a collaborative working
environment based on integrity and trust and to maintain an exemplary animal care program.
In vivo measurements of mitochondrial respiratory capacity in skeletal muscle
Terence E. Ryan1,3, Chien-Te Lin1,3, Patricia Brophy2,3, P. Darrell Neufer1,2,3
1
East Carolina Diabetes and Obesity Institute, Departments of 2Kinesiology, and 3Physiology.
Mitochondria have several roles including cellular growth and differentiation, apoptosis, and cellular
signaling, but are most known for their metabolic capability to generate chemical energy in the form of
adenosine triphosphate. The physiological importa nce of mitochondria is widely appreciated, and
dysfunctional mitochondrial energetics have been associated with a number of health problems
ranging from neuromuscular diseases and cancer to metabolic disorders such as type 2 diabetes
mellitus and insulin r esistance. In vitro and in situ approaches to measuring mitochondrial function
provide tight control over experimental conditions, however, it should be noted that these approaches
often include non-physiological conditions and do no have intact regulatory/circulatory systems. From
both a research and clinical perspective, it is of great value to assess mitochondrial function in the
intact organism using non-invasive approaches. Combining in vitro and in vivo techniques provides a
powerful approach to investigate mitochondrial bioenerg
etics. This presentation describes the
development and validation of a novel, in vivo approach to measuring mitochondrial respiratory
capacity in skeletal muscle using near infrared spectroscopy.
Research Discussions at the Tipsy Teapot
Joseph M. Chalovich
18 years ago a group of researchers interested in cell motility began meeting at regular intervals to
have critical research discussions.
The idea of those meetings was to have a “safe” forum for
introducing new ideas, for obtaining constructive criticism and for building research collaborations.
That research group has expanded to include all areas of research at ECU. Investigators from many
departments at the Brody School of Medicine and the East Campus meet monthly at the Tipsy Teapot
on Evans Street for informal discussions and presentations. Meetings are normally held on either the
4th Thursday or 4th Wednesday of each month from 4:30 until 6:00 pm. Food and beverages are
available for purchase during the meetings.
Brief (<30 minute) research presentations stress new results, research problems and opportunities for
collaboration. Everyone with an interest in biomedical research is welcome to attend. Please
send an email to chalovichj@ecu.edu to be placed on the mailing list. Please send a tentative title if
you would like to make a presentation at an upcoming meeting.
A Multidisciplinary Approach to Fight Senior Dementia: the Harriet and John Wooten
Laboratory for Alzheimer’s and Neurodegenerative Diseases Research
1
Sonja Bareiss, 2Kori Brewer, 3Stefan Clemens, 4Jamie DeWitt, 3Jian Ding, 5Hu Huang, 6Zhibin
Huang, 7Tae J. Lee, 8Qun Lu, 3Alexander K. Murashov, 9Mark D. Mannie, 4Ken Soderstrom, 10Tuan
Tran
1
Department of Physical Therapy, College of Allied Health Sciences, East Carolina University;
Departments of 2Emergency Medicine, 3Physiology, 4Pharmacology and Toxicology, 6Radiation
Oncology, 7Family Medicine, 8Anatomy and Cell Biology, 9Microbiology and Immunology, The Brody
School of Medicine, East Carolina University; 5Department of Kinesiology, College of Health and
Human Performances, East Carolina University; 10Department of Psychology, Thomas Harriot
College of Arts and Sciences, East Carolina University, Greenville, North Carolina 27834
Senior dementia has increased remarkably in recent decades due in part to the increased life
expectancy. Dementias of Alzheimer’s type and other neurodegenerative diseases such as
frontotemporal dementia (FTD) have no cure. In addition, previously over simplified therapeutic
guidance has narrowed the scope of scientific searches for effective treatments. The Harriet and John
Wooten Laboratory for Alzheimer’s and Neurodegenerative Diseases Research was established from
the vision of the Wooten family to promote multidisciplinary research in North Carolina on the
molecular and cellular mechanisms of AD and other dementias. Our strategies are to harness the
diverse talents from the neuroscience research community at the Brody School of Medicine and other
health sciences campuses to come together and work on the common platform of mouse models
expressing the most aggressive AD pathogenesis.
The Wooten Laboratory has established a triple (3xTg) transgenic core of AD mice displaying
mutations of amyloid precursor protein, presenilin-1 and microtubule-associated protein tau, three of
the most prominent molecular pathogenic hallmarks of AD. From this model, the Wooten Laboratory,
under the directorship of Dr. Qun Lu and guidance of the Wooten Advisory Board chaired by Dr.
Robert M. Lust, organizes to explore better and improved models and investigate AD pathogenic
mechanisms using a variety of molecular, genetic, biochemical, immunological, imaging, and
behavioral approaches. Our 13 principal investigators described at the author line are from 10
departments across 4 schools on East Carolina University campus and have formed a task force and
intensive research group.
For the past three years, 18 projects funded by the Wooten seed grants have been investigating the
following subjects: beneficial effects of cognitive training and physical exercise on memory and
learning, innovative small molecule modulators of synaptic remodeling on delaying
neurodegeneration and promoting neuroregeneration and neurogenesis, spinal cord function and
dopamine modulation, roles of microRNAs, early childhood exposure to environmental toxicans and
risks of AD as well as autoimmune responses as mechanisms of AD pathogenesis and dysregulation
of circadian rhythm and sleep in 3X Tg-AD mice, to name just a few. The success of these projects
will lay the groundwork for implementing senior dementia research base that is critically needed in
eastern North Carolina. Besides the Wooten seed grants, researches of our principal investigators
are currently supported in part by grants from the National Institutes of Health, the US Army,
Alzheimers North Carolina, and North Carolina Biotechnology Center.
Mitochondrial reactive oxygen species and lipid peroxidation are central to preadipocytes and adipose
tissue dysfunction. Reduced adipose tissue antioxidant capacity correlates with the systemic insulin
resistance. Two studies within the translational continuum from bench to bedside.
Carlyle Rogers1, Barbara Davis1, Jared Shine1, P. Darrell Neufer2,3,4, Michael P. Murphy5, Ethan J
Anderson1,3,4, Jacques Robidoux1,4
The Departments of 1Pharmacology and Toxicology, 2Physiology and 3Kinesiology, 4 the East Carolina Diabetes
and Obesity Institute, East Carolina University, USA, 5the MRC Mitochondrial Biology Unit, Cambridge
University, UK
Oxidative stress is a contributing factor to adipose and systemic metabolic abnormalities including insulin
resistance. Other emerging clinical correlations are that loss in preadipocyte number and adipogenic potential
correlate with obesity-associated metabolic abnormalities. Why many obese individuals experience oxidative
stress or have moribund preadipocytes while others do not is not known. As nutrient overload is the primary
cause of both obesity and its associated metabolic abnormalities, the goal of the first study was to test the
hypothesis that preadipocyte exposure to fatty acids (FAs) leads to mitochondrial permeability transition (PT)
and ATP depletion. We found that FA exposure led to progressive cyclosporin A-sensitive PT, which in turn
caused a reduction in inner membrane potential, oxygen consumption, ATP synthetic capacity, and ultimately
caused cell death. FAs also induced a subtle and transient increase in mitochondrial steady state levels of
reactive oxygen species (ROS) and lipid peroxides, which lasted 30 and 120 minutes, respectively. FAs also
promoted a delayed overt oxidative stress phase. PT and its consequences including ATP depletion and cell
death were prevented by treating the cells with the mitochondrial FA uptake inhibitor, Etomoxir; the
mitochondrion selective superoxide and lipid peroxide antioxidants, MitoTempo and MitoQ; or the lipid peroxide
and reactive carbonyl scavenger, L-carnosine. However, the beneficial effects of these treatments were lost by
delaying the treatment by 2 hours. This suggested that overt oxidative stress was a consequence, not the
cause, of mitochondrial crisis. Altogether, our data suggest that prevention of the exaggerated increases in the
steady state levels of mitochondrial ROS and lipid peroxides as well as PT are valid mechanism-based
therapeutic avenues to avoid the loss in preadipocyte health from prolonged exposure to elevated FA levels.
In a second study, we tested if the expression of genes involved in various aspects of the antioxidant response
is reduced in insulin-resistant obese (IRO) compared to insulin-sensitive obese (ISO) individuals. For this
purpose adipose tissue samples from thirty-three women covering a wide range of body mass index and
insulin sensitivity were collected and the mRNA of various antioxidant genes and the protein levels of total and
acetylated FOXO1 were quantified by real-time PCR and western blots respectively. Protein carbonyl groups
were also measured as an index of oxidative stress. Our results showed that the expression of thioredoxin
reductase 2 (TXNRD2), thioredoxin 2 (TXN2), peroxiredoxin 3 and 5 (PRDX3 and PRDX5), catalase (CAT)
and superoxide dismutase 2 (SOD2) was lower in the IRO compared to the ISO individuals. As the promoter of
these genes contains forkhead box protein response elements, we further evaluated the relationship between
FOXO1 acetylation levels and their expression. This revealed a strong inverse correlation between FOXO1
acetylation and antioxidant gene expression as well as insulin resistance.
All together, these studies supported by the Brody School of Medicine (JR), the East Carolina Diabetes and
Obesity Institute (JR), the American Heart Association (CR, JR), and the National Institutes of Health (PDN)
suggest that lower antioxidant capacity in adipose tissue may sensitize to the reducing pressure of nutrient
overload and that mitochondrion-targeted antioxidants or PT inhibitors are possible therapeutic avenues.
Fish oil differentially regulates B cell and dendritic cell activation in response to a Tindependent antigen
Heather Teague, Benjamin Drew Rockett, Mitchel Harris and Saame Raza Shaikh
Fish oil (FO) has clinical utility for regulating immunity. The functional consequences of FO on B cells
and dendritic cells (DC) are poorly studied, especially at the animal level. Here we tested the effects
of FO, modeling human pharmacological intake, on murine ex vivo B cell and DC activation in
response to the T-independent antigen lipopolysaccharide (LPS). LPS stimulation of B cells increased
CD69 surface expression and enhanced secretion of IL-6, TNFα and IFNγ with FO. In comparison,
LPS stimulation of DCs resulted in reduced CD80 surface expression and TNFα secretion with FO,
which was associated with decreased phagocytosis of E. coli bioparticles. To reconcile differences
between cell types, we measured the formation of plasma membrane lipid microdomains in both B
cells and DC. Although FO promoted an increase in EPA and DHA levels in both DCs and B cells, we
discovered FO exerted differential effects on the ability of B cells and DCs to cluster lipid
microdomains. Overall, this study shows fish oil exerts differential effects on the activation of B cells
and DCs in response to LPS, which may be due to differences in underlying membrane organization.
Our data highlight the possibility that FO can be used to exert both immune enhancing and
immunosuppressive effects, depending on the cellular target.
Retinoids prompt immune cell adhesion through RAR activity
JT Whelan, J Chen, L Wang, LC Bridges
East Carolina University and the East Carolina Diabetes and Obesity Institute
Department of Biochemistry and Molecular Biology
Greenville, NC 27834
Retinoids, biologically active oxidative metabolites of vitamin A, are essential in the proper
establishment and maintenance of immunity. Although retinoids influence immune related processes,
their role in immune cell adhesion is not well defined. Our lab recently reported that retinoids induce
immune cell adhesion through integrin-dependent and integrin-independent mechanisms across
immune lineages. Ultimately, retinoids exert transcriptional regulation by binding and activating
specific nuclear transcription factors, namely the retinoic acid receptor (RAR) and retinoid X receptor
(RXR) family of nuclear receptors. Our current work delineates which specific nuclear receptors
stimulate immune cell adhesion. By utilizing a series of isotype-specific agonists and antagonists, we
establish that integrin-independent adhesion in a human B cell line is induced upon RAR-alpha
activation. In addition, primary human PBMCs exhibit ex vivo integrin-independent adhesion upon
RAR activation. Interestingly, integrin-dependent adhesion within a human T cell line occurred
through stimulation of either RAR or RXR receptors. Our results demonstrate that retinoid exposure
can prompt immune cell adhesion through the action of distinct nuclear receptors (e.g. nonpermissive versus permissive), and suggest that the particular nuclear receptor stimulated governs
the specific adhesion receptor(s) employed.
Low Intensity Exercise Attenuates Acute Lipid Loading-Induced Alterations in Mitochondrial
Function in Rat Skeletal Muscle
Chien-Te Lin1,2,3, Kelsey H. Fisher-Wellman1,2 , Christopher G.R. Perry1,2, Rachel Kozy1,2, Daniel S.
Lark1,2,
Laura A. A. Gilliam1,2,3, Cody D. Smith1,3 and P. Darrell Neufer1,2,3
East Carolina Diabetes and Obesity Institute1, Departments of Kinesiology2, and Physiology3
Carolina University
East
A single high-fat meal acutely increases skeletal muscle mitochondrial H2O2 emitting potential
(mEH2O2), shifts the intracellular redox environment to a more oxidized state, and increases circulating
markers of oxidative stress. Bioenergetically, this implies an acute lipid load may elevate the
reducing pressure/membrane potential (ΔΨm) within mitochondria and, conversely, that even a mild
increase in energy expenditure may be sufficient to prevent these effects. To test this hypothesis,
male Sprague-Dawley rats received an oral lipid gavage (20% intralipid, 45 Kcal/kg lean body mass)
or water followed either by 2h of rest or 1h of rest plus 1h of low intensity treadmill exercise (15
m/min, 0% grade). Permeabilized fiber bundles were prepared from red gastrocnemius muscle for
testing mitochondrial function. In rats receiving lipid, ΔΨm and mEH2O2 were higher (P<0.05) and
calcium retention capacity (mCa2+RC, an index of resistance to mitochondrial permeability transition)
was lower under state IV and/or “clamped” ADP-stimulated state III conditions. All three effects were
prevented when lipid gavage was followed by low-intensity exercise. Respiratory capacity was
unaffected by any of the interventions. These findings provide evidence that mitochondrial ΔΨ m,
2+
mEH2O2, and mCa RC are acutely affected by nutritional overload in skeletal muscle, but can be
prevented by low intensity exercise.
NIH RO1 DK073488
Acute in vitro statin exposure alters mitochondrial function in permeabilized skeletal muscle
from healthy humans
Chien-Te Lin1,2, Brook L. Cathey1,2, Christopher G.R. Perry1,2, William J. Mayo1,3, Laura A. A.
Gilliam1,2, Kelsey H. Fisher-Wellman1,3, Daniel S. Lark1,3, Cody D. Smith1,2, Rachel Kozy, and P.
Darrell Neufer1,2,3
East Carolina Diabetes and Obesity Institute1, Departments of Physiology2, and Kinesiology3
Carolina University
East
Statins, widely used in the treatment of cardiovascular disease, are associated with potential adverse
side effects in skeletal muscle. Data from cell culture studies suggest statin induced myopathy may
be initiated by altered mitochondrial function. To further examine the potential impact of statins,
various aspects of mitochondrial function were assessed in human permeabilized skeletal muscle
fiber bundles (PmFB) exposed (30 min) in vitro to atorvastatin (ATOR, 10 μM) or simvastatin (SIM,
2.5 & 10 μM), and in PmFB from healthy subjects after acute (2h) and/or chronic (once/d for 7d)
treatment with ATOR (80 mg/d) or SIM (40 mg/d). In vitro exposure of PmFB to ATOR and SIM
decreased both complex I and II supported respiratory capacity (JO2), whereas only SIM decreased
complex I supported mitochondrial calcium retention capacity (mCa2+ RC). Neither acute nor chronic in
vivo treatment with ATOR or SIM altered JO2 or mCa2+ RC. Although mitochondrial H2O2 emission
potential (mEH2O2) in the presence of complex I or multiple substrates was not altered by in vitro or in
vivo exposure to either statin, both in vitro and acute in vivo exposure to SIM tended to decrease
complex II mEH2O2. These findings indicate both ATOR and SIM are capable of directly impacting
mitochondrial function in human PmFB exposed in vitro. The acute and chronic impact of in vivo
statin exposure on skeletal muscle mitochondrial function is less clear and will require further study.
NIH R01 DK074825.
How Prenatal Nutrition, Obesity and Depression Affect Pregnancy Outcomes among LowIncome Women: Health Moms Study
Principal Investigator: Dr. Juhee Kim
Study Coordinator/Contact Individual: Ayushi Shah
Sub-Investigators: Edward Newton, Jeffrey Livingston, Dennis Russo, Emily Bray
Other Study Staff: Tatiana Acosta, Katie Vang, Hemadhanvi Chagarlamudi, Shannon Heintz, Janina
Dorriety, Julia Fout, Shivajirao Patel, Sherry Jackson, Justin Raines, and Mandee Lancaster.
Background: Prenatal obesity and depression are known risk factors for adverse pregnancy
outcomes and newborn development. Obesity, depression and their implications for nutrition during
pregnancy and gestational weight gain among low income women have not been fully characterized.
However, previous research often has not considered the potential interaction between obesity and
depression at the same time even though both conditions are prevalent among low-income women.
To understand multiple pregnancy risks among low-income women, we built a prospective cohort
study to document the profile of health behaviors, medical, and social conditions from early
pregnancy (<28 weeks of pregnancy) to delivery.
Objectives: The study objectives are to determine the associations of eating behaviors and
depression with and without obesity during early pregnancy on gestational weight gain, delivery
mode,
preterm
birth,
and
infant
birth
weight
among
low-income
women.
The specific aims are:
1.
2.
3.
To validate the clinical measures of nutrition assessment during prenatal period among
low-income mothers.
To determine the associations of prenatal obesity, depression symptoms, and nutrition
among low income mothers.
To determine the effect of prenatal depression with and without the complication of obesity
during early pregnancy on pregnancy outcomes among low income mothers.
Methods: A prospective research-targeted pilot study for high risk pregnancy at the ECU Department
of Family Medicine and OB/GYN prenatal clinic has been launched since September, 2013. The
study is to follow pregnancy from the second trimester (baseline) to delivery (followed-up by the
status of obesity, BMI ≥30) and depression symptoms (Edinburgh Postnatal Depression Scale, EPDS
≥10). The study group is composed of 1) no depression/obesity 2) depression only 3) obesity only 4)
depression with obesity. Recruitment for each group (n=30-40) will be targeted for those who come to
prenatal care visit during their second trimester. The study measures included demographics,
psychosocial, health behaviors, nutrition assessment, and prenatal care experience from a prenatal
survey, a 24-hour diet recall, and an optional focus group interview (n=5-8 per group). The follow-up
pregnancy outcome data at delivery will be extracted from the ECU medical record system.
Results (recruitment update): As of October 21st we have received a total of 38 contact cards (21
from OB-GYN and 17 from Family Medicine). Of those, 11 are actively being recruited and 15 have
been excluded (due to: chronic conditions, not eligible based on set criteria, and scheduling conflicts).
From our 38 contact cards, we have recruited and consented 12 mothers, 9 non-Hispanic black and 3
non-Hispanic white. The age range is 19-28 years old. Among them, one is classified as obese
(based on pre-pregnancy BMI ≥ 30) and two have depressive symptoms (based on EPDS score ≥
10). The range of the gestational age is 17 weeks and 4 days to 29 weeks.
Implications: Not only will we document multiple pregnancy risks among low-income mothers, but
we will also document the experience and need for prenatal care. The results will be incorporated into
the development of a targeted prenatal care intervention program. The purpose of this study is to
identify prenatal nutrition, medical, and social factors that can influence pregnancy outcomes in lowincome women.
OXIDATIVE PHOSPHORYLATION EFFICIENCY DETERMINED IN REAL-TIME USING
PERMEABILIZED HUMAN AND RODENT MYOFIBERS
Daniel S. Lark2,4, P. Darrell Neufer1,2,4 and Ethan J. Anderson3,4†
Departments of 1Physiology, 2Kinesiology, 3Pharmacology and Toxicology and 4East
Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina
Novel approaches are needed to progress our understanding of mitochondrial dysfunction in
diseases such as cancer, cardiovascular disease and diabetes.
Efficiency of oxidative
phosphorylation (OxPhos), the ratio of ATP produced to oxygen consumed (ATP/O ratio), is vital to
maintaining proper myocyte energetics, and changes in ATP/O would be expected to have a
profound impact on myocyte contractile function and vitality. Previous approaches typically
report ATP/O at a single [ADP], are unable to quantify steady state ATP synthesis rates and/or
utilize isolated mitochondria. To improve the utility of ATP/O ratio as an index of mitochondrial
function, we developed a system to simultaneously measure mitochondrial respiration and ATP
production in small samples of rodent and human permeabilized myocardium and skeletal muscle
over a range of physiological [ADP] (20-500 μM) in real-time.
In permeabilized mouse
myocardium, ATP/O is constant (~2) in the presence of ADP and is not altered by the addition of
creatine or when OxPhos is supported with carbohydrate (pyruvate) or fatty acid (Palmitoyl-Lcarnitine) derived substrates. In permeabilized skeletal muscle myofibers, ATP/O ratio increases
as a function of [ADP] and our preliminary data suggest that adenylate kinase may be a
confounding factor in the determination of ATP/O ratio.
Use of this method is also
demonstrated in permeabilized myofibers prepared from human right atrium and vastus lateralis
biopsies. This report provides a method to determine mitochondrial respiratory efficiency in human
and rodent myofibers that may provide novel insights into disease paradigms.
BB-10-19-12
Impaired Homeostasis of Alveolar Macrophages from Obese Individuals: Alterations in
Scavenger Receptor and ABC lipid transporter Expression.
R.Karnekar, A. Malur, I Marshall, MC. Cashion, Z. Rehman, H. Mehta, I Huizar, BP. Barna, MJ.
Thomassen
Department of: Internal Medicine, Division of Pulmonary, Critical Care and Sleep Medicine
BACKGROUND. Obesity has been associated with numerous pulmonary co-morbidities including
sleep apnea and asthma, among others. Because alveolar macrophages are critical to pulmonary
homeostasis, our studies have focused on defining alveolar macrophage activity in obese subjects.
We recently reported reduced expression and activity of the transcription factor, peroxisome
proliferator-activated receptor-gamma (PPARγ) together with elevated expression of the proinflammatory cytokine, IL-6 in alveolar macrophages from obese subjects compared to lean, healthy
controls (Sharma et al Surgery, 2012,151:107). PPARγ is recognized as a critical regulator of
glucose and lipid homeostasis as well as a negative regulator of inflammation.
HYPOTHESIS. Based on these data, we hypothesized that alveolar macrophages from obese
patients might exhibit deficiencies in pulmonary lipid homeostasis.
METHODS. Bronchoalveolar cells (BALC) were obtained from 13 obese subjects (>40BMI) and 9
lean (BMI<28) healthy controls. Affimetrix gene array and QPCR were carried out on BALC mRNA;
QPCR was also used to analyze mRNA from healthy subject alveolar macrophages cultured in vitro.
RESULTS. Array data indicated reduced expression (-3.0 fold, p<0.05) of the ABC lipid transporter,
ABCA1 in obese subjects versus lean controls. QPCR validated array data: ABCA1 mRNA
expression was depressed 3.3-fold, (p<0.05). Array data also showed elevation of scavenger receptor
alpha subtype 3 (SCARA3) (3.6 fold, p<0.05), a key receptor in lipid endocytosis. QPCR revealed
elevated SCARA3 in BALC from obese patients with apnea (4.3-fold, p<0.05) and in addition,
elevated TNFα expression in BALC of obese subjects (2.0 fold, p<0.05) with highest levels (3.0 fold,
p<0.05) in obese with apnea compared to controls. SCARA3 stimulation by TNFα (4.0-fold, p<0.05,
n=4) was noted in alveolar macrophage cultures but TNFα did not affect ABCA1 mRNA (1.2-fold, ns,
n=5).
SUMMARY AND CONCLUSIONS. Alveolar macrophages of obese subjects exhibit alterations in
lipid regulatory components, ABCA1 and SCARA3, together with elevated TNFα, a potent proinflammatory cytokine. Findings suggest that elevation of pro-inflammatory cytokines in the obese
lung may compromise pathways of lipid homeostasis and increase pulmonary disease susceptibility.
Inflammation and disruption of the mucosal architecture in claudin-7-deficient mice
Yan-Hua Chen1*, Zhe Lu1, Lei Ding1,, Oded Foreman2, Rodney Tatum1, Qun Lu1, Randall Renegar1,
Jian Cao3, 1Department of Anatomy and Cell Biology, East Carolina University Brody School of
Medicine, Greenville, NC 27834; 2Department of Pathology, The Jackson Laboratory, Sacramento,
CA 95838; 3Department of Medicine and Pathology, Stony Brook University, Stony Brook, NY 11794
Integrity of the intestinal epithelium is required for nutrition absorption and defense against
pathogens. Multiple claudins are expressed along the gastrointestinal tract, but little is known about
their functions. We generated claudin-7-deficient (Cldn7-/-) mice and examined their intestines by
histology, molecular and cellular biology, as well as biochemistry approaches. Cldn7-/- mice had
severe intestinal defects that included mucosal ulcerations, epithelial cell sloughing and inflammation.
Intestines of Cldn7-/- mice produced significantly higher levels of cytokines, NF-B and COX-2; they
also upregulated the expressions of MMP-3 and -7. The siRNA experiments using epithelial cell lines
demonstrated that the increased expression of MMP-3 resulted directly from claudin-7 depletion,
whereas that of MMP-7 resulted from inflammation. Electron microscopy analysis showed that
intestines of Cldn7-/- mice had intercellular gaps below tight junctions (TJs) and cell-matrix loosening.
Deletion of Cldn7 reduced the expression and altered the localization of integrin α2; disrupted
formation of complexes of claudin-7, integrin α2 and claudin-1 that normally form in epithelial
basolateral compartments of intestines. We conclude that claudin-7 has non-TJ functions, including
the maintenance of epithelial cell–matrix interactions and intestinal homeostasis. This work is
supported by National Institutes of Health grant HL085752.
Divergent effects of L-carnitine on pyruvate-supported O2 consumption and H2O2 emission
within permeabilized myofibers
Kelsey H. Fisher-Wellman1,2, Laura A. A. Gilliam1,2, Chien-Te Lin1,2, Brook L. Cathey1,2 and P. Darrell
Neufer1,2,4
East Carolina Diabetes and Obesity Institute1, Departments of Kinesiology2 and Physiology4
East Carolina University, Greenville, NC 27834
Pyruvate dehydrogenase (PDH) is one of the seven potential sites for O 2-• generation within
mammalian mitochondria. PDH flux is linked to metabolic balance via the NADH/NAD+ and acetylCoA/CoA ratios, which along with pyruvate exert allosteric regulation on the complex. To determine if
PDH supported H2O2 emission is sensitive to matrix levels of acetyl-CoA, saponin permeabilized
fibers were prepared from red gastrocnemius muscle of rats and energized with pyruvate [1mM] as
the sole substrate. Under these conditions acetyl-CoA rapidly accumulates within the matrix and PDH
flux is minimal. Addition of L-carnitine removes accumulated acetyl-CoA via the activation of carnitine
acetyltransferase (CrAT) and formation/export of acetyl-carnitine. Following the addition of 5mM Lcarnitine, pyruvate supported NADH production increased ~3 fold (-Carnitine 37.75 ± 2.54, +Carnitine
113.20 ± 8.22 pmol/sec/mg dw; P<0.001, N=6) oxygen consumption increased ~ 8 fold (-Carnitine
25.92 ± 2.43, +Carnitine 222.70 ± 19.03 pmol/s/mg dw; P<0.001, N=6), and H 2O2 emission
decreased by ~50% (-Carnitine 1.82 ± 0.47, +Carnitine 0.90 ± 0.28 pmol/min/mg dw; P<0.05, N=6).
These data demonstrate the divergent relationship between PDH catalytic flux and H 2O2 emission
and suggest that PDH-supported H2O2 emission in vivo may be maximal under conditions of elevated
acetyl-CoA/CoA in conjunction with low metabolic demand (i.e., positive metabolic balance combined
with low activity level). RO1 DK073488
Title: “SNARE PROTEIN CLEAVAGE BY ANTAREASE - A NEW METALLOPROTEASE FROM THE BRAZILIAN
SCORPION Tityus serrulatus“
1
1
Senior Principal Investigator: Paul L. Fletcher, Jr. and Maryann D. Fletcher
2
2
3
4
With: Keith Weninger , Trevor E. Anderson , Thierry Galli and Brian M. Martin
1
2
Brody School of Medicine, East Carolina University, Greenville, NC 27834, North Carolina State University, Raleigh, NC
3
4
27695, INSERM-ERL Institut Jacques Monod, Paris F-75013, and National Institute of Mental Health, National Institutes
of Health, Bethesda, MD 20892 J. Biol. Chem. (2010) 285(10):7405-7416.
Abstract: Subcellular compartmental organization and function command the central role in vesicular transport. Vesicular
formation then subsequent fusion are the fundamental processes. Here we examine novel effects of cleavage of the
molecular machinery of the fusion process, the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein
receptor) proteins. Cleavage of any of the three major SNAREs is uniquely connected with lethal pathology and until now
is associated only with the botulinum and tetanus clostridial neurotoxins (CNT’s). While this pathology is restricted to
vesicles found only in the nervous system the target SNARE proteins - syntaxin, SNAP (soluble N-ethylmaleimide
sensitive attachment protein), and VAMP (vesicle-associated membrane protein) are found in all cells and there are no
reports of exogenous proteolytic disease processes in other tissues. Experimentally introduced mutations have been
found to be uniformly lethal. Our studies of the association of acute pancreatitis with accidental scorpion stings revealed
unmistakable evidence for cleavage of SNARE proteins in the exocrine pancreas. Initial recognition of a SNARE protein
targeting proteolysis led us to the isolation and characterization of the scorpion venom metalloprotease we named
Antarease. We identified the v-SNARE targets as VAMP2 (vesicle-associated membrane protein2) or synaptobrevin and
VAMP8 (endobrevin). We used recombinant SNAREs from our NCSU colleagues to provide cytoplasmic portions that
would facilitate the recovery of the cleavage products. These proteolytic peptides provided identification of the enzymatic
target sites. Recently, with Thierry Galli we made the novel finding that VAMP7 (a "longin"), also called tetanus-insensitive
(TI-VAMP), is a target for this newly described proteolytic activity.
We have extended these studies to examine a few additional North and South American scorpion venoms that are
associated with clinical reports of severe effects in humans. These experiments provided tissue for ultrastructural
examination by electron microscopy of immunolabeled tissues which demonstrated the disappearance of VAMP2 and
VAMP8.
I. SNARE Proteolysis by Various Venoms. Scorpion venom metalloprotease activity is shown to cleave the vSNAREs, VAMP2 and VAMP8, and more interestingly, TI-VAMP. These unusual proteolytic targets are likely candidates
for initiation of acute pancreatitis that is often associated with scorpion envenomation. More importantly, cleavage of
these proteins compromises major cellular functions. Our initial publication showed the precise targets of synaptobrevin
(VAMP2) and endobrevin (VAMP8) by a scorpion venom metalloprotease we named Antarease. Now we can show
additionally that other New World scorpion venoms from North and South America also can cleave the same targets. It is
important to note that this proteolysis is not characteristic of all of these venoms. Proteolytic activity was determined
using recombinant cytoplasmic v-SNAREs, tissue homogenates, and electron microscopic immunocytochemistry. Each
technology contributes additional dimensions to reveal the cleavage targets. Recombinant Antarease is now being
tested and evaluated.
II. SNARE Target Sites. In contrast to the clostridial neurotoxins (CNTs), the only previously known exogenous
proteolytic threat to vesicular fusion molecular SNARE machinery, Antarease and its scorpion venom relatives can cleave
multiple SNARE proteins. Each known serotype of CNT's is limited to single proteolytic targets. Antarease cleaves at
unique sites, not shared with CNTs. This is the only known intracellular proteolysis of TI-VAMP. SNAP25 and syntaxin,
but not SNAP23, t-SNAREs, are characteristically cleaved.
III. Cellular Effects. We observe by confocal fluorescence light microscopy that TSV-mediated intracellular damage
occurs as evidenced by widespread vacuolization - a hallmark of acute pancreatitis. Ultrastructural images reveal
zymogen granule exocytosis at the lateral plasmalemma rather than at the usual apical plasmalemmal membrane. This
observation indicates an atypical membrane fusion and misdirection of potent zymogens to the extracellular/interstitial
space. Specifically, there is a dramatic reduction of both VAMP2 and VAMP8 immunoreactivity. While there is little doubt
that Antarease exposure with envenomation results in SNARE targeting and cleavage the extent of damage in vital
organs remains unproven.
Alteration of circadian rhythm in the 3XTg mouse model of Alzheimer disease
Meina Wu1, Steve Nunn1, Ben Kassum1, Xinyi Li2, Daniel Lee3, and, Jian M. Ding1*
1
Department of Physiology, Brody School of Medicine, East Carolina University
Department of Neurology, Shanxi Grand Hospital, Taiyuan, China
3
East Carolina Neurology, Greenville, NC
2
In addition to progressive memory loss, the alteration of circadian rhythm and sleep–wake pattern are
common symptoms of Alzheimer's disease (AD). Patients afflicted with AD tend to doze off during the
day but are prone to wandering around at night. The increased daytime sleepiness worsens cognitive
performance, while the fragmented sleep may further impair memory consolidation. In addition, the
altered sleep-wake pattern increases the burden for the care givers, which often becomes the primary
reason for the patients to be institutionalized.
Although the exact mechanism of AD neural pathogenesis remains elusive, the triple-transgenic
3xTg-AD mouse model express all three genes associated with familial AD, the APPSwe,
PS1M146V, and tauP301L. The 3xTg mice exhibit both Aβ and tau pathology in the brain, which are
hallmarks of postmortem AD pathology, and develop impairment of cognitive function with aging.
The 3xTg mice and age-matched WT controls were housed in customized cages equipped with
running wheels under a 12-hour light and 12-hour dark (12:12 LD) cycle. The circadian rhythm of
wheel running activity was recorded with the ClockLab software. Consistent with previous reports
(Miller 2007; Sterniczuk 2010), the alteration of circadian rhythm in wheel running activity could be
seen as early as three months of age, and became more pronounced after six months of age.
Circadian rhythm and sleep schedule are regulated by a circadian clock in the suprachiasmatic
nucleus (SCN) of the hypothalamus. Healthy SCN can generate robust circadian rhythm in action
potential firing rate detectable with electrophysiology recording in the SCN brain slice. To investigate
whether the alteration of circadian running rhythm in the 3xTg-AD mice is associated with SCN
dysfunction, we employed the SCN brain slice recording technique, where the SCN is isolated from
feedback of other brain regions so that intrinsic properties can be probed directly. Timekeeping is
expressed as a stable, near 24-h oscillation in ensemble neuronal firing rate. The circadian rhythm of
neuronal electrical activity is roughly sinusoidal, with ensemble activity peaking at CT 7, and with a
trough at night.
In contrast to the single peak at CT 7, the SCNs of the 3xTg mice with altered running rhythm were
often found to exhibit more than one peak, with additional peaks a few hours before or after the
normal CT 7 peak. The age-matched WT mice and 3Xtg mice that did not develop altered running
rhythm occasionally had reduced amplitude in the SCN firing rate, but did not have multiple peaks.
Fourier analysis indicates that the 3Xtg mice exhibit decreased amplitude of the 24 hour rhythm, but
increased ulradian rhythm. Our findings suggest that a weakened circadian oscillation in the SCN
may underlie the altered sleep-wake schedule in familial AD.
Dopamine D3 receptor dysfunction alters the functional state of mu-opioid receptors and
induces a morphine-tolerant phenotype in the spinal cord
Grace Loeffler1, Kori Brewer PhD1, Christine Baran1 Marley Jensen2, and Stefan Clemens PhD2
Departments of Emergency Medicine1 and Physiology2
Brody School of Medicine @ East Carolina University
Mice lacking a functional D3 receptor (D3KO) have decreased withdrawal latencies from painful
thermal stimuli compared to wild type (WT) mice. We tested the hypothesis that this increased pain
sensitivity in D3KO may result from alterations in the functional opioid system.
Baseline thermal thresholds were assessed in D3KO and WT mice via the Hargreaves method.
Thresholds were reassessed after a single dose of morphine (2mg/kg, i.p.). Mu-opioid receptor
(MOR) and phosphorylated MOR (p-MOR) protein levels in the lumbar spinal cords of each group
were measured using Western Blotting. Electrophysiological methods were used to determine spinal
reflex amplitudes (SRAs) in isolated spinal cords from D3KO and WT mice before and after bathapplication of morphine.
We confirmed that D3KO exhibited lower thermal thresholds at baseline compared to WT.
Administration of morphine in vivo significantly increased thermal thresholds in WT mice, but had no
effect in D3KOs. WT and D3KO had similar protein expression levels of MOR, however, D3KO mice
had a significantly higher proportion of p-MOR than WT. In vitro, SRAs were increased in D3KO vs.
WT mice, and morphine had no effect on SRAs in D3KOs.
Our data suggest that a dysfunctional D3 receptor system promotes morphine tolerance in the spinal
cord of morphine-naïve animals. This phenomenon is associated with changes in spinal MOR
functional states in D3KO. These characteristics make the D3KO mouse a suitable animal model for
the study of chronic pain states and morphine tolerance.
Assessment of persistent neuronal morphological changes after chronic CB 1 receptor
antagonist SR141716A or antidepressant treatment in zebra finches
Tessa L. Holland, Marcoita T. Gilbert, Ken Soderstrom
Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University,
Greenville, NC, 27834
Zebra finches possess a developmental sensitive period of vocal learning in which a decrease in
dendritic spine density occurs in brain song regions. Previously, we observed that chronic
developmental treatment with CB1 receptor agonist WIN55,212-2 prevented this normal reduction, but
adult treatment had no effect, suggesting WIN55,212-2 possibly disrupted developmentally
significant endocannabinoid signaling. To evaluate the effects of CB1 receptor antagonism during
vocal development, in a similar experiment we treated developing and adult zebra finches (n=4) with
CB1 receptor antagonist SR141716A (6 mg/kg) for 25 days followed by 25 days of no treatment (until
maturation) and created 3D neuronal reconstructions using Golgi-Cox staining and Neurolucida
software. Surprisingly, both developmental and adult treatment persistently increased spine densities.
Adult treatment produced more dramatic increases, suggesting tonic inhibitory endocannabinoid
control of song region spine densities is perhaps important to maintain mature neural phenotypes.
Another interpretation is the increased spine densities are a consequence of the mood depressant
effect of SR141716A, which was used clinically to treat obesity and observed to cause depression in
patients. To discover if antidepressant treatment resulted in different effects on neuronal morphology,
we treated zebra finches (n=3) with the MAOI phenelzine (1 mg/kg) for 25 days followed by 25 days
of no treatment. Chronic phenelzine treatment had no persistent effect on spine density, and
antidepressants may help maintain stable spine populations. Preliminary results indicate coadministration of phenelzine prevents SR141716A-induced increased spine densities, suggesting an
association between spine density and the mood effects of these drugs.
Assessing the decay of heterologous tolerance to morphine after varying lengths of treatment
via intraperitoneal (I.P.) injection.
Ben Thompson, Jackie Masterson, David A. Taylor: Department of Pharmacology and Toxicology,
Brody School of Medicine, East Carolina University, Greenville, NC 27834.
The development and decay of heterologous tolerance following chronic morphine exposure via pellet
implantation is well documented in the guinea pig longitudinal muscle/myenteric plexus (LM/MP).
However, this method of administration only permits a qualitative analysis of the decay of tolerance
because the time at which morphine exposure is completed is unknown. Therefore, we assessed the
decay of tolerance in animals pretreated with twice daily i.p. injections of morphine for 4 or 7 days.
Tolerance to the analgesic effects of morphine was assessed using the paw pressure test while the
heterologous nature of tolerance was evaluated using the response of the LM/MP to DAMGO and 2CADO at 0, 1, 2, or 4 days after treatment cessation. The data indicated that the time for return to
baseline was correlated to the magnitude of tolerance that was a function of the length of treatment.
The tolerance generated in animals treated for 4 days was nearly 4-fold and decayed to baseline
within 2 days after the treatment ended while tolerance observed in animals treated for 7 days was
nearly 6-fold and returned to baseline levels 4 days after treatment cessation. The fact that the decay
of tolerance is a function of both the length of treatment and magnitude of tolerance developed
reinforces the idea that tolerance following chronic treatment with morphine is an adaptive process
produced by alterations in several cellular signaling proteins.
Environmental Effects on the Risks of Alzheimer’s Disease
Logan Webb1, Zach Elliotty1, Yi Zhu1, Christi Boykin1, and Qun Lu1, 2
1
Department of Anatomy and Cell Biology, 2The Harriet and John Wooten Laboratory for Alzheimer’s
and Neurodegenerative Diseases Research, The Brody School of Medicine, East Carolina University,
Greenville, North Carolina 27834
Alzheimer’s disease (AD) is a common form of dementia that occurs mostly among the elderly. It is a
progressive neurodegenerative disease that affects approximately 5 million people in the USA alone,
a number that is projected to increase to 12 million by 2050. Familial AD of early onset is primarily
caused by genetic factors but recent evidence shows that “environmental” effects can contribute to
the cause of the disease. Two environmental effects of interest are traumatic brain injury (TBI) and
pesticides. Studies are being done in our laboratory to find a relationship between these
environmental effects and AD.
TBI is defined as an external force to the head causing internal damage to the brain. In general, there
are two types of traumatic brain injuries: mild and severe. Research shows that TBI may cause the
overexpression of amyloid precursor proteins, which in turn causes the excessive expression of betaamyloid to be deposited into plaques in the brain. Studies also show that TBI may have the more
chronic affect of induced tauopathy. Both of these effects from TBI can be linked to the causes of AD.
On the other hand, in recent decades there has been an increased use of pesticides along with AD
cases. Therefore, there could be a possible correlation between the two. The main pesticide of
interest belongs to a family of chemicals known as organophosphates. These chemicals are wellknown for their damaging effects on the nervous system, which work by the inhibition of
acetylcholinesterase. This inhibition leads to major synaptic damages.
Collectively, these two different types of environmental effects can account for both the physical and
chemical aspects of environmental influences on AD development. We are pursuing these processes
using molecular, genetic, and imaging technologies on the wild type mice as well as the triple
transgenic mice expressing AD type mutations (3xTg-AD). Our studies are supported in part by The
Wooten Laboratory, Alzheimers North Carolina, and National Institutes of Health.
PPP1R42, a testis specific PP1 binding protein, regulates cilia biogenesis in ARPE-19 cells
Nicole DeVaul, Rong Wang, and Ann O. Sperry
Department of Anatomy and Cell Biology
Most cases of male infertility are idopathic and only 10-20% of cases can be successfully treated. In
order to design appropriate treatments for male infertility, the molecular mechanisms underlying
spermatogenesis must be better understood. Spermiogenesis, the third phase of spermatogenesis,
involves formation of unique microtubule structures including the manchette and flagellum. We have
previously identified PPP1R42 as a regulatory subunit of protein phosphatase-1 (PP1) in testis.
PPP1R42 is first associated with the manchette followed by translocation to the centrosome at the
base of the flagellum in elongated spermatids. In addition to its localization at the centrosome,
PPP1R42 is expressed in ciliated tissues suggestive of a role for this protein in flagellum biogenesis.
ARPE-19 cells serve as a model system to study how PPP1R42 regulates PP1 and downstream
targets in flagellum and cilium biogenesis. We hypothesize that PPP1R42 positively regulates PP1
and therefore, negatively regulates its downstream effectors, including Nek2, in cilium biogenesis.
Co-immunoprecipitation and immunofluorescence experiments demonstrate that PPP1R42 forms a
complex and co-localizes with γ-tubulin at the base of cilia in the polarized cell line, ARPE-19,
indicating a conserved role for PPP1R42 at the centrosome. In order to further investigate the role of
PPP1R42 in cilia biogenesis we used FLAG-PPP1R42 fusion vector or siRNA to overexpress or
knockdown the expression of PPP1R42 in these cells, respectively. Overexpression of PPP1R42
results in a significant increase in cilia number and length while knockdown of PPP1R42 results in
curled cilia morphology. These data indicate that PPP1R42 is involved in regulation of cilia length,
structure, and disassembly, likely through the activation of PP1. We then investigated the mechanism
by which PPP1R42 regulates PP1 and its downstream target, Nek2. Nek2, a serine/threonine kinase,
is negatively regulated by PP1 in cilia biogenesis. Knockdown of PPP1R42 results in a decrease of
active PP1 expression and an increase in Nek2 kinase activity. These results indicate that PPP1R42
positively regulates PP1 and, in turn, negatively regulates Nek2 activity. In summary, we have
identified a novel regulator of PP1 with a necessary role in regulation of flagellum and cilium
biogenesis.
Translational control of mRNAs by initiation factors influences germ cell fate during spermatogenesis
and oogenesis.
Henderson, MA (1), Friday, AF (1), Subash, JJ (1), Morrison, JK (1), Contreras, V (1), Song, A (2), Rhoads, RE
(2), and Keiper, BD (1)
1) Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC
27834. (contact, keiperb@ecu.edu)
2) Biochemistry and Molecular Biology, LSU Health Sciences Center, Shreveport, LA 71130
Cancers and congenital diseases share a fundamental characteristic: affected cells choose an abnormal fate
or “lifestyle” that disrupts their usual function. If we can understand how they choose altered fates and
intervene, we might redirect their decisions and reverse the diseased state. The synthesis of novel proteins
advances a cell progressively toward its final fate, be it neuron, muscle, gut, gamete (differentiation), or even
death (apoptosis). My students and I study how protein synthesis activity leads to natural fate changes in cells.
These are mechanisms that goes awry in breast, lung and many other cancers. Our NSF-funded research
uses egg and sperm stem cells (germ cells) from a simple, transgenic model animal system, C. elegans, to
study how protein synthesis drives a differentiating cell to a certain fate, including in some cases, apoptosis.
From an understanding of how germ cells choose that fate, we propose to modulate protein synthesis in
human breast cancer cells in a similar, controlled fashion. These studies hold the promise of medically
selecting, for example, the natural apoptotic fate for tumor cells, and thereby developing new cancer therapies
that are free of unwanted toxic side effects.
Germ cells rely on mRNA translational control to regulate the de novo synthesis of proteins that drive
differentiation to sperm and oocytes. Recently the role of translation initiation factors (eIF4) in mRNA
translational control has been appreciated. Our lab has described unique mRNA-selective functions for
individual isoforms of eIF4E (IFE-1, -2, and -3) and eIF4G (IFG-1) in germ cells of the nematode C. elegans.
Germline stem cells in worms differentiate sequentially into sperm and oocytes. Mutations in each eIF4E
isoform reduces fertility, but in surprisingly unique ways. Each alters a different fate of gamete differentiation,
resulting in blocked maturation steps, inefficient meiotic recombination, or even switching oocytes to a sperm
fate. We identified mRNAs that uniquely require IFE-1 or -2 for efficient initiation using biochemical
fractionation and a translation state array analysis (TSAA) from null mutant worms. Many encode proteins of
critical function in oocyte and/or spermatocyte differentiation. IFE-1 is required efficiently translation of pos-1,
gld-1, mex-1, and oma-1 mRNAs. Spermatocytes lacking IFE-1 fail in the final budding/cytokinesis step,
accumulating as multinucleated secondary spermatocytes; oocytes have slowed growth and maturation. IFE-2
is required to synthesize proteins for meiotic crossover and chromosome segregation (msh-4 and msh-5
mRNAs). Most remarkably, loss of the eIF4E isoform IFE-3 causes a fate switch in hermaphroditic worms. Late
germ cells that normally become oocytes differentiated instead into mature sperm. Thus, in reproductive cells,
eIF4 factors can select specific mRNAs for translation to drive acute developmental functions. The evidence
supports a positive regulatory network of eIF4E-eIF4G-mediated translational control specifying unique
directions in cell differentiation with very different fate outcomes.
Presenter:
Andrew Friday
Lab:
Brett D. Keiper, PhD
Dept:
Biochemistry & Molecular Biology
Retinoic Acid Stimulates Translation in the Neonatal Testis
Jonathan T. Busada1, Vesna A. Chappell1, Brett D. Keiper2, and Christopher B. Geyer1
1
Departments of Anatomy and Cell Biology and 2Biochemistry and Molecular Biology, Brody School of
Medicine, East Carolina University, 600 Moye Blvd., Greenville, NC 27834, USA. Tel: (252) 7443433, Fax: (252) 744-2850.
Transition of quiescent gonocytes to proliferating spermatogonia in the neonatal mouse testis initiates
the first wave of spermatogenesis at 3-4 days postpartum (dpp). Despite doubling of the germ cell
population during this developmental transition, mRNAs encoding only ~50 genes increase ≥2-fold in
abundance, suggesting that gene expression is not regulated at the level of transcription. In lower
organisms, germline stem cells decide to proliferate or differentiate based in large part on gene
expression changes regulated at the level of translation. We used a genome-wide approach to
determine whether changes in translation efficiency might underlie similar decisions in mice. We
found that ~3,000 unique mRNAs became ≥2-fold enriched in polysomes during this transition without
significant changes in their abundance. Retinoic acid (RA) signaling in the neonatal testis temporally
coincides with our observed increase in translation efficiency. RA provides the signal for meiotic
initiation in both sexes, yet almost nothing is known about specific pathways or genes activated
downstream of RA. We tested the hypothesis that RA stimulates translation of mRNAs in the neonatal
testis. We injected mice with DMSO ±
them 24 h later, and
harvested testes for fractionation on polyribosome profile gradients to resolve cytoplasmic translating
and non-translating mRNA complexes. Total RNA and protein were isolated from whole testis and
gradient fractions for qRT-PCR and western analysis, respectively. RA injection increased Stra8
mRNA levels ~4-fold, as expected. Total mRNA levels were relatively unchanged for a large panel of
germ cell-specific and housekeeping genes. In contrast, mRNA polysome occupancy increased
dramatically in response to RA, indicating increased translation efficiency.
We found that
accompanying protein levels for a subset of these genes increased, as predicted. Our data support a
novel role for RA in the neonatal testis, and indicates that RA signaling regulates the neonatal testis
proteome by stimulating translation of preexisting mRNAs.
Environmental developmental neuroimmunotoxicology
Jamie DeWitt
Department of Pharmacology and Toxicology
Environmental contamination is comprised of human-made and human-manipulated agents, usually
chemicals, that our environment is not able to dissipate. These agents include chemicals that are
flushed and washed down drains at home and at work, chemicals that industries are allowed to
discharge into the air or water, and chemicals that leach into food or drinking water from soil or even
the containers that hold them. On their own and in small amounts, these chemicals provide many
benefits to humankind; they repel water and stains, keep food from sticking to pans, kill unwanted or
dangerous pests, and give structure to plastic containers. However, the environment, including the
bodies of humans and other organisms, does not have an unlimited capacity to protect itself from all
of the different chemicals that it encounters. And, all of the possible health effects of these chemicals
are not well understood because not all health effects of chemicals are evaluated before they are
used in industrial or consumer products.
The DeWitt Lab is studying or planning to study health effects of fluorinated compounds,
environmental phenols, pharmaceutical and personal care products, and heavy metals. Of special
interest is communication between the immune and nervous systems during development and how
these exogenous agents may disrupt this communication. The main focus is on determining how
exposure to developmental immunotoxicants leads to changes in nervous system development.
Developmental immunotoxicity (DIT) occurs when the developing immune system experiences an
adverse outcome after exposure to an exogenous agent or other stressor. Exposure to
developmental immunotoxicants has been suggested in several neurological disorders, including
autism spectrum disorders and attention-deficit hyperactivity disorder and possibly plays a role in
neurodegenerative diseases associated with aging, including Alzheimer’s disease. Currently, the
DeWitt Lab is evaluating how microglia, resident macrophages of the central nervous system (CNS),
respond to DIT and result in altered neurodevelopment or neurodegeneration. Microglia respond to
signals from within the CNS and to signals from the periphery, so likely coordinate several brain-body
interactions during development and throughout life.
The benefit of this combined systems approach is that the complexity of development that requires
the interaction of multiple systems, processes, and events can be fully appreciated. The biggest
challenge is that evaluating multiple systems is complex. The goal of the DeWitt Lab is not to
eliminate chemicals from the environment, but to determine if exposure to these chemicals
contributes to diseases and disorders in humans and wildlife. Determining linkages between
developmental exposure and diseases/disorders, ultimately can be used to better regulate these
chemicals to reduce exposure or to develop additional safety testing guidelines for high production
volume chemicals.
The Role of PPAR in Carbon Nanotube - Elicited Granulomatous Lung Inflammation
J Patel, A Malur, I Huizar, M McPeek, L Dobbs, BP Barna, MJ Thomassen
Departments of: Internal Medicine, Division of Pulmonary and Critical Care Medicine 1; and
Pathology2, East Carolina University, Greenville, NC.
Background: Although granulomatous inflammation is a central feature of many disease processes,
cellular mechanisms of granuloma formation and persistence are poorly understood.
Carbon
nanoparticles, which can be products of manufacture or the environment, have been associated with
granulomatous disease. This paper utilizes a previously described carbon nanoparticle granuloma
model to address the issue of whether peroxisome proliferator-activated receptor gamma (PPAR), a
nuclear transcription factor and negative regulator of inflammatory cytokines might play a role in
granulomatous lung disease. PPAR is constitutively expressed in alveolar macrophages from
healthy individuals but is depressed in alveolar macrophages of patients with sarcoidosis, a
prototypical granulomatous disease. Our previous study of macrophage-specific PPAR KO mice
revealed an intrinsically inflammatory pulmonary environment with an elevated pro-inflammatory
cytokines profile as compared to wild-type mice. Based on such observations we hypothesized that
PPAR expression would be repressed in alveolar macrophages from animals bearing granulomas
induced by MWCNT instillation. Methods: Wild-type C57Bl/6 and macrophage-specific PPAR KO
mice received oropharyngeal instillations of multiwall carbon nanotubes (MWCNT) (100 µg).
Bronchoalveolar lavage (BAL) cells, BAL fluids, and lung tissues were obtained 60 days postinstillation for analysis of granuloma histology and pro-inflammatory cytokines (osteopontin [OPN],
CCL2, and interferon gamma [IFN-] mRNA and protein expression. Results: In wild-type mice,
alveolar macrophage PPAR expression and activity were significantly reduced in granuloma-bearing
animals 60 days after MWCNT instillation. In macrophage-specific PPAR KO mice, granuloma
formation was more extensive than in wild-type at 60 days after MWCNT instillation. PPAR KO mice
also demonstrated elevated pro-inflammatory cytokine expression in lung tissue, laser-microdissected
lung granulomas, and BAL cells/fluids, at 60 days post MWCNT exposure. Conclusions: Overall,
data indicate that PPAR deficiency promotes inflammation and granuloma formation, suggesting that
PPAR functions as a negative regulator of chronic granulomatous inflammation
Fesselin
Nathanial Kingsbury1, Randal R. Renegar2, Mechthild M. Schroeter3 and Joseph M. Chalovich1
1Department
of Biochemistry & Molecular Biology Brody SOM; 2Department of Anatomy & Cell
Biology, Brody SOM; 3Department of General Physiology, University of Hannover School of Medicine
Fesselin or avian synaptopodin 2 is an actin binding protein that was discovered by Barbara
Leinweber as she worked on her PhD in Physiology at the Brody School of Medicine in the Chalovich
lab. Fesselin was found to induce rapid actin polymerization by Dr. Brent Beall in his Ph.D. work in
Biochemistry. Mechthild Schroeter demonstrated that fesselin-induced actin polymerization was
strongly inhibited by Ca2+-calmodulin. Together with Randal Renegar she showed that fesselin was
strongly localized in the dense bodies of smooth muscle together with alpha actinin. Min Pham
demonstrated that fesselin binds tightly to alpha actinin forming an insoluble complex. A number of
laboratories outside of ECU demonstrated that fesselin or synaptopodin 2 levels and distributions
change in some cancers including those of the bladder and prostate. A group of investigators at ECU
including Jean-Luc Scemama, John Lehman, Margit Schmidt, Joe Chalovich, Justin Arthur and
Tamatha Baxley are continuing to investigate the translocation of fesselin/synaptopodin 2 from the
nucleus where it is cancer protective, to the cytoplasm.
This poster addresses another possible role of fesselin. We observed earlier that there is diffuse
staining for fesselin in smooth muscle cells outside of the dense bodies. This was a clue that fesselin
may also be present in contractile actin-myosin structures. Fesselin was shown to bind directly to
myosin suggesting that fesselin could tether actin and myosin together in an inactive complex. This
possibility was tested by observing the effect of fesselin on the rate of dissociation of actinmyosin by ATP in a stopped-flow device. Dissociation was measured by light scattering (a
measure of particle size) and by pyrene actin fluorescence (a specific measure of actin-myosin
binding). Fesselin reduced the multi-exponential rates of change of light scattering and pyrene
fluorescence in concentration dependent manners. Each light scattering trace had a rapid initial
transition that was not present in pyrene fluorescence traces. That rapid light scattering change was
likely due to dissociation of filamentous myosin. The reduction in rate of that rapid process could
mean that fesselin alters the structure and dissociation kinetics of smooth myosin filaments. We
examined changes in smooth muscle myosin with electron microscopy. ATP caused dissociation of
myosin filaments in both the presence and absence of fesselin. In the absence of ATP, fesselin
increased the size of myosin filaments. Furthermore, the filaments were arranged in parallel arrays.
These results suggest that fesselin may stabilize myosin filaments, as well as actin filaments.
Furthermore, fesselin may form crosslinks between myosin and actin so that these contractile units
are poised for force production.
Discovering a novel role of claudin-7 in regulating cell proliferation and maintaining cellmatrix interactions in human lung cancer cells
Zhe Lu, Qun Lu, Lei Ding, Randall Renegar, and Yan-Hua Chen
Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University,
Greenville, NC 27834, USA
Claudins are a family of tight junction (TJ) integral membrane proteins with at least 24 members in
mammals. Reduced or altered expression of claudins has been reported in various human cancers;
however, their roles in cancers are largely unknown. Claudin-7 is a unique TJ protein in that it is not
only localized at the apical TJs of epithelial cells, but also has a strong expression at the basolateral
membrane. To study the function of claudin-7, we suppressed claudin-7 expression by lentivirus
shRNA technology in a human lung adenocarcinoma cell line HCC827.
Our study showed that claudin-7 knockdown (KD) cells displayed higher proliferation rates and cell
cycle progression rates than the control cells. The expression of phospho-MAPK, survivin, and
phospho-BCL-2, which are cell survival and anti-apoptotic regulators, were all significantly increased
in claudin-7 KD cells compared to that of control cells. Realtime RT-PCR revealed that the
transcription of several collagens, such as type V α1, type XV α1, and type XVI α1, was dramatically
decreased in claudin-7 KD cells compared to that of the control cells. In addition, integrin β1, which
serves as a collagen receptor, was also decreased in claudin-7 KD cells. Immunofluorescence and
co-immunoprecipitation revealed the co-localization of integrin β1 with claudin-7. Depletion of claudin7 significantly reduced integrin β1 expression and claudin-7 KD cells were unable to attach to the
uncoated glass coverslips while the control cells adhered well and grew normally. The cell-matrix
association defect can be rescued by transfecting integrin β1 into claudin-7 KD cells. Thus, our data
suggest that suppression of claudin-7 weakened the cell-matrix interactions through integrin
β1.Transfecting claudin-7 back to KD cells slowed down cell proliferation, increased integrin β1
expression, and rescued the cell-matrix association defect. When control and claudin-7 KD cells were
inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than the control
cells. Thus, our current study highlights a novel non-TJ function of claudin-7 in regulating cell
proliferation and maintaining epithelial cell-matrix interactions. This research is supported by NIH
grant ES016888.
A new method for auto-delineation of GTV for NSCLC on PET/CT images
Yu Guo1, Wang Lin1, Ning Zhang1, Yu Sa1, Yuanming Feng3*
1Department of Biomedical Engineering, Tianjin University 2Departments of Radiation Oncology & Physics, East Carolina
University
Introduction: Combination of PET and CT provides complementary functional and anatomical information and has been
used for GTV definition in NSCLC RT planning. Auto-segmentation methods are highly desire to avoid inter- and intraobserver variability caused by manual method. In this abstract, a robust method which combines effectively the advantages
of the two image modalities is proposed for auto-delineation of GTV for the lung tumors on fused PET/CT images.
Method: The new method is based on fuzzy Markov random fields (MRF). In the model, two random fields are considered
which correspond respectively to membership degree x of a voxel to tumor class and the feature vector y extracted from
PET and CT images. The estimation of x can be expressed as

x  arg max ( log p( y /x)+ log p( x))
(1)
x
Where P(x) is Gibbs distribution, p(y/x) is the joint probability p(y1,y2/x), y1 is standardized uptake value (SUV) value and y2
is CT image intensity. It is set as
x min[ f11 ( y1 ),f 21 ( y2 )]  (1- xi ) max[ f10 ( y1 ), f 20 ( y2 )]
(2)
f11 and f21 are the probability distributions of the two features given that the studied voxel belongs to tumor class, while f10
and f20 and are the probability distributions of the two features given that the studied voxel belongs to normal tissue class.
f10, f20 and f20 are set as Gaussian functions. For f21, a uniform distribution is used, which means that voxels with a SUV
value greater than a threshold have the same possibility to be in tumor class. With (1) and (2), we get

x = arg max  i {log[ xi min[ f11 ( y1i ),f 21 ( y2i )]  (1- xi ) max[ f10 ( y1i ), f 20 ( y2i )]    jC | xi - x j |}
x
where i and j are the image indexes and Ci is the neighborhood (5x5x3) of the
gradient decent method.
i
ith voxel.
(3)
The optimization is performed with a
Results: PET and CT simulation images of 3 NSCLC patients were used. GTV delineation with the proposed method and
manual method by an experienced radiation oncologist on the fused images were performed, respectively. The manually
contoured GTVs were checked and confirmed by another experienced radiation oncologist. The robustness of MRF method
was evaluated by comparing the overlap of the two delineations using Dice’s similarity coefficient (DSC) expressed as
2x(V1∩V2)/(V1+V2), where V1 is GTVmanual and V2 is GTVauto. GTVs obtained with the two methods were similar and DSC
was 0.85±0.013.
Fig. 1. GTVs in axial
slices of Patient a, b and
c. GTVs in blue are the
results of the new method
using only PET images,
GTVs in red and green
are the results of the new
method and manual
method respectively
using both PET and CT
images.
Conclusion: Effective and automatic GTV delineation can be achieved with the new method for lung tumors located near
other organs with similar intensities in CT images (such as when the tumors extends into the chest wall or the
mediastinum).
*Corresponding author: fengyu@ecu.edu, supported by ELEKTA grant of Medical Physics Development in TJU
Generation of dual-energy CT image dataset from single-energy CT image dataset
Jie Huo1, Xuemin Wang1, Yang Dong2, Xianfeng Zhu1, Yuanming Feng3*
1
Department of Biomedical Engineering, Tianjin University
Department of Radiation Oncology, Tianjin Medical University Cancer Institute & Hospital
3 Departments of Radiation Oncology & Physics, East Carolina University
2
Introduction: The capability of obtaining tissue-specific information from dual-energy imaging has the potential to be used
in radiotherapy for gross-tumor-volume (GTV) definition and localization with enhanced accuracy in radiotherapy treatment
planning and image guided delivery. However, there are not dedicated dual energy CT (DECT) simulators in radiotherapy
clinics yet. This study is to develop a method to create a set of DECT data with the data from a conventional single-energy
CT simulator, i.e., to generate a set of CT images at energy B from a set of CT images obtained at energy A, and to
explore the feasibility of utilizing the two CT data sets to create anatomical images with higher soft-tissue-contrast.
Method: In the new method, HUs in the original images of CT A was first converted into liner attenuation coefficients at
another energy with a bilinear scaling method used for attenuation correction in SPECT and PET imaging. Photon
attenuation of tissues with higher Z-numbers has stronger energy dependence than the ones with lower Z-numbers, and
has a sharp decrease as the x-ray energy increases. Therefore, two scaling factors are required for higher Z-number
tissues and lower Z-number tissues respectively when converting CT numbers from different energy. Tissues with HUs in
the range of -1000 to 0 were assumed as the mixtures of water and air while tissues with HUs larger than 0 were assumed
as the mixtures of water and bone.
Results: CT images from 5 lung cancer patients were used in this feasibility study. The images were acquired with a
Brilliance Big Bore CT simulator at energy A (CT A, 120kVp). For each patient, one set of CT images at energy B (CT B,
30keV) was generated from CTA with a bilinear scaling method, two pairs of digitally reconstructed radiographs (DRRs)
were created from CTB and CTA respectively and then a soft-tissue-only DRR pair (DRRdualE) was obtained using in-house
software of dual-energy subtraction with DRRCTA and DRRCTB. The image quality of DRRs was quantitatively analyzed
using signal-difference-to-noise-ratio (SDNR).
The results indicate that the generated CT images showed improved contrast and the DRR dualE showed that bony structure
was eliminated, soft tissue contrast was improved. SDNR for GTV was improved from 1.94 ± 0.09 (in DRRCTA) to 4.31 ±
0.12 (in DRRdualE).
Conclusion: The feasibility study has shown the potential of utilizing a conventional single-energy CT dataset to get
another set of CT images at a different energy to achieve improved soft tissue contrast for GTV delineation or target
localization when a DECT machine is not available.
*Corresponding author: fengyu@ecu.edu, supported by ELEKTA grant of Medical Physics Development in TJU
Preliminary study of DIFC method for rapid assay of apoptotic cells
Xu Yang1, Yahui Liu1, Eleanor Harris2, Xin-Hua Hu3*, Yuanming Feng2,3*
1
Department of Biomedical Engineering, Tianjin University 2Department of Radiation Oncology, East Carolina University
3
Department of Physics, East Carolina University
Introduction: It is well established that ionizing radiation of cells produces intracellular H2O2 molecules in cells which
in turn induces cell death in the forms of apoptosis and necrosis. This study is to quantify the morphological change in
cell apoptosis induced by H2O2 and investigate a method of diffraction-imaging-flow-cytometry (DIFC) for rapid assay
of apoptotic cells.
Method: Human leukemia cells (HL-60) were cultured at 370C in humidified 5% CO2 atmosphere in RPMI1640 with
10% heat-inactivated FCS. They were divided into six equal portions (one control, five treated with 1.5mM H2O2) and
analyzed at post-treatment time of 0h, 3h, 6h, 12h and 24h, respectively, after synchronization through serum
deprivation for 6 hours. flow cytometry(FC) analysis: HL-60 cells were stained with AnnexinV-FITC/PI and analyzed by
a conventional flow cytometer(Calibur, BD Biosciences); quantitative morphology analysis: The cells were imaged
with an inverted-fluorescence-microscopy(IVFM) (IX71, Olympus) after stained with Hoechst33342 and Annexin VFITC/PI. Morphological parameters were extracted and statistically analyzed with Xcellence-Imaging-Workstation;
DIFC analysis: The cells were analyzed without any fluorescent stains in an in-house-developed DIFC system after resuspended with medium. Diffraction Images were analyzed by a GLCM based software to extract image texture
parameters as the “3D fingerprint” features.
Results: The FC data clearly differentiated the intact cells at each apoptotic stage for the experimental groups. Typical
triple-staining fluorescence microscopic images show clearly the cells at different apoptotic stage. Some of
morphological parameters are listed in Table1. Fig.1 shows cross-polarized diffraction image pairs of side scatters
acquired by two cameras denoted as camera0 and camera1. The differences in diffraction patterns or textures can be
clearly observed in the image pairs between those from the normal/early apoptotic cells of intact structures and
others from the late apoptotic/damaged cells of shrinking morphology or cellular fragments.
Conclusion: The results show that the texture parameters of the diffraction image pairs obtained from DIFC
measurements correlate well with morphological changes quantified with conventional FCM analysis and microscopic
measurement in apoptotic cells, which demonstrates that DIFC can provide fast and label-free tool for the study of
various cellular processes with underlying morphological changes.
*Corresponding authors: fengyu@ecu.edu, hux@ecu.edu. Supported by grants of NSFC (81201148 and 81171342).
Doxorubicin impairs skeletal muscle mitochondrial respiratory capacity in skeletal muscle
Laura A. A. Gilliam1,2, Kelsey H. Fisher-Wellman1,2, Chien-Te Lin1,2, Jill M. Maples1,2, and P. Darrell
Neufer1,2,3
East Carolina Diabetes and Obesity Institute1, Departments of Kinesiology2, and Physiology3
East Carolina University, Greenville, NC 27834
Debilitating muscle weakness and fatigue are common side effects of chemotherapy in cancer
patients, limiting recovery and increasing morbidity. Our previous work shows that doxorubicin, a
common chemotherapy drug, elevates reactive oxygen species (ROS) and decreases contractile
force in skeletal muscle. As mitochondria represent a primary source of oxidant generation in
muscle, we hypothesized that doxorubicin may compromise mitochondrial respiratory control and
increase ROS production. 72 hrs following a single doxorubicin injection (20 mg/kg), maximal ADPstimulated O2 consumption was decreased during respiration supported by palmitoylcarnitine (PC) (46  26 %, p<0.05) and pyruvate/malate (-43  21 %, p<0.05, n=6/group) in permeabilized fiber
bundles (PmFB) from red gastrocnemius muscle. PmFB from doxorubicin treated animals also
display increased rates of mitochondrial H2O2 emission with PC (23  2 %) and 3-phosphoglycerate
(49  2 %, p=0.1). Our data indicate that doxorubicin impairs both maximal State 3, and fatty-acidsupported respiration. In addition, doxorubicin promotes mitochondrial ROS generation, a potential
underlying cause of doxorubicin-induced muscle dysfunction.
Supported by F32-AR061946 and R01-DK073488
Stereotactic Body Radiation Therapy for Stage I Non-small Cell Lung Cancer:
Relationship between Biological Equivalent Dose and Local Tumor Control
Zhibin Huang, PhD1, Kevin Yuh2, Nina A. Mayr, MD3, Simon S. Lo, MD4,
William T. Yuh, MD, MSEE5, John C Crecula, MD2
1
Department of Radiation Oncology, East Carolina University, Greenville, NC 27834
Department of Radiation Oncology, The Ohio State University, Columbus, OH 43210
3
Department of Radiation Oncology, 5Department of Radiology, University of Washington, Seattle,
WA 89195
4
Department of Radiation Oncology, Case Comprehensive Cancer Center, Cleveland, OH 44106
2
Purpose: To investigate the relationship between tumor control probability (TCP) and biological
equivalent dose (BED) for radiation therapy in medically inoperable stage I non-small cell lung cancer
(NSCLC).
Methods and Materials: 42 studies on stereotactic body radiation therapy (SBRT) for stage I NSCLC
were published in literature, as reported in Mehta N, et al., Practical Radiat Oncol 2:288-295 (2012),
and reviewed for tumor control (TC) that was defined as crude local control ≥ 2 years. BED based on
the linear quadratic (LQ) and the generalized LQ (gLQ) models were obtained. For each dosefractionation schedule, BED was calculated at isocenter using the LQ and gLQ models. TC as a
function of BED was obtained and fitted to the standard TCP equation for both models.
Results: 1640 patients were included in the SBRT studies. An alpha-beta ratio of 8.6 was used as in
the original manuscript. Daily fraction size was 6-26 (total dose: 20-66) with SBRT. Median BED was
149.2 Gy (range, 71.4-319.1) and 103.5 Gy (range, 66.5-162.9) for the LQ and gLQ models,
respectively. Using the LQ model, BED to achieve 50% TC (TCD50) was 61 Gy (95% confidence
interval, 50-71). TCP as a function of BED was sigmoidal, with TCP ≥ 90% achieved with BED ≥ 160
Gy and 120 Gy for the LQ and gLQ models, respectively.
Conclusions: Delivered with SBRT, BED of 120 Gy by the gLQ model results in a total dose of 34.8
Gy in a single fraction, 52.4 Gy in 2 fractions and 63.9 Gy in 3 fractions estimated by the gLQ model
at the isocenter. Clinically insignificant gain in TCP may persist for dose-escalation beyond a BED of
120 Gy calculated by the gLQ model, which likely results in clinically significant toxicity.
Table: Dose and fraction derived from BED corresponding to TCP of 90% in models
Model
LQ
gLQ
BED
for Total Dose in a
TCP>90%
single
fraction
(Gy)
(Gy)
160
33.1
120
34.8
Total Dose in
2
fractions
(Gy)
44.6
52.4
Total Dose in
3
fractions
(Gy)
52.5
63.9
The use of matched pairs of tumor and normal conditionally reprogrammed primary prostate
cells in radiobiological research
Andrew W. Ju MD, Nancy Palechor-Ceron DMD, Shaan Kataria BS, Aleksandra Dakic PhD, Thomas
P. Kole MD PhD, Johng S. Rhim PhD, Xuefeng Liu MD, Olga Timofeeva PhD, Richard Schlegel MD
PhD, Anatoly Dritschilo MD
Background: A novel tissue culture technique has been developed to allow the growth of tumor and
normal primary epithelial cells in a stem-like state in the presence of the ROCK inhibitor Y-27632 and
irradiated 3T3-J2 murine fibroblasts. These “conditional reprogramming” conditions allow the cells to
grow in vitro indefinitely with a stable karyotype. Normal prostate conditionally reprogrammed cells
(CRCs) remain non-tumorigenic, whereas select prostate tumor CRCs can form tumors in SCID mice.
CRCs have the potential to serve as powerful tools for radiobiological research as they can be grown
indefinitely without the need to introduce exogenous viral or cellular genes.
Methods/Materials: Pathologically confirmed tumor (GUMC-030) and normal (GUMC-029) prostate
tissues from a patient with a Gleason 7 adenocarcinoma were grown in co-culture with irradiated 3T3J2 fibroblasts in F medium with 5 uM Y-27632. Expression of basal and luminal markers was
characterized with qRT-PCR and Western blots. The expression levels of genes involved in
oncogenesis and cell cycle were determined and compared between the tumor and normal CRCs
using microarray analysis. Prostate CRCs were isolated and grown following radiation exposure for
10-14 days in modified keratinocyte serum-free medium for clonogenic radiation survival analyses.
The linear-quadratic (LQ) cell survival parameters of tumor and normal CRCs were determined from
the clonogenic data.
Results: Prostate tumor GUMC-030 and normal GUMC-029 cells expressed basaloid markers.
CXCL11, NNMT, and CDK2 are upregulated and CDKN1C is downregulated in GUMC-030 cells as
compared to the normal GUMC-029 cells. The alpha/beta ratio for GUMC-030 CRCs was 10.9 Gy, as
compared to an alpha/beta ratio of 1.2 Gy for GUMC-029 CRCs.
Discussion: We have shown that CRCs are a potentially useful tool for radiobiological studies. This
model cell system permits comparative evaluations of the effects of radiation sensitizers on tumor and
normal derived tissues from the same patient. The alpha/beta ratio for the GUMC-030 CRCs is
comparable to the values reported for immortalized prostate cell lines, but it is higher than the values
estimated in clinical studies. Our investigation of the effects of HDAC inhibitors on the radiobiological
parameters of matched pairs of CRCs will be reported.
Two Clinical Programs to Validate a Non-Invasive Urine Biomarker for Prostate Cancer
Detection
1
Qun Lu, 2Tae Lee, 1Yan-Hua Chen, 3Heng Hong, 4Art Rodriguez, 5Michael Woods, 6Carol Otey,
5
Hong Jing Kim, 7Albert Baldwin, 8Qiang Wu and 9Jennette Bensen
Departments of 1Anatomy and Cell Biology, 2Family Medicine, and 3Pathology and Laboratory
Medicine, The Brody School of Medicine, East Carolina University; 8Department of Biostatistics,
College of Allied Health Sciences, East Carolina University; 4Department of Chemistry, Thomas
Harriot College of Arts and Sciences, East Carolina University, Greenville, North Carolina 27834;
Departments of 5Urological Surgery, 6Cell Physiology, and 9Epidemiology, and 7Lineberger
Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599
Prostate cancer is extremely common in developed countries: in the US, one in six men will be
diagnosed with prostate cancer during his lifetime, and the incidence is even higher for African
American men. Prostate tumors vary widely in their biologic behavior - some tumors are indolent and
require no treatment, whereas others are aggressive and life-threatening. Since 1991, physicians
have used PSA to screen for prostate cancer; however, the U.S. Preventive Services Task Force
(USPSTF) released a report in 2011 recommending that men no longer receive PSA blood tests as
part of routine cancer screening. This recommendation was based on an extensive review of the
literature on the performance of PSA as a screening test, as well as the outcomes and current
treatment options for prostate cancer. The Task Force concluded the harms of treatment outweigh
the small potential benefits of screening.
One of the major limitations of PSA is the inability to accurately distinguish between cancer and PSA
elevation for other reasons, mainly benign prostatic hypertrophy. The USPSTF recommendations
have created a diagnostic vacuum, generating an urgent need to develop novel screening methods
for prostate cancer. Ideally, a new screening test should be non-invasive, highly sensitive to the early
and treatable stages of the disease, and specific for cancer as opposed to benign diseases.
Two clinical pilot studies are being conducted based on the patented technologies from East Carolina
University and the collaborations with UNC Lineberger Comprehensive Cancer Center. They are
focused on new potential biomarkers for prostate cancer, including a protein called delta-catenin.
Project 1 is a clinical trial of 200 urine specimens of prostate cancer and non-cancer subjects from
North Carolina-Louisiana Prostate Cancer Project Consortium and East Carolina University Family
Medicine. Ancillary aspect of the project is the examination of our biomarker cluster expression in
other cancer types. Project 2 is a clinical trial of 200 urine specimens of prostate cancer and noncancer subjects of UNC Urological Surgery and Surgical Oncology. These studies are to determine if
the new biomarker levels are significantly and consistently elevated in the urine of patients with
prostate cancer, as compared to normal subjects. These projects are made possible because of the
close collaborations between East Carolina University and UNC core facilities including UNC
Biostatistics Core Facility and Tissue Procurement Core Facility of the Lineberger Comprehensive
Cancer Center. In addition, the going projects are supported in part by grants from the National
Institutes of Health, Golfers against Cancer Foundation, and North Carolina Biotechnology Center.
-Catenin/NPRAP/Neurojungin Mutation Promotes Prostate Tumorigenesis
In Mice Overexpressing Myc Oncogene
Jongdee Nopparat, Jiao Zhang, Yan-Hua Chen, and Qun Lu
Department of Anatomy and Cell Biology, Leo Jenkins Cancer Center, The Brody School of Medicine,
East Carolina University, Greenville, North Carolina 27834
Prostate cancer is the most common non-cutaneous malignant neoplasm in men in Western
countries. In humans, prostate cancer progresses from precursor lesions, termed prostatic
intraepithelial neoplasia (PIN), to overt carcinoma confined to the prostate, and finally to metastatic
disease that often results in lethality. Previous studies in our laboratory have shown that catenin/NPRAP/Neurojungin (gene designation: CTNND2), a primarily neural-enriched protein in the
brain of healthy individuals, is overexpressed corresponding to human prostate cancer progression.
This finding led us to investigate the roles of -catenin in prostate cancer progression. We utilized a
novel transgenic model of mouse prostate cancer with overexpression of human Myc oncogene
driven by rat-probasin promoter as the initiating event. The Myc overexpressing mice were crossed
with -catenin mutant (-cat-/-) mice whose exon 9 of -catenin gene was disrupted. In this study, we
examined the histological differences of mouse prostate from wild type and Myc overexpression with
-catenin mutant mice (Myc/-cat-/-). We observed morphological alterations characteristic of high
grade prostatic intraepithelial neoplasia (PIN) in luminal epithelial cells of transgenic mice by 6 weeks
of age. These changes included an increase in nuclear size, multi-layering, and cribriform formation.
Surprisingly, Myc/-cat-/- mice at 6 months of age displayed dramatically increased tumor size and
progression from PIN to adenocarcinomas. We also discovered an increase in both number of
proliferating cells and cells undergoing apoptosis in Myc/-cat-/- mice compared with wild type
animals. However, in Myc/-cat-/- mice, the cell proliferative index was greater than apoptotic index by
3.7 fold. Collectively, our findings suggest that mutation of -catenin contributes to tumorigenesis of
mouse prostate by enhancing the effects of Myc oncogene resulting in an increase in cell
proliferation, thereby promoting prostate tumor growth. Supported by NIHCA111891 and
NIHCA165202.
PI: MARIA J. RUIZ ECHEVARRIA
THE CONTRIBUTIONS OF TMEFF2 TO PROSTATE CANCER STUDIES
Prostate cancer (PCa) is the most common non-cutaneous cancer in men. Importantly, while the overall pattern of cancer
incidence in North Carolina is similar to that of the nation, there are several cancers and regions within the state that show
marked differences from the national rate [North Carolina State Center for Health statistics]. The high incidence of PCa,
coupled with the extended course of the disease, makes PCa a particularly appropriate candidate for prevention and early
intervention strategies. However, in its early stages when the disease is curable, prostate cancer tends to cause few or no
clinical symptoms and is difficult to detect and by the time the disease is advanced enough to cause symptoms, it is often
incurable -- referred to as castration resistant prostate cancer (CRPC). Therefore, PCa therapy will greatly benefit from
the identification of markers useful for early disease detection and from treatments for advanced disease.
The long-term goal of our studies is to develop new markers and new therapies for PCa diagnosis and treatment.
For this purpose, we have focused our efforts on studying the expression, regulation and mechanism of action of the
TMEFF2 protein during PCa development/progression and its potential role as a biomarker for this disease. Several
observations merit these studies: 1) TMEFF2 is a protein with limited distribution to adult brain and prostate and its
expression is deregulated during PCa; 2) TMEFF2 expression is regulated by the androgen receptor (AR) signaling axis
[5-7], the master regulator during all stages of the disease; 3) TMEFF2 interacts with and modulate the activity of SARDH,
the enzyme responsible for sarcosine to glycine conversion. Sarcosine and the enzymes involved in its metabolism have
been respectively described as potential biomarkers and therapeutic targets for PCa.
The overall goals from the studies in my laboratory are to define the role of TMEFF2 in PCa, elucidate its biology and
therapeutic/diagnostic potential. We have proposed three specific areas of study that will examine in vivo the tumor
suppressor/oncogenic role of two distinct TMEFF2 expressed forms, delineate downstream signaling pathways, identify
cooperating and specific effects of the TMEFF2-androgen receptor signaling axis, examine the impact of TMEFF2 in 1Cmetabolism and epigenetics, and determine the value of TMEFF2 as a non-invasive biomarker. We will use a combination
of biochemical, in vivo, and molecular biology approaches to study these events in PCa models systems (cell culture,
mouse models and human samples). The link of TMEFF2 with general cellular metabolism and the androgen receptor
axis, uncovered in our laboratory underlines the comprehensive value of these studies to PCa. Androgen receptor has an
essential role in the normal growth and development of the prostate gland and in all stages of prostate carcinogenesis,
further emphasizing the relevance of these studies.
Mechanisms of TMEFF2 function: the functional role of TMEFF2 in metabolism and epigenetics. Metabolism and
signaling are tightly linked allowing the cell to modulate its activities according to its metabolic resources and following the
microenvironment cues. It is widely accepted that altered metabolism is a hallmark of cancer and many metabolic
enzymes show altered expression in cancer. Importantly, using cell lines and a transgenic TMEFF2 mouse model we
have established that TMEFF2 influences 1C-metabolism and the methylation potential of the cell. Accordingly, TMEFF2
modulates DNA and histone methylation. Based on our preliminary data indicating differential effect of TMEFF2 on
histone methylation in the presence or absence of DHT, we hypothesize that TMEFF2 functionally interacts with the AR
(and potentially other partners) to exert epigenetic changes. The validity of this hypothesis is currently under study.
Studying the oncogenic role of TMEFF2: Characterization of the transgenic ECTO-mouse. This project has been
undertaken to determine the in vivo biological function of the extracellular portion (ectodomain) of the TMEFF2 protein,
which is produced by inflammatory molecule-mediated cleavage of the full-length protein. In vitro data from several
laboratories, including ours, suggests a growth-promoting role for the extracellularly accumulated ectodomain, which
opposes the tumor suppressor role of the full-length protein. Characterization of the ectodomain (ECTO-) transgenic
mouse will focus on the following aspects: a) size and composition of the litters; b) histological/pathological
characterization of the prostatic lobes – including gross appearance, weight, branching morphogenesis, histology and
presence and latency of tumors; c) presence of metastatic lesions to lymph nodes, bone, lungs and other organs; d)
immunohistochemical examination of cellular changes associated with expression of the transgene.
Biomarker design: Determine the potential utility of the cleaved ectodomain as a PCa marker in body fluids.
Localized, organ-confined prostate cancer (PCa) can be effectively treated. However, metastatic PCa is currently
incurable and the cause of most deaths from this disease, underlying the need for accurate diagnostic/prognostic
biomarkers. Our studies will attempt to evaluate the value of TMEFF2, a shed protein overexpressed in PCa, as a
circulating biomarker for PCa by analyzing its level in body fluids and establishing a correlation with disease grade.
Improving Community Health through Education and Access to Breast Cancer Screening:
A 3-Month Interim Report
V Jackson1, J Palchinsky2, T Finch3, A Billanti3, C Fenlon-Coda1, N Vohra2, KM Verbanac2
Leo Jenkins Cancer Center, 1Vidant Medical Center, 2Department of Surgery, 3Department of Public
Health, Brody School of Medicine at East Carolina University
Early detection to improve breast cancer survival remains the cornerstone of breast cancer control.
Studies have shown that mammography for early detection of breast cancer improves the mortality
rate and will detect a breast mass when it is too small to be detected by a physical exam. In Pitt
County, >40% of women over 40 years of age did not have a mammogram within the past year.
Minority women in Pitt County had higher rates of advanced disease at initial diagnosis compared to
their white counterparts (6.7 vs. 2.7%) and a mortality rate due to breast cancer 1.4 times greater
than the state average. Here we describe an approach to address this disparity by improving access
to breast cancer screening in Pitt County with a particular focus on African American women. This
initiative, funded as a 2013-2014 Community Health Grant, 1) works with local partners to assess
women for breast health needs and breast cancer risk; 2) links uninsured and underinsured women to
existing assistance programs and 3) provides funding for screening when women are ineligible for
existing resources or when existing resources are depleted. The program will continue through June
2014 with a goal to assess 300 women and to fund breast cancer screening for 140 women. In 3
months (July-September 2013) we have recruited 18 volunteers (9 students, 5 adults and 4 breast
cancer survivors) and have participated in multiple events at 11 outreach venues including community
free health clinics, churches, the Lucille Gorham Intergenerational Community Center and Third
Street Community Center. We have assessed the breast health of 116 women (71% African
American; 14% white; 13% Hispanic) using a 9-question tool, provided educational materials, and
offered follow-up and transportation assistance. Of these participants, 72% were in need of breast
cancer screening and 52% were uninsured. Forty uninsured women were in need of a mammogram.
Twelve women have received a screening or diagnostic mammogram funded by the Komen initiative
and 20 additional women have been scheduled for Komen-funded mammograms. Ten women have
been referred for further breast cancer risk assessment due to family history. Forty-five women have
been linked to the Breast and Cervical Cancer Control Program (BCCCP). These findings confirm
that there is an unmet need for screening in this area. Interim results suggest that this initiative will
improve community health by increasing education about breast health and access to breast cancer
screening.
[Funding by the Susan G. Komen for the Cure North Carolina Triangle-to-the-Coast Affiliate]
Pro-tumor N2 Neutrophils Contribute to the Pre-metastatic Niche
Jered Cope Meyers, H. Keith Pittman, Dare Moore Imes, Hunter Storey, George Howard IV, Kathryn
M Verbanac
Department of Surgery, Brody School of Medicine at East Carolina University
Metastasis is the primary cause of cancer-related mortalities and the tissue microenvironment that
supports tumor cell attachment and growth at metastatic sites has become an active area of
investigation. Here we present evidence that pro-tumor N2 neutrophils are one of the bone marrowderived cell types that establish this pre-metastatic niche. In the murine Lewis Lung adenocarcinoma
(LLC) in vivo metastasis model, tumor cells metastasize to the lungs with increasing primary tumor
growth over time. In these studies, immunocompetent mice injected subcutaneously with syngeneic
LLC cells are sacrificed sequentially 10-32 days later, before and after established lung metastases.
Neutrophil chemoattractants KC/CXCL1 and MIP-2/CXCL2 are significantly elevated in pre-metastatic
tissue compared to normal lungs of healthy contemporary controls. MCP-1/CCL2, a marker of protumor N2 neutrophils, increases steadily over time in pre-metastatic lung tissue of study mice and is
significantly elevated in pre-metastatic tissue and in metastases compared to normal lung tissue.
Isolated lung-infiltrating neutrophils also express N2 markers. In contrast, TNF-α, a marker for antitumor N1 cells, remains low or undetectable in lung tissue throughout metastatic tumor development.
Neither active nor total TGF-β, the putative control switch for a pro-tumor phenotype, is elevated in
pre-metastatic tissue. Notably, levels of all cytokines are low or undetectable in normal lungs from
naïve mice and in other organs of tumor-bearing mice (non-metastatic sites). Ly6G+ neutrophils were
detected at a higher density in the lungs of pre-metastatic and metastatic mice compared to normal
lungs, although the density of myeloperoxidase (MPO)-stained cells was similar in all groups. These
data are consistent with recent reports that MPO activity is low or absent from tumor-associated
neutrophils, although high in MDSC and naïve neutrophils. Neutrophil distribution in metastatic lung
tissue is most concentrated around the tumor foci and tumor vasculature. Finally, studies of lung
tissue from mice bearing LLC variants with different metastatic capacities are also consistent with our
premise that neutrophils help establish the pre-metastatic niche. Understanding the factors that
regulate the development and growth of malignant cells at metastatic sites is critical for improved
clinical strategies in the prevention, diagnosis and treatment of metastases.
Regulation of tumor cell attachment, spreading and migration by the GPR4 receptor and
related G protein pathways
Calvin R. Justus, Li V. Yang
Department of Oncology, Brody School of Medicine, East Carolina University, Greenville, NC
The tumor microenvironment has a characteristic low pH due to inadequate blood perfusion, hypoxia,
altered cellular metabolism, and inflammation. The effects of acidic pH on cancer progression are
complex. It has been shown that low pH increases the metastatic spread of cancer by assisting in the
degradation of extracellular matrix. Other studies show that acidic microenvironments decrease
metastatic potential in B16F10 melanoma cells upon activation of the GPR4 receptor and also inhibit
Akt activity in breast cancer cells. In this study we have investigated the attachment, spreading, and
migration of B16F10 cells that have been genetically modified to express the GPR4 receptor at a high
level (named B16F10/GPR4 cells). The results showed that upon stimulation of GPR4 in
B16F10/GPR4 cells with low pH, cell spreading, membrane ruffling, and migration were significantly
decreased. The phosphorylation levels of paxillin Y118 and focal adhesion kinase (FAK) Y397
following one-hour cell attachment were also decreased, and the spatial localization of the
phosphorylated paxillin and FAK was substantially altered. Further tests were performed to identify
the G-protein signaling pathway responsible for this effect. By using a G13 dominant negative
construct or inhibiting Rho activation with C3 transferase (CT04) in B16F10/GPR4 cells, the cell
spreading and membrane ruffling abilities were almost completely restored back to the level of
B16F10/vector control cells, whereas the inhibition of the Gs and Gq pathways had little effect. These
results suggest that activation of the GPR4 receptor and subsequently the G13/Rho downstream
pathway are responsible for decreased cell attachment, spreading and membrane ruffling, which may
cause decreased metastatic potential of B16F10/GPR4 cells found previously.
Gene expression profiling reveals acidosis/GPR4-induced inflammatory responses in vascular
endothelial cells
Lixue Dong, Li V. Yang
Department of Oncology, Brody School of Medicine, East Carolina University, Greenville, NC
Due to deficient blood perfusion, hypoxia, and glycolytic metabolism, acidic tissue microenvironments
commonly exist in inflammation, ischemia, solid tumors, and many other pathological conditions.
However, the molecular mechanisms by which cells respond to acidic microenvironments are not well
understood. GPR4 has recently been identified as a novel proton-sensing receptor expressed in
endothelial cells, cancer cells, and other cell types. GPR4 is activated by acidic extracellular pH but
has lower activation at the physiological pH 7.4 and minimal activation at more alkaline pH. To
delineate the function and signaling pathways of GPR4 in endothelial cells under acidic conditions,
we performed gene expression microarray analyses. Primary human umbilical vein endothelial cells
(HUVEC) that express endogenous GPR4 were stably transduced with the MSCV-IRES-GFP control
vector (designated as HUVEC/Vector cells) or the MSCV-GPR4-IRES-GFP construct (designated as
HUVEC/GPR4 cells). HUVEC/Vector and HUVEC/GPR4 cells were treated with pH 6.4 (400 nM H+)
for 5 hours to activate GPR4 or with pH 8.4 (4 nM H+) as a negative control. RNAs were isolated,
labeled, and hybridized with the Agilent Whole Genome Microarray Chip to compare gene
expression. The results demonstrated that the acidic pH treatment of HUVEC/Vector cells stimulated
the expression of a number of inflammatory genes, which represent a predominant signature in the
microarray dataset. The induction of most of the acid-responsive inflammatory genes were further
enhanced by GPR4 overexpression in HUVEC/GPR4 cells at pH 6.4, suggesting these genes are
responsive to varying levels of GPR4 signaling. The inflammatory genes with substantial upregulation by acidosis/GPR4 include chemokines, cytokines, adhesion molecules, genes involved in
the TNFα and NF-κB pathways, prostaglandin-endoperoxidase synthase, and EGR transcription
factors. The up-regulation of the genes at pH 6.4, compared to pH 7.4 and pH 8.4, was further
confirmed by realtime RT-PCR. Taken together, our results show that activation of GPR4 by acidosis
stimulates the expression of a wide range of inflammatory genes in vascular endothelial cells.
Antiestrogens amplify antitumor impact of nanoformulated ceramide--- pan efficacy in a
myriad of tumor types.
Samy A.F. Morad 1, Mark Kester 2, Myles C. Cabot 1.
1 Department of Biochemistry & Molecular Biology, Brody School of
Medicine; 2 Penn State Hershey Medical Center, Hershey, PA.
Although ceramide has potent tumor suppressor properties, its use in cancer therapeutics has
been limited; this is mainly due to rapid metabolism of ceramide by cancer cells, the inability to
attain therapeutic levels when ceramide-generating agents are employed, and the inherent
insolubility of ceramide as a therapeutic. Our approach circumvents these disadvantages and
provides advantages over existing methodologies. For example, rather than promoting intracellular
ceramide generation by application of exogenous drugs that activate de novo and
sphingomyelinase pathways, we have chosen to administer ceramide exogenously in the form of
short-chain C6-ceramide nanoliposomes; this overcomes solubility issues and improves uptake. In
addition, nanoliposomes are efficient platforms for delivering other hydrophobic
chemotherapeutics. The antiestrogen tamoxifen was selected for our studies because of its
unusual “off-target” actions, which include inhibition of ceramide metabolism at glycosylation and
hydrolysis. Blocking these junctures increases C6-ceramide intracellular residence time and
diminishes production of sphingosine 1-phosphate, a mitogenic lipid derived via the action of
ceramidase and sphingosine kinase. Hence, C6-ceramide and tamoxifen make up a versatile drug
duo. Using in vitro models of human colon and breast cancer, acute mylogenous leukemia (AML),
and melanoma, we show that simultaneous administration of nanoliposomal C6-ceramide and
tamoxifen potently and synergistically decreased cell viability, compared to the single agents. The
mix induced caspase-dependent apoptosis, cell cycle arrest at G1 and G2, upregulation of JNK,
p38, and p53, and downregulation of Akt and survivin. In triple negative, hormone-insensitive
breast cancer cells, C6-ceramide-tamoxifen induced cell cycle arrest, caspase-dependent
apoptosis, and lysosomal and mitochondrial membrane permeability. Evaluation of single agents
showed that tamoxifen elicited lysosomal membrane permeability and inhibited acid ceramidase.
We have obtained similar results in in vitro models of human melanoma and AML. In an in vivo
retroviral transduced murine AML model, the combination lowered leukemia burden in spleen and
marrow. The combination also perpetuated synergistic increases in long-chain ceramides, likely
compounding the overall ceramide effect. Of interest regarding in vivo utility, the major
metabolites of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen, were effective in
combination with C6-ceramide. Our findings show that tamoxifen magnifies the antiproliferative
effects of C6-ceramide via aggregate mechanisms. This proficient, ceramide-based nanoliposomal
drug regimen may have potential as an effective anticancer therapeutic.
Supported by NIH 1P01 CA 171983 ; NIGMS 77391; ABC’s, Los Angeles; Fashion Footwear
Association of New York Charitable Foundation.
Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of Adult T-cell Leukemia (ATL), a
fatal malignancy associated with uncontrolled proliferation of infected CD4+ T-cells. ATL is divided
into multiple subtypes based on clinical features, with indolent subtypes progressing to aggressive
subtypes for which there is no effective treatment. While approximately twenty million people are
infected with HTLV-1 worldwide, less than five percent develop ATL. The basis for this outstanding
feature remains unknown as do the precise cellular events caused by viral infection that ultimate lead
to ATL. As a complex retrovirus, HTLV-1 is integrated into the host genome. Integration is random, so
the provirus does not alter the expression of a specific cellular proto-oncogene or tumor suppressor
through proximity of integration. Furthermore, none of the viral genes resemble cellular protooncogenes. However, two viral genes, Tax and HBZ, display oncogenic properties. Our group is
currently interested in characterizing molecular events that HBZ modulates that, in turn, may
contribute to cellular transformation or an enhanced survival capacity of ATL cells. HBZ is a nuclear
protein that was initially found to repress transcription from the HTLV-1 proviral promoter, and our first
studies aimed at characterizing the mechanism of HBZ-mediated repression. We showed
biochemically and in cells that HBZ is able to interact with a number of cellular transcription factors
that are involved in activation of HTLV-1 transcription, and when bound by HBZ, these factors are
unable to associate with DNA and regulate transcription. We then showed that HBZ-mediated
repression of HTLV-1 transcription additional involves an interaction with the homologous
coactivators p300 and CBP. These results relate to the currently held model that active viral
transcription enhances elimination of HTLV-1-infected T-cells by the host immune system, while
repression of viral transcription allows infected cells to evade immune surveillance. Importantly, the
HBZ gene is uniquely encoded on the antisense strand of the provirus, and its expression is
controlled by a separate promoter to that regulating expression of all other viral genes. Therefore,
repression of HTLV-1 transcription does not influence expression of HBZ. Based on its interaction
with multiple cellular transcriptional regulators, we developed clonal cell lines that stably express HBZ
and, through microarray analysis, found that HBZ modulates expression of multiple cellular genes
both positively and negatively. Interestingly, we found that HBZ can upregulate gene expression
through its interaction with p300 and CBP. These coactivators contain multiple protein docking sites.
By binding tightly to one of these sites, HBZ blocks certain transcription factors from accessing the
same site, which in the case of the HTLV-1 provirus, inhibits transcription. However, other protein
docking sites become more accessible when HBZ is bound to the coactivators, and transcription
factors that interact with these sites can more efficiently recruit p300 and CBP for transcriptional
activation. Several HBZ-target genes identified from our microarray analysis have potential functions
related to pathological effects associated with ATL, and we have been directing our research towards
these observations. For example, a prominent clinical feature of ATL is the infiltration of infected Tcells into the skin, lungs and other organs. Our data revealed a subset of HBZ-target genes that
functions in cell migration and infiltration, and we have found that our HBZ-expressing cell lines
exhibit phenotypes related to these functions. Therefore, we expect HBZ to play an important role in
the T-cell infiltration that is associated with ATL.
Vaccinia Virus A35R Virulence Gene and Leukocyte Migration
Smallpox killed an estimated 500 million people in the twentieth century alone. Although this fatal
infectious disease was eradicated from the world over thirty years ago, it remains an important
concern as a bioterrorism agent. Vaccinia virus, the live virus vaccine for smallpox, is extremely
dangerous for immunocompromised individuals. Since this cohort comprises a significant portion of
the world’s population, a safer vaccine is needed. The vaccinia virus A35R gene is highly conserved,
and our lab has shown that it increases virulence by inhibiting the body’s anti-viral immune
responses. When A35R is removed from the virus to create an A35R deletion mutant, the virus
becomes attenuated, and immune responses are improved. This study compares the responses of
lung leukocyte populations between WR wild type virus infected mice and A35R deletion mutant
infected mice to understand the mechanism of A35R immunosuppression. Mice were infected with
vaccinia virus (WR and A35RDel), lungs were harvested three days later, and cell populations were
quantified using flow cytometric analysis. Initial data showed increased recruitment and/or
proliferation of cell populations in response to vaccinia infection, and the A35R gene slightly
perturbed populations of B220+ cells (activated B and T cells), macrophages, dendritic cells, and
granulocytes. Further repeats of the experiment will be required to look at the kinetics of the immune
cell responses and to confirm findings in order to better understand A35R immunosuppression.
Shayna N. Mooney, Gwendolyn B. Jones, Rachel L. Roper
Vaccinia virus A35R virulence gene and Leukocyte Migration
Vaccinia virus A35R and the elicitation of lymphocyte subpopulations
CHANGE IS GOOD FOR THE LYME DISEASE SPIROCHETE,
BORRELIA BURGDORFERI
S. Sultan, E. Novak, K. Moon, A. Yerke, A. Manne, and MD MOTALEB
Department of Microbiology and Immunology
Image is adapted from
Google
Borrelia burgdorferi is the causative agent of Lyme disease. It is the most prevalent
arthropod borne infection in the United States. The disease is a multiple- systemic
disorder with various clinical manifestations such as erythema migrans rash, arthritis,
cardiac, and neurological manifestations. While the disease is rarely associated with
fatalities, the persistent and debilitating nature of the disease leads to $1-2 billion in
direct medical expenses and lost productivity each year in the United States. There is
currently no commercially available vaccine to prevent Lyme disease.
B. burgdorferi exists in nature in an enzootic cycle. Ixodes scapularis ticks (commonly
known as deer ticks) acquire the infection when they feed on an infected host, mainly
rodents. During subsequent tick feeding, which lasts for several days, B. burgdorferi
migrate from the tick midgut, pass through the salivary glands, and are then transmitted
to the mammal through the saliva. B. burgdorferi is highly invasive. After being
deposited in the skin following a tick bite, the spirochetes can invade many tissues
including the joints, heart, and nervous system.
Cyclic-di-GMP, motility, and chemotaxis are critical for bacterial survival and adaptation
in diverse environmental conditions. One of our immediate goals is to determine the
extent to which c-di-GMP, motility and chemotaxis systems of Borrelia burgdorferi are
involved in transmission and disease progression. Our long term goal is to identify a
molecule or a pathway (e.g., chemotaxis signal transduction pathway) in the organism
for which a novel pharmacological agent can be developed to better treat or prevent the
multi-systemic Lyme disease.
Brucella – a highly evolved intracellular pathogen.
R. Martin Roop II, Department of Microbiology and Immunology
Brucella spp. are Gram-negative bacteria that cause abortion and infertility in their natural hosts, and
a chronic debilitating febrile illness in humans known as undulant fever. B. melitensis, B. abortus, and
B. suis are major pathogens of goats and sheep, cattle, and swine, respectively. Brucella infections in
these food animals have a significant economic impact in areas of the world where they are not
controlled by effective eradication programs. They also pose a considerable public health threat in
these areas because the brucellae are easily transmitted to humans through exposure to infected
animals or their products. In fact, brucellosis is considered to be one of the world’s leading zoonoses.
B. melitensis, B. abortus, and B. suis strains are also classified as ‘non-Tier 1’ Select Agents by the
CDC and are included in NIAID’s Biodefense Research Agenda because they possess characteristics
that make them attractive for use as agents of biological warfare or bioterrorism. Specifically, they are
easy to aerosolize and highly infectious by the aerosol route, the disease they produce in humans is
debilitating and difficult to treat with antibiotics, and there is no vaccine available to prevent human
brucellosis.
The capacity of Brucella strains to survive and replicate for prolonged periods within macrophages is
critical for their capacity to produce in their mammalian hosts. During their residence in host
macrophages, these bacteria are exposed to a variety of environmental stresses including exposure
to acidic pH, oxidative stress and nutrient deprivation. The research being performed in our laboratory
is directed at understanding how the brucellae adjust their patterns of gene expression to successfully
withstand the environmental stresses they encounter during their intracellular replication in host
macrophages. Results obtained from these studies will not only help us gain a better understanding
of the mechanisms these important zoonotic agents employ to produce disease, but they will also
provide information that can be used to develop improved strategies for preventing and treating
brucellosis in humans.
Virulence Factors of an Anaerobic, Opportunistic Pathogen Bacteroides fragilis
C. Jeffrey Smith, Edson Rocha, Anita Parker, Ivan Ndamukong, Yanlu Cao, Michael Betteken
Department of Microbiology & Immunology
Introduction:
The human microbiome or indigenous microflora inhabits every mucosal surface of the human body and
outnumbers the human cells by more than 10 to 1. This normal bacterial flora has a mutualistic association
with the host which influences many of the host’s physiological, nutritional, and immunological activities. The
beneficial relationship is maintained by physical barriers and immunological processes that keep the microbial
populations in check. However, disruption of these defense mechanisms leads to opportunistic infections,
which can have a serious impact on human health. So how does our indigenous microflora cause disease? In
order to address this question we study intra-abdominal abscesses which are formed when the peritoneal
cavity is contaminated with indigenous intestinal bacteria following perforation of the bowel (appendicitis,
diverticulitis, carcinoma, surgery). These are polymicrobic infections that result in a multifactorial host
response designed to wall off and contain the invading microbes. Bacteroides fragilis is the predominant
anaerobe associated with intra-abdominal infections and overall is the most frequently isolated anaerobe from
clinical samples.
Hypothesis:
We hypothesize that both resistance to oxidative stress and nutritional factors give B. fragilis a survival
advantage during the development of these infections. In order to test these hypotheses we use a variety of
approaches such as genomics, transcriptomics, and proteomics to measure gene expression and protein
profiles of organisms with specific genetic mutations during the course of infection in a rat abscess model.
Results:
Oxidative stress.
Relative to the colon, the peritoneal cavity is an oxygenated environment. This and the
recruitment of PMNs to the site of infection will result in substantial exposure of B. fragilis to reactive oxygen
species during colonization of the peritoneum. Our work has documented that B. fragilis induces an oxidative
stress response that encompasses about 25% of the genome. This response includes an acute oxidative
stress response which is designed to minimize the immediate effects of oxygen radicals. We have shown that
this rapid response is mediated by the LysR-family regulator OxyR which is required for abscess formation.
Following the acute phase, there is a novel widespread induction of genes associated with metabolism which
occurs when there is prolonged exposure to oxidative stress. We are currently working to understand the
genetic mechanisms that regulate this global response to prolonged oxidative stress and how this response
enhances persistence in the abscess milieu of necrotic cell debris, viable PMNs, and host serum factors.
Nutritional factors.
B. fragilis is saccharolytic and in the colon it derives energy by catabolism of
polysaccharides that escape the human digestive system. The transition from growth in the colon to the
peritoneal cavity requires rapid adaption to a new nutrient supply. Gene expression studies with cells grown in
an artificial abscess in vivo suggest that B. fragilis quickly induces a novel set of polysaccharide utilization
operons that reflect the availability of new nutrient sources that are rich in glycoproteins. These enzymes allow
B. fragilis to harvest N-linked glycans from a range of glycoproteins that are abundant in serum. We ultimately
wish to learn more about the regulatory mechanisms that control how the organism remodels its catabolic
metabolism to survive for extended periods in the abscess.
Investigation of the Immunosuppressive Function of the Vaccinia Virus Protein O1L
Anastasia Weeks, Gwendolyn Jones, and Rachel Roper
Poxviruses cause a wide range of mammalian diseases, and there are many poxviruses that
are emerging human pathogens. Although smallpox was declared eradicated from nature due
to the success of the Vaccinia virus vaccine, poxviruses still pose a bioterrorism threat.
Vaccinia is also currently used successfully as a vector in recombinant vaccines that target
diseases such as HIV, malaria and cancer. However, Vaccinia virus retains significant
virulence in mammals and is unsafe in approximately 25% of the US population. In order to
understand poxvirus pathogenesis, our lab is investigating poxvirus virulence genes.
In this study, we show that the previously uncharacterized, highly conserved Vaccinia virus
gene O1L is a virulence factor in mammals. Prior data from this lab indicated that the O1L
protein was not required for replication, therefore we hypothesized that O1L functions in some
immunomodulatory capacity. To ascertain the mechanism of action of the O1L protein, we
investigated the immune response in mice infected with Vaccinia virus or O1L deletion mutant
viruses. Data from an ELISA assay demonstrated that O1L suppressed antiviral antibody
production, supporting our hypothesis that O1L is immunoregulatory. Currently we are
investigating the effects of O1L on T cell activation and differentiation by intracellular cytokine
staining and analysis of secreted cytokines.
Removal of immunosuppressive virulence genes may allow the creation of more safe and
immunogenic vaccines. In addition, immunosuppressive viral genes have the potential to be
used clinically to reduce undesirable immune responses.
Prohibitin coordinates an anti-inflammatory/antioxidant feedback loop from mitochondria to
nucleus to protect the heart from severe inflammatory stress
Taylor A. Mattox, Kathleen Thayne, Ethan J. Anderson
Department of Pharmacology & Toxicology, Brody School of Medicine
Cardiovascular and mitochondrial dysfunctions are well-known complications of sepsis. Prohibitin
(PHB), a mitochondrial protein, has been implicated in experimental models involved in coordinating
inflammatory signaling; its mechanisms are unknown. We investigated the role of PHB in the heart
during sepsis using an in vivo model (LPS) and in vitro model (TNFα/IL1β) in HL1 cardiomyocytes
(HL1c). Sepsis caused reductions in mitochondrial function in both models. In rats, LPS reduced
PHB expression in whole heart at 4 and 24 hrs post exposure. While the total cardiac PHB
decreased, serum levels spiked 3-fold and the remainder became concentrated in nucleus. In vitro
overexpression of PHB (oPHB) in HL1c attenuated the cytokine-induced reduction in O2
consumption, ATP generation, and Ca2+ retention, and prevented raises in H2O2 in response to these
cytokines. oPHB protected the HL1c from TNFα/IL1β-induced cytotoxicity. oPHB and recombinant
PHB to HL1c reduced TNFα/IL1β-induced NFκB activation and transcription of its downstream
targets. These findings uncover a novel role for PHB and suggest that PHB can act as a mobile
signal transducer which can suppress inflammation and oxidative stress during sepsis. We anticipate
that these findings will lead to novel therapies that exploit the pleiotropic functions of PHB, protecting
the heart from diseases in which inflammation and oxidative stress are common.
An RpiR-Family Transcriptional Regulator Modulates PQS Concentration in Pseudomonas
aeruginosa
Kyle A. Tipton, James P. Coleman, and Everett C. Pesci
Department of Microbiology and Immunology
Pseudomonas aeruginosa is a gram negative, opportunistic pathogen that can cause disease in
varied sites within the human body and is a significant source of morbidity and mortality in those
afflicted with cystic fibrosis. P. aeruginosa is able to coordinate group behaviors, such as virulence
factor production, through the process of cell-to-cell signaling. There are three intercellular signaling
systems employed by P. aeruginosa and one of these systems utilizes the small molecule 2-heptyl-3hydroxy-4-quinolone [Pseudomonas quinolone signal (PQS)]. PQS is required for virulence in multiple
infection models and has been found in the lungs of cystic fibrosis patients colonized by P.
aeruginosa. In this study, we have identified a transcriptional regulator, QapR (for quinolone alteration
pathway regulator), that is an autoregulatory repressor. We found that mutation of qapR caused
overexpression of the qapR operon. We characterized the qapR operon to show that it contains
genes qapR, PA5507, PA5508, and PA5509, and that QapR directly controls the operon’s expression
in a negative manner. We also show that overexpression of this operon greatly reduces PQS
concentration in P. aeruginosa. Our results suggest that qapR affects PQS concentration by
repressing an enzymatic pathway that either acts on PQS or a PQS precursor to lower PQS
concentration which in turn reduces the activation of the PQS biosynthetic operon. We believe that
this operon comprises a novel mechanism to regulate PQS concentration in P. aeruginosa.