Retreat 2009 M.D./Ph.D. Student Abstracts
EFFECTORS SECRETED BY THE BRUCELLA TYPE IV SECRETION SYSTEM INTERFERE WITH MACROPHAGE ANTIGEN PROCESSING AND PRESENTATION
PSTP Student: Vidya Atluri
Mentor: Dr. Renee Tsolis
Brucella is an intracellular bacterium that infects phagocytic cells and causes a chronic disease called brucellosis. The World Health Organization estimates 500,000 new cases of brucellosis a year. Death occurs in less than 2% of cases but it is a large drain on resources due to the chronic nature of the disease. The mechanism of persistence is not well understood. Brucella enters the host and is taken up by phagocytic cells such as macrophages. After the first five days of infection the type IV secretion system (T4SS), encoded by the virB operon, is required for the bacteria to persist in its host. Since this process is required for persistence rather than initial infection and colonization, it might subvert the adaptive immune response. The proteins secreted by the T4SS direct the phagosome to a compartment associated with the endoplasmic reticulum (ER) rather than to the lysosome, where proteins are processed for presentation on MHC class II. We hypothesize that Brucella might interfere with antigen processing and presentation. To test this, macrophages will be differentiated from hematopoietic precursors isolated from the femurs of mice. Then, they will be infected with various strains of Brucella. The infected macrophages will be exposed to ovalbumin followed by formalin fixation and exposure to OVA specific CD4+ T cell hybridomas. If the macrophages present OVA on MHC II then the hybridomas will respond by secreting IL-2, which we will measure using ELISA. Preliminary results show that macrophages infected with the T4SS deficient Brucella strain present more OVA than those infected with wild type Brucella. Complementing the mutations leads to the wild type phenotype. Further studies will be done to confirm these results and elucidate which part of the antigen processing and presentation pathway is being inhibited by the wild type Brucella strain.
DEPRESSIVE SYMPTOMS AND CONTRACEPTIVE USE AMONG LATINAS IN AN AGRICULTURAL COMMUNITY
PSTP Student: Teresa L. Barcellos
Mentor: Dr. Marc Schenker
Collaborators: Drs. Maria T. Stoecklin-Marois and Tamara E. Hennessy-Burt
OBJECTIVE: To evaluate the effects of depressive symptoms, perceived stress, and family support on rural Latinas’ contraceptive choices. METHODS: A subsample of all 326 premenopausal women not currently pregnant or attempting pregnancy from a larger study of farm worker households in Mendota, California, was used to evaluate contraceptive use in women at risk for unintended pregnancy. The survey assessed reproductive health and history, including current contraceptive use, and validated scales evaluating depression, perceived stress, and family support. Logistic regression analyses were performed to evaluate the relationships between depression risk, stress, or family support and contraceptive use. RESULTS: Participants had an average age of 34.9 years and were overwhelmingly (96.0%) born in Mexico or Central America. Of these, 79.1% reported using some form of contraception. Preliminary analyses showed a nonsignificant trend of decreasing odds of contraceptive use with increasing risk of depression. Women with the highest risk of depression were less likely to use any contraceptive method than women with the lowest risk (odds ratio 0.085, 95% confidence interval 0.009–0.824). Additionally, women scoring above the screening cutoff for depression were less likely to use reversible methods (odds ratio 0.386, 95% confidence interval 0.178–0.835). Neither perceived stress nor family support had a significant impact on the odds of using contraception or the method used. CONCLUSION: Latinas with depressive symptoms, who are likely to be especially vulnerable to the adverse effects of an unintended pregnancy, may be at increased risk of becoming pregnant through diminished contraceptive use. Further evaluation of the role of depression in contraceptive decision making is needed.
IN VITRO EFFECTS OF PARTICULATE MATTER ON HUMAN ENDOTHELIAL ADHESION MOLECULE EXPRESSION
VSTP Student: Alissa Blum
Mentor: Dr. Dennis W. Wilson
Collaborators: Drs. Michael Lamé and Fern Tablin
Evidence suggests that inhalation of environmental fine particulate matter (PM 2.5) effects not only the lung, but the cardiovascular system as well. PM inhalation results in systemic inflammatory effects with increases in white blood cell and fibrinogen concentrations as well as plasma viscosity. Clinical studies have shown PM related vascular responses including reduced flow-mediated dilation in conducting vessels and nitroglycerine-mediated dilation in resistance vessels, with decreases in vascular response to both endothelium-dependent (bradykinin, acetylcholine) and independent (sodium nitroprusside and verapamil) vasodilators. Reactive oxygen species (ROS) produced by PM interfere with nitric oxide (NO) production and bioavailability. In vitro studies with human monocytes and granulocytes have shown altered expression of cell surface adhesion molecules such as CD54, CD18, CD49d and CD11b. Intratracheal instillation of diesel exhaust particles in Animal studies results in increased coagulability. Recent studies from our laboratory suggest that inhalation of concentrated ambient PM results in platelet activation as demonstrated by increased aggregation and degranulation. We hypothesize that endothelial cells exposed to PM will have altered expression of cell-surface adhesion molecules such as VCAM-1, p-selectin, and e-selectin that directly interact with platelets. Expression of these molecules in cultured human aortic endothelial cells treated with fine particulate matter collected from the San Joaquin Valley of California will be examined with flow cytometry and immunocytochemistry.
IDENTIFICATION AND CHARACTERIZATION OF ANTIGENIC PROTEINS OF THE ETIOLOGIC AGENT OF EPIZOOTIC BOVINE ABORTION
VSTP Student: Roxann Brooks
Mentor: Dr. Jeffrey L. Stott
Epizootic Bovine Abortion (EBA) commonly referred to as Foothill Abortion, is the leading cause of beef cattle abortion in California. Each year it causes an estimated 45,000 to 90,000 abortions and substantial economic losses. First reported in California in the 1950s, EBA has since been identified in Oregon and Nevada. The management and grazing practices of beef cattle contribute to the incidence of this disease by facilitating their exposure to the vector, an Argasid tick, Ornithodoros coriaceus. Recently, a deltaproteobacterium, most closely related to the order Myxococcales, has been identified as the etiologic agent of EBA. Naïve heifers are susceptible to infection during the first six months of pregnancy, and if exposed will abort in the third trimester or have a weak calf. Dams develop immunity following repeated exposure to the bacterium, making a vaccine possible. Additionally, an EBA infected fetus produces antibody specific for the bacterium because of the lack of passive transfer across the bovine placenta. We intend to identify the bacterial antigenic proteins against which an EBA infected fetus mounts an antibody reponse by screening an expression library with EBA-positive polyclonal fetal antisera. We will then produce the proteins recombinantly to test whether they can be used as an effective subunit vaccine in the dam.
IDENTIFICATION OF ENDOCYTIC MOTIFS IN RECEPTORS IMPORTANT FOR SYNAPTIC STABILITY
PSTP Student: Jolene Chang
Mentor: Dr. Michael Ferns
Postsynaptic receptor insertion and maintenance is a crucial component of synapse formation in both the central and peripheral nervous systems. At the neuromuscular junction in the peripheral nervous system, there is a precise overlap in the colocalization of the presynaptic motor nerve terminal and postsynaptic acetylcholine receptor clusters in order to facilitate efficient synaptic transmission. Postsynaptic levels of acetylcholine receptors are regulated and maintained by trafficking of the receptor to and from the synapse, either through insertion of newly synthesized receptor, or removal through endocytosis. Under normal conditions, the acetylcholine receptor half life at the neuromuscular junction is fairly stable, with a half life of about 11-14 days. However, abnormal turnover of receptor at the neuromuscular junction underlies certain diseases, including myasthenia gravis. Myasthenia gravis is an autoimmune neuromuscular disorder, where an immune response is mounted against the nicotinic acetylcholine receptor at the neuromuscular junction in skeletal muscle. Symptoms include generalized muscle weakness. Autoimmune antibodies impair synaptic transmission at the neuromuscular junction in several ways, including crosslinking of receptor to induce endocytosis and degradation of the receptor. We know that receptor endocytosis is important for the symptoms of myasthenia gravis, but very little is known about the specific mechanism for the endocytosis of the acetylcholine receptor in both normal and diseased skeletal muscle. This is the basis of my project. My aim is to identify and map the endocytic motifs in the acetylcholine receptor subunits by using an in vitro endocytic assay and recombinant DNA constructs of subunit receptor intracellular loops, in order to determine which segments of the intracellular loops are important for antibody-induced endocytosis. Using this assay, I was able to identify a 33 amino acid sequence in the b subunit loop (b 333-369) and a 34 amino acid sequence in the d subunit loop (d 337-370) that mediates endocytosis in each subunit expressed on the surface of a heterologous cell. Mutations were made in a YXXL endocytic motif in the b subunit, a common endocytic motif found in other surface proteins, and preliminary data suggests that the endocytic motif in the b subunit is mediated through this motif. Further studies will determine specific endocytic motifs in the d subunit, as well as the function of these endocytic motifs in full receptor and possible regulatory mechanisms in antibody-induced receptor turnover.
ILEAL INTERPOSITION (IT) SURGERY INCREASES CIRCULATING GLP-1 AND PYY RESPONSES AND DELAYS DIABETES ONSET IN A NOVEL RAT MODEL OF TYPE 2 DIABETES MELLITUS, THE UCD-T2DM RAT
VSTP Student: Bethany P. Cummings
Mentor: Dr. Peter J. Havel
Collaborator: Dr. April D. Strader
Bariatric surgery often results in resolution of type 2 diabetes mellitus (T2DM). While the mechanisms for diabetes resolution remain undefined, studies suggest that endocrine mechanisms have an important role. Previous work has shown that the reduction in length of the proximal intestine and the resulting increase in delivery of incompletely digested nutrients to the distal intestine can increase the production of gastrointestinal hormones (namely GLP-17-36 and PYY3-36) that further promote weight loss and improve glucose homeostasis. IT surgery involves transposing a 10 cm segment of ileum into the proximal intestine and provides a model whereby the effects of bariatric surgery on gut hormones can be isolated. We hypothesize that IT surgery will delay the onset T2DM in UCD-T2DM rats, a model of T2DM more similar to T2DM in humans than other existing rat models, in part by increasing nutrient-stimulated GLP-1 and PYY release. IT (n=11) or sham surgery (n=13) was performed on 4 mo old UCD-T2DM rats weighing ~580 g. Diabetes onset was determined by weekly non-fasted blood glucose (<200 mg/dl). Oral glucose tolerance (OGTT), insulin tolerance (ITT) and oral fat tolerance tests (OFTT) were conducted at 1, 2.5 and 3.5 months after surgery, respectively. At this time, sham-operated animals have higher diabetes incidence (75%) and earlier onset (9.5±0.3 mo) compared with IT (43% and 11±0.6 mo; P<0.05). Body weight and food intake do not differ between groups. During OGTT, IT animals exhibited lower glucose (AUC: Sham= 17759±1599, IT= 11696±1096 mg/dl x 120 min; P<0.01) and insulin excursions (AUC: Sham= 135±25, IT= 105±12 ng/ml x 120 min; P<0.05) and greater active GLP-1 secretion (AUC: Sham= 60±20, IT= 205±39 pM x 60 min; P<0.01). IT animals also exhibit improved insulin sensitivity. Plasma glucose levels fall by 19±3% in IT animals compared with 8±4% in Sham animals 45 min after ITT (P<0.05). Total PYY excursions during OFTT were 4-fold greater in IT animals (AUC: Sham= 4704±1175, IT= 19670±3367 pg/ml x 180 min; P<0.01). Thus, IT surgery delays T2DM onset by improving glucose tolerance and insulin sensitivity, which may be in part due to increased nutrient-stimulated release of GLP-1 and PYY.
BIOMIMETIC SCAFFOLDS FABRICATED FROM APATITE-COATED POLYMER MICROSPHERES
PSTP Student: Hillary E. Davis
Mentor: Dr. J. Kent Leach
Strategies to engineer bone have focused on the use of natural or synthetic biodegradable materials to support cell transplantation or as substrates to guide bone regeneration. The deposition of a biomineral on the surface of synthetic polymer scaffolds results in the formation of hybrid biomaterials with desired biodegradability and enhanced osteoconductivity. However, current methods of fabricating such composite scaffolds use a prolonged incubation process, which permits scaffold deformation and premature loss of incorporated macromolecules. We hypothesized that the fabrication of biomineralized polymer scaffolds could be achieved using premineralized polymer microspheres generated through incubation in a modified simulated body fluid (mSBF). We explored the material characteristics of these substrates and characterized the in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) when cultured on these novel scaffolds. Unlike scaffolds prepared using the conventional approach, premineralized scaffolds maintained their initial conformation after fabrication, achieved improved mineral distribution throughout the substrate, and enabled significantly greater incorporation efficiency of a model protein. We did not detect differences in osteogenic differentiation as determined by alkaline phosphatase activity and osteopontin secretion. However, we did observe a significant increase in cell-secreted calcium by hMSCs seeded on scaffolds prepared from premineralized polymer. These results demonstrate that the use of premineralized polymeric materials to fabricate biodegradable polymer scaffolds is an improved method for composite scaffold formation and may have advantages for use in bone tissue engineering.
THE EFFECTS OF LIPOPOLYSACCHARIDE ON EGG YOLK CLEARANCE BY CHICKEN MACROPHAGES
VSTP Student: Ingrid Cornax Edwards
Mentor: Dr. Kirk C. Klasing
Collaborator: Dr. Rosemary Walzem
The interaction between lipoproteins and inflammation is an area of great interest to human health. Chickens provide an interesting new model to study this interaction in a disease called egg yolk peritonitis (EYP). Normally, yolk material in the body cavity is cleared by blood-derived macrophages and returned to the liver, however, in the presence of an inflammatory stimulus yolk clearance fails. To explore how inflammation prevents yolk clearance we grew chicken macrophages in vitro with egg yolk in the presence or absence of LPS and measured cell cholesterol content and changes in expression of lipoprotein receptor genes (CD36, VLDLR and MARCO), lipid efflux genes (SRB1 and ABCA1) and apoprotein genes (ApoA1 and SAA1). LPS appeared to inhibit yolk-mediated rises in cell cholesterol (p=0.0842) and inhibited the expression of VLDLR and CD36 (p=0.0047 and p=0.0221, respectively). Egg yolk increased the expression of MARCO (p=0.0158) and MARCO may mediate yolk and LPS clearance. LPS significantly reduced the expression of SRB1 and ABCA1 (p<0.0001 and p<0.0001, respectively) so probably reduces reverse cholesterol transport. Chicken macrophages expressed appreciable levels of SAA1 and ApoA1 so may be capable of producing HDL for reverse cholesterol transport. Lipid tracer studies employing radiolabeled egg yolk should be conducted to gain a stronger understanding of movement of yolk lipid through chicken macrophages.
MONOCHROMATIC X-RAY CANCER PHOTOTHERAPY AND CHARACTERIZATION OF THE COMPTON LIGHT SOURCE
PSTP Student: William J. Frederick III
Mentors: Drs. Jonathan P. Heritage and Neville C. Luhmann
Collaborators: Drs. Arnold E. Vlieks, Christopher A. Destefano, and Dandan D. Zheng
The Compton Light Source (CLS) promises to provide a compact source of high intensity, well-collimated, small spot size, hard x-rays with tunable monochromatic energy. By colliding relativistic electrons with picosecond photon bunches, x-rays are generated through inverse Compton scattering (ICS). The tunable energy range of these x-ray photons (between 10-120 keV) is well suited for diverse medical applications allowing work, which is now possible only at synchrotron sources, to take place in a clinical or local research setting. High quality images and better tumor control with lower dose on normal tissue may be achieved with x-rays tuned just above the k-edge characteristic energies of the contrast agents. The first part of this work considered design, initial beam alignment and x-ray detection efforts at the CLS. In particular, methods to characterize the system performance and to detect x-rays generated by inverse Compton scattering are discussed. A differential energy x-ray detector to resolve ICS x-rays between 20 and 120 keV against a bremsstrahlung background of up to 50 MeV was designed, calibrated with a synchrotron source, and employed at the CLS in initial detection efforts. Imaging of scintillation crystals placed in the beamline was also employed. Although these detection methods showed that a significant background bremsstrahlung field was produced during ICS x-ray generation, a small signal (~9.1% above noise) of ICS x-rays was measured against the background during initial alignment efforts. The second part of this work considered the in vitro evaluation of PC3 prostate cancer cells treated with radiosensitizing agents such as Iohexol and tin protoporphyrin IX (SnPPIX) to determine the dose enhancement effect under irradiation by both conventional and synchrotron x-ray sources. A dose enhancement factor at 10% cell survival (DEF10%) of up to 1.66 was measured for cells treated with Iohexol at 14 mg/ml using a hardened conventional x-ray source. Synchrotron experiments at the Stanford Synchrotron Radiation Laboratory confirmed the predicted energy dependence of the dose enhancement factor for cells in solution with 28 mg/ml Iohexol. The relative relationship between DEF10% and energy was reproduced experimentally for energies between 25 and 42 keV. Although irradiation below the k-edge of iodine showed only small dose enhancement (DEF10% of 1.16, 1.2 and 1.37 at 25, 28, and 32 keV, respectively), irradiation above the k-edge increased dose enhancement (DEF10% of 1.7, 2.12, and 2.24 at 33.4, 34, and 42 keV, respectively). Experiments with extracellular SnPPIX at a conventional x-ray source showed modest dose enhancement (DEF10%=1.17), but the inherent toxicity of SnPPIX precluded evaluation at distant sources.
ENVIRONMENTAL FACTORS THAT ENHANCE THE SURVIVAL AND REPLICATION OF VIRULENT RHODOCOCCUS EQUI BY INDUCTION OF VIRULENCE PLASMID GENE EXPRESSION
VSTP Student: Denise F. Gonzalez
Mentor: Dr. Barbara Byrne
Rhodococcus equi is a pleomorphic, Gram-positive rod that causes a severe pyogranulomatous bronchopneumonia in foals that if left untreated can lead to death. Infection in other species is rare, but is usually associated with immunosuppression, such as in human AIDS patients. The primary source of infection is thought to be inhalation of aerosolized R. equi. In the mammalian host, it survives in macrophages as a facultative intracellular pathogen. There are avirulent and virulent strains. Only the virulent strain has the potential to cause disease in foals due to an 85-kb plasmid that contains genes encoding virulence-associated proteins (vap A,C,D,E,F,G and H) that are found on two of the plasmid’s pathogenicity islands. The effect of environmental factors on vap gene expression was first examined using vapA. Maximum expression of vapA was found to be at high temperature (37°C) and low pH (< 6.5). The expression of the vap genes on the plasmid’s pathogenicity island have been characterized under different conditions within the macrophage. Temperature, low iron, and low magnesium seem to be important regulators of vap gene expression in vivo and in vitro. We hypothesize that virulent R. equi expresses virulence plasmid genes in soil and has enhanced survival and/or replication when compared to avirulent strains in this environment. This hypothesis will be tested by doing side-by-side and competition soil studies with avirulent and virulent strains. We aim to (1) identify environmental factors that induce virulence plasmid gene expression in virulent R. equi, (2) determine if virulent strains have enhanced replication over avirulent strains, (3) determine if expressed virulence genes enhance survival and replication of the virulent strain, and (4) determine the efficacy of current management strategies used on farms by identifying which factors can be manipulated in order to reduce the survival, replication, and/or ability of the virulent R. equi to infect foals. Investigating the expression of vap genes and their influence on R. equi survival and replication in the environment is important in order to determine the most effective management strategies that can reduce virulent R. equi concentration and thus prevent infection of foals on farms.
THE PHARMACOKINETICS AND PHARMACODYNAMICS OF MEDETOMIDINE IN THE HORSE FOLLOWING INTRAVENOUS ADMINISTRATION
VSTP Student: Kristin N. Grimsrud
Mentor: Dr. Scott Stanley
Collaborators: Dr. Khursheed R. Mama
Medetomidine is a potent sedation and analgesic agent labeled for use in dogs, but has proven to be efficacious for sedation in standing procedures, as a premedication for anesthesia and in combination infusions for total intravenous anesthesia in the horse. Medetomidine is an α2-agonist that exerts its affects by stimulation of the central and peripheral α2-adrenergic receptors resulting in decreased sympathetic outflow. It is a lipophilic compound that is rapidly absorbed and distributed, which allows for immediate effects. The biotransformation of the parent compound via the liver is believed to mediate its elimination from the blood; however, extrahepatic elimination is also believed to contribute to elimination since the rate of clearance described in sheep and ponies exceeds the blood flow rate of the liver. The pharmacokinetics of medetomidine has been reported in rats, dogs, cats, sheep, and ponies. Although the clinical effects of medetomidine have been well documented, the correlation between these effects and the pharmacokinetics of the parent compound has not been well described in the horse. The aim of the present study is to describe the pharmacodynamic effects of medetomidine in conjunction with the pharmacokinetics at a clinically relevant dose. The current study demonstrates that following intravenous administration of a 10ug/kg bolus to 8 adult healthy horses, there is a rapid onset of sedation which positively correlates with circulating drug levels. There is a resulting increase in blood glucose concentrations due to an inhibition of insulin release that is seen with a lag effect. A decrease in heart rate, respiratory rate, and gastric motility was observed. The horses demonstrated a rapid decrease in head height, a decreased response to noise and noxious stimuli and were immobile due to increased ataxia. Medetomidine has a fairly short half life and rapid clearance with drug concentrations presenting as either a one and two compartment pharmacokinetic model.
GENETIC TEST AND DETERMINATION OF ALLELE FREQUENCY FOR CANINE HYPERURICOSURIA
VSTP Student: Nili Karmi
Mentor: Dr. Danika Bannasch
All mammals, with the exception of great apes, humans and Dalmatian dogs excrete allantoin, a soluble compound, in their urine as the end product of purine metabolism. Great Apes, humans and Dalmatians excrete uric acid instead of allantoin, making them more susceptible to uric acid crystal formation. Hyperuricosuria is the excess excretion of uric acid in urine that may lead to the formation of uric acid stones and consequently urinary blockage. This condition has been studied in Dalmatians for nearly a century and was determined to have an autosomal recessive mode of inheritance. The mutation that is responsible for hyperuricosuria in Dalmatians has recently been identified. Although individuals from other dog breeds have been reported to form uric acid stones, the cause was unknown and was presumed to be different than that of Dalmatians. We identified two breeds of dog, the Bulldog and Black Russian Terrier, in which hyperuricosuric individuals were also homozygous for the mutation identified in Dalmatians. The phenotype of dogs affected with hyperuricosuria from these two breeds was determined based on uric acid levels in the urine and found to be similar to that of Dalmatians. A genetic test for the mutation has been designed and allele frequencies within these two breeds have been determined. In Bulldogs, the mutated allele frequency was found to be 0.16 and in Black Russian Terriers the mutated allele frequency was 0.51. Although the high mutated allele frequency may indicate selection for the trait, selection was not observed based on comparison to the expected frequencies in Hardy Weinberg equilibrium. The high mutation rate in these two breeds indicates a need for genetic counseling and the formation of a breeding strategy in order to decrease the number of affected individuals.
ARGININE DEIMINASE AS A NOVEL THERAPY FOR PROSTATE CANCER INDUCES AUTOPHAGY AND CASPASE-INDEPENDENT APOPTOSIS
PSTP Student: Randie Kim
Mentors: Drs. Hsing-Jien Kung and Dr. Richard Bold
Arginine deprivation as an anti-cancer therapy has historically been met with limited success. The development of pegylated arginine deiminase (ADI-PEG20) has renewed interest in arginine deprivation for the treatment of some cancers. The efficacy of ADI-PEG20 is directly correlated with argininosuccinate synthetase (ASS) deficiency. CWR22Rv1 prostate cancer cells do not express ASS, the rate-limiting enzyme in arginine synthesis, and are susceptible to ADI-PEG20 in vitro. Interestingly, apoptosis by 0.3 mg/mL ADI-PEG20 occurs 96 hours post treatment and is caspase-independent. The effect of ADI-PEG20 in vivo reveals reduced tumor activity by microPET as well as reduced tumor growth as a monotherapy and in combination with docetaxel against CWR22Rv1 mouse xenografts. In addition, we demonstrate autophagy is induced by single amino acid depletion by ADI-PEG20. Here, autophagy is an early event that is detected within 1 to 4 hours of 0.3 mg/mL ADI-PEG20 treatment and is an initial protective response to ADI-PEG20 in CWR22Rv1 cells. Significantly, the inhibition of autophagy by chloroquine and Beclin1 siRNA knockdown enhances and accelerates ADI-PEG20-induced cell death. PC3 cells, which express reduced ASS, also undergo autophagy and are responsive to autophagy inhibition and ADI-PEG20 treatment. In contrast, LNCaP cells highly express ASS and are therefore resistant to both ADI-PEG20 and autophagic inhibition. These data point to an interrelationship among ASS deficiency, autophagy, and cell death by ADI-PEG20. Finally, a tissue microarray of 88 prostate tumor samples lacked expression of ASS, indicating ADI-PEG20 is a potential novel therapy for the treatment of prostate cancer.
STUDIES OF ANTIMICROBIAL AND CHEMOTACTIC ACTIVITIES OF CANINE B-DEFENSIN 103
VSTP Student: Brian C. Leonard
Mentor: Dr. Charles L. Bevins
Skin has traditionally been thought to serve solely as a physical barrier to damage and infection. However, recent studies have focused on ability of the epidermis to coordinate innate immune responses. Various antimicrobial peptides, including defensins, are key effector molecules produced by the epidermis, which play a significant role in protection from bacterial invasion. While most defensins have potent antibacterial activity, permitting them to function as endogenous antibiotics, some defensins can function also as chemoattractants through high affinity binding to chemokine receptors, such as CCR6. Two recent studies have identified a defensin termed canine b-defensin 103 (k9bD-103), as an abundant peptide produced from the skin of the dog. Interestingly, a common variant of k9bD-103 peptide that lacks encoding of a glycine at the amino terminus of the mature defensin, has the ability to govern coat color in the dog through high affinity binding to the melanocortin receptor, a non-canonical role not seen in any other species. To date, nether the antimicrobial or chemotactic activities of the wild type k9bD-103, or its common variant, are unknown. A recent study by Taylor, et al has shown that the amino terminus of the human ortholog of k9bD-103 is important for antimicrobial activity, and furthermore, loss of the first two amino acids from the mature peptide results in elimination of chemotaxis (unpublished data). With this knowledge, the common variant k9bD-103, lacking the amino-terminal glycine residue, might have significantly altered antimicrobial and/or chemotactic activity. Ultimately, dogs harboring this variant allele may be more susceptible to bacterial infection than dogs expressing wild type k9bD-103. Hypothesis: K9bDl03 possesses broad-spectrum antimicrobial activity and facilitates the chemotaxis of CCR6-expressing cells to sites of inflammation, but the common N-terminal variant may have attenuated activity. Significance: Characterization of the antimicrobial activity and chemotactic properties of k9bD-103 will help provide a framework for understanding innate immunity in the skin. Our unpublished studies indicate that k9bD-103 is expressed in skin at high levels and is present in some breed as a copy-number variable gene. Altered activity of this peptide may alter innate immune function in the skin.
DIVERSE ROLES FOR CLASS A PLEXIN SIGNALING DURING THE DEVELOPMENT OF THE CORTICOSPINAL TRACT
PSTP Student: Lawrence K. Low
Mentor: Dr. Hwai-Jong Cheng
Collaborators: Drs. Regina Faulkner, Xiao-Bo Liu, Jeff Coble, and Edward Jones
During development of the central nervous system (CNS), the formation of a network of appropriate and functional connections is initiated by the general guidance of axonal projections to their targets followed subsequently by a phase of remodeling to stereotypically remove any exuberant axons. A classic example of the intricate pairing of these two phenomena occurs during the early development of the corticospinal tract (CST). The CST originates from layer V neurons of the sensorimotor and visual cortices and represents the longest bundle of subcortically projecting axons in the CNS. During early development, CST axons from both cortices are initially guided to overlapping targets in the superior colliculus and spinal cord. In later development, distinct stereotypical pruning events result in the remodeling of layer V motor CST axons from the superior colliculus and the remodeling of layer V visual CST axons from the spinal cord. While the cellular events associated with the development of the CST have been reported in great detail, much less is known about the molecules that directly regulate its development. In the present study, we use a candidate-based approach to identify two class A plexins, PLXA3 and PLXA4, that play critical yet distinct roles in regulating the guidance and stereotypical pruning of motor and visual CST projections, respectively. We first demonstrated that PLXA3 and PLXA4 were expressed in layer V corticospinal neurons both during the guidance and stereotypical pruning of CST axons. Mice with knockouts for PLXA3 and PLXA4 exhibited no patterning defects within the cortex and no initial axonal guidance defects. However, in knockout mice lacking expression of PLXA3, PLXA4, and PLXA3/PLXA4, anterograde and retrograde neuronal tracing studies revealed that layer V motor CST axons exhibited an abnormal CST in the ventrolateral regions of the brainstem and spinal cord; however, stereotypical pruning of motor CST axon collaterals from the superior colliculus was retained. On the contrary, similar tracing studies revealed that PLXA3 and PLXA4 were both required for the stereotypical pruning of layer V visual CST axons from the spinal cord; however, the initial guidance of these CST axons was unaffected by loss of PLXA3 and PLXA4. Together, these results indicate a requirement for class A plexins in regulating multiple axon guidance events associated with the development of the CST.
EARLY POSTNATAL PLP-PROMOTER EXPRESSING PROGENITORS PRODUCE MULTILINEAGE CELLS IN VIVO
PSTP Student: Joyce H. Ma
Collaborator: Dr. Fuzheng Guo
Mentor: Dr. David E. Pleasure
Multiple Sclerosis (MS) is a chronic immune-mediated demyelinating disorder characterized by loss of myelin and oligodendrocytes, as well as axonopathy, within demyelinated plaques scattered throughout the central nervous system (CNS) white matter. Current treatments for MS have largely focused on controlling the inflammatory response, rather than encouraging regeneration of damaged tissues. The human CNS harbors two known neural stem cell niches: the subventricular zone of the lateral ventricle, and the dentate gyrus of the hippocampus. Recently a novel class of cells with progenitor properties has been characterized in the CNS. These so-called polydendrocytes are a cell population distinct from astrocytes, mature oligodendrocytes, and neurons. Polydendrocytes are capable of giving rise to oligodendrocytes in vitro, thus traditionally have been described as oligodendrocyte progenitors. Unlike the two known niches, polydendrocytes are distributed throughout the adult mammalian CNS. Thus one foreseeable strategy for the treatment of MS could involve the remyelination of plaques via polydendrocyte differentiation. Pursuing this strategy requires further characterization of the pluripotency potential of polydendrocytes in vivo in different anatomic regions of the CNS. Proteolipid promoter (plp-promoter) activity in the newborn mouse CNS is restricted to polydendrocytes. Polydendrocytes are multipotent in vitro and after transplantation, but it is not known whether plp-promoter expressing polydendrocytes (PPEPs) have the potential to generate multiple cell lineages during normal development in vivo. We addressed this issue by fate mapping plp-Cre-ERT2/Rosa26-EYFP double transgenic mice. In addition to generating mature myelinating oligodendrocytes, these cells give rise to a subset of immature postmitotic protoplasmic astrocytes in gray matter of the spinal cord and ventral forebrain, but not in white matter. Small numbers of neurons in the ventral forebrain, dorsal cerebral cortex and hippocampus were also generated from postnatal PPEPs. EYFP-labeled representatives of each of these lineages survived to adulthood. These findings indicate regional differences in the fates of neonatal PPEPs, which are multipotent in vivo, giving rise to oligodendrocytes, astrocytes and neurons. To determine whether PPEPs could serve to repair MS lesions, we intend to fate-map adult PPEPs in an animal model of MS and pursue transplantation studies to assess the repair of lesions in vivo.
IMMUNOLIPOSOMAL / IMMUNO-NANOMICELLE THERAPY OF B CELL NON-HODGKIN’S LYMPHOMA
PSTP Student: Shiloh Martin
Mentor: Joseph Tuscano
Collaborators: Drs. Jason Kato, Juntao Luo, Yunpeng Ma, Robert O’Donnell, and Kit Lam
Non-Hodgkin’s Lymphoma (NHL) is the sixth most common cause of cancer death in the US. Chemotherapy is initially effective, but often limited by toxicity and relapse is common due to resistance. Thus, new agents for the treatment of NHL are needed. HB22.7 is a mouse monoclonal antibody (mAb) that binds CD22 (a B cell specific glycoprotein), blocking ligand binding. Previous studies in the lab have demonstrated that HB22.7 reduces human NHL xenograft volume in nude mice. Piceatannol (3,4,3’,5’-tetrahydroxy-trans-stilbene) is a metabolite of resveratrol (3,5, 4’-trihydroxy-trans-stilbene), a polyphenolic compound found in wine, grapes, mulberries, cranberries, and other foods. Resveratrol has been shown to inhibit growth and induce apoptosis in many tumor cell lines and has been investigated as a chemopreventative agent. Piceatannol has been reported to be a selective inhibitor for Syk and STAT3. We are developing an immunoliposome with piceatannol (or resveratrol) encapsulated inside the liposome and HB22.7 conjugated on the liposome surface. HB22.7 will target the liposomes to the CD22-expressing B cell tumor. Upon reaching the tumor, the liposome can fuse with the target cell’s plasma membrane, releasing the piceatannol (or resveratrol) into the cell, ultimately causing cell death. The use of immunoliposomes allows more drug to be delivered to the target site, while lowering potential toxicities by avoiding release of drug at non-targeted sites. We are also developing immuno-nanomicelles, encapsulating piceatannol (or resveratrol) with HB22.7 conjugated to the surface. Immuno-nanomicelles are much smaller than liposomes (20nm versus 100nm) and are able to encapsulate hydrophobic agents (such as piceatannol/resveratrol) much more readily than liposomes. Thus, when using hydrophobic drugs, immuno-nanomicelles may be more efficacious than immunoliposomes for NHL therapy.
THE ROLE OF CAVEOLAE IN OSTEOBLAST MECHANOTRANSDUCTION
VSTP Student: Jessica M. Morgan
Mentor: Dr. Clare Yellowley
The skeletal system is known to adapt to mechanical loading by altering the balance between bone formation and bone resorption. Bone cells, including osteoblasts and osteocytes, are thought to be the primary mechanosensors in bone. It is these cells that can detect mechanical loads and transduce them into either an anabolic or catabolic response in the bone tissue. Studies suggest free cholesterol and sphingolipid rich microdomains of the cell membrane, such as caveolae, may be instrumental in bone cell mechanotransduction. Further studies indicate shear flow induces a reorganization of these membrane domains through re-localization of caveolin-1. Shear stress induces a variety of responses in osteoblasts including transient calcium responses, and activation of the Wnt/βcatenin signaling pathway. Activation of nuclear β-catenin subsequently leads to osteoblastic differentiation. We propose that the activation of a calcium response and Wnt/βcatenin signaling in response to fluid flow requires caveolae. To test the hypothesis that shear stress responses are caveolae dependent, we will disrupt caveolae via cholesterol depletion and caveolin-1 siRNA. Osteoblast response will be quantified with real-time intracellular calcium imaging using the radiometric dye fura-2 and Western blot to detect activation of the Wnt/βcatenin pathway. Identifying the role of caveolae in mechanotransduction will provide potential target sites for intervention when bone mineral density is perturbed in conditions such as osteoporosis, weightlessness, or stress fracture.
STOICHIOMETRY OF CLC-K AND BARTTIN AS DETERMINED BY TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF)
PSTP Student: Karen J. Mu
Mentors: Drs. Ebenezer Yamoah and Tsung-Yu Chen
Barttin is an auxiliary subunit of the ClC-K channel and its functional importance in sound transduction is underlined by the fact that mutations result in Bartter Syndrome type IV, of which deafness is a component. Barttin has been identified as a necessary subunit of the ClC-K channel, crucial for translocation and function. However, the stoichiometry of the assembly between Barttin and the ClC-K dimer is not known. The photobleaching of a single GFP is a discrete process, thus the fluorescence intensity of a protein complex with one or several GFP molecules drops in a stepwise fashion. The number of bleaching steps reveals the number of GFP-tagged subunits in the channel complex on a single-molecule level. We employed Total Internal Reflection Fluorescence (TIRF) in order to determine the composition of ClC-K channels on the surface membrane of Xenopus laevis oocytes. TIRF enabled the selective illumination and analysis of fluorescent proteins expressed on the membrane in the absence of background auto fluorescence from the cytoplasm. ClC-K1-EGFP and Barttin-EGFP fusion protein constructs were generated using a modified pGEM-HE vector, with every tagged subunit carrying exactly one fluorophore. Barttin-EGFP co-expressed with untagged ClC-K revealed a predominance of spots exhibiting single-bleaching steps, implying that the ClC-K channel complex is composed of a ClC-K dimer and a single Barttin subunit. This is consistent with evidence that Barttin plays a role in the trafficking of the ClC-K dimer to the surface. Indeed, a single Barttin binding to the ClC-K dimer may be sufficient to ensure surface expression and function of the mature channel.
A SYNTHETIC, CHEMICALLY DEFINED, THREE-DIMENSIONAL CELL ADHESION MATRIX AS A TOOL FOR THE STUDY OF CANCER CELL PHENOTYPES AND THE TUMOR MICROENVIRONMENT
PSTP Student: Ekama Onofiok
Mentor: Dr. Kit S. Lam
In recent tumor biology studies, manipulation of tumor cell-extracellular matrix interactions in three-dimensional (3-D) culture using laminin- or collagen-rich gels, has been shown to influence cancer cell phenotypes such as tumor growth properties, angiogenesis, response to cytotoxic and apoptosis- inducing agents, and remarkably, a reversion of the malignant phenotype and re-establishment of normal architecture in 3-D breast cancer models. 3-D cancer cell culture has thus emerged as a superior ex vivo system for studying cancer biology, as it captures more of the relevant complexity of the tumor microenvironment than traditional 2-D culture on plastic substrata can achieve. In efforts to study the influence of individual and select combinations of extracellular matrix components on cancer cell behavior in culture, in this study, we develop a synthetic, polyvinyl alcohol-based cell adhesion scaffold, comprised of well-defined integrin-targeting matrix components identified in our lab using the one-bead-one-compound (OBOC) combinatorial library method. This scaffold can be cross-linked to form a hydrogel suitable for 3-D culture of cancer cells expressing cognate integrin heterodimers, for further dissection of integrin signaling pathways in cancer, and effects exerted by cell-matrix adhesion on morphological and functional phenotypes of a number of epithelial tumors in culture.
RENAL VOLUME DETERMINED BY COMPUTED TOMOGRAPHY
PSTP Student: Nicolas D. Prionas
Mentor: Dr. John M. Boone
Collaborators: Drs. Marijo Gillen and Richard W. Katzberg
Radiologic determination of renal size is a critical step in establishing a differential diagnosis in renal parenchymal disease. Renal length is currently used to measure renal size, but there is a broad push toward three-dimensional, quantitative techniques. Thirteen patients from our institution who received multiple computed tomography (CT) scans (average number of CT scans = 20.8, range 5 to 45) for symptoms unrelated to renal disease were identified retrospectively. CT scans were performed at 120 kV with a slice thickness of 5 mm, although some scans had up to 10 mm slices. For each scan, the left kidney (LK) and right kidney (RK) volumes were assessed by hand segmentation (window = 400 HU, level = 30 HU). In total, volume assessment was performed on 253 abdominal CT scans, 104 of which specifically evaluated renal function using contrast medium. The effect of contrast medium (iohexol-350 and iodixanol-320) on kidney volume was established by comparing LK and RK volume before and after contrast administration. The mean LK and RK volumes were 179.1 ± 39.7 [standard deviation] cm3 and 172.7 ± 37.1 cm3, respectively. The LK and RK volumes for each patient were found to fluctuate proportionally over time with a mean range of fluctuation of 48.0 cm3 and 47.4 cm3, respectively. In response to contrast medium, the mean increase in the LK volume was 8.6 cm3 (4.7%) and the mean increase in the RK volume was 9.1 cm3 (5.0%). These increases were found to be statistically significant with p = 0.00019 and p = 0.0013, respectively. The anticipated LK and RK volumes and volume relationships between LK and RK as well as between men and women were reinforced by this study. The findings emphasize the utility of CT as a three-dimensional, quantitative tool not only for the assessment of normal physiologic volume fluctuations, but also in the diagnosis of pathologic changes to the renal parenchyma.
EFFECTS OF OXYGLOBIN VERSUS HESPAN AND SHED BLOOD RESUSCITATION ON THE MICROCIRCULATION IN A CANINE HEMORRHAGIC SHOCK MODEL: CORRELATION WITH SYSTEMIC FUNCTION AND OXYGENATION
PSTP Student: Patricia To
Mentor: Dr. Anthony Cheung
Collaborators: Drs. Linda Talken and Robert Gunther
The care of rural and combat trauma victims is complicated by difficult to address issues of greater injury severity and delays to definitive care, leaving the area of prehospital care the one viable area to focus efforts on. Prehospital care may be improved by the development of an effective blood substitute, which does not currently exist. Previous studies on the effectiveness of blood substitutes focused primarily on restoration of systemic function (e.g., arterial pressure, cardiac output) and oxygenation and largely ignored the microcirculation, the organ system where blood performs its tissue level functions. The hypotheses of this study are that the microcirculation changes in a predictable manner simultaneously with systemic function and oxygenation during hemorrhage and resuscitation and that microvascular parameters (venular diameter, red-cell velocity) correlate with systemic function and oxygenation parameters. Following instrumentation for monitoring of systemic function and oxygenation, dogs were splenectomized, allowed to stabilize, hemorrhaged, and randomly assigned to three resuscitation groups: Hespan/colloid (n=3), shed blood (n=3), and Oxyglobin/blood substitute (n=3). Computer-assisted intravital microscopy was used to non-invasively videotape and quantify microvascular changes in the conjunctival microcirculation during pre-hemorrhage, post-hemorrhage, and resuscitation phases to correlate with systemic function and oxygenation changes. The shed blood group showed decreased venular diameter and increased red-cell velocity during hemorrhagic shock along with systemic function and oxygenation changes followed by restoration to pre-hemorrhage values after resuscitation. The Hespan and Oxyglobin groups followed this pattern up to immediately post-resuscitation, but by three hours post-resuscitation, microvascular parameters began approaching post-hemorrhage values while systemic function and oxygenation parameters remained relatively stable. These results suggest that microcirculatory parameters may change before systemic function and/or oxygenation in non-blood products and that the microcirculation can be used to distinguish between blood products and non-blood products with an effective blood substitute being indistinguishable from blood in the microcirculation hours after its infusion. Strong correlations between many systemic function and oxygenation parameters with microvascular parameters show that there is a strong link between what occurs in the microcirculation and what occurs systemically and that the microcirculation can be used to follow parameters that otherwise may require invasive procedures to monitor.
EXACERBATED DISEASE IN SIMIAN IMMUNODEFICIENCY VIRUS (SIV) AND PLASMODIUM FRAGILE CO-INFECTED RHESUS MACAQUES
VSTP Student: Kristin A. Trott
Mentors: Dr. Kristina Abel
Collaborator: Drs. Shirley Luckhart and Jennifer Y. Chau
Epidemic HIV and malaria co-occur in Africa and Asia. From available human data, we hypothesized that P. fragile infection during acute SIV infection enhances immune activation and thereby virus replication and, conversely, that SIV infection enhances parasitemia and parasite transmission in co-infected macaques. We developed a rhesus macaque model of SIV and Plasmodium fragile infection to model human co-infection. Adult male rhesus macaques were inoculated with SIVmac251 (n=2), or P. fragile infected erythrocytes (n=2), or both (n=2). Immunological, parasitological and virological parameters were assessed throughout the course of infection by multiparameter flow cytometry, blood smears, and PCR. For transmission studies, freshly drawn blood from P. fragile infected macaques was fed to naïve Anopheles freeborni and after 10-12 days, midguts were dissected for oocyst development to determine infectivity. P. fragile-only infection resulted in increased CCR5 and Ki67 expressing CD4+ T cells, potential target cells for SIV during peak parasitemia. Indeed, acute malaria infection was associated with increased viremia in co-infected animals. Further, the SIV-induced immunosuppression correlated with severe anemia and uncontrolled parasitemia in co-infected animals. Importantly, mosquitoes that fed on blood from co-infected animals had higher mean oocysts per midgut than those fed on blood from P. fragile-only animals suggesting that co-infection will lead to higher transmission rates. The underlying immunological mechanisms likely include reduced dendritic cell function and increased regulatory T cell function. The data suggest that co-infection results in a dysfunctional immune response which will potentially increase the rate of transmission of both SIV and malaria in co-infected individuals. The established model can be used to test drug and vaccine effectiveness and elucidate the mechanisms of increased transmission and the immunological response to HIV-P. falciparum co-infection.
ROLE OF INNATE SIGNALING IN B-1 CELL RESPONSES TO VIRAL INFECTION
VSTP Student: Elizabeth Waffarn
Mentor: Dr. Nicole Baumgarth
Collaborator: Dr. Youn Soo Choi
B-1 cells represent a unique subset of B lymphocytes acting in an autonomous T-independent manner as primary sources of IgM following influenza virus infection. Little is known about the mechanisms controlling B-1 cell function. Our previous work revealed that B-1 cells redistribute from various tissues, such as pleural cavity, spleen and bone marrow to the regional lymph nodes after infection with Influenza virus. The major subset of B-1 cells, known as B-1a, is unable to signal through their B cell receptor (BCR) due to expression of the inhibitor CD5, yet they were the major subset that responded to infection. We aimed to test our hypothesis that infection-induced innate signals regulate the migration of B-1 cells to sites of infection and differentiation to IgM secreting effectors. Noting that IL-1 increases both locally and systemically following infection, we first investigated the role of IL-1 in B-1 cell activation. Peritoneal lavage cells as source of B-1 cells from uninfected wild-type mice were used in a transwell migration assay that allowed identification of cells migrating towards relevant chemokines by multi-color flow cytometry. Ongoing in vivo experiments utilize IL-1R-/- mice and chimeric mice, in which only B-1 cells lack IL-1R expression, to determine the need for IL-1R signaling in B-1 cell activation following influenza virus infection. IL-1 did not directly induce chemotactic B-1 cell migration, but 10 hour pre-incubation of B-1 cells with IL-1 inhibited migration towards the chemokine CXCL12 but not towards CCL21 or CXCL13. These results indicate a role for IL-1 in directing receptor-dependent homing mechanisms and guiding chemotactic migration of B-1 cells between peritoneum and other lymphoid organs. Future in vivo studies will define the need for the IL-1R and/or other innate receptors in regulating B-1 cell responses to influenza and other pathogens.
ROLE OF GLIAL SODIUM-DEPENDENT CO-TRANSPORTERS IN AN IN VITRO TRAUMATIC BRAIN INJURY MODEL
VSTP Student: Eric West
Mentor: Dr. Bruce Lyeth
Collaborator: Dr. Gene Gurkoff
Traumatic brain injury (TBI) stimulates a massive elevation in extracellular glutamate and the neuropeptide N-acetyl-aspartyl-glutamate (NAAG). NAAG is rapidly broken down into NAA and glutamate by peptidases located on nearby astrocytes. Glutamate and NAA are then removed from the synapse by Na+ dependent co-transporters located on nearby astrocytes. Previous work has shown that astrocyte death precedes neuronal death in the rat in vivo model of TBI; and that a main cause of this is from excessive intracellular Na+ ([Na+]i) that purportedly leads to [Ca++]i stimulated cell death. Our current research is examining the relative contribution of the Na+/glutamate co-transporter and the Na+/NAA co-transporter to the elevated levels of [Na+]i and [Ca++]i in astrocytes. Differentiation of each co-transporter is accomplished with pharmacological inhibitors; TBOA to block Na+/glutamate transporter and ZJ-43 to block NAA co-transporter via inhibition of the NAAG peptidase. Primary mixed neuronal-glial cultures grown in 6-well plates with flexible well bottoms will be imaged at 10-14 days. Prior to injury, the selective epiflouresent sodium indicator SFBI-AM or the selective calcium indicator FURA-2-AM will be loaded into the cells, followed by a drug inhibitor or vehicle. The cells are then mechanically strain injured by flexing the well bottoms and monitored for 1 hour. Cell death will be measured at 24-hours to assess neuronal protection. Solely, the inhibitors should cause varying reductions to the ions, thus representing the contributions of each specific Na+ co/transporter. When applied together, the inhibitors are expected to drastically reduce both [Na+]I, [Ca++]I, and cell death.