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					Schedule at a Glance
Monday, June 14
             Room                 Event
8:00-9:00    PBB 156/157          Welcome reception with refreshments
9:00-10:15   PBB 156/157          Keynote address: Dr. Steven N. Blair, “Exercise is Medicine”
10:30-11:45 See session matrix    New investigator presentations
            (pp. 6–7)
12:00-1:00 PBB 160                Featured speaker: Dr. Kristina Thiagarajan, “Post-doctoral
                                  Opportunities”
1:30-4:45    See session matrix   New investigator presentations
             (pp. 6–7)




Tuesday, June 15
             Room                 Event
8:00-9:00    PBB 156/157          Refreshments
9:00-10:00   PBB 156/157       Keynote address: Dr. Arnold I. Caplan, “Adult Mesenchymal Stem
                               Cells and Their Use in Various Therapies”
10:15-12:00 See session matrix New investigator presentations
            (pp. 8–9)
12:00-1:00 PBB 160             Plenary address: Dr. Asha Kallianpur, “Progress and Promise in Iron
                               Biology: A Balanced Perspective”
1:30-4:45   See session matrix New investigator presentations
            (pp. 8–9)
6:00        Hollingsworth      Awards banquet & after-dinner address: Sam Venable, “Warning!
            Auditorium, EPS Living Can Be Hazardous to Your Health”

PBB, Plant Biotechnology Building; EPS, Ellington Plant Sciences Building (see map)




                                                                              THE

                                                                                                     OF




                                                   4
  UT Agriculture Campus Map                                     Location Information

                                                                    Room Locator
                                              CEMPH Symposium



                                  cl e
                               Cir
                           n
                       Morga




                              d    Bldg.   Lot 66
                       Vet Me


 University of Tennessee Agricultural Campus
 All parking will off site. Shuttle service
 will be provided from the UT welcome
 center (formally the UT Faculty
 Club).Parking Passes will be necessary
Key:
 each day. Parking charges are included
PBB, Plant Biotechnology Building                                                  (EPS)
 in registration fees.
EPS, Ellington Plant Sciences Building




Parking
All parking in campus lots is by permit only. Faculty & staff with a valid
parking permit for a main (east) campus lot may park in lot 66–across from
Ellington Plant Sciences–at no additional cost or ride the “T” (free for all
UT faculty, staff, & students with ID). The T: East to West circles every 15
minutes between 7:00 am and 6:00 pm. Students with valid permits should
park in designated student parking areas.



                                                                5
Session Matrix                                                  (Abstracts on pp. 14-49)
                                                                                                            Monday, June 14

                  Public Health                                                     Pharmacology & Pharmacokinetics
                  Rm. PBB* 156/157                                                  Rm. PBB 160
                  1. Organizational Cultural Competence of Post-Secondary           10. Ponazuril Pharmacokinetics in Healthy Llamas (Lama
    10:30 am




                  Health-Related Academic Units (Dotson)                            glama) Following Single Oral Administration (Prado)


                  2. Youth Can! Grow Healthy! (Carberry)                            11. The Effect of Ketamine on the MACBAR of
                                                                                    Sevoflurane in Dogs (Love)
    10:45




                  3. Investigation of Geographic Access to Emergency                12. A Comparison of AUC24/MIC Pre- and Post-
                  Stroke and Heart Attack Care in Middle Tennessee                  conversion from Intermittent Infusion Vancomycin to
    11:00




                  (Busingye)                                                        Continuous Infusion Vancomycin (Burke)

                  4. From 911 to ER: Analysis of Ambulance Transport of             13. Interscalene Anesthesia for Arthroscopic Shoulder
                  Stroke and Heart Attack Patients (Pedigo)                         Surgery: Failures and Complications (DiRuzzo)
    11:15




                  5. A Novel Processing Approach for the Inactivation of            14. Pharmacokinetics and Bioavailability of Carvedilol in
                  Salmonella enteritidis in vitro with Potential Applications       Cats (Durtschi)
    11:45 11:30




                  for Liquid Whole Eggs (Techathuvanan)


                  BREAK for Lunch--See schedule on p. 4

                                                                                    Bacterial Virulence & Transmission
                  6. Second Cut of the Rough: Revisiting the Site of an             15. Genomic Sequencing of Methicillin-resistant
    1:30 pm




                  Ehrlichiosis Outbreak in Middle Tennessee (Harmon)                Staphylococcus pseudintermedius from Canine Pyoderma
                                                                                    (Eberlein)

                  7. Application of Spatial Analysis, at Three Geographic           16. Variation of mecA Expression Among Regional Clones
                  Scales, to Detect Clustering of Humans with                       of Methicillin-resistant Staphylococcus pseudintermedius
                  Campylobacteriosis Reported to the Tennessee Department           and Characterization of mec and bla Regulatory Elements
    1:45




                  of Health (Weisent)                                               (Black)
                  8. Prevention of Pancreatic Cancer by Inhibition of Beta-         17. The Selection of New Gene Candidates for the
                  adrenergic Signaling (Al-Wadei)                                   Improvement of the Current MLST Scheme for
                                                                                    Staphylococcus pseudintermedius (Solyman)
    2:00




                  9. Formation of Nicastrin and Aph1 Subcomplex is                  18. Gene Expression Profile of Bovine Primary Mammary
                  Necessary for Recruitment of Amyloid Precursor Protein            Epithelial Cells Infected with Escherichia coli Associated
                  but Not for Interaction with Presenilin (Mao)                     with Acute or Chronic Bovine Mastitis (Kerro Dego)
    2:15




                  BREAK                                                             BREAK
    2:30




*
 PBB, Plant Biotechnology Building



                                                                                6
Monday, June 14

          Viral Pathology &                                                              Innovative Biomedical
                                                 Nutrition & Metabolism
          Immunity                                                                       Technologies
          Rm. PBB 113                            Rm. PBB 160                             Rm. PBB 156/157
          19. Robo 4 Activation Intercepts       26. Size of Food Bowl and Scoop         34. Synthesis and Characterization
          Herpes Simplex Virus 1-induced         Affects Amount of Food Owners Feed      of Diamond-like Carbon Films for
          Angiogenesis and Immunopathology       their Dogs (Murphy)                     Biomedical Applications (Leonard)
2:45 pm




          in the Eye (Mulik)

          20. An Imbalance in sVR-1              27. Nutritional Ergogenic Aids: The     35. In vitro Evaluation of Artificial
          and VEGF-A Mediates Corneal            Effects of Carbohydrate-protein         Membrane Biocompatibility (Beeler)
          Neovascularization after HSV-1         Supplementation During Endurance
          Ocular Infection (Suryawanshi)         Exercise (Coletta)
3:00




          21. Regulatory T Cell Expression       28. Effects of Leucine and Calcitriol   36. Optimizing the Performance of
          of Herpes Virus Entry Mediator         on Monocyte-Vascular Endothelial        ZBLAN Glasses as Image Plates (Vu)
          (HVEM) Following HSV-1                 Cell Adhesion (Curry)
          Infection and Its Functional
3:15




          Significance (Sharma)
          22. Deficient Virus-specific IgA       29. Assessing Potential for DNA         37. Fibroblast and Bacterial Growth
          Production Following Murine            Methylation Patterns to Affect          on Infection-resistant Materials
          gammaherpesvirus 68 Infection of the   Adipocyte Metabolic Gene Expression     (Minkin)
          Respiratory Tract (Sundararajan)       (Gouffon)
3:30




          23. In Vitro Time Dependence of        30. Dairy Attenuation of Oxidative      38. Enhanced Nerve Cell Attachment,
          Water-Soluble Chitosan Against         and Inflammatory Stress in Metabolic    Proliferation, and Differentiation
          Foodborne Viral Surrogates at 37ºC     Syndrome (Stancliffe)                   on Hydrogels Grafted with Photo-
          and Room Temperature (Ganapathy)                                               polymerizable Poly-L-Lysine (Cai)
3:45




          24. Antiviral Effects of Chitosan as   31. Microarray Profiling of             39. Predictive Ophthalmic Simulation
          Affected by its Molecular Weight and   Chicken Adipose Tissue under            and Applications (Y.-L. Chen)
          Degree of Acetylation (Davis)          Insulin Neutralization and Fasting
                                                 Conditions (Ji)
4:00




          25. Combination of Lecithin and        32. Mechanisms Linking                  40. Design of an Automatic Cover-
          High Pressure Homogenization for       Overproduction of Angiotensinogen       Uncover Test for Diagnosing
          the Inactivation of Human Norovirus    by Adipose Tissue to Inflammation,      Strabismus and Ocular Alignment
          Surrogates in Milk and Orange Juice    Glucose Intolerance and Insulin         Impairments (Shi)
4:15




          (Horm)                                 Resistance (Kalupahana)
          -                                      33. Eicosapentaenoic Acid (EPA)         41. Injectable Blends Consisting
                                                 Regulates Hypertensive, Inflammatory    of Poly(propylene fumerate) and
                                                 and Adipogenic Mediators in             Methacryl-Polyhedral Oligomeric
                                                 Mouse and Human Adipocytes              Silsesquioxane for Regulating Material
                                                 (Siriwardhana)                          Properties and Bone Cell Behavior
4:30




                                                                                         (Wang)




                                                             7
                                                                                              Tuesday, June 15
                                                                                            Oncology & Cancer Cell
             Clinical Sciences                        Nutrition & Metabolism
                                                                                            Biology
             Rm. PBB 156/157                          Rm. PBB 160                           Rm. PBB 113
             42. An Acute Reversible Experimental     49. Regional Differences in Adipose   56. Anticancer Activity of
             Model of Pneumonia in Pigs: Time-        Tissue in the Sinclair Minipig        Damnacanthal from Noni (Morinda
10:15 am




             related Histological and Clinical Sign   (Booker)                              citrifolia) in Colorectal Cancer Cells
             Changes (Villarino)                                                            (Nualsanit)

             43. Evaluation of Tyrosinase             50. Transient Silencing of Atp10c     57. Early Growth Response-1 and
             Expression in Canine and Equine          mRNA in C2C12 Myotubes                Krüppel-Like Factor 4 Regulate
             Melanocytic Tumors (Lembcke)             Decreases Glucose Uptake              Resveratrol-induced Activating
                                                      Potentially via MAPK Signaling        Transcription Factor 3 Expression
10:30




                                                      Pathway (Hurst)                       (Whitlock)
             44. Effects of Season on                 51. Anti-inflammatory and             58. Targets for Dietary Grape Seed
             Adrenocorticotropin Hormone              Metabolic Mechanisms by which         Extract-Induced Suppression of
             (ACTH) Concentrations in Horses          Omega-3 Fatty Acids Improve Insulin   Breast Cell Chronic Carcinogenesis
             with Pituitary Pars Intermedia           Resistance in High Fat Diet-induced   (Rathore)
             Dysfunction (PPID) from 15               Obese Mice (Kalupahana)
             Different Locations in East
10:45




             Tennessee (Elliott)
             45. Novel Endocrine Disruptor in         52. Effects of Bisphenol A on         59. Medicinal Herbs Inhibit
             Personal Care Products (J. Chen)         Adipocyte Growth, Metabolism, and     Prostate Cancer (PCa) Through p 21
                                                      Endocrine Functions (Wyatt)           via Epigenetic Regulation (Huang)
11:00




             46. Effects of Metformin                 53. Leucine Stimulation of Fatty      60. Capsaicin Inhibits β-catenin
             Hydrochloride on Glucose                 Acid Oxidation via an mTOR-           Pathway in Colorectal Cancer
             Dynamics During Transition to            independent Mechanism (Smart)         (Richardson)
             Grass Paddocks in Insulin-Resistant
11:15




             Horses (Chameroy)
             47. Assessment of Two Experimental       54. Quercetin Overcomes               61. Sulindac Sulfide Suppresses
             Models for Prolonged (48 Hours)          Rosiglitazone-associated Adiposity    Epithelial Cell Adhesion Molecule
             Endotoxemia in Horses (Tadros)           and Adiposity- associated             (EpCAM) Expression in Colorectal
                                                      Inflammation (Siriwardhana)           Cancer Cells (Liggett)
11:30




             48. Tear, Cornea, and Aqueous            55. Enhancing Male Reproductive       62. Anti-cancer Activity of
             Humor Concentrations of                  Potential by Herbal Tea Therapy       Cyclooxygenase Inhibitors in
             Ciprofloxacin and Moxifloxacin after     (Gomes)                               Colorectal Cancer (Lee)
             Topical Application in Normal Horses
Noon 11:45




             (Westermeyer)

             BREAK for Lunch--See schedule on p. 4




                                                                        8
Tuesday, June 15
          Clinical Sciences                                             Oncology & Cancer Cell Biology
          Rm. PBB 156/157                                               Rm. PBB 113
          63. Magnetic Resonance Imaging of the                         75. Role of Reactive Oxygen Species (ROS) in
          Temporomandibular Joint in Normal Dogs (Macready)
1:30 pm




                                                                        Histone Deacetylase Inhibitor (HDACI)-mediated
                                                                        Selective Apoptosis of Oncogenic H-Ras-expressing
                                                                        Cells (Choudhary)
          64. Effect of Hyperbaric Oxygen Treatment on Canine           76. Therapeutic Peptides Block the Immunosuppression
          Mesenchymal Stem Cells (Cekanova)                             of Fibrin and its Effects on B16 Melanoma and
1:45




                                                                        Metastases in vivo (McCartt)
          65. Equine Progenitor Cells – Potential Role in Basic         77. Alternative Splicing of Krüppel-like Factor 4 Plays a
          and Translation Research (Dhar)                               Role in Colorectal Tumorigenesis (Bahn)
2:00




          66. Quantitative Analysis of Inflammatory Responses           78. Modeling the Effect of Melanoma Tumor Cell
          from Extracorporeal Membrane Oxygenation Device               Growth in the Presence of Natural Killer Cells (A.
2:15




          Implantation in an Animal Model (P. Brown)                    Wells)
          67. Effect of Order Set Utilization on Outcomes in Septic     79. Safety and Efficacy of a Novel Method of
          Shock in the Intensive Care Unit (Malcom)                     Localized Hyperthermia Delivery in Conjunction with
                                                                        Chemotherapy for the Treatment of Non-resectable
2:30




                                                                        Canine and Feline Sarcomas: A Pilot Study (Smrkovski)
          68. Labor Epidural Fever and Pyrogenic Cytokines              80. Expression of KIT and PDGFR in Canine Anal Sac
          (Erickson)                                                    Adenocarcinoma Using Tissue Immunohistochemistry
2:45




                                                                        (R. Brown)
          69. Air vs Saline Loss of Resistance for Epidurals–CA1        81. Serial 18FDG-PET/CT Can Assess Response to
          Residents During Primary Obstetrical Rotation (Owens)         Receptor Tyrosine Kinase Inhibition in Canine Cancer
3:00




                                                                        (LeBlanc)

          BREAK                                                         Bovine Diseases & Theriogenology
3:15




          70. Ultrasonographic Observation of Secretin-induced          82. Genetic Approach to Improve Fertility in Cattle
          Pancreatic Duct Dilation in Healthy Cats (Baron)              (Di Croce)
3:30




          71. A Description of Motion of the Canine Pelvic Limb         83. Effect of Reproductive Tract Size on Conception
          in 3 Planes Using a New Inverse Dynamics Model                Rates in Lactating Dairy Cows Utilizing a
3:45




          (Headrick)                                                    Reproductive Tract Scoring System (Young)
          72. Evaluation of Veterinary Nitinol Stents, Vet Stent-       84. Real-Time Polymerase Chain Reaction for
          Trachea, for the Palliation of Tracheal Collapse in Dogs      Simultaneous Detection of Mastitis Pathogens Directly
4:00




          (Durant)                                                      from Milk (Gillespie)
          73. From Cell Culture to Living Mice–The Use of               85. Characterization of the Immune Response Following
          Mammalian-adapted Bacterial Luciferase as a Target for        Vaccination of Dairy Cows with Recombinant
          Bioluminescent Imaging (Close)                                Streptococcus uberis Adhesion Molecule (rSUAM)
4:15




                                                                        (Almeida)
          74. Biodistribution of a Novel Radiolabeled Fibril-reactive   86. pGhost9:ISS1 Transpositional Mutations in
          Monoclonal Antibody In Patients with Systemic Light           Streptococcus uberis UT888 Causes Reduced Bacterial
          Chain (AL) Amyloidosis (K. Wells)                             Adherence to and Internalization into Bovine Mammary
4:30




                                                                        Epithelial Cells (Kerro Dego)




                                                                9
Abstracts
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              ‫تعاون‬
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                1. Organizational Cultural Competence of                      consume fruits and vegetables. The after-school program
Public Health   Post-Secondary Health-Related Academic                        was implemented in collaboration with University of
                                                                              Tennessee Extension and an urban demonstration farm,
                Units                                                         Beardsley Community Farm. An intervention school was
                                                                              chosen for the combined after-school garden program and
                Laura Dotson                                                  youth-based participatory action research intervention,
                Department of Nutrition, Public Health Program                and a comparison school was chosen for the after-school
                                                                              garden program. Data were collected at 2 times (pre-post)
                According to the US Office of Minority Health and             and included anthropometric data as well as survey mea-
                Health Disparities, one way to eliminate health disparities   sures of fruit and vegetable consumption, preferences for
                is by providing educational opportunities in cultural         fruits and vegetables, self-efficacy to consume fruits and
                competence to health care professionals. Quality health       vegetables, self-efficacy to garden and school connected-
                care professionals graduate from accredited universities      ness. Preliminary data showed that 72% (8/11) students in
                or colleges. Therefore, academic units have a unique          the program had a BMI above the 95th percentile. Prelimi-
                opportunity to provide cross-cultural training. Academic      nary results from a food frequency questionnaire showed
                units can institute organizational cultural competence in     that 53% of students ate vegetables one or fewer times
                terms of policies, programs, and activities. Organizational   and 61% ate fruit one or fewer times. The results of this
                cultural competence in a post-secondary, health-related       pilot project are important to the development of school
                academic unit refers to the teaching, research, service,      gardens that are as effective in changing students’ dietary
                outreach, administrative and personnel policies, and          behaviors as they are in changing their preferences. The
                practices that provide students the cultural competencies     areas of youth development and youth-based participa-
                needed to work in health care delivery systems. This study    tory action research can inform and improve future school
                was designed to test the Krause model’s domains and           garden interventions.
                criteria statements for organizational cultural competence
                of post-secondary, health-related academic units. The
                Krause model was developed by an expert panel who             3. Investigation of Geographic Access
                provided input on domains and criteria statements that        to Emergency Stroke and Heart Attack
                are important and relevant for academia. To test this         Care in Middle Tennessee
                model, administrators and tenured/tenure-track faculty
                of post-secondary, health-related academic units from         Doreen Busingye, Agricola Odoi
                family medicine, nursing, psychology, public health, and      Department of Comparative Medicine (Busingye, Odoi),
                nutrition will rate the essentialness of the Krause model’s   Comparative and Experimental Medicine Program (Busingye)
                criteria statements through an online survey. Principle
                component analysis with varimax rotation will be              Heart disease and stroke are, respectively, the first
                performed to identify factors with highly correlated items.   and third leading causes of death in Tennessee and the
                Factor loadings above 0.40 will be considered. Final          United States. These conditions require time-sensitive
                factors will be reviewed to determine their respective        treatments, and therefore timely access to care is critical
                common themes.                                                to minimize complications and death. The objective of
                                                                              this study was to investigate the timeliness of access to
                                                                              emergency care for stroke and heart attack patients in
                2. Youth Can! Grow Healthy!                                   middle Tennessee. Street network and hospital data were
                                                                              obtained from Streetmap USA and the Joint Commission
                                                                              on Accreditation of Health Organizations, respectively,
                Andrew Carberry                                               and used in network analysis. Network Analysis,
                Department of Nutrition
                                                                              implemented in ArcGIS, was used to compute travel time
                                                                              from different neighborhoods to the nearest emergency
                This pilot project was conducted to test the effectiveness
                                                                              room (ER), cardiac and stroke centers. Neighborhoods
                of an after-school, school garden program combined with
                                                                              within 30, 60 and 90 minutes driving time to the nearest
                a youth development curriculum involving participa-
                                                                              ER, cardiac and stroke centers were identified. Population
                tory action research in improving the fruit and vegetable
                                                                              data were obtained from the 2000 census and used to
                consumption of 4th and 5th graders. The main outcome
                                                                              estimate the percentage of population with/without
                of interest was fruit and vegetable consumption, which
                                                                              access. Most (96.2%) of the population had access to an
                was hypothesized to increase along with self-efficacy to




                                                                     14
ER within 30 minutes. Additionally, 73%, 26% and 1%           5. A Novel Processing Approach for the
of the population had access to a cardiac center within       Inactivation of Salmonella enteritidis




                                                                                                                            Public Health
30 minutes, 30–60 minutes, 0–90 minutes, respectively.
In contrast, only 42.6% of the population had access          in vitro with Potential Applications for
to stroke centers within 30 minutes, and 9.2% had no          Liquid Whole Eggs
access within 90 minutes. Most of the population lacking
access were in the rural north. The identified geographic     Chayapa Techathuvanan, Doris Helen D’Souza
disparities is vital for guiding planning and policy          Department of Food Science and Technology
decisions aimed at reducing/eliminating health disparities
at the neighborhood level with the view to improving          High intensity ultrasound (HIU) is a non-thermal
health of the entire population.                              inactivation technology that is attractive to the food
                                                              industry. HIU can inactivate microbes and yet maintain
                                                              the nutritional value, sensory quality, and freshness of
4. From 911 to ER: Analysis of                                foods. Research is ongoing to determine the feasibility
                                                              of this technology for food applications. Frequent
Ambulance Transport of Stroke and                             outbreaks of salmonellosis are linked to liquid whole egg
Heart Attack Patients                                         (LWE) products associated with Salmonella enteritidis
                                                              contamination. These outbreaks result in significant
Ashley S. Pedigo, Lisa Escalante, Tim Aldrich, Agricola       socio-economic impacts on industry and public health.
Odoi                                                          This study aimed to investigate the effect of HIU on
Comparative and Experimental Medicine Program (Pedigo),       S. enteritidis inactivation in pure culture and in LWE.
Physician Assistant Studies, South College (Escalante),
                                                              Overnight S. enteritidis and LWE spiked with S.
Department of Epidemiology, East Tennessee State University
(Aldrich), Department of Comparative Medicine (Odoi)
                                                              enteritidis were treated with 20 kHz of HIU for 0, 1, 5,
                                                              10 and 30 minutes in a temperature-controlled system
Stroke and heart attack are serious health conditions         at < 20°C. Samples were enumerated by spread plating
with specific time-sensitive treatments. Patients who         on XLT4 agar at each time point. S. enteritidis was
use Emergency Medical Services (EMS) transport have           significantly reduced by 2.2 and 2.3 log CFU/ml after
higher odds of receiving treatments within the medically      30-minutes HIU treatment in pure culture and LWE,
recommended times. Our goal was to investigate the use        respectively (p<0.05). After 5 and 10-minute treatment,
of EMS in East Tennessee (TN) and describe ambulance          S. enteritidis reductions of 1.0 and 1.3 log CFU/ml in
transport time for stroke and heart attack patients. Paper    pure culture, and 1.4 log CFU/ml in LWE for both times,
or electronic records of suspected stroke and heart attack    respectively, were obtained (p<0.05). Even at 1 minute,
911 calls and ambulance dispatches made from 2006 to          HIU showed inactivation of 0.6 log CFU/ml in pure
2009 were obtained from EMS in East TN. Confidential          culture (p<0.05); however, no reduction was observed
patient data were removed from the records before             in LWE. HIU shows promise for Salmonella control in
being released to the investigators. Data manipulations       LWE and potentially for other liquid food products as an
and analyses were conducted in SAS 9.2. Descriptive           alternative pasteurization method in hurdle approaches.
statistical analyses were performed to investigate use of
EMS, patient-related information (e.g. dispatch reasons
and choice of hospital), as well as ambulance travel time     6. Second Cut of the Rough: Revisiting the
to the patient location, time spent on scene, and travel      Site of an Ehrlichiosis Outbreak in Middle
time to the hospital. Preliminary results show that the
travel times to patients were relatively short while the
                                                              Tennessee
time from scene to the hospital tended to be longer, likely
due to the distribution of ambulances throughout the          Jessica Harmon, Cathy Scott, Graham Hickling, Carl
community. The time spent on scene had more variability       Jones
than other time components. The majority of heart attack      Entomology and Plant Pathology (Harmon, Jones), Center for
                                                              Wildlife Health (Harmon, Scott, Hickling)
patients were transported to a cardiac specialty hospital.
In contrast, fewer stroke patients were transported to a
                                                              In 1993, 4 residents of a retirement community in a forest-
stroke specialty hospital. In conclusion, EMS provides
                                                              ed area of middle Tennessee were hospitalized with symp-
rapid emergency care and has a critical role in improving
                                                              toms of ehrlichiosis. This case cluster triggered a Centers
outcomes for stroke and heart attack patients in East TN.




                                                                15
                for Disease Control outbreak investigation, reported by        subpopulations based on geography to improve the cost
Public Health   Standaert and Dawson (1995). Anecdotal reports indicate        effectiveness of disease control. Results were pending at
                that residents of the outbreak community have perceived        the time of publication.
                resurgence in tick-related infections in recent years. The
                motivation for this study was to revisit the retirement
                community 15 years later and assess the risk that ticks and    8. Prevention of Pancreatic Cancer by
                their associated pathogens may still pose for residents. In
                a pilot assay of 16 adult Amblyomma americanum ticks,
                                                                               Inhibition of Beta-adrenergic Signaling
                we detected 1 tick positive for Ehrlichia chaffeensis (6%)
                and 3 ticks positive for E. ewingii (19%). The observed        Hussein A. Al-Wadei, Mohammed H. Al-Wadei,
                rate of infection is well over the established 2–5% infec-     Hildegard M. Schuller
                tion rate for an endemic area, suggesting that this com-       Department of Pathobiology
                munity represents a hot spot for Ehrlichia infection.
                Additionally, the detection of E. ewingii in sampled ticks     Pancreatic ductal adenocarcinoma (PDAC) is among the
                indicates that serum test results of community residents       leading causes of cancer deaths and is unresponsive to ex-
                need to be interpreted with caution, as E. chaffeensis and     isting therapy. Smoking and alcohol-induced pancreatitis
                E. ewingii antibodies are likely cross-reactive. We specu-     are among the risk factors for PDAC. We have previously
                late that other pathogens could also be contributing to        reported that beta-adrenergic receptors (β-ARs) stimulate
                tick-associated illness in this community. For example,        the proliferation and migration of human PDAC cells in
                Rickettsia amblyommii was found in a high proportion of        vitro via cAMP-dependent signaling and that the nicotine-
                ticks, but its potential for pathogenicity is unclear.         derived nitrosamine NNK activates this pathway directly
                                                                               in vitro while additionally stimulating the release of
                                                                               noradrenaline/adrenaline by binding to α7 nicotinic ace-
                                                                               tylcholine receptors in hamsters. In the current study, we
                7. Application of Spatial Analysis, at Three                   tested the hypothesis that the β-AR antagonist propranolol
                Geographic Scales, to Detect Clustering                        prevents the development of PDAC induced in hamsters
                of Humans with Campylobacteriosis                              with ethanol-induced pancreatitis by NNK. Western blots
                                                                               of pancreatic duct cells and PDAC cells harvested by laser
                Reported to the Tennessee Department of
                                                                               capture microscopy showed significant upregulation of the
                Health                                                         α7nicotinic acetylcholine receptor (α7nAChR) associated
                                                                               with significant inductions of p-CREB, p-ERK1/2 and
                Jennifer Weisent, Agricola Odoi, Barton Rohrbach               increases in EGF and VEGF in PDAC cells of hamsters
                Comparative and Experimental Medicine Program (Weisent),       not treated with propranolol. These effects were reversed
                Department of Comparative Medicine (Odoi, Rohrbach)            by treatment with propranolol. Our data suggest that
                                                                               propranolol may prevent the development of PDAC by
                                                                               blocking cAMP-dependent intracellular signaling, cAMP-
                Campylobacteriosis is the leading cause of bacterial           dependent release of EGF and PKA-dependent release of
                gastroenteritis in the United States and many other de-        VEGF while additionally downregulating α7nAChR via
                veloped countries. The objective of this study is to use       inhibition of cAMP-mediated subunit assembly. Increased
                Geographic Information Systems (GIS) to investigate the        cAMP signaling is an important factor that drives the
                spatial distribution of Campylobacter infection in the state   development and progression of PDAC, and the inhibition
                of Tennessee at 3 levels: county subdivision, zipcode and      of cAMP formation is a promising new target for the pre-
                census tract. The study aims to test for the existence of      vention and adjuvant therapy of PDAC. Our current study
                disease clusters and determine whether differences exist in    shows that γ-aminobutyric acid GABA also blocks the
                the risk of Campylobacter infection between geographical       signaling of NNK by inhibiting the activation of adenylyl
                levels. Population data were obtained from the U.S. Cen-       cyclase. On the other hand, GABA production by these
                sus Bureau, and distribution of Campylobacter cases was        cells was reduced by a single dose of NNK via desensi-
                supplied by the Tennessee Department of Health. Disease        tization of α4β2 nAChR. Supported by R01 CA042829,
                data were aggregated at 3 levels and stratified by age and     National Cancer Institute.
                gender. Statistical techniques for spatial clustering were
                then applied for the analysis. Study results are intended to
                provide information that will enable public health person-
                nel to focus disease prevention and control strategies on




                                                                      16
9. Formation of Nicastrin and Aph1                              10. Ponazuril Pharmacokinetics in Healthy
Subcomplex is Necessary for Recruitment                         Llamas (Lama glama) Following Single
of Amyloid Precursor Protein but Not for                        Oral Administration
Interaction with Presenilin
                                                                Maria E. Prado, Josiah T. Ryman, Tomas Martin-
Guozhang Mao, Mei-Zhen Cui, Tong Li, Bart De                    Jimenez, Bernd Meibohm
Strooper, Huaxi Xu, Xuemin Xu                                   Department of Large Animal Clinical Sciences (Prado), Depart-
                                                                ment of Comparative Medicine (Martin-Jimenez), College of




                                                                                                                                Pharmacology & Pharmacokinetics
Department of Pathobiology (Mao, Cui, X Xu), Johns Hop-
kins University, Baltimore, MD (Li), Katholieke Universiteit    Pharmacy (Ryman, Meibohm)
Leuven, Leuven, Belgium (De Strooper), Sanford-Burnham
Institute for Medical Research, La Jolla, CA (H Xu)              Eimeria macusaniensis is a coccidian parasite that
                                                                causes severe diarrhea in New World camelids (NWCs).
Accumulating evidence supports the hypothesis that              Currently, there are no approved drugs for treating
the abnormal accumulation of β-amyloid peptide (Aβ)             infected NWCs against E. macusaniensis. Thus treatments
in the brain is a causative event in the development of         are empirical and extrapolated from other species.
Alzheimer’s disease. Amyloid precursor protein (APP)            Ponazuril is a triazine coccidial drug related to toltrazuril
is first cleaved by β-secretase to produce a membrane-          that has been shown to be effective against different
associated C-terminal fragment, CTFβ. CTFβ is                   members of the phylum apicomplexa. The purpose of
subsequently cleaved within its transmembrane domain            this study was to determine the pharmacokinetics of oral
by γ-secretase to produce the full-length Aβ. Thus,             ponazuril in healthy llamas. Ponazuril was administered
γ-secretase-mediated processing of APP is a crucial step in     once orally to 6 llamas (20 mg/kg of body weight), and
the formation of Aβ. In the present study, using knockout       serum was collected on days 0, 0.5, 1, 2, 3, 4, 5, 6, 7,
cell lines in which some components of the γ-secretase          9, 11, 14, 21, 28, 35, 42 and 49. Serum samples were
complex have been eliminated, we determined some                analyzed by a validated reversed phase high performance
aspects of how the multisubunit γ-secretase complex             liquid chromatography (HPLC) assay with UV absorbance
is assembled and how it interacts with APP. Our data            detection. Ponazuril was readily absorbed by healthy
clearly show that, in the absence of Aph1α, a major             llamas following a single oral dose. The mean elimination
component of the complex, nicastrin (NCT), another              half-life, Tmax, Cmax and area under the curve for
major component of the γ-secretase complex, can be co-          Ponazuril were 135±16.7 hours, 84±25.2 hours, 23.6±6.03
immunoprecipitated with presenilin, the catalytic subunit       mg/L and 7515±2750 hr*mg/L, respectively. Plasma
of the γ-secretase complex, but, under the same condition,      concentrations of ponazuril peaked at 23.60±6.0 µg/mL at
the NCT cannot be co-immunoprecipitated with CTFβ;              84 (range 48–120) hours post-administration. Thereafter,
vice versa, in the absence of NCT, APH1aL can also be           plasma concentrations gradually decreased but remained
co-immunoprecipitated with presenilin, but not CTFβ.            detectable up to 35 days post-administration. In summary,
Furthermore, in the presenilin-knockout cells, NCT and          results from this study showed that oral administration of
APH1aL form a subcomplex that binds CTFβ with high              ponazuril to healthy llamas at 20 mg/kg resulted in plasma
affinity. These results indicate that presenilin can interact   concentrations that were high enough to be effective
with NCT without the involvement of Aph1α, and vice             against different apicomplexans based on data for other
versa, presenilin can interact with Aph1α without the           species The effective minimal inhibitory concentration
involvement of NCT. However, the presence of both               of ponazuril for E. macusaniensis in NWCs is currently
NCT and Aph1α are required for their interaction with the       unknown.
substrate CTFβ. These results provide new insight into
the molecular mechanism of γ-secretase-mediated APP
processing.




                                                                  17
                                  11. The Effect of Ketamine on the                               MIC ratio ≥ 400 to achieve clinical effectiveness with
                                  MACBAR of Sevoflurane in Dogs                                   vancomycin. To our knowledge, no study has compared
                                                                                                  intermittent (IIV) to continuous (CIV) infusion
                                                                                                  vancomycin with regards to attainment of target AUC24/
                                  Lydia Love, Christine Egger, Barton Rohrbach, Sherry            MIC ratio, and as such we designed this study. Most
                                  Cox, Meredith Hobbs, Thomas Doherty                             literature describes weight-based dosing of CIV; however,
                                  Department of Large Animal Clinical Sciences (Love, Doherty),
                                                                                                  vancomycin peak and trough levels are commonly
                                  Department of Small Animal Clinical Sciences (Egger),
                                  Department of Comparative Medicine (Cox, Rohrbach), College
                                                                                                  available at our institution prior to conversion from IIV
                                  of Veterinary Medicine (Hobbs)                                  to CIV. Therefore, we also sought to evaluate whether a
Pharmacology & Pharmacokinetics




                                                                                                  pharmacokinetic-based or weight-based approach would
                                  Our objective was to determine the effect of intravenous        more reliably predict CIV dose requirement. IRB approval
                                  ketamine on the minimum alveolar concentration                  was obtained before commencement of this retrospective
                                  of sevoflurane needed to block autonomic response               analysis. Adults ≥ 18 years old were eligible for inclusion
                                  (MACBAR) to a noxious stimulus in dogs. Six adult dogs          if they received at least 3 doses of IIV followed by
                                  (11.2–16.1 kg) were anesthetized twice with sevoflurane         conversion to CIV for at least 24 hours. The primary
                                  1 week apart, and baseline MACBAR (B-MACBAR)                    outcome was a comparison of AUC24/MIC ratio before
                                  was determined. MACBAR was defined as the mean of               and after conversion from IIV to CIV in patients with
                                  the end-tidal sevoflurane concentrations that prevented         a documented gram-positive pathogen. As a secondary
                                  and allowed an increase (≥15%) in heart rate or invasive        analysis, patient-specific pharmacokinetic values (Ke, Vd,
                                  mean arterial pressure in response to a noxious electrical      CLvanc) were calculated in those with evaluable peak
                                  stimulus. Dogs were then randomly given either a low-           and trough levels while receiving IIV. Several mg/kg/
                                  dose (LDS) or high-dose series (HDS) of ketamine, and           day doses were then compared to pharmacokinetic-based
                                  treatment MACBAR (T-MACBAR) was determined. The                 dosing to determine which method more reliably predicted
                                  LDS included a loading dose (LD) of 0.5 mg kg-1 and             initial plateau level and CIV dose requirement. Results
                                  constant rate infusion (CRI) at 6.25 µg kg-1 minute-1,          and conclusions were pending at the time of printing.
                                  followed by a second LD (1 mg kg-1) and CRI (12.5 µg
                                  kg-1 minute-1). The HDS had an initial LD (2 mg kg-1)
                                  and CRI (25 µg kg-1 minute-1) followed by a second LD           13. Interscalene Anesthesia for
                                  (3 mg kg-1) and CRI (50 µg kg-1 minute-1). Data were            Arthroscopic Shoulder Surgery: Failures
                                  analyzed with a mixed-model ANOVA and are presented
                                  as LSM ± SEM. The B-MACBAR was not significantly                and Complications
                                  different between treatments. Ketamine at 12.5, 25, and
                                  50 µg kg-1 minute-1 decreased sevoflurane MACBAR.               Jonathan S DiRuzzo, Daniel R Bustamante, Roger C
                                  The percentage change in MACBAR was not correlated              Carroll, Carolyn G Snyder, Jerry Epps
                                  with either the log plasma ketamine or norketamine              Department of Anesthesiology, UT Medical Center
                                  concentration. Ketamine at clinically relevant doses of
                                  12.5, 25, and 50 µg kg-1 minute-1 decreased sevoflurane         The purpose of this study is to determine the failure and
                                  MACBAR, although the reduction was neither dose-                complication rates for this anesthetic technique at our in-
                                  dependent nor linear.                                           stitution. After IRB approval, we retrospectively reviewed
                                                                                                  100 consecutive patients of a single surgeon for whom ar-
                                                                                                  throscopic shoulder surgery under interscalene block was
                                                                                                  planned. Records from anesthesia, postanesthesia recov-
                                  12. A Comparison of AUC24/MIC Pre- and                          ery, and surgical follow-up were reviewed; 9 interscalene
                                  Post-conversion from Intermittent Infusion                      blocks were judged failures. One conversion to general
                                  Vancomycin to Continuous Infusion                               anesthesia (GA) was judged not to be a block failure as
                                  Vancomycin                                                      the conversion GA occurred at the request of the surgeon
                                                                                                  because of a change in surgical plans. Of the 9 cases
                                                                                                  judged to be failures, 6 were converted to GA after the
                                  Christopher F. Burke, Barbara E. Faircloth, Edwin H.            interscalene block was placed, 2 had repeat blocks (one
                                  Varnadoe                                                        preoperatively and one immediately postoperatively), and
                                  Department of Pharmacy, UT Medical Center
                                                                                                  in 1 case the block was aborted when satisfactory nerve
                                                                                                  stimulation was not obtained. The primary limitation of
                                  Recently published guidelines recommend an AUC24/




                                                                                         18
this retrospective review revolves around the definition of   of about every 5 hours. Pharmacodynamic studies
a failed block and by implication the definition of a suc-    investigating beta-adrenergic blockade duration may
cessful block. As we had no documentation of a detailed       provide a more accurate dosing interval of carvedilol.
evaluation of the blocks based upon specific sensory and      Also, further studies are needed to assess steady-state and
motor examination, we used the clinical end points of         population.
conversion to GA, performance of a repeat block or any
notation that a block was inadequate to define a failure.
While these endpoints are clinically relevant, a rigor-       15. Genomic Sequencing of Methicillin-
ous motor and sensory examination would be preferable.
This retrospective review of 100 consecutive interscalene     resistant Staphylococcus pseudintermedius
blocks for arthroscopic shoulder surgery, revealed a 9%       from Canine Pyoderma
failure rate, a 4% rate of postoperative complications and
1 immediate complication, systemic local anesthetic toxic-    Laura Eberlein
ity.                                                          Department of Comparative Medicine

                                                              Staphylococcus pseudintermedius is the most common
14. Pharmacokinetics and Bioavailability                      bacterium associated with canine bacterial skin infections.
                                                              Antibiotic resistance in this organism is a growing con-
of Carvedilol in Cats                                         cern in veterinary medicine. For example, the prevalence
                                                              of methicillin-resistant S. pseudintermedius (MRSP) has
April Durtschi, Tomas Martin-Jimenez, Sherry Cox,             risen to 30% at the University of Tennessee College of
Rebecca Gompf                                                 Veterinary Medicine, and resistance to other antibiotics
Department of Small Animal Clinical Sciences (Durtchi,        also follows an upward trend. Because of this resistance
Gompf, Bartges), Department of Comparative Medicine
                                                              problem, many antibiotics are now ineffective against S.
(Martin-Jimenez, Cox)
                                                              pseudintermedius infections. Factors such as the overuse
Carvedilol, a 3rd-generation non-selective, beta-blocker      of antibiotics and the acquisition of resistance genes have
with ancillary alpha1-blocking and antioxidant may            facilitated the spread of MRSP. It has also been sug-
have therapeutic implications for multiple diseases in        gested that using certain combinations of drugs together
cats; however, pharmacokinetics and bioavailability           may increase the likelihood of resistance. Whole genome
of commercially prepared oral carvedilol has not been         sequencing of S. pseudintermedius can provide invaluable




                                                                                                                             Bacterial Virulence & Transmission
determined. HPLC for carvedilol measurement in feline         information in regards to antibiotic resistance mechanisms
plasma was validated and standardization curves created.      and the relationship between resistance genes. In this
The pharmacokinetics (PK) of carvedilol was evaluated         project, sequence information for S. pseudintermedius was
in 5 apparently healthy male neutered adult cats (average     determined using Solexa and 454 DNA sequencing tech-
weight of 5 kg) following single dose intravenous (IV)        nology. Contig assembly software (Lasergene) was used
of 0.5 mg/kg and single dose oral administration of 1.6       to assemble thousands of sequence fragments containing
mg/kg. Concentrations of the active parent compound,          the millions of bases that make up the bacterial chromo-
carvedilol, were detected in plasma using HPLC analysis.      some. Large portions of the genome are currently assem-
Lower limit of quantification was 5 ng/ml.                    bled, but additional sequencing is required to complete the
The mean peak concentration after IV administration           project. Upon preliminary annotation, genes responsible
of carvedilol was 8639 ng/mL (range, 901–8648),               for resistance to tetracycline, beta lactams, flouroquino-
elimination half- life was 2.9 hours (range, 2.0–5.4), and    lones, bacitracin, and other antibiotics have been found.
clearance was 0.35 L/hr/kg. The volume of distribution        Investigation of these genes in relation to the complete ge-
was 1.18 L/hr. After a single oral administration of          nome may shed light on resistance mechanisms and help
carvedilol, the time to peak plasma concentration was 60      combat the problem of antibiotic resistance in dogs.
minutes (range, 30–90 minutes) and the mean residual
time was 4.8 hours. The half life was 4.44 hours. Maximal
concentration 294 ng/mL, and the mean bioavailability
was 15.7% with a median of 9.97% (range, 4.7% to 46%).
These data demonstrate a low bioavailability of oral
carvedilol and a wide variation in cats. Also, a mean
residual time of 4.8 hours suggests a dosing interval




                                                                19
                                     16. Variation of mecA Expression Among                        dates to increase the number of genes used in the current
                                     Regional Clones of Methicillin-resistant                      multilocus sequence type scheme from 4 genes to 7 genes.
                                                                                                   S. pseudintermedius is the most common cause of canine
                                     Staphylococcus pseudintermedius                               pyoderma and occasionally causes zoonotic infections of
                                     and Characterization of mec and bla                           humans. Twenty different sequence types were identified
                                     Regulatory Elements                                           among the regional S. pseudintermedius isolates examined
                                                                                                   using the current scheme, and methicillin-resistant isolates
                                                                                                   were predominantly sequence type 68, which suggests it
                                     Chad Black, Laura Eberlein, Samar Solyman, David
                                                                                                   is a regional clone originated from a single source and is
                                     Bemis, Stephen Kania
                                     Department of Comparative Medicine
                                                                                                   different from clones in the other regions. Increasing the
                                                                                                   number of genes in the current scheme will increase the
                                     Regional clones of methicillin-resistant Staphylococcus       discrimination power between genetically-related strains
                                     pseudintermedius (MRSP) affecting veterinary clinical         and will provide an insight into the epidemiology of the
                                     practice have recently been identified in Europe and North    species.
                                     America–multilocus sequence types (MLST) 71 and 68,
                                     respectively. One notable difference between the two
                                     clones is a deletion in the mec regulatory elements of ST     18. Gene Expression Profile of Bovine
                                     68. This deletion in analogous methicillin-resistant Staph-   Primary Mammary Epithelial Cells
                                     ylococcus aureus (MRSA) results in more responsive and
                                     greater expression of the mecA encoded penicillin-binding
                                                                                                   Infected with Escherichia coli Associated
                                     protein 2a, a characteristic associated with community-       with Acute or Chronic Bovine Mastitis
                                     acquired MRSA lineages. The aim of this study was to
                                     characterize mec and bla regulatory apparatuses in MRSP.      Oudessa Kerro Dego, Raul Almeida, Douglas Luther,
                                     Twenty wild-type S. pseudintermedius isolates repre-          Stephen Oliver
                                     senting the 7 known methicillin-resistant lineages were       Department of Animal Science
                                     screened via PCR for the presence of the repressors blaI
                                     and mecI and sensors blaR1 and mecR1. The bla and mec         Acute Escherichia coli mastitis is associated with rapid
                                     operons for each isolate were sequenced and compared          onset of clinical signs including mammary gland swell-
                                     for homology between the repressor open-reading frames        ing and abnormal milk with rapid elimination of bacteria
                                     (ORF), sensor ORFs, and mecA promoter regions. A              from milk. Some strains of E. coli are associated with
Bacterial Virulence & Transmission




                                     real-time reverse transcriptase PCR expression assay was      chronic mastitis, which often begins with mild clinical
                                     developed, validated and applied to 8 isolates analyzing      symptoms that disappear soon after onset of infection
                                     the effect of oxacillin induction on mecA transcript pro-     only to flare-up again during lactation, usually resulting
                                     duction. Comparisons of mecA expression induction and         in mild clinical mastitis. Recently published data suggests
                                     volume are made between isolates with a full regulatory       that adhesion to and internalization into bovine mam-
                                     complement (mecI/mecR1 and blaI/blaR1) and those with         mary epithelial cells (BMEC) and subsequent intracellular
                                     truncated and/or absent regulatory elements.                  survival might be important virulence attributes of strains
                                                                                                   of E. coli associated with chronic mastitis. The aim of this
                                                                                                   study was to compare gene expression profiles of BMEC
                                     17. The Selection of New Gene                                 co-cultured with acute and chronic strains of E. coli, and
                                                                                                   to delineate expression profiles of virulence genes in these
                                     Candidates for the Improvement                                strains before and after co-culture with BMEC to identify
                                     of the Current MLST Scheme for                                virulence factors of E. coli involved in internalization into
                                     Staphylococcus pseudintermedius                               and intracellular persistence in BMEC. Gene expression
                                                                                                   patterns in BMEC infected with acute or chronic strains
                                     Samar Solyman, Stephen Kania, David Bemis                     were significantly different from those of uninfected cells.
                                     Comparative and Experimental Medicine Program (Solyman),      Expression of genes in acute strain-infected BMEC was
                                     Department of Comparative Medicine (Kania, Bemis)             higher than that of chronic strain-infected BMEC. Genes
                                                                                                   that showed increased expression of transcript were pri-
                                     In order to understand the population genetic structure of    marily proinflammatory cytokines and complement. The
                                     the newly emerging methicillin-resistant pathogen Staphy-     chronic strain of E. coli had lower cell activation than the
                                     lococcus pseudintermedius we selected new gene candi-         acute strain of E. coli, potentially explaining mild clinical




                                                                                          20
symptoms observed in natural chronic E. coli intramam-       20. An Imbalance in sVR-1 and VEGF-A
mary infection and increased intracellular persistence of    Mediates Corneal Neovascularization
chronic E. coli strains in vitro.
                                                             after HSV-1 Ocular Infection
                                                             Amol Suryawanshi, Sachin Mulik , Shalini Sharma,
19. Robo 4 Activation Intercepts Herpes                      Sharvan Sehrawat, Barry T. Rouse
Simplex Virus 1-induced Angiogenesis                         Department of Pathobiology
and Immunopathology in the Eye
                                                             The normal cornea is transparent, which is essential for
Sachin Mulik, Shalini Sharma, Tamara Veiga Parga,            normal vision, and although the angiogenic factor VEGF-
Amol Suryawanshi, Pradeep Reddy, Naveen Rajasagi,            A is present in the cornea, its angiogenic activity is imped-
Barry T. Rouse                                               ed by being bound to a soluble form of the VEGF recep-
Comparative and Experimental Medicine Program (Mulik,        tor-1 (sVR-1). This study investigates the mechanisms
Parga, Suryawanshi), Department of Pathobiology (Reddy,      by which herpes simplex virus (HSV) infection of the
Rajasagi, Rouse)                                             eye results in neovascularization, an essential step in the
                                                             pathogenesis of the vision-impairing lesion, stromal kera-
Roundabout proteins have a well characterized function       titis (SK). We demonstrate that HSV-1 infection results
in neuronal migration in nervous system. However,            in an imbalance in the production of VEGF-A and sVR-1.
activation of the endothelial-specific Robo 4 receptor       Levels of VEGF-A were increased, but the production of
by Slit 2 inhibits migration of endothelial cells,           sVR-1 was decreased. Moreover, the sVR-1 protein made
tube formation and permeability in vitro and vessel          was degraded by the metalloproteinase (MMP) enzyme
leakage in vivo induced by proangiogenic factors,            MMP-7 and MMP-9 produced by infiltrating neutrophils.
especially VEGF-A. Herpes simplex virus 1 (HSV-              The outcome was that the VEGF-A produced was able
1) ocular infection results in notable pathological          to induce neovascularization. Inhibition of sVR-1 break-
neovascularization, an essential step in vision-impairing    down with the MMP inhibitor (MMPi), marimostat, or
lesion stromal keratitis (SK), where VEGF-A plays an         the provision of exogenous recombinant sVR-1 protein,
important role in mediating angiogenesis. In this study,     resulted in reduced angiogenesis. Our results demonstrate
we found evidence of the presence of a Slit 2-Robo 4         that HSV infection causes ocular neovascularization by
antiangiogenic network during SK. Endothelial cells          changing the balance between VEGF-A production and its
expressing Robo 4 receptor were detectable as early as       soluble inhibitory receptor. Future therapies aimed to in-
day 1 post HSV-1 infection with heavy invasion noted         crease the production and activity of sVR-1 protein could
during the clinical phase of SK. We also detected a sharp    benefit the management of SK.
rise in mRNA transcripts of Robo 4 during the clinical
phase, but the ligand, Slit 2 was minimal during this
stage. In line with this, provision of recombinant Slit 2
protein during the clinical phase of SK activated Robo
                                                             21. Regulatory T Cell Expression of
4 receptor and blocked activation of Arf 6 and Rac 1         Herpes Virus Entry Mediator (HVEM)
(both downstream targets of VEGF-A and integrins) as         Following HSV-1 Infection and Its


                                                                                                                             Viral Pathology & Immunity
was demonstrated by GTPase activation assays. Finally,       Functional Significance
subconjunctival administration of Slit 2 protein (starting
from day 1/day 6 post HSV-1 infection) in mice reduced       Shalini Sharma, Aarthi Sundararajan, Sachin Mulik,
angiogenesis and HSK lesion scores. In conclusion, we        Barry T. Rouse
provide evidence for the existence of an anti-angiogenic     Comparative and Experimental Medicine Program (Sharma,
Slit 2-Robo 4 network during SK; manipulation of this        Mulik), Department of Microbiology (Sundararajan), Depart-
axis could provide therapeutic potential to treat Herpes     ment of Pathobiology (Rouse)
stromal keratitis (HSK), an important cause of human
blindness.                                                   In many infections, especially those that are chronic, the
                                                             outcome may be influenced by the activity of one or more
                                                             types of regulatory T cells (Tregs), particularly those
                                                             that express FOXP3. In many instances, for example
                                                             the response that follows HSV infection, the majority of




                                                               21
                             expanded Tregs are not antigen specific. In this report, we   naïve mice, since mice primed i.n. with inactivated MHV-
                             demonstrate one possible mechanism by which HSV-1             68 and then challenged with live virus generated a strong
                             infection could act to signal and expand the Treg popu-       secondary IgG and IgA response. An IgA response in
                             lation. We show that HVEM, a tumor necrosis factor            IFNgR -/- mice following i.n. MHV-68 infection indicated
                             receptor superfamily member that interacts with the gD        a role for IFNg in opposing IgA production, perhaps by
                             protein of HSV-1 to facilitate cell entrance, is expressed    establishing a strongly polarizing Th1-type environment.
                             by FOXP3+ T cells and that engagement of HVEM by its          Down-regulation of IgA production by MHV-68 may
                             ligand expands the Treg population. To demonstrate this,      reflect a high level of viral adaptation to the respiratory
                             FOXP3-GFP knock in mice were infected with HSV kos,           tract that favors viral survival and transmission.
                             and change in cell numbers of Tregs and their expres-
                             sion levels of HVEM were recorded in draining popliteal
                             lymph node (DLN). Treg expansion occurred following           23. In Vitro Time Dependence of Water-
                             HSV-1 infection, peaking at around day 8 compared to the
                             peak numbers of CD8+T cells (day 5 p.i). Both FOXP3+
                                                                                           Soluble Chitosan Against Foodborne
                             and FOXP3- CD4 T cells expressed HVEM in the resting          Viral Surrogates at 37ºC and Room
                             state, but Tregs upregulated HVEM reaching highest MFI        Temperature
                             at day 7 p.i. In vitro stimulation of DLN cells with UV-
                             inactivated HSV kos further up regulated HVEM on Tregs        Radha Ganapathy, Svetlana Zivanovic, Doris H.
                             (possibly through gD) and down regulated on FOXP3_            D’Souza
                             cells suggesting a possibility that engaging this recep-      Department of Food Science and Technology
                             tor with gD expressed by infected cells results in Treg
                             activation and expansion, which in turn modulates CD8+        Chitosan is a non-toxic, natural, biodegradable polymer
                             effector responses.                                           with antimicrobial activity, recently approved by the EPA.
                                                                                           Chitosan’s effects on foodborne viruses have not been
                                                                                           investigated in detail. We evaluated the time dependence
                             22. Deficient Virus-specific IgA Production                   of water-soluble chitosan against foodborne viral surro-
                             Following Murine gammaherpesvirus 68                          gates, murine norovirus (MNV-1) and feline calicivirus
                                                                                           (FCV-F9) in vitro. Water-soluble chitosan (53kDa) was
                             Infection of the Respiratory Tract                            mixed with equal volume of virus at ~7log10PFU/ml to
                                                                                           provide final concentrations of 0.7% and 0.35% chitosan,
                             Aarthi Sundararajan, Hye Mee Joo, Mark Y. Sangster            and samples were incubated at 37°C and room tempera-
                             Department of Microbiology                                    ture (RT) for 2 hours, 1 hour, and 30, 15, and 5 minutes.
                                                                                           Duplicate treated and untreated viruses were enumerated
                             The B cell response to viruses that replicate in the          using standardized plaque assays. At both temperatures,
                             respiratory tract typically includes a substantial IgA        increasing contact time enhanced reduction of FCV, but
                             component that localizes in the respiratory tract. Murine     there was no significant effect on MNV-1 (p<0.05). Chito-
                             gammaherpesvirus 68 (MHV-68) is a naturally occurring         san at 0.7% decreased FCV titers by ~3 and ~1 log10PFU/
                             virus of small rodents and many features of MHV-68            ml after 2 hours at 37°C and RT, respectively. Chitosan at
Viral Pathology & Immunity




                             infection in mice closely parallel Epstein-Barr virus         0.35% decreased FCV by ~0.6 and ~0.1 log10PFU/ml after
                             infection in humans. Following intranasal (i.n.) infection,   2 hours at 37°C and RT, respectively. Chitosan at 0.7% for
                             infectious MHV-68 is cleared from the lung within             1 hour, and 30, 15 and 5 minutes showed FCV reductions
                             2 weeks, but the virus also spreads systemically and          of 1.6, ~0.8, ~0.2, and ~0.2 log10PFU/ml, respectively, at
                             establishes lifelong latency in B cells, macrophages and      37°C, with 0.8 log10PFU/ml reduction at RT after 1 hour
                             dendritic cells. The respiratory mucosa is thus a critical    and insignificant reduction at ≤30 minutes. Chitosan at
                             component of MHV-68 pathogenesis, but little is known         0.35% for ≤1 hour showed reductions of <0.2 and ~0.1
                             about B cell-mediated mucosal immunity to the virus.          log10PFU/ml at 37°C and RT. The results indicate that
                             Our analysis revealed a strong MHV-68-specific IgG            0.7% chitosan at 37°C for 2 hours is needed for significant
                             response in draining lymph nodes, but few IgA ASCs            activity. Chitosan shows potential as a natural antiviral to
                             were detected. IgA was prominent in the response to i.n.      control foodborne viral infections.
                             immunization with UV-inactivated MHV-68, indicating
                             that the IgA response was modulated by factors associated
                             with virus replication. This effect was most evident in




                                                                                  22
24. Antiviral Effects of Chitosan as                          high demand. High pressure homogenization (HPH)
Affected by its Molecular Weight and                          is one novel technology that needs research for viral
                                                              inactivation in fluids. In the absence of culturable human
Degree of Acetylation                                         noroviruses, cultivable murine norovirus (MNV) and
                                                              feline calicivirus (FCV) were used for infectivity. The
Robert Davis, Doris H. D’Souza, Svetlana Zivanovic            objectives of this research were to determine the effects
Department of Food Science and Technology                     of HPH alone or with an emulsifier (lecithin) on MNV
                                                              and FCV inactivation in skim milk and orange juice (OJ).
Chitosan is a cationic polysaccharide that has been ap-       Experiments were conducted in duplicate using pressures
proved as a biopesticide due to demonstrated improve-         of 0, 100, 200, 250, and 300 MPa for <2 seconds and
ment of plant resistance to fungal and viral attack.          plaque assayed in duplicate. For FCV in milk, reductions
Although foodborne viruses are the leading cause of           of ≥ 4 log10 PFU/ml at 300 MPa and ~1.3 log at 250 MPa
nonbacterial gastroenteritis, information on chitosan         with no reduction at ≤200 were obtained. For OJ, FCV
efficacy against human enteric viruses is limited. The ob-    was reduced by ≥4 and ~1 log at 300 and 250 MPa only.
jectives of this work were to determine the effectiveness     MNV in milk showed reductions of ~1.3 and ~0.1 log
of chitosan on 3 enteric viral surrogates and to further      at 300 and 250 MPa, respectively, and none below 200
investigate the effect of molecular weight and degree of      MPa. MNV in OJ was reduced by~0.8 and ~0.4 log at 300
acetylation (DA) of chitosan on its antiviral efficiency.     and 250 MPa, respectively. Lecithin had minimal effects,
Four chitosans (53kDa, 9.1%DA; 222kDa, 32.5%DA;               where MNV in milk or OJ containing lecithin showed
307kDa, 20.2%DA; 421kDa, 30.3%DA) were tested                 1.32 or 2.5 log reduction, respectively, at 300 MPa. FCV
against feline calicivirus (FCV), bacteriophages MS2




                                                                                                                             Nutrition & Metabolism
                                                              showed complete inactivation in OJ or milk combined
and phiX174. Purified chitosans were dissolved either in      with lecithin at 300 MPa. At 250 MPa, FCV in milk or
water (53kDa) or 1% acetic acid (other chitosans), filtered   OJ with lecithin showed reductions of 1.77 and 0.78 log,
through 0.2μm membrane, and mixed with equal volume           respectively. These results show that HPH has potential
of 5 log PFU/ml virus in phosphate buffered saline (PBS).     for commercial use to inactivate foodborne viruses.
The final concentration of chitosan was 0.7%, and virus-
chitosan suspensions were incubated for 3 hours at 37°C.
Untreated viruses in PBS or PBS with 0.5% acetic acid
were tested as controls. Water-soluble chitosan (53kDa)
                                                              26. Size of Food Bowl and Scoop Affects
reduced FCV, MS2 and phiX174 titers by 2.15, 2.33 and         Amount of Food Owners Feed their Dogs
0.65 log PFU/ml, while 421kDa chitosan reduced 2.22,
3.69 and 1.01 log PFU/ml, respectively, compared to the       Maryanne Murphy, Claudia A. Kirk, Joseph W. Bartges,
PBS control, with intermediate reductions for the other       Angela L. Lusby
two chitosans. To date, this is the first report on chito-    Department of Small Animal Clinical Sciences
san’s effects against phiX174. Overall, chitosan showed
a greater log reduction on FCV and MS2 than phiX174,          Canine obesity is increasing at a dramatic rate, and under-
with 421kDa causing the greatest effect on both viruses.      standing factors impacting the amount of food pet own-
Methods based on chitosan application show promise for        ers provide their dogs may improve weight management.
inactivating foodborne viruses.                               Human research has shown the size of food bowls, plates
                                                              and utensils can significantly impact the amount of food
                                                              portioned and consumed. This effect can be attributed to
25. Combination of Lecithin and                               the Delboeuf optical illusion in which a circle enclosed by
                                                              a larger ring appears bigger than the circle alone. To in-
High Pressure Homogenization for                              vestigate the existence of a similar effect with dog owners,
the Inactivation of Human Norovirus                           46 dogs and their owners were recruited for a 4-treatment,
Surrogates in Milk and Orange Juice                           randomized prospective trial. Owners scooped out a nor-
                                                              mal kibble-based meal using a small bowl (4.75 cups) and
Katie Horm, Doris D’Souza, Federico Harte                     small scoop (1.5 cups), small bowl and large scoop (3.6
Department of Food Science and Technology                     cups), large bowl (10 cups) and small scoop, or a large
                                                              bowl and large scoop. Each treatment was used once per
Novel processing technologies that inactivate viruses         owner over 4 visits. Repeated measures ANOVA revealed
in foods and retain nutritional attributes remain in          the mean amount of food portioned using the small bowl




                                                                23
                         and small scoop was significantly less than the large bowl-   runners during TT runs.
                         large scoop combination (150.7 g vs 184.5 g, respectively,
                         p<0.05). The small bowl and large scoop combination
                         did not differ from large bowl and small scoop (171.5 g vs    28. Effects of Leucine and Calcitriol on
                         172.7 g, respectively, p>0.05). Owners were more likely
                         to portion a larger amount of food with a large bowl and
                                                                                       Monocyte-Vascular Endothelial Cell
                         large scoop. Results are consistent with human data and       Adhesion
                         emphasize the need for owners to use standard measur-
                         ing cups. Results also suggest owner compliance dur-          Benjamin Curry, Michael Zemel, John Biggerstaff
                         ing weight loss programs may be improved with smaller         Department of Nutrition
                         bowls and serving scoops.
                                                                                       We previously demonstrated that calcitriol and
                                                                                       leucine modulate adipocyte and macrophage cytokine
                         27. Nutritional Ergogenic Aids: The Effects                   production. Since several of the affected cytokines may
                                                                                       modulate vascular infiltration by monocytes, we tested
                         of Carbohydrate-protein Supplementation                       the hypothesis that calcium (via calcitriol suppression)
                         During Endurance Exercise                                     and leucine attenuate vascular infiltration. Adipocytes
                                                                                       were treated with vehicle, calcitriol (to mimic calcium
                         Adriana Coletta, Hollie Raynor                                deprivation), leucine or both for 48 hours and conditioned
                         Department of Nutrition                                       medium (CM) collected and incubated with fluorescently-
Nutrition & Metabolism




                                                                                       labeled U937 monocytic cells perfused across monolayers
                         This study examined performance benefits from ingesting       of EA hy.926 endothelial cells; adherence was then
                         a carbohydrate (CHO), carbohydrate-protein (CHO-P),           quantified by microscopy and image analysis. Results are
                         and double carbohydrate (CHO-CHO) compared to a               presented as a monocyte : endothelial cell ratio (M:E).
                         placebo (PLA) supplement, during a time trial (TT) run.       Calcitriol (10 nM) increased the M:E from 0.088+0.048
                         Twelve male recreational runners (age = 32.4±9.5yrs;          to 0.201+0.112 (228% increase, p = 0.0001). Conversely,
                         BMI = 22.7±1.5kg/m2; VO2max = 59.68 ±7.53mL/kg                leucine (0.5 mM) markedly decreased the M:E to
                         body weight; 100% white) individually completed 4, 12-        0.013+0.011 (p value = 0.001); moreover, addition of
                         mile TT runs at 75% of their race pace. Dietary consis-       leucine CM to the calcitriol CM treatment abolished
                         tency within the 24-hour time period prior to each run was    the effects of calcitriol. A parallel experiment using
                         controlled. Throughout each run, participants consumed        flow cytometry of CD11b expression on U937 cells
                         625mLs of 1 of the 4 supplements aforementioned. A            under parallel conditions showed an identical trend to
                         Latin square design counterbalanced supplement order.         the microscopy data. These data show that monocyte
                         Endurance performance was measured by time to com-            adhesion to endothelial cells is decreased in the presence
                         plete the 12-mile run and time to complete the last 1.2       of leucine and increased by calcitriol, suggesting that
                         miles of the run, where participants were instructed to run   foods rich in calcium and leucine may be beneficial in
                         at maximal effort, 100% race pace. A main effect of time      reducing monocyte adhesion. Supported by the National
                         occurred during the TTs for heart rate (HR) and perceived     Dairy Council.
                         exertion (RPE). HR significantly increased from the
                         start to the start of the maximal effort and was higher at
                         completion of the effort (84.4±14.5bpm, 166.0±8.3bpm,         29. Assessing Potential for DNA
                         178.8±7.4bpm, p<0.001); RPE (Borge 10-point scale)
                         significantly increased from mid-point to completion of
                                                                                       Methylation Patterns to Affect Adipocyte
                         TT (4.7±0.7, 9.7±0.9, p<0.001). No significant difference     Metabolic Gene Expression
                         was found in overall time to complete the 12-mile run
                         (PLA = 88.6±11.6min; CHO = 89.1±11.3min; CHO-P =              Julia Stair Gouffon, Michael Zemel
                         89.1±11.8min; and CHO-CHO = 89.6±11.9min) or maxi-            Comparative and Experimental Medicine Program (Gouffon,
                         mal effort (PLA = 8.3±1.2min; CHO = 8.2±1.2min; CHO-          Zemel), Department of Nutrition (Zemel), Affymetrix Core Lab
                         P = 8.2±1.2min; and CHO-CHO = 8.4±1.5 min) between            (Gouffon)
                         supplements. These findings suggest that type of supple-
                         mentation (CHO, CHO-CHO, CHO-P) has no effect on              Recent experiments have shown the importance of
                         enhancing endurance performance in male recreational          epigenetics as a means of intermediate gene expres-




                                                                              24
sion control, serving as a kind of rheostat allowing for        35% decrease in MDA evident at 7 days (p<0.01), while
modulation or complete restriction of gene expression.          LD had no effect. Similarly, inflammatory markers
Epigenetic modifications include changes in histones,           were suppressed by AD, with decreases in TNF-α
methylation of cytosine residues in CpG islands and             evident at 7 days and further reductions through 12
control by small RNAs. Changes in the epigenetic status         weeks (35%, p<0.05), IL-6 (21%, p<0.02), MCP-1 (14%
of a cell can effect differentiation and determine the fate     drop at 4 weeks, 24% drop at 12 weeks, p<0.05), and a
of stem cells and fibroblasts. Maternal nutritional status,     corresponding 55% increase in adiponectin (55% at 12
as well as current nutritional inputs, are critical for sup-    weeks, p,0.01). LD exerted no effect on these markers.
plying methyl groups in pyrimidine synthesis and have           Thus, increasing dairy intake results in attenuation of
been demonstrated to alter the phenotype of offspring.          oxidative and inflammatory stress in metabolic syndrome.
Therefore, we predict that specific nutrient availability       Supported by the National Dairy Council.
will affect adipocyte development and thereby influence
subsequent chronic disease risk. Accordingly, our present
work seeks to determine if the methylation level of CpG         31. Microarray Profiling of Chicken
islands can be influenced in adipocytes and if so, how
this might affect gene signaling in mature adipocytes and
                                                                Adipose Tissue under Insulin
a mouse model system. Methylated regions of adipocyte           Neutralization and Fasting Conditions
genomic DNA was obtained using a methylation-specific
antibody and hybridized against a whole genome CpG              Bo Ji, Arnold M. Saxton, Jean Simon, Suchita Das,
island array (Nimblegen). We found methylated regions           Shawn R. Campagna, Jessica R. Gooding, Brynn H. Voy




                                                                                                                                Nutrition & Metabolism
in or near transcription start sites for acetyl CoA carbox-     Department of Animal Science (Ji, Saxton, Das, Voy), Institut
ylase A, acetyl CoA carboxylase B, adiponectin receptor         National de la Recherche Agronomique (Simon), Department of
2, cytochrome C oxidase, DNA methyltransferase 3A,              Chemistry (Campagna, Gooding)
folate receptors 1 and 2, hormone sensitive lipase, resistin,
retinoid X receptor beta and vitamin D receptor. Work is        Excessive fat accumulation in today’s commercial
ongoing to determine if these regions can be influenced         broiler chickens due to intensive genetic selection for
by addition of folate or a methyltransferase inhibitor using    rapid growth is a problem for both the broiler industry
sequencing after bisulfate conversion and amplification of      and consumers. Developing approaches to reduce fat
regions of interest.                                            deposition requires improved understanding of avian
                                                                adipose tissue development and physiology. We used
                                                                microarray profiling to characterize the molecular
                                                                response to energy manipulation in chicken adipose tissue
30. Dairy Attenuation of Oxidative                              and to test its sensitivity to insulin, a key hormone in
and Inflammatory Stress in Metabolic                            energy use. Approximately 3 week-old commercial broiler
Syndrome                                                        chickens (ISA915) were fed ad libitum, fasted for 5 hours,
                                                                or fed but deprived of insulin by injections of anti-insulin
Renée A. Stancliffe, Michael B. Zemel                           serum. Plasma was used to measure circulating levels
Department of Nutrition                                         of insulin, glucose, tri-iodothyronine (T3), glucagon,
                                                                non-esterified fatty acid (NEFA) and α-amino-non-
We previously demonstrated that dairy components                protein nitrogen (α NH2NPN). Pair-wise contrasts of
(calcium, leucine, & dairy peptides) suppress adipocyte         expression data using the Affymetrix GeneChip Chicken
and macrophage-mediated oxidative and inflammatory              Genome Array identified a total of 1016 genes that were
stress. Since oxidative and inflammatory burdens are            differentially expressed within different contrasts after
magnified in metabolic syndrome, we have assessed the           correction for multiple testing, with the vast majority
effects of low (<0.5 servings) vs. adequate (3.5servings)       of differences due to fasting. Gene ontology analysis
dairy (LD vs. AD) diets on oxidative and inflammatory           indicated that genes responsive to fasting were enriched
stress in the absence of caloric restriction in 40 metabolic    for many functions related to cellular metabolism. Gene-
syndrome subjects with habitually low dairy intakes. Diet       phenotype correlation with plasma hormone levels
had no effect on body weight over the 12-week study,            suggests significant relationships between gene expression
but AD significantly reduced waist circumference and            in adipose tissue and plasma T3 and NEFA levels.
DXA-measured trunk fat (p<0.01 for both), while LD              Ongoing measures of global metabolite profiles using
had no effect. AD attenuated oxidative stress, with a           mass spectrometry will be integrated with transcriptomics




                                                                  25
                         and physiological data. These data support that concept        adipo/cytokine levels are potential mechanisms for insulin
                         that insulin action in chicken adipose differs significantly   resistance in this model. Supported by UT-ORC, AHA,
                         from that in mammals and provide a foundation for              AHA pre-doctoral fellowship, and USDA-NRI.
                         further study into molecular bases for adipose expansion
                         in poultry.
                                                                                        33. Eicosapentaenoic Acid (EPA)
                                                                                        Regulates Hypertensive, Inflammatory
                         32. Mechanisms Linking Overproduction
                                                                                        and Adipogenic Mediators in Mouse and
                         of Angiotensinogen by Adipose Tissue to
                                                                                        Human Adipocytes
                         Inflammation, Glucose Intolerance and
                         Insulin Resistance                                             Nalin Siriwardhana, Sarah Fletcher, Wenting Xin, Avik
                                                                                        Mukherjee, Nishan Kalupahana, Suzanne Booker, Patrick
                         Nishan S Kalupahana, Sarah J Fletcher, Taryn Stewart,          Wortman, Arnold M. Saxton, Naima Moustaid-Moussa
                         Brynn H Voy, Annie Quignard-Boulange, David                    Department of Animal Science (Siriwardhana, Fletcher, Xin,
                         Wasserman, Naima Moustaid-Moussa                               Mukherjee, Booker Wortman, Saxton, Moustaid-Moussa), UT
                         Obesity Research Center and Department of Animal Science       Obesity Research Center (Siriwardhana, Kalupahana, Moustaid-
                         (Kalupahana, Fletcher, Stewart, Voy, Moustaid-Moussa),         Moussa)
                         UMR914 INRA-AgroParisTech, Paris, France (Quignard-
                                                                                        Excessive secretion of angiotensinogen (Agt) and pro-
Nutrition & Metabolism




                         Boulange), Department of Molecular Physiology and
                         Biophysics, Vanderbilt University (Wasserman)                  inflammatory cytokines has been linked to obesity and
                                                                                        associated metabolic disorders with a common feature
                         Activation of the adipose tissue renin angiotensin             being inflammation. While n-3 and n-6 polyunsaturated
                         system (RAS) has been implicated in obesity and co-            fatty acids (PUFAs) are known to modulate adiposity
                         morbidity. We hypothesized that adipose overproduction         and inflammatory status, PUFA-mediated regulation of
                         of angiotensin II (Ang II) and angiotensinogen (Agt) will      the adipose renin-angiotensin system and the specific
                         exacerbate obesity, inflammation and insulin resistance.       mechanisms by which PUFAs modulate the endocrine
                         To test this hypothesis, we bred mice overexpressing           functions of adipose tissue remain unclear. Thus, we
                         Agt in the adipose tissue via the adipose-specific             investigated the effects of eicosapentaenoic acid (EPA,
                         promoter aP2 (aP2-Agt mice). Plasma Agt levels in the          n3-PUFA) and arachidonic acid (AA, n6-PUFA) on
                         aP2-Agt mice were 25% higher than the wild type, in            expression and secretion of Agt and pro-inflammatory
                         agreement with earlier reports supporting the endocrine        cytokines and expression of adipogenic and lipogenic
                         role for adipose-derived Agt. These mice were glucose          markers in 3T3-L1 adipocytes. Treatments lasted up to 48
                         intolerant and exhibited a near doubling of visceral           hours, and culture media as well as cell extracts (RNA and
                         adiposity, concomitant with adipocyte hypertrophy when         proteins) were prepared. Agt expression and secretion was
                         compared to their wild-type littermates. When subjected        detected only in differentiated mature adipocytes. Further,
                         to a euglycemic-hyperinsulinemic clamp, aP2-Agt mice           non-differentiated pre-adipocytes were not responsive
                         exhibited lower insulin-stimulated glucose clearance           to PUFA. Both fatty acids increased intracellular Agt
                         when compared to B6 mice. This difference was largely          protein expression in 3T3-L1 adipocytes, and AA dose-
                         accounted for by reduced muscle glucose uptake, in sharp       dependently increased Agt secretion into culture media
                         contrast with sustained insulin-stimulated glucose uptake      while EPA dose-dependently decreased Agt. In agreement
                         by fat. Proteomic studies of adipose tissue showed that        with its pro-inflammatory effects, EPA (not AA)
                         P2-Agt mice exhibited significantly lower expression           significantly reduced Interleukin-6 (IL-6) and monocyte
                         of key antioxidant enzymes (catalase and superoxide            chemotactic protein-1 (MCP-1) secretion. Similarly,
                         dismutase) and higher expression of lipogenic proteins         EPA-treated, differentiated human adipocytes from both
                         (glycerol-3-phosphate dehydrogenase) when compared             lean and obese subjects significantly reduced secretion of
                         to the wild-type. In conclusion, overproduction of Agt         MCP-1, IL-6, and IL-8. EPA dose dependently decreased
                         from adipose tissue results in increased visceral adiposity,   the NF-kB activation measured in 3T3-L1 adipocytes
                         adipocyte hypertrophy, glucose intolerance and insulin         stably transfected with NF-kB responsive reporter (NF-
                         resistance, at least in part due to reduced skeletal muscle    kB-Luc) while AA treatment reported the opposite.
                         glucose uptake. Further, our data indicate that increased      These studies demonstrate that EPA down-regulates the
                         oxidative stress in adipose tissue and pro-inflammatory        secretion of inflammatory and hypertensive mediators




                                                                                26
from adipocytes, and those activities were at least partially   the blood. In the early days, major complication with
mediated via repression of NF-kB activation.                    this technique was excessive bleeding due to complete




                                                                                                                            Innovative Biomedical Technologies
                                                                anticoagulation (heparinization). Today, heparin-
                                                                coating of ECMO equipment surfaces has improved the
34. Synthesis and Characterization                              performance of the technique, allowing ECMO patients to
                                                                receive minimal or no systemic heparinization. However,
of Diamond-like Carbon Films for                                treated surfaces are still not fully blood-compatible,
Biomedical Applications                                         and clot formation and immune activation within the
                                                                ECMO circuit eventually reduce the performance of
Russell Lee Leonard, Saad A. Hasan, Alexander                   the membrane oxygenator and can lead to disseminated
Terekhov, Robert A. Erck, James H. Dickerson, Jacqueline        intravascular coagulation and embolisms. To address
A. Johnson                                                      this problem, methods are being developed to improve
Department of Materials Science and Engineering (Leonard,       the quality of heparin surface-coatings for use in ECMO
Terekhov, Johnson), Vanderbilt University Department of Ma-     equipment. In this study, we report the use of a new and
terials Science and Engineering (Hasan, Dickerson), Argonne     improved methodology for in vitro evaluation of surface
National Laboratory (Erck)                                      biocompatibility, using temperature-controlled perfusion
                                                                system with human blood. We evaluated 2 heparin-
The objective of this research is to produce diamond-like       coatings (0.15M and 0.5M EDA/MES + heparin) on
carbon (DLC) films by pulsed laser deposition (PLD)             the biocompatibility of gas-exchange membranes. We
to be used as coatings for biomedical materials. To this        report a substantial improvement in the biocompatibility
end, films have been deposited on a variety of substrates       of the heparin-coated membranes compared to control
including silicon, fused silica, and silicon nitride. The       membranes, including reductions in thrombin generation
films have been characterized by Raman spectroscopy,            (90+/- 1.2%) and complement activation (51+/- 1.4%) in
atomic force microscopy, ball-on-flat tribometer, and           circulating blood and reduced deposition of fibrin (89+/-
contact angle machine. Film adhesion has been checked           1.9 %), fibrinogen (99+/- 1%), thrombin (85 +/- 4%),
by soaking the films in simulated body fluid (SBF) and          platelets (90+/- 5%), and leukocytes (95 +/- 0.3%) on the
monitoring the quality of the film surface at varying time      membrane surface (p<0.05). This new dynamic in vitro
intervals using an optical microscope. DLC coatings             biocompatibility testing system is an improvement on
have been produced with an average surface roughness            static systems because of its ability to more accurately
of less than 1 nm and a lubricated friction coefficient of      simulate the fluid dynamics of the circulatory system.
0.7. Contact angles for the films vary between 79 and
88 degrees. The DLC coatings on fused silica show
no delamination after 9 weeks of immersion in SBF.
Research is ongoing. Current results will be presented.




35. In vitro Evaluation of Artificial                           36. Optimizing the Performance of
Membrane Biocompatibility                                       ZBLAN Glasses as Image Plates
Michael Beeler, Steve Minkin, Patricia Brown, Benjamin
                                                                Manh Vu, Jackie Johnson, Stefan Schweizer
Curry, Linda Cahalan, Patrick Cahalan, John Biggerstaff         Department of Materials Science, UT Space Institute (Vu,
Center for Environmental Biotechnology (Beeler, Minkin,         Johnson), Fraunhofer Center for Silicon Photovoltaics,
Brown, Biggerstaff), Department of Nutrition (Curry), Ension,   Germany (Schweizer)
Inc. (Cahalan, Cahalan)
                                                                ZBLAN (zirconium, barium, lanthanum, aluminum,
Extracorporeal membrane oxygenation (ECMO) is a
                                                                and sodium fluorides) glass-ceramic materials are being
technique for providing life support to patients whose
                                                                developed as scintillators and storage phosphors mainly
lungs are unable to provide sufficient oxygenation to
                                                                for applications in medicine. The materials are doped




                                                                  27
                                     with europium and chlorine. Upon heat treatment,               Staphylococcus aureus, and Pseudomonas aeruginosa
Innovative Biomedical Technologies   barium chloride nanoparticles containing optically             to the material surfaces. Levels of GS release from GS-
                                     active europium (II) form within the glass matrix.             loaded materials were determined by measuring inhibition
                                     X-ray diffraction (XRD) has shown a particular phase           of E. coli growth in liquid and solid media. The results
                                     of the barium chloride nanoparticles to be allied with         show that addition of hydrogel inhibits bacterial adhesion
                                     either storage or scintillation. The hexagonal phase is        and addition of collagen promotes fibroblast adhesion.
                                     synonymous with scintillation, and the orthorhombic            Gentamicin-loaded materials were actively bactericidal
                                     phase is synonymous with the storage mechanism. In             while allowing for robust attachment of fibroblasts.
                                     single crystals, it has long been thought that oxygen
                                     defects gave rise to the storage of electron-hole pairs,
                                     so these were artificially introduced into the glass           38. Enhanced Nerve Cell Attachment,
                                     ceramic described here in the hope of enhancing storage.
                                     However, by serendipity, it was discovered that in fact
                                                                                                    Proliferation, and Differentiation
                                     the reducing conditions gave rise to storage, the complete     on Hydrogels Grafted with Photo-
                                     opposite to what happens in single crystals. Extensive         polymerizable Poly-L-Lysine
                                     characterization of the materials for x-ray imaging
                                     applications had taken place to determine the exact            Lei Cai, Shanfeng Wang
                                     composition and structure/property relationships by ion        Department of Materials Science and Engineering
                                     chromatography, inductively coupled plasma spectroscopy
                                     and secondary ion mass spectroscopy as well as x-ray           Poly(ethylene glycol) diacrylates (PEGDAs) are injectable
                                     diffraction, differential scanning calorimetry and many        and photo-crosslinkable materials that can form hydrogels
                                     large facility techniques. In situ transmission electron       with degradability to mimic extracellular matrices in
                                     microscopy is ongoing to explore the interface kinetics        brain and nerve tissues. Three photo-polymerizable
                                     between the nanoparticles and glass matrix. A summary of       PEGDAs with different molecular weights synthesized
                                     medical applications and scientific development method         using K2CO3 as the proton scavenger other than the
                                     will be presented on ZBLAN glass ceramics for imaging          widely used triethylamine have been modified with a
                                     applications.                                                  crosslinkable poly-L-Lysine (PLL) with 1 double bond
                                                                                                    at the end synthesized using allylamine as the initiator.
                                                                                                    PEGDAs with different molecular weights exhibited
                                     37. Fibroblast and Bacterial Growth on                         controllable mechanical properties that greatly influenced
                                     Infection-resistant Materials                                  the behavior of nerve cells, such as PC12 cells and neural
                                                                                                    stem cells (NSCs). For both pristine PEGDA-based
                                                                                                    and PLL-modified hydrogels, PC12 cells were found
                                     Steven Minkin, Michael Beeler, Patrick Cahalan, Linda          consistently to favor softer substrates by demonstrating
                                     Cahalan, John Biggerstaff                                      higher attachment, faster proliferation, and more neurite
                                     Center for Environmental Biotechnology (Minkin, Beeler,
                                                                                                    extension in the presence of nerve growth factor. Tethered
                                     Biggerstaff), Ension Inc. (Cahalan, Cahalan)
                                                                                                    PLL chains dramatically enhanced PC12 cell attachment,
                                     Sites of transdermal medical implants (e.g., for indwelling    proliferation and neurite extension in differentiation.
                                     catheters, ports) are at increased risk of infection.          The same preference on mechanical stimuli was found in
                                     Materials destined for long-term transdermal implantation      NSCs, while grafted PLL chains with charges significantly
                                     should encourage local wound healing but not serve as          improved the microenvironment for NSCs to proliferate
                                     a site for bacterial biofilm formation. Thus, infection-       and differentiate. These results suggest that PEGDA
                                     resistant materials should inhibit bacterial adhesion,         hydrogels with grafted PLL segments with optimal
                                     or be actively bactericidal, while promoting fibroblast        mechanical properties and cell behavior have excellent
                                     adhesion. In this work, we evaluate fibroblast and             potential as injectable materials for nerve regeneration.
                                     bacterial growth on 29 synthetic materials: four material
                                     types (polyurethane, silicone, polyester, and titanium)
                                     with various combinations of surface modifications             39. Predictive Ophthalmic Simulation and
                                     (hydrogel, collagen, gentamicin [GS], and heparin).            Applications
                                     Fluorescence microscopy was used to measure adherence
                                     of human foreskin fibroblasts (HFF-1), Escherichia coli,
                                                                                                    Ying-Ling Chen, J. W. L. Lewis, Lei Shi




                                                                                               28
Department of Physics                                         vision and to decrease the risk of amblyopia. However,
                                                              uncooperative children (49%, n = 377) and the time-




                                                                                                                             Innovative Biomedical Technologies
Recently, high precision ophthalmic patient data have         consuming current methods (23%) deter mass pediatric
become available to characterize surfaces of the cornea       screening [Kemper 2006]. The objective of this study
and crystalline lens, wavefront aberrations, and ocular       is to design a child friendly, objective, and automatic
element biometry in microscopic accuracy. These               method to diagnose strabismus and ocular alignment
measurements can be incorporated into the creation of         impairments. This method utilizes a dynamic Hirschberg
optically functional and analytical, personal-tailored,       test to monitor the binocular orientation, gazing angle, and
eye models. Computational personalized eye modeling           convergence while the examinee’s eyes pursue a moving
techniques are beneficial since they allow the integrated     bulls-eye target on a screen in a 10-second trajectory.
information to be quantitatively evaluated without            The novel design also uses a nasal divider to produce a
repetitive examination of human subjects. Simulating          binocular viewing at the center region of the screen and
ocular device measurements using eye modeling is also         2 monocular viewing zones at either side of the screen.
useful to aid medical personnel training and for evaluating   While the examinee follows the target’s trajectory through
ocular instrument sensitivity. The objectives of this         the 3 viewing zones, test results of cover-uncover and
project are to investigate the feasibility of personalized    smooth pursuit are automatically obtained from both
eye modeling on normal and diseased conditions and            eyes. Dynamic testing allows us to obtain the Hirschberg
to demonstrate the applications of realistic ophthalmic       ratio and fovea kappa angle for each eye to calibrate the
simulation. Specifically, contemporary optical design         result. Since the eye position is instantaneously captured
programs, ZEMAX and ASAP, are used with clinically-           and used in the analysis, eye movement does not affect
acquired patient data to construct the 3-dimensional          the performance of test. The pilot study results of 40
optical models. These customized models have identical        optometry students show interesting insights of eye
clinical measured details as well as optical performance      movement behavior. This rapid diagnostic method is
and visual acuity. Using optical parameters of a common       even more sensitive when the parameters of 2 eyes are
ophthalmic streak retinoscope and the eye models, the         compared.
clinical observations of the diseased and normal retinal
reflex patterns were simulated successfully. A comparison
of the retinal reflex images between the laboratory           41. Injectable Blends Consisting of
measurements and simulations were also obtained.
These results show the viability of predictive medicine       Poly(propylene fumerate) and Methacryl-
and possibly a revolutionary approach in developing           Polyhedral Oligomeric Silsesquioxane for
ophthalmic optical instruments with eye modeling              Regulating Material Properties and Bone
technique.
                                                              Cell Behavior

                                                              Kan Wang, Shanfeng Wang
                                                              Department of Materials Science and Engineering

                                                              Polyhedral oligomeric silsesquioxane (POSS) has a
                                                              cage-like hybrid inorganic–organic nanostructure.
                                                              Incorporating POSS cages into polymers has been
40. Design of an Automatic Cover-Uncover                      reported to improve the mechanical and viscoelastic
Test for Diagnosing Strabismus and Ocular                     properties of the matrix polymer. Because of their non-
Alignment Impairments                                         toxicity, POSS-based polymeric nanocomposites are
                                                              suitable for biomedical applications. Poly(propylene
                                                              fumarate) (PPF) is a linear unsaturated polyester and an
Lei Shi, Ying-Ling Chen, J.W.L. Lewis                         excellent injectable, degradable material for bone tissue
Department of Physics
                                                              engineering applications. PPF can be crosslinked though
With a 2–5% prevalence rate in the United States,             its carbon-carbon double bonds and can be degraded by
amblyopia plays the key role of childhood monocular           simple hydrolysis of ester bonds. The degradation time
blindness. Pediatric strabismus must be detected/             depends on PPF molecular weight, type of cross-linker,
treated early to optimize the potential for binocular         and cross-linking density. In this work, we have blended
                                                              PPF with 2 molecular weights and methacryl-POSS at




                                                                29
                    different compositions and have studied their rheological     The administration of LPS-E-coli, under the conditions
                    and thermal properties, and the mechanical, thermal, and      evaluated, can be used to induce an acute reproducible
                    surface characteristics after crosslinking using photo-       lung inflammatory model in growing commercial pigs.
                    initiation. Methacryl-POSS was found to speed up the
                    photo-crosslinking and decrease the viscosity of PPF,
                    which were both important in the formation of PPF             43. Evaluation of Tyrosinase Expression in
                    networks in bone surgeries. In addition, the density of
                    crosslinkable segments and mechanical properties could
                                                                                  Canine and Equine Melanocytic Tumors
                    be regulated by blending methacryl-POSS with different
                                                                                  Luis Lembcke, Jeff Phillips, Stephen Kania, James
                    compositions. In vitro cell studies have been performed
                                                                                  Blackford
                    to evaluate the biocompatibility of this series of hybrid
                                                                                  Comparative and Experimental Medicine Program (Lembcke),
                    polymer networks using mouse MC3T3 cells and                  Department of Small Animal Clinical Sciences (Lembcke,
                    investigate the correlation between material properties and   Phillips), Department of Comparative Medicine (Kania),
                    cell behavior.                                                Department of Large Animal Clinical Sciences (Blackford)

                                                                                  DNA vaccination against proteins specifically or
                    42. An Acute Reversible Experimental                          preferentially expressed by tumors is a strategy for cancer
                                                                                  therapy. To have a successful vaccine, one must identify
                    Model of Pneumonia in Pigs: Time-related
                                                                                  tumor proteins that have restricted expression patterns
                    Histological and Clinical Sign Changes                        and can facilitate an appropriate immune response while
                                                                                  not generating immune tolerance or significant auto-
                    Nicolas Villarino, Tomas Martin-Jimenez                       immunity. To this end, a vaccine was developed for use
                    Comparative and Experimental Medicine Program (Villarino),    in dogs with melanoma that targets tyrosinase, an antigen
                    Department of Comparative Medicine (Martin-Jimenez)           preferentially expressed in melanoma tumors (Oncept).
                                                                                  Furthermore, pending definition of the expression pattern
Clinical Sciences




                    Pneumonia represents one of the most important diseases       of this antigen, the vaccine may also be considered in
                    both in veterinary and human medicine. A considerable         other affected species such as the horse. Tyrosinase is
                    amount of information about pneumonia is generated            preferentially expressed in canine and equine melanocytic
                    from experimental animal models. However, there is            tumors and is associated with tumor histology. A total of
                    limited information about pneumonia models involving          42 canine and 8 equine melanocytic tumor samples were
                    growing commercial pigs. The objective of this study was      used in this study. RNA was isolated from formalin-fixed,
                    to evaluate long-term survival rates, clinical response,      paraffin-embedded tissues using standard techniques.
                    and lung gross and microscopic changes in growing             TaqMan RT-PCR assays were designed to amplify the
                    commercial pigs treated intra-tracheally with 2 dose          exon 1/2 boundary of canine and equine tyrosinase.
                    levels of lipopolysaccharide of Escherichia coli (LPS-        Relative expression levels were determined and compared
                    E-coli). Fourteen healthy pigs were randomly allocated        to both pigmented and non-pigmented cutaneous tissues.
                    to 3 groups: (i) no-LPS (n = 1), (ii) (LPS 1 mg/mL, 10        High tyrosinase expression was found in both pigmented
                    mL/pig, n = 7) and (iii) (LPS 0.5 mg/mL, 10 mL/pig, n =       and non-pigmented melanocytic tumors with insignificant
                    6). Pigs were evaluated during a period of 0–144 hours        levels in normal skin. No significant difference in
                    post-LPS-E-coli administration following a terminal           tyrosinase expression was found between different canine
                    schedule design. Two pigs from each dose level were           or equine histologic variants of melanocytic tumors.
                    euthanized at 24, 48 and 144 hours post-LPS. Clinical,        Melanocytic tumors expressed significantly higher levels
                    gross and histological changes were evaluated using a         of tyrosinase than non-melanocytic tumors and/or control
                    score system. None of the animals died as a consequence       samples. This RT-PCR methodology is highly sensitive
                    of the treatments. All the LPS-treated animals showed         and specific for the detection of tyrosinase expression
                    lung gross changes. A consistent acute lung inflammatory      in both fresh and fixed tumor samples and demonstrates
                    response was induced by both LPS dose levels. A               the preferential expression of this antigen in melanoma
                    consistent reversible recruitment of macrophages and          tumors.
                    neutrophils was observed. The highest cellular infiltration
                    level was observed at 24 hours after challenge. A dose-
                    dependent cellular infiltration was evident. The highest
                    clinical scores were evident in both experimental dose
                    levels within 3 and 5 hours after LPS administration.



                                                                          30
44. Effects of Season on                                      45. Novel Endocrine Disruptor in
Adrenocorticotropin Hormone (ACTH)                            Personal Care Products
Concentrations in Horses with Pituitary
                                                              Jiangang (Jay) Chen, Bill L. Lasley, Antoni J. Duleba
Pars Intermedia Dysfunction (PPID)                            Department of Nutrition (Chen), Center for Health and the En-
from 15 Different Locations in East                           vironment, University of California Davis (Lasley), Department
Tennessee                                                     of Obstetrics and Gynecology, University of California Davis
                                                              Medical Center (Duleba)
Sarah Beth Elliott, Nicholas Frank, Robin McClamroch,
                                                              Many xenobiotics have been associated with endocrine
Kelly Ann Chameroy, Ferenc Toth, Eric Etheridge
Department of Large Animal Clinical Sciences                  effects in a wide range of biological systems. Triclocarban
                                                              (TCC; 3,4,4’-trichlorocarbanilide), an antimicrobial
We have previously reported a seasonal increase in            compound used in personal care products, was evaluated
adrenocorticotropin hormone (ACTH) concentrations in          for its endocrine disrupting property. Recent evidence
horses from a single farm in Tennessee, with significantly    demonstrates that TCC is a common contaminant of the
higher concentrations detected in September and Octo-         environment and has been detected in high concentrations
ber. Since all the horses included in the previous study      in US water systems as well as in agricultural soils subject
were from the same farm, this study was undertaken to         to land application of biosolids. In this report, cell-based
determine whether seasonal alterations in ACTH con-           sex hormone receptor-mediated bioassays were used to
centrations occur in horses from different locations. We      demonstrate that TCC has little or no endocrine activity
evaluated 18 horses, 12 horses with pituitary pars inter-     when tested alone, but acts to enhance sex hormone-
media dysfunction and 6 unaffected animals, located on        induced steroid receptor-mediated transcriptional activity
15 different farms in East Tennessee to test the hypothesis   in vitro. This amplification effect of TCC was also
that seasonal alterations in ACTH concentrations occur        apparent in vivo when TCC was added to the diet of
with varying diets and husbandry methods. Eighteen            castrated male rats that were given exogenous testosterone




                                                                                                                               Clinical Sciences
horses (7 mares, 11 geldings) with a median (range) age       treatment for 10 days. All male sex accessory organs
of 19.8 (8 to 45) years were included in the study. Horses    increased significantly in size after the T+TCC treatment,
were sampled under fed (hay or grass) conditions every        compared with T or TCC treatments alone. The data
month across a 12-month period (August 2007 to July           presented here suggest that the bioactivity of endogenous
2008). Blood concentrations of hormones and metabolites       hormones may be amplified by exposure to commercial
were measured. Data were analyzed by mixed model for          personal care products containing sufficient levels of
repeated measures. Plasma ACTH concentrations were            TCC and TCC represents a new category of endocrine-
significantly affected by season (time effect; p< 0.001),     disrupting substance.
with a significantly higher mean ± SD concentration
(155.5 ± 292.6 pg/mL) detected in August, compared with
all other months. There was no effect of season on serum      46. Effects of Metformin Hydrochloride
insulin concentrations. Mean ACTH concentration was           on Glucose Dynamics During Transition
higher (p = 0.0202) in horses with PPID, but disease
status did not significantly alter the seasonal effect. We
                                                              to Grass Paddocks in Insulin-Resistant
conclude that horses from different locations, with varying   Horses
diets and husbandry practices, have significantly higher
plasma ACTH concentrations in August.                         Kelly Chameroy, Nicholas Frank, Sarah B. Elliott
                                                              Department of Large Animal Clinical Sciences

                                                              Metformin hydrochloride is used as a treatment for
                                                              insulin resistance (IR) in equids. This drug may also be
                                                              useful for controlling IR when insulin-resistant horses
                                                              are reintroduced to pasture. We therefore tested the
                                                              hypothesis that metformin alters glucose dynamics in
                                                              insulin-resistant horses undergoing reintroduction to
                                                              grazing. Six adult horses with chronic IR were transferred
                                                              to grass paddocks after spending >15 months confined




                                                                31
                    in stalls with attached gravel turnout areas. Glucose         With the exception of 1 horse, all animals treated with
                    dynamics were assessed using the combined glucose-            LPS exhibited tachycardia. Laminitis was not detected.
                    insulin test (CGIT) before and at 1, 2, 4, 6, 8, and 10       A significant time effect was detected for area under the
                    weeks after horses were transferred to grass paddocks.        glucose curve, but groups did not differ significantly. No
                    After horses were housed in grass paddocks for 2 weeks,       differences were detected between the 2 models, and
                    they were treated with metformin (15 mg/kg q12h) for 2        most horses appeared to be more tolerant of LPS after 24
                    weeks, taken off treatment for 2 weeks, and then treated      hours. These models increased the duration of systemic
                    with metformin at a high dosage (30 mg/kg q12h) for 2         inflammation, but did not match the severity of naturally
                    weeks. Horses were also reevaluated 2 weeks later. Time       occurring disease.
                    effects were significant (p < 0.001) for resting plasma
                    glucose concentrations and area under the glucose curve
                    (AUCg) values. Insulin results are pending. Mean glucose      48. Tear, Cornea, and Aqueous Humor
                    concentrations increased from 81.2 ± 3.4 to 99.4 ± 6.7
                    mg/dL over 1 week and then decreased to 81.9 ± 4.0 mg/
                                                                                  Concentrations of Ciprofloxacin and
                    dL by 10 weeks. No differences were detected between          Moxifloxacin after Topical Application in
                    pre- and post-metformin mean values for resting glucose       Normal Horses
                    concentrations or AUCg at either dosage level. However,
                    mean AUCg significantly decreased (18.6%) across the          Hans D. Westermeyer, Diane V.H. Hendrix, Daniel A.
                    10-week study period. Metformin did not affect glucose        Ward, Sherry K. Cox
                    dynamics in this study, but AUCg decreased over time          Department of Small Animal Clinical Sciences (Westermeyer,
                    when insulin-resistant horses were transferred to grass       Hendrix, Ward), Department of Comparative Medicine (Cox)
                    paddocks.
                                                                                  Our objective was to determine ocular tissue drug
                                                                                  concentrations after topical administration of 0.3%
                    47. Assessment of Two Experimental
Clinical Sciences




                                                                                  ciprofloxacin and 0.5% moxifloxacin in 28 adult
                                                                                  horses with normal ophthalmic examinations. Both
                    Models for Prolonged (48 Hours)
                                                                                  antimicrobials (0.1 ml) were delivered to the ventral
                    Endotoxemia in Horses                                         conjunctival fornix as follows: Group 1 (n = 8) hours 0,
                                                                                  2, 4, 6; Group 2 (n = 8) hours 0, 2, 4, 6, 10; and Group 3
                    Lisa Tadros, Nicholas Frank                                   (n = 8) hours 0, 2, 4, 6, 10, 14. Tears, cornea, and aqueous
                    Department of Large Animal Clinical Sciences                  humor (AH) were collected at 8, 14, and 18 hours for
                                                                                  groups 1, 2, and 3, respectively. Drug concentrations were
                    Lipopolysaccharide (LPS) is detected in the blood of
                                                                                  determined by high performance liquid chromatography.
                    horses after experimental alimentary carbohydrate
                                                                                  Concentration (µg/ml) of ciprofloxacin (median [25%–
                    overload and may contribute to the development of
                                                                                  75%]) for groups 1, 2, and 3 in tears was 53.67 (25.47–
                    laminitis by inducing systemic inflammation. Models for
                                                                                  88.75), 48.52 (19.65–74.66), and 24.35 (15.43–67.05),
                    endotoxemia have focused upon single bolus infusions
                                                                                  respectively; in cornea (µg/g), 0.945 (0.599–1.02), 0.367
                    of LPS in the past, but clinical endotoxemia has a greater
                                                                                  (0.323–0.474), and 0.480 (0.340–0.952), respectively; in
                    magnitude and duration. The objective of this preliminary
                                                                                  AH, they were below quantification levels in all groups.
                    study was therefore to assess 2 models of prolonged (48
                                                                                  Concentrations of moxifloxacin for groups 1, 2, and 3
                    hours) endotoxemia. Twelve healthy adult horses were
                                                                                  in tears were 188 (44.5–669.2), 107 (41.7–296), and
                    randomly assigned to receive a continuous rate infusion
                                                                                  178 (70.1–400), respectively; in cornea, 1.841 (1.440–
                    (CRI) of LPS (80μg total over 48 h; n = 4), a saline CRI
                                                                                  2.109), 0.777 (0.552–0.980), and 0.769 (0.647–0.966),
                    (80 mL/h for 48 h; n = 4), or 8 boluses of LPS (10μg q6h
                                                                                  respectively; in AH (µg/ml), 0.0550 (0.037–0.075),
                    for 48 h; n = 4). Physical parameters were recorded every
                                                                                  0.0340 (0.0208–0.0475), and 0.0195 (0.006–0.043),
                    4 hours, and intravenous glucose tolerance tests were
                                                                                  respectively. Group 1 corneal moxifloxacin concentrations
                    performed at –24, 8, 24, 32, and 48 hours. A treatment ×
                                                                                  were significantly higher than groups 2 and 3 (P = 0.003).
                    time effect (p< 0.001) was detected for rectal temperature,
                                                                                  Topical fluoroquinolones reach therapeutic levels in tears
                    with LPS groups differing significantly from the saline
                                                                                  and cornea even when there is an intact epithelium.
                    group. Mean rectal temperature was significantly higher
                    than baseline from 4 to 12 hours and again at 20 hours
                    but did not increase significantly again after this point.




                                                                         32
49. Regional Differences in Adipose                             50. Transient Silencing of Atp10c mRNA
Tissue in the Sinclair Minipig                                  in C2C12 Myotubes Decreases Glucose
                                                                Uptake Potentially via MAPK Signaling
Suzanne Booker, Cheryl Kojima, Shirley Newman, Julia
Gouffon, Steven Minkin, John Biggerstaff, Hank Kattesh,
                                                                Pathway
Naima Moustaid-Moussa
Department of Animal Science (Booker, Kojima, Kattesh,          Sarah Hurst, Madhu Dhar
Moustaid-Moussa), Department of Pathobiology (Newman),          Comparative and Experimental Medicine Program (Hurst),
Comparative and Experimental Medicine Program (Gouffon),        Department of Large Animal Clinical Sciences (Dhar)
Center for Environmental Biotechnology (Minken, Biggerstaff),
UT Obesity Research Center (Booker, Kojima, Moustaid-           Atp10c, a putative phospholipid translocase, appears to
Moussa)                                                         be a strong candidate for diet-induced obesity and type II
                                                                diabetes. Skeletal muscle is responsible for the majority
Obesity has reached epidemic proportions worldwide, and         of insulin-stimulated glucose clearance via the stimula-
mounting evidence has implicated adipose tissue endo-           tion of glucose uptake into targeted body tissues. Defects
crine and inflammatory function in obesity-associated           in this action contribute to the development of insulin
co-morbid conditions. Further, different adipose depots,        resistance and type II diabetes. Studies revealed that dur-
visceral (VF) and subcutaneous (SQF), exhibit differential      ing myogenesis, Atp10c expression increases significantly
physiological properties and metabolic profiles. In this        along with increased MyoD expression (p<0.05). Using
study, we sought to elucidate and characterize depot-spe-       siRNA technology, we were able to achieve a greater than
cific differences in the Sinclair minipig, a porcine model      90% knockdown of Atp10c in myotubes derived from a
with similar physiologic properties to humans. RNA              murine C2C12 skeletal muscle cell line. In vitro studies
was isolated from 8-month-old minipigs and processed            revealed that when Atp10c gene expression was knocked
using the Porcine Affymetrix Gene Chip oligonucleotide          down, glucose uptake significantly decreased (>2 fold),
array. Only transcripts for which expression differed           results of which compliment in vivo data obtained from
between treatments by 2-fold or greater with Step-up, p         Atp10c /ATP10C heterozygous mice. As signal transduc-
value < 0.05 were noted. Using this cut off, we identi-         tion is crucial for glucose uptake and many pathways are
fied 598 transcript IDs whose expression changed by >2          involved, we sought to identify Atp10c’s effect on known
fold. From this subset of the transcriptome, 471 IDs were       signal transduction pathway proteins. Our results indi-
up-regulated vs. 127 down- regulated in SQF compared            cate that Atp10c may work in either one or more of the
to VF. Interestingly, the genes that that were up-regulated     MAPK pathways as seen in changes of protein expression.
in SQF vs. VF included both pro- and anti-inflammatory          Transient siRNA transfection of myotubes significantly
genes such as ESR 1, while those down-regulated includ-         (p<0.05) altered the level of expression of several proteins
ed pro-inflammatory and lipogenic genes such as IL-18           involved in signal transduction, notably the proteins p38,
and FASN, respectively. Histological analysis of adipose        p44/42 and JNK. We also observed significant (p<0.05)
tissue from minipig and commercial pigs revealed that           changes in Actin and MyoD expression, which were pos-
minipig but not commercial pig SQF contained significant        sibly affected by changes in the MAPK proteins. As such,
connective tissue compared to VF. Moreover, VF but not          further characterization of this protein is ongoing.
SQF from commercial pigs expressed significant levels
of calprotectin, a monocyte and immature macrophage

                                                                                                                               Nutrition & Metabolism
membrane marker, as assessed by immunofluorescence,
compared to VF. In conclusion, these studies support the
relevance of the porcine model for human obesity and
studies of adipocyte biology and inflammation. Supported
by USDA AFRI, UT AgResearch, and the UT-ORC.




                                                                  33
                         51. Anti-inflammatory and Metabolic                           52. Effects of Bisphenol A on Adipocyte
                         Mechanisms by which Omega-3 Fatty                             Growth, Metabolism, and Endocrine
                         Acids Improve Insulin Resistance in High                      Functions
                         Fat Diet-induced Obese Mice
                                                                                       Brantley Wyatt, Jessica Gooding, Shawn Campagna,
                                                                                       Brynn Voy
                         Nishan S Kalupahana, Kate Claycombe, Taryn Stewart,           Genome Science and Technology Program (Wyatt, Voy),
                         Rachael Hadidsaz, Sarah J Fletcher, Suzanne Booker,           Department of Animal Science (Voy), Department of Chemistry
                         Nalin Siriwardhana, Naima Moustaid-Moussa                     (Gooding, Campagna)
                         Obesity Research Center and Department of Animal
                         Science (Kalupahana, Stewart, Hadidsaz, Feltcher, Booker,     While diet and sedentary lifestyle remain important fac-
                         Siriwardhana, Moustaid-Moussa), Michigan State University     tors in the development of obesity, recent findings have
                         (Claycombe)                                                   shown the possible involvement of environmental obe-
                                                                                       sogens, chemicals that can disrupt homeostatic energy
                         In this study, we investigated the effects of
                                                                                       balance and increase adiposity. Bisphenol A (BPA) is
                         eicosapentaenoic acid (EPA) on prevention (P) and
                                                                                       a compound ubiquitous in the environment due to its
                         reversal (R) of high-fat (HF) diet-induced obesity and
                                                                                       widespread use in manufacturing of plastics. BPA has
                         metabolic alterations. Male C57BL/6J mice were fed a
                                                                                       been shown to be an endocrine disruptor through its
                         low-fat (LF), HF or a HF-EPA-P diet for 11 weeks. A
                                                                                       ability to mimic estrogen, which is now known to play
                         fourth group was initially fed the HF diet for 6 weeks
                                                                                       important roles in adipose tissue growth and metabolism.
                         followed by the HF-EPA-R diet for 5 weeks. As
                                                                                       In fact, a number of studies have shown that mice ex-
                         expected, mice fed the HF diet developed obesity and
                                                                                       posed to BPA in utero or postnatally are fatter as adults.
                         glucose intolerance. In contrast, mice fed HF-EPA-P diet
                                                                                       In humans, BPA is detectable in urine of over 90% of the
                         maintained normal glucose tolerance despite weight gain
                                                                                       population, and higher levels of BPA are associated with
                         when compared to the LF group. While the HF group
                                                                                       increased incidence of heart disease. We hypothesize that
                         developed fasting hyperglycemia and hyperinsulinemia,
                                                                                       BPA exposure exerts effects on adipose tissue, promot-
                         both HF-EPA groups (P and R) exhibited normal glycemia
                                                                                       ing adipogenesis and inflammation, and altering energy
                         and insulinemia, demonstrating EPA’s ability to prevent
                                                                                       homeostasis that promotes obesity and insulin resistance.
                         and improve insulin resistance associated with high fat
                                                                                       We treated adipose tissue ex vivo with BPA and looked at
                         feeding. Analysis of adipokines revealed that plasma
                                                                                       the effects on metabolite profiles and expression of select
                         adiponectin levels were lower in the HF group, but were
                                                                                       genes. Metabolomics data indicate that BPA-treatment
                         comparable between LF and HF-EPA groups, regardless
                                                                                       altered adipose glycolysis and promoted de novo lipogen-
                         of the high fat content of the diet. Further analysis of
                                                                                       esis. BPA treatment also increased the expression of the
                         adipose tissue adipokine levels and proteomic studies
                                                                                       pro-inflammatory adipokines tumor necrosis factor alpha
                         in cultured adipocytes demonstrated that EPA reduces
                                                                                       and interleukin-6. We also found that BPA increased
                         adipose inflammation and lipogenesis, while increasing
                                                                                       proliferation in rat mesenchymal stem cells, suggesting
                         markers of fatty acid oxidation and mitochondrial content.
                                                                                       that BPA may promote adipogenesis through increasing
                         These studies demonstrate that beneficial effects of EPA in
                                                                                       the number of adipocyte precursors. These data support
                         improving insulin resistance are in part mediated by EPA’s
                                                                                       the theory that BPA may disrupt energy homeostasis in
                         lipid oxidizing and anti-inflammatory effects.
Nutrition & Metabolism




                                                                                       adipose, promoting adipocyte expansion and proliferation,
                                                                                       as well as inflammation.


                                                                                       53. Leucine Stimulation of Fatty Acid
                                                                                       Oxidation via an mTOR-independent
                                                                                       Mechanism

                                                                                       Kenny Smart, Michael Zemel, Xiaocun Sun
                                                                                       Department of Nutrition

                                                                                       Leucine as a nutrient has been highlighted recently for




                                                                               34
its effects on mammalian target of rapamycin (mTOR)             will reduce adiposity-associated inflammation. Quercetin
and subsequent stimulation of protein synthesis.                is one of the most widely available dietary flavonoids
Because leucine affects a key energetic system involving        (found in apples, onions, teas, red wines, citrus, garlic,
anabolism of protein, catabolic systems are also                parsley and many other foods). It is a multifunctional
stimulated to fuel this process, resulting in increased fatty   natural compound with potent anti-obesity, anti-
acid oxidation. This project was designed to determine          inflammatory, anti-diabetic, anti-cancer, anti-hypertensive
the mechanisms through which leucine stimulates fatty           and anti-oxidant activities. Therefore, we have selected
acid oxidation on skeletal muscle cells. As first steps,        quercetin to test against RZ-induced adiposity. Also we
we sought to determine inhibition of mTOR signaling             further extended our investigations to understand the
with rapamycin or of translational elongation in protein        mechanisms by which quercetin can reduce RZ-induced
synthesis with cyclohexamide. Rapamycin (20nM) or               adiposity. Co-treatment of quercetin with RZ significantly
cycloheximide (0.7 uM, 0.1 uM, and 0.07 uM) were                suppressed the RZ-induced differentiation of 3T3-L1
used in the presence or absence of leucine (0.5 mM) for         preadipocytes into mature adipocytes. Further, quercitin
4, 24, or 48 hours in C2C12 myotubes. Valine was used           significantly decreased RZ-induced lipid accumulation
as a control for non-specific effects of branched chain         in mature differentiated adipocytes. Moreover, quercetin
amino acids in all experiments. Fatty acid oxidation with       significantly reduced the release of pro-inflammatory
3H-Pamitic acid and subsequent protein correction was           cytokines (especially monocyte chemoattractant protein
performed for each treatment. Leucine stimulated fatty          [MCP-1], IL-6, resistin and plasminogen activator
acid oxidation by 50% (p = 0.03); addition of rapamycin         inhibitor-1[PAI-1]) and adipogenic protein markers
inhibited fatty acid oxidation but did not prevent              (specially fatty acid synthetase [FAS]) from differentiating
leucine stimulation of fat oxidation. Similarly, leucine        preadipocytes. In summary, quercetin is an effective anti-
stimulated fatty acid oxidation by 40% (p<0.001) above          obesity and anti-inflammatory compound that can be used
cycloheximide control. Thus, leucine stimulation of fatty       as an effective co-treatment with RZ to overcome RZ-
acid oxidation appears to be independent of stimulation of      induced adiposity and adiposity-associated inflammation.
mTOR signaling and protein synthesis, and the pathway(s)
activated remain to be determined.
                                                                55. Enhancing Male Reproductive
                                                                Potential by Herbal Tea Therapy
54. Quercetin Overcomes Rosiglitazone-
associated Adiposity and Adiposity-                             Marcos Gomes, Juliana Monteiro, Heidi Dolder,
associated Inflammation                                         Chamindrani Mendis-Handagama
                                                                Department of Comparative Medicine (Gomes, Mendis-Handa-
                                                                gama), Department of Anatomy, Cell Biology and Physiology,
Nalin Siriwardhana, Wenting Xin, Nishan Kalupahana,
                                                                State University of Unicamp, Brazil (Monteiro, Dolder)
Avik Mukherjee, Suzanne Booker, Arnold M. Saxton,
Naima Moustaid-Moussa                                           Heteropterys aphrodisiaca, a native Brazilian plant
Department of Animal Science (Siriwardhana, Xin,
                                                                popularly known as “nó-de-cachorro,” is commonly used
Jukherjee, Booker, Moustaid-Moussa), UT Obesity Research
(Siriwardhana, Kalupahana, Moustaid-Moussa)                     as an herbal tea to treat nervous and muscular disorders.
                                                                Previous studies have shown that infusion of this plant
                                                                results in increasing seminal vesicle mass, as well as the

                                                                                                                                Nutrition & Metabolism
The high prevalence of obesity, the most serious
escalating global health problem, leads to increased risk       Leydig cell population in adult rats, suggesting that it has
for metabolic, cardiovascular and inflammatory diseases,        the potential to increase testicular testosterone production.
including type 2 diabetes (T2D). Currently used effective       Therefore, the objective of the present study was to test
T2D treatments include rosiglitazone (RZ), a potent             whether H. aphrodisiaca can be used as an herbal tea ther-
ligand for the adipogenic transcription factor peroxisome       apy to males, in order to enhance their testicular testoster-
proliferator-activated receptor gamma (PPARγ). Despite          one production using the rat model. Two groups of adult
effectiveness of this drug in treating T2D, its side effects    rats, i.e. controls and tea-treated, were gavaged water or
are of concern and include increasing adiposity as well as      tea daily, respectively, for 56 days (duration of spermato-
consequently worsening inflammation. We hypothesized            genic cycle in rats). Blood and testis tissue were collected
that a carefully selected multifunctional natural bioactive     for hormonal analysis and light microscopic morphometry
compound may counteract RZ’s negative effects and also          of testis, respectively. Results revealed that the individual
                                                                Leydig cell volume (or size), the circulating testosterone




                                                                  35
                                 levels and spermatogenic yield in tea-treated rats were         57. Early Growth Response-1 and
                                 significantly higher compared to those in controls. These       Krüppel-Like Factor 4 Regulate
                                 findings indicate that H. aphrodisiaca, when given as a
                                 daily herbal tea therapy, results in increasing the Leydig      Resveratrol-induced Activating
                                 cell volume and their testosterone producing potential          Transcription Factor 3 Expression
                                 together with improving the spermatogenesis yield.
                                                                                                 Nichelle C. Whitlock, Jae Hoon Bahn, Seung Joon Baek
                                                                                                 Department of Pathobiology
                                 56. Anticancer Activity of Damnacanthal
                                                                                                 Resveratrol, a dietary phytoalexin readily available in the
                                 from Noni (Morinda citrifolia) in                               diet, is reported to possess anti-tumorigenic properties
                                 Colorectal Cancer Cells                                         in several cancers, including colorectal. However, the
                                                                                                 underlying mechanism(s) involved is not completely
                                 Thararat Nualsanit, Seung Joon Baek                             understood. In the present study, we investigated the
                                 Department of Pathobiology                                      effect of resveratrol treatment on gene modulation in
                                                                                                 HCT-116 human colorectal cancer cells and identified
                                 Morinda citrifolia (Noni) grows extensively throughout          activating transcription factor (ATF) 3 as the most highly
                                 the South Pacific and was at one time the most widely           induced gene after treatment. We confirm resveratrol up-
                                 used medicinal plant in the region. In addition, this           regulates ATF3 expression, both at the mRNA and protein
Oncology & Cancer Cell Biology




                                 evergreen shrub grows especially well in the rich               level, and showed resveratrol involvement in ATF3
                                 volcanic ash of Hawaii. Damnacanthal, an anthraquinone          transcriptional regulation. Analysis of the ATF3 promoter
                                 compound isolated from the root of Morinda citrifolia           revealed the importance of early growth response-1
                                 L. shows beneficial effects on several diseases. Although       (Egr-1; located at -245 to -237) and Krüppel-like factor
                                 it has been shown to inhibit Ras oncogene functions in          4 (KLF4; located at -178 to -174) putative binding sites
                                 cancer, the molecular mechanism by which this compound          in resveratrol-mediated ATF3 transactivation. Specificity
                                 affects tumorigenesis has not been elucidated. In the           of these sites to the Egr-1 and KLF4 protein was
                                 present study, we characterized comprehensive anti-             confirmed by electrophoretic mobility shift and chromatin
                                 cancer activity of damnacanthal on human colorectal             immunoprecipitation assays. Resveratrol increased Egr-1
                                 carcinoma cells. Damnacanthal significantly inhibited the       and KLF4 expression, which preceded ATF3 expression;
                                 proliferation of colorectal cancer cells in a dose- and time-   this result further suggests Egr-1 and KLF4 involvement
                                 dependent manner as assessed by cell proliferation assay.       in resveratrol-mediated activity. Furthermore, we
                                 Damnacanthal also showed inhibitory activity on cell            found that Egr-1 and KLF4 act synergistically in ATF3
                                 migration and colony formation in HCT-116 cells. Finally,       transactivation and provide evidence for their interaction,
                                 we investigated the expression of several proteins that are     both of which are increased after resveratrol treatment.
                                 involved in tumorigenesis and apoptosis. Our data reveal        Egr-1 and KLF4 interaction may facilitate ATF3
                                 that damnacanthal induces nonsteroidal anti-inflammatory        transcriptional regulation by resveratrol. Taken together,
                                 drug (NSAID)-activated gene one (NAG-1) expression              these results provide a novel mechanism by which
                                 and reduces cyclin D1 expression in human colorectal            resveratrol induces ATF3 expression and represent an
                                 cancer cells, whereas p53, p21 and ATF3 expression do           additional explanation of how resveratrol exerts its anti-
                                 not change. Overall, our data indicate that damnacanthal        tumorigenic effects in human colorectal cancer cells.
                                 shows a unique feature of protein expression compared
                                 to other phytochemicals, and provide a rationale for the
                                 use of damnacanthal in the treatment of colorectal cancer
                                 prevention.




                                                                                        36
58. Targets for Dietary Grape Seed                             59. Medicinal Herbs Inhibit Prostate
Extract-Induced Suppression of Breast                          Cancer (PCa) Through p 21 via
Cell Chronic Carcinogenesis                                    Epigenetic Regulation
Kusum Rathore, Xiaoyu Song, Nalin Siriwardhana,                E-Chu Huang, Guoxun Chen, Seung Baek, Michael
Hwa-Chain Robert Wang                                          McEntee, Jay Whelan
Department of Comparative Medicine (Rathore, Song, Siri-       Department of Nutrition (Huang, Chen, Whelan), Department of
wardhana, Wang), Genomic Science and Technology Program        Pathobiology (Baek, McEntee)
(Rathore, Wang)
                                                               In the last few years, prostate cancer (PCa) became the
Breast cell carcinogenesis is a multiyear, multistep, and      second leading cause of cancer-related deaths in the
multipath disease process. Epidemiologic and experimen-        United States. Advanced PCa is the last stage of PCa
tal evidence suggests that dietary constituents in fruits      where cancer cells metastasize to other organs, mostly to
and vegetables prevent human cancers; dietary preven-          bone. The typical therapy for advanced PCa is hormone
tion is a cost-efficient strategy to reduce the risk of this   ablation therapy (HAT); however, following regression,
disease. To identify dietary components for prevention         the tumor eventually re-grows and is invariably fatal.
of chronic human breast cell carcinogenesis, we studied        Therefore, novel forms of adjuvant therapy to enhance
biological, biochemical, and molecular target endpoints        the effectiveness of HAT need to be investigated. Some




                                                                                                                               Oncology & Cancer Cell Biology
that were induced by low doses of carcinogens and sup-         experimental studies showed that medicinal herbs inhibit
pressed by dietary grape seed proanthocyanidin extract.        prostate cancer growth, but the benefits in clinical trials
Immortalized, non-cancerous human breast epithelial            are unclear. In recent studies, a combination of 10 me-
MCF10A cells were repeatedly exposed to carcinogens            dicinal herbs, Zyflamend, has been shown to reduce the
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and/            aggressiveness of early stages of PCa in patients. This
or benzo[a]pyrene, each at picomolar concentrations, to        clinical evidence provides sufficient rational for the use of
induce cancer-associated properties in cells. We then used     Zyflamend in advanced PCa. Using experimental models
these changes as target endpoints to reveal the activity of    of advanced PCa, we observed that Zyflamend reduced
GSPE in suppression of NNK- and B[a]P-induced cellu-           tumor cell growth by increasing cell cycle inhibitors via
lar carcinogenesis. We identified that cells acquiring the     epigenetic regulation. p21, a protein that inhibits cell
biological properties of reduced dependence on growth          cycle, was up-regulated in the presence of Zyflamend.
factors, anchorage-independent growth, and acinar-con-         Increase in p21 was associated with decreases of histone
formational disruption were reduced by treatment with          deacetylases, key mediators of cancer promotion and
dietary catechins. We identified that hydroxysteroid (11-      progression by regulating Erk1/2 signaling. Other media-
beta) dehydrogenase 2 (HSD11B2) played a novel role in         tors of advanced PCa, IGF1R, AKT and androgen recep-
NNK- and B[a]P-induced cellular carcinogenesis, and its        tor, also were down-regulated by Zyflamend. In addition,
expression acted as a molecular target endpoint in GSPE-       these effects were not observed with the individual herbs.
induced suppression of cellular carcinogenesis. The            In summary, advanced prostate cancer is very aggressive
ability of GSPE to reduce gene expression of cytochrome        due to the complicated survival mechanisms, and Zyfla-
P450 enzymes CYP1A1 and CYP1B1, which can bioac-               mend, consisting of polyherbal components, appears to be
tivate NNK and B[a]P, may be involved in the preventive        effective by coordinately modulating multiple signaling
mechanism for GSPE in suppression of cellular carcino-         pathways that negatively affect tumor growth.
genesis.

                                                               60. Capsaicin Inhibits β-catenin Pathway
                                                               in Colorectal Cancer
                                                               Raphael L. Richardson, Seong-Ho Lee
                                                               Comparative and Experimental Medicine Program
                                                               (Richardson), Department of Pathobiology (Lee)

                                                               Capsaicin, a compound in the chili peppers, is used as a
                                                               pain reliever by blocking or interfering with neurotrans-




                                                                 37
                                 mitter substances; however, recent data suggest that           cancers including colon, breast, pancreas, and prostate.
                                 capsaicin also modulates tumor cell growth. We have            EpCAM was recently described to undergo 2 cleavage
                                 previously reported that capsaicin induces apoptosis and       events, an extracellular cleavage that frees the potentially
                                 cell growth arrest in colorectal cancer cells. To elucidate    oncogenic outer portion of the protein, and the release of
                                 the molecular mechanisms by which capsaicin induces            the intracellular region of EpCAM that complexes with
                                 anti-tumorigenesis, we examined the β-catenin pathway.         beta-catenin and, once freed, translocates to the nucleus to
                                 Capsaicin decreased the amount of β-catenin and Cyclin         initiate transcription of beta-catenin target genes. NSAID
                                 D-1 expression most dramatically at a dose of 100 μM.          down regulation of pro-tumorigenic EpCAM could
                                 When the GFP-β-catenin construct was transfected into          provide a novel mechanism to further understand COX-
                                 SW480 cells, capsaicin reduced the expression of both en-      independent NSAID anti-cancer activity.
                                 dogenous and exogeneous β-catenin, in both the nucleus
                                 and cytosol. When the proteasome inhibitor MG-132 was
                                 used, β-catenin degradation by capsaicin was not ob-           62. Anti-cancer Activity of
                                 served, indicating that capsaicin affects a protein degrada-
                                 tion pathway, thereby reducing β-catenin expression. This
                                                                                                Cyclooxygenase Inhibitors in Colorectal
                                 result was confirmed by experiments with puromycin, a          Cancer
                                 protein synthesis inhibitor. Finally, TOPflash construct
                                 containing 4 copies of β-catenin response elements trans-      Seong-Ho Lee, Jae Hoon Bahn, Nichelle C. Whitlock,
Oncology & Cancer Cell Biology




                                 fected and luciferase activity was measured. Capsaicin         Seung Joon Baek
                                 decreases TOPflash activity, suggesting that capsaicin         Department of Pathobiology
                                 decreases not only protein but also activity of β-catenin,
                                 overall. Our results imply that capsaicin may be a chemo-      Tolfenamic acid (TA) is a non-steroidal anti-inflammatory
                                 preventive compound to prevent colorectal cancer through       drug associated with anti-tumorigenic and pro-apoptotic
                                 the inhibition of the β-catenin pathway.                       properties in animal and in vitro models of cancer. How-
                                                                                                ever, the underlying cellular mechanisms by which TA
                                                                                                exerts its effects are only partially understood. Activat-
                                                                                                ing transcription factor 3 (ATF3) is a member of the
                                 61. Sulindac Sulfide Suppresses Epithelial                     ATF/CREB subfamily of the basic region-leucine zip-
                                 Cell Adhesion Molecule (EpCAM)                                 per family and has been known as a tumor suppressor
                                 Expression in Colorectal Cancer Cells                          in human colorectal cancer cells. The present study was
                                                                                                performed to observe whether ATF3 mediates TA-induced
                                                                                                apoptosis and to elucidate the molecular mechanism of
                                 Jason L. Liggett, Seung J. Baek
                                 Department of Pathobiology                                     ATF3 transcription induced by TA. TA treatment and
                                                                                                ectopic expression of ATF3 increased apoptosis, whereas
                                 Non-steroidal anti-inflammatory drugs (NSAIDs) are             knockdown of ATF3 resulted in significant repression of
                                 extensively used over the counter to treat headaches           TA-activated apoptosis. The TA treatment induced ATF3
                                 and inflammation as well as clinically to prevent              promoter activity. Internal deletion and point mutation of
                                 cancer among high-risk groups. The inhibition of               the predicted ATF/C/EBP binding site in ATF3 promoter
                                 cyclooxygenase (COX) activity by NSAIDs plays a role           abolished luciferase activation by TA. Overexpression of
                                 in their anti-tumorigenic properties. NSAIDs also have         ATF2 resulted in significant increase of ATF3 promoter
                                 COX-independent activity, which is not fully understood.       activity, and EMSA identified this region as a core se-
                                 Our lab has previously shown that NSAIDs induce other          quence to which ATF2 binds. TA treatment resulted in an
                                 anti-tumorigenic genes including the NSAID-activated           increase of ATF2 phosphorylation, which was followed
                                 gene (NAG-1) and activating transcription factor 3             by a subsequent increase of ATF3 transcription. Knock-
                                 (ATF3). In this study, we report that sulindac sulfide         down of ATF2 abolished TA-induced ATF3 expression.
                                 (SS), a conventional NSAID, down-regulates epithelial          We further provide evidence that TA leads to increases of
                                 cell adhesion molecule (EpCAM) protein expression in           phospho-p38 MAPK, JNK, and ERK levels. Inhibition
                                 a COX-independent manor; interestingly, this occurs            of these pathways using selective inhibitors ameliorated
                                 independent of transcriptional regulation. EpCAM               TA-induced ATF3 expression and promoter activities.
                                 is a type I transmembrane glycoprotein that has been           The current study demonstrates that TA stimulates ATF3
                                 implemented as an over-expressed oncogene in many              expression and subsequently induces apoptosis. These




                                                                                       38
pathways are mediated through phosphorylation of ATF2,         64. Effect of Hyperbaric Oxygen
which is mediated by p38 MAPK, JNK, and ERK-depen-             Treatment on Canine Mesenchymal Stem
dent pathways.
                                                               Cells

63. Magnetic Resonance Imaging of the                          Maria Cekanova, Dennis Geiser, Darryl Millis, Dianna
                                                               Whitlock, Alfred Legendre, Madhu Dhar




                                                                                                                               Clinical Sciences
Temporomandibular Joint in Normal Dogs                         Department of Small Animal Clinical Sciences (Cekanova,
                                                               Millis, Whitlock, Legendre), Department of Large Animal
Dawn Macready, Silke Hecht, Linden Craig, Gordon               Clinical Sciences (Geiser, Dhar)
Conklin
Department of Small Animal Clinical Sciences (Macready,        Mesenchymal stem cells (MSC) originate from adult
Hecht, Conklin), Department of Pathobiology (Craig)            organs, such as bone marrow, or fat and have a capability
                                                               to self regenerate and differentiate into specific cells
Evaluation of the canine temporomandibular joint (TMJ)         of bone, cartilage, muscle, or fat. MSC are used to
is important in the clinical diagnosis of animals presenting   replace and repair damaged tissue and to induce the
with dysphagia, malocclusion and jaw pain. In human            healing process. Hyperbaric oxygen therapy treatment
medicine, MRI is the imaging modality of choice for            (HBOTx) has been shown to be effective in improving
evaluation of the TMJ. The objectives of this study            and accelerating the healing process in many diseased
were to establish a technical protocol for performing          tissues; this effect is achieved through several different
MRI on the TMJ in dogs and describe the MRI anatomy            mechanisms. In our study we proposed to evaluate the
and appearance of the normal canine TMJ. Ten dogs (1           canine MSC from fat, bone marrow and peripheral blood
fresh cadaver and 9 healthy live dogs) were examined by        and to study the effect of HBOTx on the production
MRI. MR images were compared to pathologic sections            of circulating canine MSC into peripheral blood and
in the cadaver. T1-W transverse closed-mouth, T1-W             adipose tissue isolated from adult dogs. This study used
sagittal closed mouth, T1-W sagittal open-mouth and            6 healthy dogs where each dog was its own control.
T2-W sagittal open-mouth sequences were obtained. The          Venous blood was withdrawn; bone marrow and fat
condyloid process of the mandible and mandibular fossa         samples were surgically removed pre- and post- HBOTx
of the temporal bone were hyperintense to muscle and           with 100% oxygen at 2.5 atmospheres of pressure for
isointense to hypointense to fat on T1 weighted images,        60 minutes for 3 days from each dog. Canine MSC
mildly hyperintense to muscle on T2 weighted images,           population was isolated and characterized by capacity
and were frequently heterogeneous. The articular disc          of MSC differentiation into specific type of cells. Our
was visible in 14/20 (70%) TMJs on T1-W images and             data indicate that HBOTx caused a significant increase
13/20 (65%) TMJs on T2-W images. The disc was                  of the production of MSC in bone marrow and then
isointense to hyperintense to muscle on T1-W images            released canine MSC into peripheral blood circulation.
and varied from hypointense to hyperintense to muscle          Increased circulating pluri-potent MSC may be one of the
on T2-W images. The lateral collateral ligament was            mechanisms by which HBOT improves tissue healing.
not identified in any joint. MRI allows evaluation of the
osseous and certain soft tissue structures of the TMJ in
dogs.                                                          65. Equine Progenitor Cells – Potential
                                                               Role in Basic and Translation Research
                                                               Madhu Dhar, Nancy Neilsen, Shannon Eaker, Steve
                                                               Adair, Dennis Geiser
                                                               Department of Large Animal Clinical Sciences (Dhar, Adair,
                                                               Geiser), Department of Pathobiology (Neilsen), Millipore Cor-
                                                               poration, Billerica, MA (Eaker)

                                                               Equine regenerative medicine is an exciting but relatively
                                                               new research field. Adult stem cells, also referred to as
                                                               multipotent mesenchymal stromal cells, are plastic-ad-
                                                               herent cells that express CD antigen surface markers and




                                                                 39
                    demonstrate tri-lineage differentiation potential towards        control animal. Percent of activated lymphocytes in the
                    osteoblasts, adipocytes, and chondroblasts. Adipose              implanted animal were also elevated in comparison to the
                    tissue, bone marrow and peripheral blood are the com-            control animal by a difference of 26.09%. The differences
                    monly used sources of adult mesenchymal stromal cells.           in the amount of fibrin and fibrinogen showed a 0.22%
                    We have established protocols to isolate progenitor stem         and 0.11%, respectively, in comparison to the control
                    cells from horse adipose tissue and peripheral blood. We         animal. These results show that implantation of ECMO
                    validated them by immunophenotyping using human CD               induced a severe inflammatory response in the treated
Clinical Sciences



                    antigen surface markers. After proliferation and expan-          animal, and confirm that we have developed a sensitive
                    sion, the progenitor cells stained positive for CD34,            quantitative assay for the measurement of inflammatory
                    CD45, CD51 and CD90. We have optimized protocols                 and clotting parameters.
                    for their in vitro differentiation into osteocytes, adipo-
                    cytes and chondrocytes. This is confirmed by alizarin red
                    (oesteocytes), alcian blue (chondrocytes) and oil-red-o          67. Effect of Order Set Utilization on
                    (adipocytes) staining, as well as by semiquantitative PCR
                    for lineage-specific expression of genes. Immunofluo-
                                                                                     Outcomes in Septic Shock in the Intensive
                    rescence assays are currently being optimized to identify        Care Unit
                    protein markers specific to the differentiated cell types.
                                                                                     Daniel R. Malcom, Barbara E. Faircloth
                                                                                     Department of Pharmacy, UT Medical Center
                    66. Quantitative Analysis of Inflammatory
                                                                                     Severe sepsis and septic shock continue to have high
                    Responses from Extracorporeal Membrane                           morbidity and mortality in emergency departments and
                    Oxygenation Device Implantation in an                            intensive care units worldwide. With the publication of
                    Animal Model                                                     the 2008 Surviving Sepsis guidelines, our institution
                                                                                     implemented a septic shock order set that included early,
                                                                                     goal-directed therapy and many high-level guideline
                    Patricia Brown, Steve Minkin, Michael Beeler, Benjamin
                                                                                     recommendations. No study to date has evaluated the
                    Curry, Linda Cahalan, Pat Cahalan, John Biggerstaff
                    Center for Environmental Biotechnology (Brown, Minkin,           2008 guidelines specifically included in an order set and
                    Beeler, Biggerstaff), Department of Nutrition (Curry), Ension,   their potential impact on mortality outcomes. The purpose
                    Inc. (Cahalan, Cahalan)                                          of this study is to determine if using a sepsis order set for
                                                                                     directed care affects outcomes, and to evaluate secondary
                    Extracorporeal membrane oxygenation (ECMO) can                   predictive factors for mortality in the total population that
                    activate circulating blood, resulting in significant             may be influenced by the order set.
                    inflammation and coagulation in downstream                       Our study is a retrospective cohort analysis comparing
                    organs. This study sought to design a methodology                patients who received order set-directed care for the
                    to quantitatively assess these parameters in several             treatment of septic shock to those who did not. All
                    tissues obtained from untreated and ECMO-exposed                 patients admitted to the Medical Critical Care service
                    animals using 3-dimensional fluorescence microscopy              from July 2009 through December 2009 with a diagnosis
                    and image analysis. After implantation, the animal               of septic shock will be included. Exclusion criteria include
                    was observed for 72 hours, and tissue samples were               age less than 18 years or known terminal diagnosis at time
                    collected from 5 organs from the control animal and              of admission. The primary endpoint is all-cause mortality,
                    4 organs from the experimental animal. The samples               with secondary endpoint of hospital length of stay. We
                    were immunofluorescently stained for fibrin, fibrinogen,         will also use a multivariable logistic regression analysis to
                    platelets and leukocytes and leukocyte activation markers.       determine potential predictive factors of mortality in these
                    Cells were quantified using mean fluorescence volumes            patients. Results and conclusions were incomplete at the
                    leukocytes using a seeded segmentation algorithm                 time of printing.
                    designed in our laboratory. In comparison to the control
                    animal, there was minimal difference in the accumulation
                    of platelets in the lungs. However, the monocytes
                    and polymorphs in the lungs of the implanted animal
                    displayed a difference in percentage of activated cells
                    14.32% and 21.93% higher, respectively, compared to the




                                                                             40
68. Labor Epidural Fever and Pyrogenic                          study has evaluated if either method results in more suc-
Cytokines                                                       cessful epidural placement and fewer complications with
                                                                CA 1 physician residents during their primary obstetrical
                                                                rotation. This study evaluated the number of attempts for
Cara Erickson, Stephen Patteson, Carolyn Snyder                 epidural placement, incidence of intravascular catheters,
Department of Anesthesiology (Patteson, Erickson), Department
                                                                inadvertent dural puncture, epidural replacement rate for
of Anesthesiology Research (Snyder)
                                                                labor analgesia and conversion from epidural to general




                                                                                                                                 Clinical Sciences
An association between epidural analgesia in laboring           anesthesia for cesarean section and bilateral tubal ligation.
women and maternal fever has been identified. A                 After obtaining IRB approval and patient consent, the
variety of human cytokines have been identified with            CA 1 resident assigned to the obstetrical unit sequentially
inflammation and fever. This study investigates cytokine        alternated between the air or saline LOR technique. Anes-
levels in laboring women prior to and after placement           thesia records were completed by the residents in compli-
of epidurals. The null hypothesis is that labor epidurals       ance with standard medico-legal documentation. Patient
do not elevate inflammatory cytokine levels causing             anesthesia charts were then reviewed by an independent
fever. After IRB approval and patient consent, blood            reviewer. Twelve patients were consented and admitted to
was drawn prior to epidural placement and every 4 hours         the study. Four patients had LOR with air and 8 patients
until delivery. Maternal temperature was assessed.              LOR with saline. No patient required ephedrine for hypo-
Quantitative IL-1β, IL-6, IL-8, TNF-α, and GM-CSF               tension. No patient required cesarean section for deliv-
levels were measured using the Millipex MAG human               ery. One patient in the saline group required more than 1
cytokine panel (Millipore; Billerica, MA). Six patients         attempt at epidural placement. One patient in the saline
were consented. Mean age was 25.0 years ± 7.8, mean             group experienced an intravascular catheter placement.
gestational age was 38.9 weeks ± 1.0, pre-epidural white        One patient in the air group required general anesthesia
blood cell count (WBC) was 10,700 ± 3.1, post-delivery          for bilateral tubal ligation for failed epidural reactivation.
WBC was 14,800 ± 4.2. The assay kit failed to measure           There were no statistical differences found between num-
data in 1 patient. All 6 patients had increased temperature     ber of attempts at placement, unintentional dural puncture,
from baseline. The range of temperature (T) increase            epidural replacement rate, intravascular catheters, ephed-
was between 0.9–2.2° F. None of the patients developed          rine use, BMI or age.
a fever >100.4°. There was no statistical significance
between baseline and cytokine levels 4 hours after
epidural placement. There appeared to be a positive             70. Ultrasonographic Observation of
correlation between GM-CSF and maximum temperature              Secretin-induced Pancreatic Duct Dilation
after delivery. This is the presentation of the initial
patients consented. As the study progresses, we expect          in Healthy Cats
to have patients with fever > 100.4° F. Human cytokine
levels as measured by IL-1β, IL-6, IL-8, TNF-α, and             Matthew Baron, Silke Hecht, Andrea Matthews, Jennifer
GM-CSF do not appear to be elevated by labor epidural           Stokes
placement.                                                      Department of Small Animal Clinical Sciences

                                                                Secretin is a polypeptide hormone that stimulates
                                                                secretion of bicarbonate from the exocrine pancreas
69. Air vs Saline Loss of Resistance                            and, in healthy human subjects, has been shown to
for Epidurals–CA1 Residents During                              cause transient pancreatic duct dilation observable via
Primary Obstetrical Rotation                                    abdominal ultrasonography. In humans afflicted with
                                                                chronic pancreatitis, secretin administration fails to
Tony Owens, Stephen K. Patteson                                 cause pancreatic duct dilation, theoretically due to the
Department of Anesthesia                                        restrictive effects of periductal fibrosis. This diagnostic
                                                                test has been shown to be highly sensitive and specific
Studies have been conducted using experienced anesthe-          for the diagnosis of chronic pancreatitis in humans. The
siologists with and without CSE to determine if the air vs.     purpose of this study was to establish the potential effects
saline loss of resistance (LOR) method for identifying the      of exogenous secretin administration on the width of the
epidural space resulted in more favorable outcomes. No          pancreatic duct in healthy domestic cats. Nine healthy
                                                                cats were administered a commercially available secretin




                                                                  41
                    product (ChiRhoStim) while the pancreatic duct was               These descriptions of motion in 3 planes allow for a
                    monitored by percutaneous ultrasonography. Maximum               greater understanding of the biomechanics of the canine
                    pancreatic duct diameter after secretin stimulation was          pelvic limb and may lead to a better understanding and
                    compared to measurements made prior to injection, and            treatment of pathologic processes.
                    a percentage increase over basal width was calculated.
                    Mean pancreatic duct diameter increased from 0.77 ±
                    0.33 mm to 1.42 ± 0.40mm after secretin administration,          72. Evaluation of Veterinary Nitinol
Clinical Sciences



                    which was statistically significant (p = 0.0017). The
                    mean percent increase in pancreatic duct diameter over
                                                                                     Stents, Vet Stent-Trachea, for the
                    basal diameter for all time points 1 to 15 minutes post          Palliation of Tracheal Collapse in Dogs
                    secretin administration was 101.9 ± 58.8%. Exogenously-
                    administered secretin causes significant pancreatic              April M. Durant, Patricia Sura, Barton Rohrbach
                    duct dilation in healthy cats that can be observed via           Department of Small Animal Clinical Sciences (Durant, Sura),
                    percutaneous ultrasound. Applicability of this technique         Department of Comparative Medicine (Rohrbach)
                    in cats with chronic pancreatitis will need to be
                    investigated in future studies.                                  This study was designed to retrospectively evaluate the
                                                                                     use of the Vet Stent-Trachea for palliation of clinical
                                                                                     signs related to tracheal collapse. Our theory was that the
                                                                                     Vet Stent would result in improvement of the patient’s
                    71. A Description of Motion of the Canine                        clinical signs with minimal complications. The medical
                    Pelvic Limb in 3 Planes Using a New                              records database of the UT Veterinary Teaching Hospital
                    Inverse Dynamics Model                                           was searched for a diagnosis of tracheal collapse.
                                                                                     We evaluated 116 medical records for radiographic
                                                                                     documentation of tracheal collapse and placement of
                    Jason Headrick, Songning Zhang, Darryl Millis
                    Department of Small Animal Clinical Sciences (Headrick,          Vet Stent. Data were collected from 18 medical records.
                    Millis), Comparative and Experimental Medicine Program           All data comparisons were tested for normality using
                    (Headrick), Department of Exercise, Sport, and Leisure Studies   the Shapiro-Wilk test. Tracheal measurements were
                    (Zhang)                                                          compared by a paired t test if normally distributed
                                                                                     and a signed rank test if abnormally distributed. Stent
                    A new inverse dynamics model was created that derives            measurements immediately after placement and at follow
                    net joint moments and power from a calculated joint              up were also compared by a paired t test. There was a
                    center. The authors hypothesized that this new model             significant difference in the minimum tracheal width
                    would produce an accurate description of motion in               when initial and post deployment tracheal widths were
                    the sagital plane and that it would allow for description        compared (p = 0.0005). There was a significant difference
                    of motion in the frontal and transverse planes, thus             in stent length at follow up when compared to immediate
                    producing the first description of motion in the canine          deployment (p = 0.001). Owner outcome assessment
                    pelvic limb in all 3 planes. Six dogs were outfitted             was available in 18/18 patients, with 11.1% morbidity
                    with medial and lateral reflective markers. A static             within 60 days and 16.6% morbidity at 10 months. Use
                    calibration was collected that allowed for identification        of the Vet Stent-Trachea as a palliative therapy for
                    of joint centers. Radiographs helped identify the hip            tracheal collapse is associated with an overall fair to
                    joint center. Kinetic and kinematic data were collected          good outcome. The chance of stent fracture is infrequent
                    as the dogs were trotted across a calibrated spaced. An          with this type of nitinol stent, and this stent appears to be
                    inverse dynamics method was used to describe the joint           effective in relieving clinical signs of tracheal collapse.
                    net moment and power across the pelvic limb in 3 planes.
                    In the sagital plane, the hock experiences the greatest
                    moments and power of all 3 joints. In the frontal plane,
                    the hock and stifle abductors and adductors share equal
                    time in the stance phase and the hip abductors cause the
                    net moment. The moments and power generated in the
                    frontal and transverse planes are one half to one tenth that
                    produced in the frontal plane. Inverse dynamics results
                    derived from joint centers allow for more accurate data.




                                                                            42
73. From Cell Culture to Living Mice–                          74. Biodistribution of a Novel Radiolabeled
The Use of Mammalian-adapted                                   Fibril-reactive Monoclonal Antibody In
Bacterial Luciferase as a Target for                           Patients with Systemic Light Chain (AL)
Bioluminescent Imaging                                         Amyloidosis
Dan Close, Stacey Patterson, Steven Ripp, Seung Joon           Karen Wells, Jonathon Wall, Yitong Fu, Bjoern




                                                                                                                              Clinical Sciences
Baek, John Sanseverino, Gary Sayler                            Jakoby, Steve Kennel, Alan Solomon
Center for Environmental Biotechnology (Close, Patterson,      Department of Radiology (Wells, Fu), Department of Medicine
Ripp, Sanseverino, Sayler), Genome Science and Technology      (Wall, Kennel, Solomon), Molecular Imaging and Translational
Program (Close), Department of Pathobiology (Baek)             Research Group (Jakoby)

The definitive shortcoming of bacterial luciferase (lux)
                                                               The objective of this study was to characterize the
is that it is believed to be functional only in prokary-
                                                               biodistribution of I-124 11-1F4, a novel radiolabeled
otes. However, this preconception is erroneous. With an
                                                               murine monoclonal antibody that binds to deposited fibrils
emission spectra that is virtually background-free when
                                                               in AL amyloidosis, using organ-specific PET time activity
expressed in a mammalian host and the ability to generate
                                                               curves (TAC) of patients with AL amyloidosis. Eighteen
light autonomously, the sensitivity and real-time imaging
                                                               patients with AL amyloidosis underwent PET/CT after
capacity of bacterial luciferase far surpasses that of the
                                                               IV administration of I-124 11-1F4. Initially, 3 patients
current mainstays of molecular imaging. Since its incep-
                                                               were imaged at multiple time points, 1 minute to 14 days
tion as a prokaryotic reporter protein nearly 2 decades
                                                               post-radiotracer (RT). Subsequent patients were imaged
ago, the overarching goal of bacterial luciferase reporter
                                                               2 and 5 days post-RT. Biodistribution was assessed with
technology has been to expand its sensing repertoire to
                                                               TAC using decay corrected mean activity in regions of
include eukaryotes. We report here our achievement of
                                                               interest defining blood pool and individual organs. Ratios
this goal by using codon-optimization and bicistronic ex-
                                                               of organ specific activity to blood pool activity and to
pression strategies to induce bioluminescence in HEK293
                                                               organs not clinically involved with AL amyloidosis (lung
cells expressing the full lux cassette at a rate ~1000 times
                                                               and skeletal muscle) were calculated. TAC of I-124 11-
higher than untransfected cells. These bioluminescent
                                                               1F4 in AL amyloidosis patients demonstrated variability
reporter cells were shown stable over prolonged periods,
                                                               in radiotracer biodistribution between patients. Variability
making them ideally suited for general laboratory use. On
                                                               of biodistribution likely reflects heterogeneity of clinical
average, bioluminescent output was 5,400% (± 1,300)
                                                               disease manifestations. Ratio of radiotracer uptake to
greater in HEK293 cells expressing the entire lux cassette
                                                               blood pool activity proved helpful to define uptake in
than those transfected with only the luxA and luxB genes.
                                                               organs and for comparison of RT uptake between patients.
To demonstrate the real-time, reagent-free nature of this
                                                               We demonstrated marked and persistent increased I-124
system, a bioluminescent mouse model was developed;
                                                               11-1F4 organ-specific uptake in 8 patients including the
it was consequently confirmed that bioluminescence was
                                                               spleen, liver, lymph nodes, and bone marrow on TAC,
both rapidly detectable (<10 seconds following injection)
                                                               suggesting binding of RT to pathologic sites of fibril
and free from background over prolonged time periods
                                                               deposition. Characterization of the biodistribution of I-124
(>30 minutes post injection). These results demonstrate
                                                               11-1F in patients with AL amyloidosis (1) demonstrated
the potential applications of a mammalian-compatible
                                                               variability between patients and (2) defined organ-specific
lux reporter for use in both cell culture and whole animal
                                                               uptake in some patients. Results reflect heterogeneity
imaging applications in order to fill the niche created by
                                                               of clinical disease manifestations and indicate potential
the shortcomings of the currently available systems.
                                                               usefulness of a chimeric form of this antibody for
                                                               immunotherapy.




                                                                 43
                                 75. Role of Reactive Oxygen Species                            76. Therapeutic Peptides Block the
                                 (ROS) in Histone Deacetylase Inhibitor                         Immunosuppression of Fibrin and its
                                 (HDACI)-mediated Selective Apoptosis of                        Effects on B16 Melanoma and Metastases
                                 Oncogenic H-Ras-expressing Cells                               in vivo
                                 Shambhunath Choudhary, Hwa-Chain Robert Wang                   Bill McCartt, Patricia N. Coan, Joleen K. Adams, Steven
                                 Department of Comparative Medicine                             Minkin, John Biggerstaff
                                                                                                The Center for Environmental Biotechnology (McCartt,
                                 Oncogenic induction of the ras genes is widely involved        Minkin, Biggerstaff), The Office of Laboratory Animal Care
                                 in human cancers. Expression of oncogenic H-Ras in-            (Coan, Adams)
                                 creases susceptibility of human and mouse cells to HDA-
                                 CIs, such as FK228 and trichostatin A, for inducing cas-       Hypercoagulability is common in many cancers, including
                                 pase activation and selective apoptosis. To understand the     melanoma. Melanoma cells and leukocytes express
                                 mechanisms for HDACI-induced differential activation           fibrin receptors, and it is hypothesized that a fibrin
                                 of caspases and apoptosis in oncogenic H-Ras-expressing        blockade may prevent interactions between leukocytes
                                 cells, we investigated the role and regulation of ROS in       and melanoma, leading to immunosuppression. This
                                 HDACI-induced cell death. We detected that expression          pilot study examined the efficacy and toxicity of peptides
                                 of oncogenic H-Ras resulted in potentiating intracellular      designed to prevent fibrin binding to cognate receptors
Oncology & Cancer Cell Biology




                                 ROS machinery, increasing the susceptibility to HDACIs         on melanoma cells and leukocytes in C57BL/6 mice.
                                 for enhancing the production of ROS, and depleting glu-        The peptide sequences bind to fibrin and inhibit its
                                 tathione, leading to differential activation of caspases and   subsequent binding to MAC-1 and ICAM-1, receptors
                                 cell death. Furthering our understanding of how oncogen-       important for adherence and the induction of cytotoxicity
                                 ic H-Ras augmented the generation of ROS, we detected          in leukocytes and their target tumor cells. In order to
                                 that expression of oncogenic H-Ras was able to increase        determine toxicity of the peptides, 4 mice were injected
                                 the level of Nox1, a homologue of the catalytic subunit of     with 1.5 mg of fluorescently-labeled peptide or 1.5 mg
                                 the superoxide-generating NADPH oxidase. Suppression           of vehicle. A half-life of 24 hours was determined by
                                 of Nox1 by specific inhibitor or specific siRNAs signifi-      measuring plasma separated from blood removed from
                                 cantly reduced ROS production induced by oncogenic             the submandibular vein using a fluorescence plate reader.
                                 H-Ras and HDACIs as well as HDACI-induced cell death           Therapeutic efficacy of the peptides was determined
                                 of oncogenic H-Ras-expressing cells. Our results led us        by measuring tumor growth and metastases in mice.
                                 to a suggestion that Nox1 played an important role in the      Primary tumor tissue was cryosectioned and measured for
                                 ability of oncogenic H-Ras to potentiate ROS-producing         microvessel density by immunohistochemistry for von
                                 machinery for enhancing cell susceptibility to HDACIs          Willebrand Factor. GFP expressing B16 melanoma cells,
                                 and, in turn, increasing ROS contents leading to caspase       and metastasis in the pulmonary tissue were detected
                                 activation and selective apoptosis. Differential increases     by fluorescence microscopy. The primary tumor growth
                                 of intracellular ROS by oncogenic H-Ras to potentiate cell     of the peptide-treated group was inhibited by 69.3%
                                 death should be seriously considered as a potential target     (+/- 28%), in addition to a 47.9% (8.3%) reduction in
                                 in developing therapeutic strategies to control oncogenic      metastases of the lungs, when compared to the vehicle.
                                 H-Ras-involved human cancers by HDACIs.                        These results clearly show that fibrin inhibitory peptides
                                                                                                were effective in reducing tumor growth and metastasis
                                                                                                in vivo, and suggest their possible use as adjuvant
                                                                                                therapeutic agents in cancer immunotherapy.




                                                                                       44
77. Alternative Splicing of Krüppel-                           this model and included MMPs, soluble MICA, tumor
like Factor 4 Plays a Role in Colorectal                       cells, NK cells and ECM. During the assay, cells were
                                                               followed by time-lapse fluorescence microscopy. We
Tumorigenesis                                                  used image analysis software to obtain quantitative and
                                                               qualitative data (cell number, cell density, cell viability,
Jae Hoon Bahn, Seong-Ho Lee, Seung Joon Baek                   cell migration pathways, velocity, etc.). The experimental
Department of Pathobiology                                     system yielded results derived from the interaction of
                                                               NK cells with a growing tumor mass over time, and
Most human genes undergo alternative splicing, and             showed how changes in cell-surface molecule expression
many abnormal splicing processes are associated with           and ECM can moderate these interactions. This system
human diseases. However, the molecular relationship            simulates the progression of tumor invasion by immune
between alternative splicing and tumorigenesis is not well     cells in vivo. Based on our studies, we expect that by
understood. Here, we found novel Krüppel-like factor 4         changing selected biological parameters, we can verify the
(KLF4) splicing variants produced by exon skipping in          mathematical model or modify it to be reassessed through
several human cancer cell lines. One of splicing variants,     the experimental setup.
small KLF4 (KLF4S), localizes in the cytoplasm and
nucleus, and antagonizes transcription factor activity of
wildtype KLF4 (KLF4L). Interestingly, sulindac sulfide
treatment restored KLF4L expression, and this is mediated      79. Safety and Efficacy of a Novel Method




                                                                                                                              Oncology & Cancer Cell Biology
by dephosphorylation of RNA binding motif 5 (RBM5).            of Localized Hyperthermia Delivery in
Overexpression or knockdown of RBM5 altered the                Conjunction with Chemotherapy for the
splicing ratio of KLF4. Our data suggest that RBM5 plays
a pivotal role in the alternative splicing of KLF4, and        Treatment of Non-resectable Canine and
these splicing variant forms may impact tumorigenesis.         Feline Sarcomas: A Pilot Study

                                                               Olya Smrkovski, Aknine Jerard, Jeffrey Phillips
78. Modeling the Effect of Melanoma                            Department of Small Animal Clinical Sciences
Tumor Cell Growth in the Presence of                           Soft tissue sarcomas are a locally invasive group of
Natural Killer Cells                                           cancers that occur in both veterinary and human patients.
                                                               These tumors are treated with aggressive surgery,
Ann Wells, Sharon Bewick, John Biggerstaff, Kara               radiation, and chemotherapy. In some cases, this approach
Kruse, Richard C. Ward                                         is not possible because of tumor location, size, or both. In
Department of Nutrition (Wells, Biggerstaff), NiMBIOS (Be-     addition, tumors that are refractory to aggressive therapy
wick), Oak Ridge National Laboratory, Sciences and Engineer-   represent a particular therapeutic challenge. Therefore, a
ing Division (Kruse, Ward)
                                                               need exists for novel therapeutic approaches. Recent and
                                                               historic literature has shown that the use of hyperthermia
A major challenge in the mathematical modeling of              (tumor exposure to temperatures over 42° C) as an
biological processes is the development of appropriate,        adjuvant to chemotherapy results in improved tumor
physiologically-relevant experimental systems that             control in a variety of locally advanced malignancies.
can yield results readily usable as input parameters           However, the delivery of the precise amount of heat
for mathematical models. We therefore designed an              to the tumor while sparing normal tissues has been
experimental system to investigate the interactions of         challenging. This problem was solved with the discovery
immune cells with tumors. This yielded a defined set of        of metamaterials, dielectrics that can interact with and
numerical parameters for input into a simple mathematical      control electromagnetic radiation. Recent technologic
model of cancer immunity, using human melanoma                 advances allowed us to develop a hyperthermia delivery
cells, natural killer (NK) cells, cell receptors and the       device (iTherm 2000) that uses the unique properties
extracellular matrix (ECM). Tumor growth tends to              of metamaterials. This device contains a conformal
increase in the presence of soluble MICA (a tumor cell         ultrawideband metamaterial lens applicator that is able
receptor) but decreases when high levels of MICA bind          to provide efficient, focused, deep-penetrating low-cost
to the tumor cell. MMPs putatively induce tumor cell           clinical hyperthermia. We have assessed the safety and
shedding of MICA and facilitate degradation of the ECM.        activity of chemotherapy and hyperthermia in a pilot
Our novel cell migration system was devised based on




                                                                 45
                                 study in 11 pet animals (6 dogs and 5 cats). We observed       81. Serial 18FDG-PET/CT Can Assess
                                 clinical responses in all patients. Treatment-related          Response to Receptor Tyrosine Kinase
                                 toxicity was observed in 3 out of 6 dogs and 1 out of
                                 5 cats. These findings suggest that the combination of         Inhibition in Canine Cancer
                                 chemotherapy and hyperthermia delivered with iTherm
                                 2000 has a promise as a novel therapeutic approach for         Amy K. LeBlanc, Federica Morandi, Ashley N. Miller,
                                 non-resectable sarcomas.                                       Jonathan S. Wall, Gina D. Galyon, Tammy Moyers, Misty
                                                                                                Long, Alan Stuckey
                                                                                                Department of Small Animal Clinical Sciences (LeBlanc,
                                                                                                Morandi, Miller, Galyon, Moyers), Molecular Imaging and
                                 80. Expression of KIT and PDGFR in                             Translational Research Program, Graduate School of Medicine
                                 Canine Anal Sac Adenocarcinoma Using                           (LeBlanc, Wall, Long, Stuckey)
                                 Tissue Immunohistochemistry
                                                                                                PalladiaTM (toceranib phosphate, Pfizer Animal Health)
                                 Rebecca J Brown, Shelley J. Newman, Amy K. LeBlanc             is a novel receptor tyrosine kinase (RTK) inhibitor
                                 Department of Small Animal Clinical Sciences (Brown, LeB-      approved for treatment of canine mast cell disease.
                                 lanc), Department of Pathobiology (Newman)                     Dysregulated RTKs induce tumor growth, progression,
                                                                                                and metastasis. PalladiaTM has been shown to target
                                 Apocrine gland adenocarcinoma is a highly invasive and         vascular endothelial growth factor receptor (VEGFR-2/
                                                                                                Flk-1/KDR), platelet derived growth factor receptor,
Oncology & Cancer Cell Biology




                                 metastatic malignancy, and life-threatening paraneoplastic
                                 hypercalcemia occurs in 25-50% of cases. PalladiaTM            and Kit. Positron emission tomography/computed
                                 (toceranib phosphate) is a receptor tyrosine kinase (RTK)      tomography (PET/CT) is commonly used to diagnose
                                 inhibitor that targets several members of the split kinase     and monitor response to anti-neoplastic therapy in
                                 RTK family, including VEGFR-2 (Flk-1/KDR), PDGFR,              cancer patients. In this study, serial PET/CT imaging
                                 and c-Kit. Anecdotally, dogs with measurable anal sac          with 18F-fluorodeoxyglucose (18FDG) was used to
                                 adenocarcinoma have responded to treatment with Pal-           assess response to PalladiaTM therapy in 7 dogs with
                                 ladia. The objective of this study was to evaluate archived,   measureable solid malignancies. Dogs underwent whole
                                 paraffin-embedded canine anal sac adenocarcinomas for          body contrast-enhanced PET/CT with 18FDG (4.547±0.77
                                 immunohistochemical detection of c-KIT and PDGFR.              mCi, mean ±SD) using a 64-slice PET/CT scanner. Six
                                 The UT Department of Pathobiology histopathology               dogs were imaged at baseline with PET/CT; one dog
                                 archives were searched for anal sac adenocarcinoma tis-        underwent CT only at baseline. All dogs underwent PET/
                                 sue samples. A total of 77 samples were examined by            CT at a median of 5.5 weeks post-baseline. Two dogs
                                 immunohistochemistry for c-kit and PDGFR expression            underwent additional PET/CT scans at 14 weeks, and at
                                 and assigned a grade based on the intensity and pattern        10 and 15 weeks post-baseline, respectively. Images were
                                 of staining with: (0) <50% of cells positive with weak         correlated to treatment response. One dog demonstrated
                                 expression, (1) <50% of cell positive with intense expres-     significant clinical improvement at first recheck and
                                 sion, (2) >50% of cells positive with weak expression, (3)     was maintained on drug despite dose modification
                                 >50% of cells positive with intense expression. Two of the     due to toxicity; measureable clinical and image-based
                                 77 samples positively expressed c-Kit, with Grade 1 ex-        response was seen after 12 weeks of therapy. All other
                                 pression. Fifteen of the 77 samples positively expressed       dogs demonstrated stable or progressive disease based on
                                 PDGFR, 14 had Grade 1 expression and 1 had Grade 3             follow-up clinical staging and PET/CT at first recheck.
                                 expression. These results suggest that c-Kit and PDGFR         Scan results agreed with clinical assessment of externally
                                 may not have a significant role in mediating the response      measurable disease. Palladia therapy was continued in
                                 of anal sac adenocarcinomas to Palladia. To further ana-       patients with stable disease and acceptable drug tolerance
                                 lyze the role of Palladia for the treatment of this cancer,    and was discontinued in non-responsive patients. PET/CT
                                 studies should be done to investigate the presence of these    with 18FDG can be used to monitor clinical response to
                                 markers in biopsy material and their eventual response to      Palladia.
                                 therapy, and the role of VEFGR-2 in this cancer.




                                                                                       46
82. Genetic Approach to Improve                                   83. Effect of Reproductive Tract Size
Fertility in Cattle                                               on Conception Rates in Lactating Dairy
                                                                  Cows Utilizing a Reproductive Tract
Fernando Di Croce, Arnold Saxton, Daniel Casanova,
Neal Schrick
                                                                  Scoring System
College of Veterinary Medicine, University of the Center of the
Buenos Aires Province (Di Croce, Casanova), Department of         Charles D. Young, Fernando A. Di Croce, David A.
Animal Science (Di Croce, Saxton, Schrick)                        Roper, Jessica P. Harris, F. Neal Schrick
                                                                  Department of Animal Science, UTIA Reproductive Research
In spite of the economic importance of fertility and              Program (Young, Di Croce, Roper, Harris, Schrick)
increased knowledge of reproductive biology in the cow,           Tennessee AgResearch (Young)
efficiency of reproductive performance has continued to
                                                                  Reproductive tract scoring (RTS) systems have been
decline in dairy cattle. Improvements in fertility through
                                                                  effective in evaluating fertility of heifers. A similar
genetic selection may be a possible approach to increase
                                                                  RTS system has not been reported in lactating dairy
reproductive efficiency in dairy cattle. The objectives
                                                                  cows (LDC) pertaining to fertility. Objectives of this
of this study were to (1) estimate genetic parameters
                                                                  study include the development of an RTS system for
and breeding values for fertility traits from a Holstein
                                                                  LDC and determining if RTS has an effect on artificial
population and (2) develop a multi-trait selection index
                                                                  insemination (AI) success with semen type (conventional
(dollar fertility value, $F) based on estimated breeding
                                                                  [CS] vs. sexed [SS]). Prior to estrus, cows were assigned
values. Data representing 3,282,843 lactations and
                                                                  an RTS (1, 2, or 3) by at least 2 experienced palpators.
1,622,088 animals were used for the analysis and obtained
                                                                  Reproductive tracts for RTS 1 were small, compact, and
from official records maintained by the Argentinean
                                                                  resting within the pelvic cavity; RTS 2 were intermediate
Holstein Association. Days open (DO), calving interval
                                                                  in size and resting partially outside the pelvic cavity; RTS
(CI), age to first calving (AFC), and daughter pregnancy
                                                                  3 were large, deep tracts resting nearly entirely outside
rate (DPR) were traits chosen for genetic parameter
                                                                  the pelvic cavity. Cows (n = 1822 inseminations) were
estimation. Multiple-trait, derivative-free restricted
                                                                  artificially inseminated with either CS (n = 1621) or SS
maximum likelihood (MTDFREML) was used to estimate
                                                                  (n = 201). Pregnancy was determined by transrectal
covariance components. The proposed selection index
                                                                  palpation or ultrasonography at 28–45 days post-AI for
($F) included AFC and CI, with economic weights from
                                                                  determination of conception rate (CR). The highest
-0.28 and -4.9 $/year per cow, respectively. Heritabilities
                                                                  numerical CR with respect to RTS was observed in RTS
for DO and DPR ranged from 2 to 7%; CI ranged from 3
                                                                  1, followed by RTS 2 and RTS 3 (29.7%, 26.5%, 26.6%,
to 8%; and AFC was 16%. Predicted transmitting ability
                                                                  respectively, p = 0.5771). Regardless of RTS, conception
values across different lactations ranged from -18.5 to
                                                                  rates of CS (31.4%) were higher than SS (23.4%, p =
15.8 days, -23.15 to 16.0 days, -4.0 to 4.6%, and -120.3
                                                                  0.0525). Conception rates were higher for RTS 1 (38.4%)
to 76.2 days for DO, CI, DPR, and AFC, respectively.
                                                                  compared to RTS 3 (23.8%) with CS (p = 0.0176). RTS




                                                                                                                                 Bovine Diseases & Theriogenology
Values for $F ranged from -$89.16 to $147.12 in this
                                                                  1 conception rates were higher for CS (38.4%) compared
Holstein population. Results indicate substantial variation
                                                                  to SS (22.3%, p = 0.0272). No differences were detected
in fertility traits, suggesting that genetic selection may be
                                                                  in conception rates using SS. These results indicate the
effective in improving declines in fertility.
                                                                  detection of smaller tracts can improve fertility in LDC
                                                                  when using CS.




                                                                    47
                                   84. Real-Time Polymerase Chain Reaction                       85. Characterization of the Immune
                                   for Simultaneous Detection of Mastitis                        Response Following Vaccination of Dairy
                                   Pathogens Directly from Milk                                  Cows with Recombinant Streptococcus
                                                                                                 uberis Adhesion Molecule (rSUAM)
                                   Barbara E. Gillespie, Megan L.Maxwell, Daniel Scholl,
                                   Stephen P. Oliver                                             Raul A. Almeida, Maria E. Prado, Douglas A. Luther,
                                   Department of Animal Science (Gillespie, Maxwell, Oliver),    Susan I. Headrick, Mark J. Lewis, Hugh Moorehead,
                                   Canadian Bovine Mastitis Research Network, Quebec, Canada
                                                                                                 Stephen P. Oliver
                                   (Scholl)
                                                                                                 Department of Animal Science (Almeida, Prado, Luther,
                                                                                                 Headrick, Oliver), Dairy Research and Education Center,
                                   Mastitis is the most common and most costly disease           Lewisburg, TN (Moorehead)
                                   of dairy cattle in the United States. A real-time PCR
                                   multiplex assay for detection of mastitis pathogens has       Research conducted in our laboratory on the pathogenesis
                                   been developed in our laboratory. The objective of the        of Streptococcus uberis mastitis led to the discovery of
                                   present study was to evaluate this real-time PCR assay        a novel virulence factor, referred to as SUAM. Further
                                   for detection of Staphylococcus aureus, Streptococcus         research showed that antibodies directed against rSUAM
                                   agalactiae, Streptococcus uberis and Escherichia coli         inhibited adherence to and internalization of S. uberis
                                   directly from milk. The real-time PCR multiplex assay         into mammary epithelial cells, placing rSUAM as a
                                   targeted the following genes: cfb gene encoding the           promising antigen for the control of S. uberis mastitis.
                                   CAMP factor for S. agalactiae, nuc gene for S. aureus, the    To design an effective vaccine formulation, we compared
                                   plasminogen activator gene for S. uberis, and the wec A       the safety and humoral immune response (IgG) induced
                                   gene targeting E. coli. Quarter milk samples from clinical    by rSUAM emulsified with commercially available
                                   (n = 1471) and subclinical (n = 1307) mastitis previously     adjuvants (Emulsigen, Seppic VG70, Seppic ISA760, and
                                   evaluated by the Canadian Bovine Mastitis Research            Seppic ISA780). For this experiment, 5 cows per each
                                   Network by conventional microbiological methods               formulation and a control group (n = 4) were vaccinated
                                   were used to evaluate the real-time PCR procedure.            28 days before dry off (D-28), at dry off (D0) and 28
                                   Milk samples were enriched with trypticase soy broth          days after dry off (D+28). Serum samples were obtained
                                   and incubated overnight. Isolation of bacterial DNA           every 2 weeks from D-28 to 28 days after calving (C+28).
                                   from milk used a technique modified in our laboratory.        Rectal temperature and local reaction measurements
                                   Multiplex real-time PCR was performed in a Bio-Rad iQ5        were obtained 3 times during the 1st week and every 2
                                   Thermocycler. The sensitivity (78.02%) and specificity        weeks after each subsequent vaccination. Result obtained
                                   (97.28%) of real-time PCR to detect mastitis pathogens        showed that IgG response induced by SV70-rSUAM was
                                   from clinical mastitis samples was much lower than            significantly higher than the control (rSUAM alone) or
                                   the sensitivity (90%) and specificity (98.01%) to detect      the rest of the vaccine formulations tested. Local reaction
                                   mastitis pathogens from subclinical mastitis samples.         induced by SV70-rSUAM was no different than the rest of
Bovine Diseases & Theriogenology




                                   Results of this study indicate that the multiplex real-       the vaccine formulations with the exception of the second
                                   time PCR procedure has the potential to be a valuable         vaccination, where the local reaction of SV70-rSUAM
                                   diagnostic technique for simultaneous identification of       was significantly higher than the rest. Results of this study
                                   commonly isolated mastitis pathogens directly from            led to the selection of SV70-rSUAM for an upcoming
                                   quarter milk samples. Even with overnight enrichment,         vaccination study, which includes challenge of vaccinated
                                   real-time PCR can reduce identification time by at least 24   and control cows with homologous and heterologous S.
                                   hours compared to conventional microbiological methods.       uberis strains.




                                                                                        48
86. pGhost9:ISS1 Transpositional
Mutations in Streptococcus uberis UT888
Causes Reduced Bacterial Adherence to
and Internalization into Bovine Mammary
Epithelial Cells

Oudessa Kerro Dego, Maria Prado, Raul Almeida, Xue
Chen, Douglas Luther, Stephen Oliver
Department of Animal Science

Streptococcus uberis is an important mastitis pathogen
that affects dairy cows worldwide. Pathogenic strategies
and associated virulence factors of S. uberis are not well
understood. S. uberis attaches to and internalizes into
mammary epithelial cells and can survive inside cells for
extended periods. We hypothesize that early attachment
to and internalization into mammary epithelial cells is a
critical step for the establishment of infection. The aim
of this study was to identify and characterize chromo-
somally encoded virulence factors of S. uberis that are
involved in early attachment to and internalization into
mammary epithelial cells. We identified virulence factors
by generating random transposon insertion mutants that
are defective in adherence and internalization using the
pGhost9:ISS1 mutagenesis system. Random transposon
insertion mutant libraries of S. uberis UT888 were created
using thermosensitive plasmid pGhost9:ISS1 carrying
ISS1 transposon. Of 1000 random chromosomal mutants
of S. uberis UT888 screened, 32 had significantly reduced
ability to adhere to and internalize into mammary epi-
thelial cells. Integration of the transposon into the chro-
mosome of these mutant clones was confirmed by PCR
and Southern blot. Chromosomal mapping of transposon
integration sites and identification and characterization of




                                                                    Bovine Diseases & Theriogenology
insertionally inactivated genes are currently being done
using improved methods of inverse PCR that uses prim-
ers on either end of the transposon. By sequencing the
inverse PCR products, we found that some mutant clones
had transposon integration into penicillin binding protein
2A (pbp2A), phosphoribosylaminoimidazole carboxylase
catalytic subunit (purE), lactose phosphotransferase and a
few other genes.




                                                               49

				
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