Retreat 2008 M.D./Ph.D. Student Abstracts
CORRELATIONS BETWEEN ACCULTURATION AND ADOLESCENT PREGNANCY AMONG LATINAS
PSTP Student: Teresa Barcellos
Mentor: Dr. Marc Schenker
Collaborator: Dr. Laurel Beckett
Purpose: To elucidate the relationship between acculturation and risk of adolescent pregnancy among Latina teens in California and to evaluate whether acculturation primarily correlates with pregnancy risk via an effect on age at sexual debut.
Methods: A secondary analysis was performed using data from 572 participants in a study of pregnant Latinas. Acculturation was measured using the ARSMA-II and proportional hazards regression models were employed to determine the risk of adolescent pregnancy while controlling for the effects of age at immigration.
Results: Overall, 354 women (62.4%) had experienced their first pregnancy before age 20. Relative to women who delayed childbearing until after adolescence, women who had adolescent pregnancies were less acculturated and more likely to be foreign-born and have immigrated during late childhood or adolescence. High acculturation was associated with an earlier onset of sexual activity, but this did not translate to an increased risk of adolescent pregnancy.
Conclusions: Low-acculturated and recently-immigrated Latina teens are at high risk of pregnancy. In contrast to current beliefs, the correlation between high acculturation and early sexual debut does not equate with an increased likelihood of pregnancy; it is probable that more acculturated teens are better able to practice effective contraception. Future study and interventions should focus on strategies for increasing contraceptive use and promoting delayed childbearing among less acculturated Latina adolescents.
RECOMBINANT RIFT VALLEY FEVER VIRUS LACKING THE NSs AND NSm GENES IS HIGHLY ATTENUATED, CONFERS PROTECTIVE IMMUNITY FROM VIRULENT VIRUS CHALLENGE AND ALLOWS FOR DIFFERENTIAL IDENTIFICATION OF INFECTED AND VACCINATED ANIMALS
VSTP Student: Brian H. Bird
Mentor: Dr. N. James MacLachlan
Collaborator: Dr. Stuart T. Nichol
Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping “abortion storms” and high mortality among young animals. Human infection results in self-limiting febrile disease that in ~1-2% of patients progresses to more serious complications including hepatitis, encephalitis, retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs protein and the M segment-encoded NSm proteins are important virulence factors. The development of safe effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here we report the successful reverse genetics generation of multiple recombinant eGFP tagged RVF viruses containing either full length complete virus genome or precise deletions of the NSs alone or NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated with no detectable viremia or clinical illness observed at high challenge dosages (1.0x104 PFU) in a rat lethal disease model. A single dose immunization regimen induced robust anti-RVF virus IgG antibodies (titer ~1:6400) and mean PRNT50 neutralization titers (>1:640) by day 26 post-immunization. All pre-immunized animals subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF virus vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following natural or intentional introduction into previously unaffected areas.
A COMPARISON OF TWO DNA EXTRACTION METHODS OF TOXOPLASMA GONDII OOCYSTS FROM TURBAN SNAILS (TEGULA SPP.)
VSTP Student: Roxann S. Brooks
Mentors: Dr. Patricia Conrad and Dr. Christine Kreuder-Johnson
Collaborator: Dr. Heather M. Fritz
Infection with the terrestrial protozoal pathogen, Toxoplasma gondii, is associated with encephalitis in the California Southern Sea otters (Enhydra lutris nereis) and thought to be a major contributor to their population stagnation. Toxoplasma is a zoonotic pathogen that rarely causes a problem in healthy adults but can be fatal for immuno-compromised individuals, and the fetuses of pregnant women. The development of sensitive and specific detection techniques are necessary to determine potential sources of infection in the environment and are critical for preserving the sea otter population and protecting public health. Detection of T. gondii has proven difficult because of the low concentrations of oocysts persisting in the environment and the presence of inhibitors in the samples. T. gondii oocyst-spiked Turban snails (Tegula spp.), a common prey item of the otters, were used to evaluate the sensitivity of two DNA extraction methods by PCR. Snail tissue was spiked with serial dilutions representing 500, 50, 5, 1, and 0 oocysts. Oocyst DNA was then extracted using either a Cetyl trimethylammonium bromide (CTAB) extraction protocol or a Qiagen DNeasy kit. DNA was amplified using a nested PCR protocol with three different T. gondii specific primers. The CTAB protocol was the more sensitive extraction method, with a detection limit of the 50 oocysts per sample using three different primer sets to amplify DNA as compared to the Qiagen kit with a detection limit of 500 oocysts per sample. Neither extraction method was sensitive enough for environmental sampling, warranting continued work to develop a more sensitive detection method.
IDENTIFYING THE NEURONAL RECEPTOR FOR AGRIN INVOLVED IN INTERNEURONAL SYNAPSE FORMATION
PSTP Student: Jolene M. Chang
Mentor: Dr. Michael Ferns
Collaborator: Dr. Jacinthe Gingras
Synapse formation is a complex process that is essential for the normal development of a functional nervous system. Synapse formation is regulated by bidirectional signalling between pre and postsynaptic neurons which in turn induce pre and postsynaptic differentiations to align together to mediate efficient synaptic transmission. Synaptogenesis has been studied extensively in the neuromuscular junction, where an extracellular matrix protein called agrin is deposited by the presynaptic motoneuron into the basal lamina of the muscle cell, where it signals through MuSK receptor tyrosine kinase to cluster acetylcholine receptors at the postsynaptic site. Outside the neuromuscular junction, agrin has also been studied in the superior cervical ganglion (SCG). Interneuronal synapses in the SCG system shares some similarities to the neuromuscular junction – it is also a fast cholinergic synapse. Agrin knockout mice have fewer synapses in SCG and impaired synaptic transmission. Thus, agrin regulates synaptic differentiation in the peripheral nervous system as well as at the neuromuscular junction. The mechanism through which agrin acts to stabilize and maintain synapses in interneuronal cholinergic synapses is unknown. Most importantly, the receptor for agrin in neurons has not yet been identified. My project is to characterize and identify the agrin receptor in neurons. One approach is to use recombinant agrin fragments to to map agrin’s interaction domain with the receptor, in order to find a receptor that binds to this active agrin region. Another approach is to take a candidate receptor, MuSK, and examine for similar synaptic defects to agrin knockouts. Preliminary results show that there is a qualitative decrease in the overlap of pre and postsynaptic staining in vivo in MuSK knockout SCG. The decreased colocalization of pre and postsynaptic markers mirrors the agrin knockout SCG phenotype, which is consistent with the idea that MuSK is agrin’s receptor in neurons. These findings suggest that MuSK may be the receptor for agrin in the SCG. Future studies will have to show a direct correlation of agrin activation of MuSK, which can be shown by looking at agrin effects of MuSK tyrosine phosphorylation of neurons in vitro.
SRC TYROSINE KINASE ONCOGENIC POTENTIAL AND PATHWAY AS REVEALED BY SELECTIVE INHIBITOR AZD0530
PSTP Student: Yu-Ming Chang
Mentor: Dr. Evans Christopher and Dr. Kung Hsing-Jien
Prostate cancer is the most frequently diagnosed cancer in American men. We have previously demonstrated that Src mediates androgen-independent proliferation in prostate cancer. We sought to investigate the Src-mediated oncogenic pathways and resultant tumor biology using AZD0530, a novel Src inhibitor that is entering phase II clinical trials. We show that Src is expressed and activated in all prostate cancer cell lines and that AZD0530 inhibits Src activation in a rapid and dose-dependent manner. In addition, AZD0530 inhibits cell proliferation in DU145 and PC3 cells through increased G0/G1 and decreased S phase cell populations. AZD0530 inhibits the binding of beta-catenin to the promoters of G1 phase cell cycle regulators cyclin D1 and c-Myc. AZD0530 may also inhibit c-Myc at the protein level via the Src-ERK1/2-c-Myc pathway. Furthermore, cell motility factors FAK, p130CAS, and paxillin activation in DU145 and PC3 cells were inhibited by AZD0530. AZD0530 treatment reduced orthotopic DU145 tumor growth in mice by 45% (p<0.01). We conclude that AZD0530 inhibits growth of prostate cancer in vivo and may be an effective therapy in the treatment of prostate cancer.
EFFECTS OF EICOSAPENTAENOIC ACID, FISH OIL AND LIPOIC ACID IN A NOVEL RAT MODEL OF TYPE-2 DIABETES
VSTP Student: Bethany Cummings
Mentor: Dr. Peter J. Havel
Collaborators: Drs. Kimber Stanhope, James Graham, and Steven Griffen
Due to the increasing prevalence of Type-2 Diabetes Mellitus (T2DM) there is a need to identify effective new strategies for diabetes prevention, which could include the use of nutritional supplements. We investigated the efficacy of several widely used nutritional supplements to prevent T2DM in a novel diabetic rat model developed in our laboratory (UCD/T2DM rat). Eicosapentaenoic acid (EPA) and fish oil (containing EPA and DHA) are activators of peroxisome proliferator-activated receptors. Alpha-lipoic acid (LA) is a potent anti-oxidant which may reduce oxidative stress associated with insulin resistance/diabetes. In Study 1 we investigated the effect of EPA, fish oil and LA on the time to diabetes onset and longitudinally assessed circulating lipids and hormones. Animals received supplements added to their food starting at 1 month of age for 11 months: EPA (1g/kg/d), fish oil (3g/kg/d), LA (40mg/kg/d), Diabetes onset was determined by weekly blood glucose measurements. Following Study 1, LA and fish oil were chosen for further study in Study 2. To focus on the anti-oxidant actions of LA, animals were challenged with dietary fructose (20% of energy). Animals received fish oil (3g/kg/d) or LA (80mg/kg/d) in food along with fructose starting at 2 months of age. Blood glucose was monitored and an intravenous glucose tolerance test (IVGTT) was performed at 3.5 months of age (prior to diabetes onset). There were no significant differences in energy intake or body weight gain between treatment groups in either study. Average ages of onset in Study 1 were: control- 6.7±2.5 (n=17), EPA- 6.0±2.3 (n=13), fish oil- 6.0±3.1 (n=15), LA- 7.4±3.5 (n=15) months. In Study 1, fasting plasma free fatty acids were 23±6% lower (p< 0.05) in EPA-treated animals and fasting triglycerides were 40±14% lower (p< 0.01) in fish oil-treated animals from 1 month before to 1 month after diabetes onset. Fasting glucose, insulin, leptin and adiponectin concentrations were similar between treatment groups before and after diabetes onset. In Study 2, fructose feeding alone increased glucose excursions during IVGTT by 13±7% (p=0.07). Glucose excursions were 25±3% lower in fructose-fed animals receiving LA (p<0.05). In conclusion, EPA and fish oil did not delay the onset of T2DM, but improved lipid dysregulation before and after diabetes onset in this rat model. Supplementation with LA improved glucose homeostasis in the pre-diabetic state in fructose-fed insulin-resistant rats and these animals are being studied to determine if this leads to a delay in diabetes onset.
THE EFFECT OF EGG YOLK AND LPS ON MACROPHAGE CHEMOTAXIS AND THE INFLAMMATORY RESPONSE
VSTP Student: Ingrid Cornax Edwards
Mentor: Dr. Kirk C. Klasing
After ovulation in the egg-laying chicken, the lipid-rich egg yolk is captured by the oviduct and moved by peristalsis down the reproductive tract where egg white and shell deposition occur. Occasionally, reverse peristalsis occurs and the egg yolk and some bacteria from the distal cloaca are moved out of the oviduct into the body cavity. This process of “internal lay” causes blood-derived macrophages to invade the body cavity to clear the yolk and invading bacteria. Most cases of internal lay lead to egg yolk peritonitis and death due to sepsis. OBJECTIVES: Determine how egg yolk and lipopolysaccharide (LPS) affect macrophage chemotaxis and macrophage IL1, IL10 and nitric oxide (NO) production. HYPOTHESIS: Yolk and LPS will cause a synergistic increase in cell chemotaxis, IL1, IL10 and NO production. MATERIALS and METHODS: Monocytes were isolated from chicken blood and matured into macrophages in vitro. Macrophage chemotaxis due to yolk, LPS or yolk and LPS was measured using a commercial fluorescent chemotaxis assay kit. Macrophage IL1 and IL10 production due to yolk, LPS or yolk and LPS exposure were measured by quantitative real-time PCR after 4 and 12 hours of incubation. NO production was measured by Griess assay kit after 24 hours of incubation with yolk, LPS or yolk and LPS. RESULTS: Yolk significantly increases macrophage chemotaxis (p=0.0003), LPS does not affect yolk chemotaxis (p=0.1939) and there is no interaction between yolk and LPS (p=0.4158). Yolk and LPS do not significantly affect IL1 messenger level at 4 hours (p=0.8876 and p=0.1887 respectively), but yolk does decrease LPS-induced IL1 message levels (p=0.0341). At 12 hours, yolk and LPS do not significantly affect IL1 message levels (p=0.3106 and p=0.3626, respectively) and yolk does not affect LPS-induced IL1 message levels (p=0.1113). At 4 and 12 hours of incubation, only LPS significantly affects IL10 message levels (p<0.0001 and p=0.0009, respectively). Yolk significantly reduces LPS-induced NO-production (p<0.0001). CONCLUSIONS: Yolk is chemotactic and may be the main stimulus for blood-derived macrophage invasion during internal lay. Yolk decreases LPS-induced IL1 and NO production by macrophages and does not affect LPS-induced IL10 production so is anti-inflammatory in nature.
A MONOCHROMATIC X-RAY SOURCE FOR CANCER DIAGNOSIS AND PHOTOTHERAPY
PSTP Student: William J. Frederick III
Mentors: Dr. Jonathan P. Heritage and Dr. Neville C. Luhmann
Collaborators: Drs. Arnold Vlieks, Dandan Zheng, and Christopher Destefano
The Compton Light Source (CLS) under development as collaboration between UC Davis, UC Los Angeles, and the Stanford Linear Accelerator Center (SLAC), promises to provide a source of high intensity, well-collimated and small spot size hard x-rays with monochromatic energy tunable between 10-120 keV. This broad energy range is well suited for medical applications ranging from mammography, contrast-enhanced radiography, to x-ray phototherapy. Development of such a source should allow work now possible only at synchrotron sources to take place at smaller facilities in a clinical environment. Development of this source will employ Monte Carlo simulation, in-vitro cancer cell radiation, in-vivo tumor radiation, and imaging with phantoms and patients. Nanoparticle targeted contrast agents will deliver high-Z metals directly to tumor cell nuclei, and higher quality images or better tumor control with lower dose on normal tissue may be achieved with the x-rays tuned to the k-edge characteristic energies of the contrast agents. This proposal focuses on the in vitro evaluation of the dose enhancement effect of radiosensitizing agents irradiated with both conventional and synchrotron x-ray sources. Current radiosensitizing agents under consideration include Iohexol, metalloporphyrins including Tin Protoporphyrin IX, and metal oxides nanoparticles such as Titanium Oxide. For 28 mg/ml Iohexol, a dose enhancement factor (DEF) of 1.5 has been measured using a hardend conventional x-ray source. Synchrotron experiments at the Stanford Synchrotron Radiation Laboratory have confirmed energy dependence of the dose enhancement factor for this radiosensitizer, with the DEFs of 1.2, 1.7, and 2.3 for irradiation energies of 32, 34 and 42 keV, respectively.
TEST FOR HYPERURICOSURIA
VSTP Student: Nili Karmi
Mentor: Dr. Danika Bannasch
All mammals, with the exception of great apes, humans and Dalmatians excrete allantoin (a soluble compound) in their urine as the end product of purine metabolism. Great apes, humans and Dalmatians excrete uric acid rather than allantoin as the end product of purine metabolism. Hyperuricosuria is excess excretion of uric acid in urine (which can lead to the formation of uric acid stones that can cause dangerous blockage of the urinary tract). Since Dalmatian dogs have been studied with regard to hyperuricosuria for nearly a century, it is presumed that the breed is fixed for this trait. We plan to correlate the genotype at this locus with the hyperuricosuria phenotype in other breeds of dogs that are known to have a predisposition to uric acid stones. Since not all dogs with hyperuricosuria form uric acid stones, it is necessary to have a test to determine if individuals from the predisposed breeds are hyperuricosuric. To accomplish this, a non-invasive test to phenotype adult individuals for hyperuricosuria has to be established. In Dalmatians, it has been shown that a 24 hour urine collection is necessary to quantitate uric acid levels in order to monitor drug therapy for this disorder. However, a urine spot test has been used to distinguish puppies with hyperuricosuria from puppies with normal urine metabolism. Urine samples from Dalmatian, non-Dalmatian, and interbreed backcross adult dogs were collected. An Analysis of Variance (ANOVA) determined a significant difference in the uric acid to creatinine ratio between the three groups representing different states of the condition, in both life stages. The Dalmatian group was chosen to represent dogs suffering from hyperuricosuria. The interbreed backcross group was chosen to represent individuals that are heterozygous at the hyperuricosuria locus. The non-Dalmatian group represents dogs not affected by hyperuricosuria. Based on the statistical analysis results, the urine spot test can be used to determine whether a dog is hyperuricosuric or normal. This test will be used to phenotype individuals from non-Dalmatian breeds to allow for the correlation of phenotype to genotype in research pertaining to the genetic cause of hyperuricosuria.
THE ROLE OF AUTOPHAGY WITH ARGININE DEIMINASE AS A NOVEL PROSTATE CANCER THERAPY
PSTP Student: Randie Kim
Mentors: Dr. Richard Bold and Dr. Hsing-Jien Kung
Arginine deprivation by arginase as an anti-cancer therapy has historically been met with limited success. With the development of arginine deiminase (ADI), there has been a renewed interest in arginine deprivation for the treatment of some cancers such as prostate cancer. The efficacy of ADI is correlated with the lack of expression of argininosuccinate synthetase (ASS), a ubiquitous enzyme involved in the two-step synthesis of arginine from citrulline. In this study, we demonstrate ASS expression in different prostate cancer lines correlate with cell survival after ADI treatment. In particular, CWR22Rv1 expresses reduced amounts of ASS mRNA and protein and is most sensitive to ADI. FACS analysis shows ADI-induced apoptosis occurs in CWR22Rv1 after 4 days. Western blot analysis suggests that this apoptosis is caspase-independent. Caspase-independent cell death has been confirmed with the use of z-VAD-fmk, a pan-caspase inhibitor. The search for alternative cell death pathways has led to the autophagy, a process of lysosomal-mediated self-digestion of cellular proteins and organelles. Autophagy has been detected in as early as 4 hours in CWR22Rv1 cells after ADI treatment through pEGFP-LC3 translocation under fluorescence microscopy. Autophagy depends on the integration of all cellular inputs and paradoxically may lead to either cell death or cell survival. For instance, arginine deprivation possibly triggers autophagy as an early response to promote cell survival under starvation conditions. Prolonged autophagy, however, may lead to cell death. Understanding the context under which autophagy is occurring in prostate cancer may lead to the development of new chemotherapeutics. Co-administration of an autophagy inhibitor can greatly enhance the apoptotic efficacy of ADI treatment.
A CHARACTERIZATION OF b -DEFENSINS IN THE CANINE GASTROINTESTINAL TRACT
VSTP Student: Brian C. Leonard
Mentors: Dr. Charles L. Bevins and Dr. Stanley L. Marks
Innate immunity is responsible for providing ever-present or immediately inducible defense mechanisms that greatly diminish the likelihood of host infection. Antimicrobial peptides, including as defensins, are emerging as key effector molecules of innate immunity and important links to disease susceptibility are emerging in several organ systems. Defensins are antibiotic peptides with a broad-spectrum of activity, produced by phagocytes and epithelial cells, where constitutive and inducible expression has been observed. Defensins can be categorized into three families, a, b, and q based on their secondary structure and disulfide array. Variation in b-defensin expression exists in humans, cows and mice, and little is known about b-defensin expression in other species. Our interest in companion animal medicine has led us to characterize b-defensin expression in the dog and begin to explore possible association with canine disease. A computational analysis of the canine genome revealed 43 putative b-defensin gene sequences; however, canine tissue expression patterns remain to be investigated. Using a relatively comprehensive screening process we analyzed the constitutive expression of b-defensins in selected tissues with emphasis on the gastrointestinal tract. To date, have made PCR amplification primers to all 43 putative b-defensins and qualitatively evaluated their expression. A more generalized pattern of constitutive expression was found for canine b-defensin (k9BD)-1, -108 and -123, being present in nearly all of the tissues examined, while k9BD-103, -109, -112, -119, -120, -122, -139 and -140 appear to be expressed in an tissue-specific manner. PCR products have been or will be sequence verified to prove identity with the predicted sequences. Current studies include real-time quantative PCR (qPCR) assays for evaluating constitutive and inducible b-defensin expression. Canine intestinal and skin explants will be cultured and challenged with inflammatory stimuli, such as IL-1b, to induce expression of tissue specific b-defensins. Our future goal is to evaluate the possible role of b-defensins in the development of intestinal disorders including canine inflammatory bowel disease and boxer histiocytic ulcerative colitis.
ESTABLISHMENT OF A VIRAL VECTOR SYSTEM FOR EXPRESSION OF ANDES VIRUS PROTEINS IN TARGET CELLS
VSTP Student: Jessica Levine
Mentor: Dr. Tilahun Yilma
Collaborators: Dr. Heinz Feldmann and Dr. Hideki Ebihara
Within the family of Bunyaviridae is the genus Hantavirus, which is comprised of rodent-borne viruses that are found worldwide. Hantavirus infection is known to result in two major forms of disease in humans, hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) depending on the hantavirus type. Andes virus (ANDV), a HCPS-causing hantavirus, was first characterized in Argentina in 1995. Wild sigmodontine rodents are the known primary rodent reservoir of ANDV, specifically Oligoryzomys longicaudatus (long-tailed pygmy rice rat). Similar to other hantaviruses, rodents are considered to be the infectious source of ANDV for humans. However, a novel route of infection has been demonstrated for ANDV: person-to-person transmission. HCPS-associated hantaviruses have been shown to replicate predominantly in pulmonary endothelial cells, but macrophages and dendritic cells can also support replication of hantavirus in vivo and in vitro. Although there is agreement on the target cells, the mechanism for the changes in vascular permeability resulting in the observed clinical syndrome remains unclear. A few possibilities include viral replication, cytokine expression, activation of the acquired immune response, or a combination of the aforementioned. We hypothesize that hantavirus cardiopulmonary syndrome (HCPS) causing hantaviruses, such as Andes Virus (ANDV), cause capillary leakage directly through infection/activation of endothelial cells and indirectly through infection and activation of cells of the mononuclear phagocytotic system. To investigate this hypothesis we are developing a HIV-1 derived lentivirus vector system for the expression of hantavirus proteins in target cells. With this system we aim to characterize and compare the modification of cellular responses by protein expression of highly human pathogenic (ANDV), less human pathogenic (Laguna Negura) and apathogenic (Maporal and Prospect Hill) hantaviruses.
DIVERSE ROLES FOR CLASS A PLEXIN SIGNALING DURING THE DEVELOPMENT OF THE CORTICOSPINAL TRACT
PSTP Student: Lawrence K. Low
Mentor: Dr. Hwai-Jong Cheng
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
TRAFFICKING IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS: THE EFFECT OF ASTROGLIAL CHEMOKINES CCL2 AND CXCL10 ON INFILTRATION OF TH17 LYMPHOCYTES INTO THE BRAIN
PSTP Student: Joyce H. Ma
Mentor: Dr. David E. Pleasure
Multiple Sclerosis 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. Experimental allergic encephalomyelitis (EAE) is a murine model of multiple sclerosis that recapitulates many aspects of the disease.
Effector Th17 cells have been implicated in the innate immune response in the CNS in human multiple sclerosis as well as in murine EAE. Th17 cells express several pro-inflammatory cytokines such as IL-17, IL-21, IL-22, TNF-a and IFN-g, which have known effects suggestive of possible mechanisms for demyelination in EAE. Of note, IL-17 signaling has been shown to mediate autoimmunity by promoting spontaneous development of germinal centers which lead to increased production of pathogenic autoantibodies; while IFN-g has been shown to have cytotoxic effects on proliferating oligodendrocytes. Chemokines play an important role in attracting cells of the immune system as well as resident adult stem and progenitor cells to sites of injury in the central nervous system. Preliminary data from our lab shows that two chemokines, CCL2 and CXCL10 are specifically upregulated greater than 100 fold early in EAE, with the source of the chemokines being astroglial in origin. While CCL2 is chemotactic for monocytes as well as Th17 cells, and CXCL10 is important for general T cell chemotaxis, the receptors for both chemokines are expressed by Th17 cells. To test the hypothesis that astroglial production of CCL2 and CXCL10 early in EAE promote chemotaxis of Th17 cells into the CNS we constructed via recombineering conditional knockout targeting vectors for each chemokine. While studies from other groups confirm that constitutive ablation of CXCL10 and of CCL2 individually attenuate susceptibility to EAE, the question still remains as to the progression of EAE upon targeted ablation of astroglial production of CCL2 and CXCL10. Thus, studying the extent of injury in EAE under conditions of targeted ablation of astroglial CCL2 and CXCL10 could elucidate a possible mechanism of injury, and could indicate therapeutic strategies for reducing inflammatory cell infiltration past the blood brain barrier after injury to the adult CNS.
IMMUNOLIPOSOMAL THERAPY OF B CELL NON-HODGKIN’S LYMPHOMA
PSTP Student: Shiloh Martin
Mentor: Dr. Joseph Tuscano
Collaborators: Dr. Yunpeng Ma and Dr. Robert O’Donnell
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. Syk is a tyrosine kinase involved in B cell receptor (BCR)-mediated signaling, while STAT3 is a transcription factor involved in growth factor and cytokine signaling, including IL-10. The activation of Syk affects several downstream signaling pathways in the B cell, overall leading to B cell growth and proliferation. The STAT-3/IL-10 signaling pathway enhances B cell growth while suppressing T helper type I responses. We are developing an immunoliposome which encapsulates piceatannol (or resveratrol) inside and expresses HB22.7 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. The piceatannol (or resveratrol) can then inhibit Syk and/or STAT3 in the tumor cell, leading to 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.
VISUAL FIXATION PATTERNS DURING RECIPROCAL SOCIAL INTERACTION DISTINGUISH A SUBGROUP OF 6-MONTH-OLD INFANTS AT-RISK FOR AUTISM FROM COMPARISON INFANTS
PSTP Student: Noah M. Merin
Mentor: Dr. Sally J. Rogers
Autism is a neurodevelopmental disorder that profoundly affects social development and language. Genetic factors are thought to play an important role in the risk of developing the disorder; twin-concordance studies have estimated heritability at 60-90%, one of the highest for any psychiatric disorder. It is presently diagnosed using a structured clinical observation of young toddlers who show signs of abnormal development. This clinical observation assesses behaviors that do not emerge until the second year of life (such as language, peer interaction, pointing, etc.). Therefore, autism cannot currently be diagnosed until after these behaviors typically appear. If we could identify the presence of behavioral markers of the disorder at an earlier age, there would be more time to intervene before the onset of symptoms. Knowing the age of onset also has theoretical importance—it would help in the search for environmental triggers and focus attention on specific neurodevelopmental processes. Infant gaze behavior, particularly during reciprocal social interaction, may be a useful behavioral marker for autism in young infants. In the present study, we tested 31 six-month-old infants at elevated risk for developing autism, and 24 typically-developing controls. We recorded eye-tracking data during a live reciprocal interaction between the infant and the mother. In the at-risk group, we found 10 infants who made poor eye contact, and focused visual attention predominantly on the mother’s mouth. Only one infant in the control group showed this pattern of gaze. Ongoing longitudinal work will determine if the subgroup of at-risk infants who do not look at their mother’s eyes later develop autism or autism-related disorders.
STOICHIOMETRY OF CLC-K AND BARTTIN AS DETERMINED BY TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF)
PSTP Student: Karen J. Mu
Mentor: Dr. Tsung-Yu Chen
Collaborator: Dr. Ebenezer Yamoah
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
Collaborator: Dr. Juntao Luo
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 culture of cancer cells 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.
THE ROLE OF VEGETATION DENSITY AND FLOW CONDITIONS ON REMOVAL OF CRYPTOSPORIDIUM PARVUM OOCYSTS FROM SURFACE WATERS IN COASTAL CALIFORNIA WETLANDS
VSTP Student: Amber Roegner
Mentor: Dr. Patricia Conrad
Collaborators: Drs. Woutrina Miller, Kristen Arkush, Barbara Byrne, Melissa Miller, Dane Hardin, Karen Shapiro, John Largier, Stefan Wuertz, and Dave Jessup
Wetlands have been shown to reduce the fecal pathogen load of nonpoint source pollution flowing from land to freshwater and marine ecosystems, and are being recognized within the US and internationally as a promising and practical secondary and tertiary wastewater treatment method for both agricultural and municipal run-off. However, the major mechanisms by which this reduction occurs within wetlands are not well characterized. Preliminary work has demonstrated the role of sedimentation in the removal of protozoal pathogens in constructed wetlands, but under tightly controlled, non-variable environmental conditions. Periods of heavy rainfall or flooding have been correlated with increased concentrations of Cryptosporidium parvum oocysts in surface waters (hypothesized to be associated with increased agricultural run-off or sewer overflows), so it is crucial to investigate the efficacy of secondary removal under these high flow conditions. Understanding these mechanism of oocyst removal under variable environmental circumstances will help to evaluate wetland function and could help public health officials anticipate times of greater potential public health risk of waterborne cryptosporidiosis. Controlled laboratory flume experiments will use natural coastal waters to investigate the impact of flow rates and variable vegetation density on oocyst removal and sedimentation within the water column. Natural substrate (sand) and artificial plant life will be used to simulate native central California coast wetland conditions. Known concentrations of inactivated C. parvum oocysts will be introduced into the enclosed flumes with re-circulating coastal waters of pre-determined ranges of turbidity, salinity and temperature. Oocyst removal efficiency over time will be quantified through periodic water samples at inlet and outlet sampling points of the flume under low, mid and high flow conditions and under variable vegetation densities. Cryptosporidium oocysts will be detected using water filtration and direct fluorescent antibody (DFA) quantification.
MICROVASCULAR CHANGES FOLLOWING HEMORRHAGE AND RESUSCITATION IN A CANINE HYPOVOLEMIA MODEL
PSTP Student: Patricia L. To
Mentor: Dr. Anthony T. Cheung
Collaborator: Dr. Robert A. Gunther
Serious concerns associated with the use of allogeneic blood transfusion as treatment for acute blood loss and anemia have led to the development of hemoglobin-based oxygen carriers (HBOC) with emphasis placed on restoration of systemic function, biochemistry, and oxygenation variables rather than on the behavior of the HBOC in the microcirculation, the organ system in which blood performs its tissue-level function. Previous studies in our lab have shown that volume replenishment restores systemic function and microvascular but not oxygenation changes following moderate hypovolemia. In this study, we hypothesized that only shed blood resuscitation will restore and maintain microvascular changes beyond an initial one-hour observation period. We further hypothesized that other resuscitants will initially restore microvascular changes, but will not maintain these improvements as their volume replenishment effect wears off over time. Animals were instrumented for monitoring of systemic function and oxygenation characteristics, splenectomized, hemorrhaged (MAP reduced to 50mmHg; ~40% blood loss=32-36ml/kg), and randomly assigned to four resuscitation groups: shed blood (n=3), crystalloid/normal saline (n=3), colloid/hetastarch (n=3), and HBOC/Oxyglobinâ (n=3). Computer-assisted intravital microscopy was used to non-invasively videotape and quantify microvascular changes in the conjunctival microcirculation during prehemorrhagic, posthemorrhagic, and resuscitation phases of the study to correlate with systemic function and oxygenation changes. Prehemorrhagic microvascular characteristics were similar in all animals: venular diameter=42±4µm, red-cell velocity=0.55±0.50mm/s. All animals showed similar significant (P<0.05) posthemorrhagic changes including a 19% decrease in venular diameter (34±7µm) and a 27% increase in red-cell velocity (0.70±0.50mm/s). Shed blood resuscitation restored microvascular changes to prehemorrhagic baseline values (venular diameter=39±6µm, red-cell velocity=0.60±0.40mm/s). Normal saline, hetastarch, and Oxyglobinâ restored microvascular changes close to prehemorrhagic values (normal saline venular diameter=41±4µm, red-cell velocity=0.65±0.40mm/s; hetastarch venular diameter=38±7µm, red-cell velocity=0.50±0.40mm/s; Oxyglobinâ venular diameter=38±3µm, red-cell velocity=0.60±0.40mm/s). Three hours post-resuscitation, microvascular variables in all groups except shed blood showed a trend towards the post-hemorrhagic state. These results suggest that volume replenishment serves best as a short-term treatment for moderate hypovolemia to buy time until more definitive treatment can be given and emphasize the continuing need to develop a blood substitute that can maintain microvascular improvements following hemorrhage for a longer period of time.
A RHESUS MACAQUE MODEL FOR HUMAN CO-INFECTION WITH HIV-1 AND MALARIA
VSTP Student: Kristin Trott
Mentors: Dr. Kristina Abel and Dr. Shirley Luckhart
HIV-malaria co-infection is a major problem in sub-Saharan Africa where there is a geographical overlap of stable malaria and generalized HIV epidemics. In fact, Plasmodium falciparum, the major malaria parasite in sub-Saharan Africa is responsible for 500 million clinical infections every year resulting in more than a million deaths, and HIV prevalence is greater than 10% amongst adults. Because both HIV and malaria infections have profound effects upon the immune system, it is expected that co-infection will intensify those effects due to interacting pathology. Specifically, HIV infection has been shown to increase the risk of malaria infection and the development of clinical malaria. Equally, malaria infection has been shown to enhance HIV-1 replication. Mathematical simulations have predicted that the associated increases in viremia and parasitemia have historically increased the transmission rates of HIV-1 and P. falciparum, respectively. We hypothesize that the enhanced pathology seen with co-infection is due to altered immunological response mechanisms. To test this hypothesis, we are developing a rhesus macaque model of co-infection with the simian immunodeficiency virus (SIV) and the simian parasite Plasmodium fragile as a model of co-infection with HIV-1 and P. falciparum in sub-Saharan Africa. We aim to determine (1) whether P. fragile infection in SIV-infected macaques enhances immune activation and delays the establishment of viral set-point, and (2) whether SIV infection results in enhanced parasitemia in SIV-P. fragile infected macaques.
ANNEXIN 2 PLAYS A ROLE IN FLOW-INDUCED PGE2 RELEASE AND COLOCALIZED WITH RAFT LIKE STRUCTURES IN OSTEOBLASTS
VSTP Student: Jessica M. Wade
Mentor: Dr. Clare Yellowley
Collaborators: Drs. Damian Genetos, Christina Lee, and Majken Wadum
Biophysical signals induced by mechanical loading elicit a variety of cellular responses in bone cells, however there is no consensus regarding the underlying mechanotransduction mechanism. Members of the annexin family, a group of structurally related Ca2+ binding proteins, have been implicated as potential players in the mechanotransduction signaling cascade (1,2). In particular, annexin 2 (ANXA2) has been shown to play a role in mechanical stress-induced increases in proliferation in osteoblasts (2). ANXA2, like all annexins, associates with phospholipid membranes in a Ca2+ dependent manner. Interestingly, ANXA2, has been found in association with lipid rafts (3), potential sites of signal transduction and membrane trafficking. In this study we determined the effect of ANXA2 gene knockout on OFF induced prostaglandin E2 (PGE2) responses and the localization of ANXA2 in relation to lipid rafts in osteoblasts. Attenuation of the OFF-induced PGE2 response in ANXA2 deficient cells suggests an important role for this annexin in cellular mechanotransduction. Furthermore, our data suggest that this process is not calcium dependent. We also demonstrate co-localization of ANXA2 with lipid raft domains. Further studies will elucidate the specific role of ANXA2 in mechanosignaling and the possibility that the mechanosensor and signaling complex are in raft like structures.
THE ROLE OF IL-1 IN DIRECTING THE ANTIVIRAL B-1 CELL RESPONSE
VSTP Student: Elizabeth Waffarn
Mentor: Dr. Nicole Baumgarth
B-1 cells represent a unique subset of B lymphocytes able to produce significant amounts of IgM, IgG3, and IgA antibodies in an autonomous T-independent manner, possibly representing a bridge between innate and adaptive viral immune responses. They function to secrete antigen specific antibodies at early time points in infection before T-dependent B cell activation and plasma cell formation by B-2 cells. Little is known about the mechanisms underlying their migration out of the peritoneal cavity, the sites at which they secrete natural antibody, or the factors controlling these mechanisms. The Baumgarth lab has recently shown that B-1 cells are the primary source of local IgM responses following influenza virus infection. Work by another group, focusing on the involvement of IL-1 in directing the IgM response after viral infection, indicates that peak IgM production levels are decreased in IL-1 receptor (IL-1R)-deficient mice. These findings indicate a connection between IL-1R signaling and B-1 cell activation, specifically in the magnitude of the B-1 cell-derived IgM response. Consistent with that, systemic IL-1 levels increase transiently after viral infection, perhaps directly or indirectly promoting migration of B-1 cells to lymph nodes and their subsequent activation and differentiation to IgM producers. IL-1 may alter receptor dependent homing mechanisms and guide chemotactic migration of B-1 cells between peritoneum and other lymphoid organs. Specifically, IL-1 may activate chemokine receptor pathways involving CXCR4, CXCR5, and CCR7, present on the surface of B-1 cells. We aim to determine the mechanisms by which IL-1 regulates B-1 cell activation and secretion of IgM, and thereby the protective antiviral B cell response. IL-1 may function as a direct chemotactic stimulus inducing changes in expression of surface receptors such as CXCR4, CXCR5, and CCR7 critical for B-1 cell migration to local lymph nodes. IL-1 may also guide activation of B-1 cells and secretion of IgM upon arrival in the lymph node environment. Thus, we hope to identify the modes of action of IL-1 in moderating lymph node recruitment and activation of B-1 cells neighboring mucosal infection sites and in shaping frontline innate IgM responses to mucosal viral infection.
MOLECULAR PHYLOGENETIC ASSESSMENT OF A SYSTEMIC IRIDOVIRUS FROM THE BANGGAI CARDINALFISH (PTERAPOGON KAUDERNI)
VSTP Student: Thomas Waltzek
Mentor: Dr. Ronald P. Hedrick
Collaborators: Drs. E. Scott Weber, Devon A. Young, Erica L. Twitchell, Amy E.
Gates, Alejandro Vagelli, Guillermo Risatti, and Salvatore Frasca Jr.
In 2003-2005, a systemic iridovirus was identified in Banggai cardinalfish (Pterapogon kauderni)that were dying after transport and apparent acclimation to aquarium conditions. Throughout multiple tissues of affected fish, often recognizable beneath endothelium, were cytomegalic cells with basophilic granular cytoplasmic inclusions, which were determined by transmission electron microscopic examination to consist of arrays of viral particles of a size and shape consistent with those of previously described iridoviruses causing systemic infections of ornamental freshwater and marine fish. Partial DNA fragments of the DNA polymerase (DNApol), major capsid protein (MCP), as well as the full length adenosine triphosphatase (ATPase) genes were amplified by PCR from total genomic DNA extracted from fresh frozen tissues or formalin-fixed paraffin-embedded tissues. Molecular data permitted riboprobe generation toward in situ detection of virus in research and diagnostics, as well as molecular phylogenetic studies to gain insight into the epidemiology and evolution of this virus. Phylogentic analysis revealed the virus belongs to the genus Megalocytivirus within the family Iridoviridae.
PHARMACOKINETICS OF DETOMIDINE AND ITS METABOLITES FOLLOWING INTRAVENOUS AND INTRAMUSCULAR ADMINISTRATION IN HORSES
VSTP Student: Kristin N. Whitley
Mentor: Dr. S.D. Stanley
Collaborators: Dr. K.R. Mama, and Dr. S.M. Thomasy
Detomidine is commonly used intravenously for sedation and analgesia in horses, but the pharmacokinetics and metabolism of this drug have not been well described. The purpose of this study was to describe the pharmacokinetics of detomidine and its metabolites, 3-hydroxy-detomidine (OH-detomidine) and detomidine 3-carboxylic acid (COOH-detomidine), after intravenous (i.v.) and intramuscular (i.m.) administration of detomidine to eight healthy horses (3 mares, 5 geldings) age 5.3 ± 3.2 years and weight of 531 + 31 kg. A balanced crossover design was used. During Treatment 1, horses received a single dose of intravenous detomidine (30 μg/kg bwt) and during treatment 2, they received a single dose of intramuscular detomidine (30 μg/kg bwt). Plasma detomidine, OH-detomidine, and COOH detomidine were measured at various time points using liquid chromatography-mass spectrometry. Following i.v. administration, detomidine rapidly distributed and was eliminated with a half-life of approximately 30 min. Pharmacokinetic analysis determined that i.v. administration had a faster absorption and elimination rate, while i.m. administration had a greater volume of distribution. This trend held true for both the parent compound and the metabolites. OH-detomidine was detected much early than COOH-detomidine, however COOH-detomidine had a much greater area under the curve and mean residence time. This finding coincides with the metabolic behavior of detomidine being broken down initially to OH-detomidine which undergoes further dehydrogenation to COOH-detomidine. These pharmacokinetic parameters provide information necessary for determination of suitable detomidine loading and infusion doses in horses. Characterization of the metabolites provides valuable information for regulatory purposes and allows for the detection of detomidine after the parent compound has been eliminated.