2013 CTRI Pilot Projects - year 4

Clinical Research Pilot Projects

Funding Period 4/1/2013 through 3/31/2014

 

Dawn Schiehser, MD/PhD                     UCSD Department of Psychiatry                                    $25,000
Title: Neurocognitive and neural changes associated with cognitive rehabilitation in Parkinson’s disease

Project Description: Individuals with Parkinson's disease (PD) frequently have cognitive deficits, particularly in attention/executive function. These impairments often represent some of the most debilitating features of the disease and have been associated with poor quality of life (QoL) in these individuals. At present, there are few treatment options to help with this aspect of the disease. However, recent evidence suggests that cognitive rehabilitation (CR) may be an effective treatment for cognitive impairment in PD. Functional magnetic resonance imaging (fMRI) has recently emerged as a promising tool to assess neural changes associated with CR. One published study provided preliminary evidence that training on puzzles improved executive function and reduced (improved) cortical activation patterns in trained vs. untrained PD patients (Nombela et al., 2011). While promising, this study contained an unrandomized, small sample (n=5 per group) and the training paradigm was limited in content.

Innovation: To date, no study has examined cognitive and neural changes associated with a CR program that targets the PD-specific cognitive deficits; such a study is critically important in order to obtain essential data on program efficacy and the neural underpinnings of CR. Cognitive Symptom Management and Rehabilitation Therapy for Parkinson’s disease (CogSMART-PD) is a novel 10-week CR program predicated on the validated CogSMART program for schizophrenia that teaches compensatory strategies to improve memory and attention/executive function. The aim of this current study is to determine the neurocognitive and neural changes associated with CR in PD (CogSMART-PD).

Hypotheses: We hypothesize that compared to the non-intervention (“care-as-usual”) PD participants, CogSMART-PD completers will demonstrate 1) improvement on executive functioning and memory tasks as well as QoL, and 2) reduction in the dorsolateral prefrontal gyrus (due to increased cognitive efficiency) during an fMRI task of executive functioning.

Approach/Methods: Twenty PD patients with documented cognitive impairment will be recruited for this study. Participants will be assessed with a comprehensive neuropsychological battery and undergo fMRI while performing the Matrix Reasoning task (f-MRT), a well-developed and normed test of executive functioning for fMRI (Allen & Fong, 2008). Half of the participants will be randomly assigned to the intervention group (n=10) and participate in 10 weeks of CogSMART-PD for one hour per week. The remaining participants (n=10) will be assigned to the waitlist (non-intervention) group. Participants will be retested by a group assignment-blinded examiner on the neuropsychological battery and imaging protocol following the 10 weeks of intervention or waitlist. Statistical analysis will be conducted with a 2 Group (intervention vs. no intervention) x 2 Condition (pre vs. post) repeated measures mixed model ANOVA with f-MRT accuracy, brain activity, and QoL serving as dependent variables.

Results/Conclusions: To our knowledge, this will be the first randomized and largest study of CR in PD. Results of this study will provide important information about the cognitive and neural changes associated with a CR intervention (CogSMART-PD) that targets cognitive deficits in PD; and will provide essential pilot data for potential VA MERIT Award and/or R01 submissions.

 

Pam Taub, MD                                        UCSD Department of Medicine (Cardiology)                           $25,000*
Title: Effects of epicatechin on functional capacity, skeletal muscle structure and diastolic function in patients with heart failure with preserved ejection fraction (HFPEF)

Project Description: There are currently no effective therapies for patients with HFPEF (previously termed diastolic heart failure). The fundamental pathophysiology of HFPEF is thought to be due to mitochondrial dysfunction and there are no targeted therapies for HFPEF that focus on improving mitochondrial structure, function and number. Diastolic function is an energy-dependent process requiring ATP. It is estimated that relaxation of the myocardium requires up to 15% of the total energy cost of the cardiac cycle. In patients with HFPEF there is a decrease in the ratio of mitochondria to myofibrils and the function of the mitochondria is impaired. In animal models of diastolic heart failure mitochondrial dysfunction with decreased ability to upregulate ATP synthesis is present.

Our preliminary data suggests the natural compound epicatechin (Epi) found in chocolate has the capacity to improve mitochondrial structure and function in skeletal muscle. The improvements seen in skeletal muscle may also occur in cardiac muscle and improve diastolic function. In prior studies we have demonstrated that Epi enhances skeletal muscle (SkM) and endothelial cell mitochondrial mass/volume and increases maximal mitochondrial respiratory rate. We have also demonstrated that in SkM biopsies of patients with heart failure (HF) and diabetes (DM2) there is a severe reduction in mitochondria volume/cristae and these changes likely contribute to fatigability. Our preliminary results also indicate that the protein and/or activity levels of molecules involved in mitochondrial biogenesis (MiB) are severely reduced in these patients. We implemented a proof-of-concept intervention in which 5 HF/DM2 patients were provided a Epi enriched chocolate for 3 months. Results indicate a significant recovery of SkM mitochondrial cristae as well as structural and signaling indicators for enhanced MiB.

Hypothesis: Based on these results we hypothesize that pure Epi can enhance SkM structure/function leading to improved diastolic function, exercise performance and quality of life in patients with HFPEF. The mechanistic underpinnings of these functional improvements will likely be due to the upregulation of key MiB signaling pathways.

Aims: We propose to test this hypothesis in a proof-of-concept double blind placebo-controlled randomized clinical trial in which 8 HFPEF patients will be given 3 months of Epi capsules or placebo.

The primary endpoint will be functional Capacity as assessed byVO2 max/ exercise capacity.
The secondary endpoints will be 1)Structural (SkM capillarity, fiber isotypes, mitochondrial proteins/structure, MiB indicators from SkM biopsy) 2) Quality of life (by questionnaire) 3)Cardiac: We will assess by echocardiography if parameters of diastolic function (E/A ratio, E/E’ etc.) improve after three months of Epi treatment.

 

Adriana Tremoulet, MD         UCSD & Rady Children’s Hospital – Department of Pediatrics        $25,000*
Title: Immunoglobulin G sialylation in patients with treatment-resistant Kawasaki disease

Project Description: Kawasaki disease (KD), an acute, self-limited vasculitis in childhood, is the leading cause of acquired heart disease in children in the United States and Japan. While therapy with intravenous immunoglobulin (IVIG) can reduce the incidence of coronary artery aneurysms (CAA) from 25% to 5%, between 10-20% of children with KD are resistant to IVIG and are at an increased risk of CAA compared to IVIG-responsive patients.

Although IVIG is the standard therapy for KD, its anti-inflammatory mechanism is poorly understood. Ravetch and colleagues demonstrated in a mouse arthritis model that the anti-inflammatory action of IVIG was determined by the α2,6 linked sialic acid on the Fc portion of the IgG molecule by inducing expression of the inhibitory Fcgamma receptor 2B. To evaluate the role of IgG sialylation in children with KD, we measured α2,6 linked sialic acid on the infused IVIG and on the endogenous IgG from 20 patients with KD (10 responsive and 10 resistant patients) before IVIG treatment and 1 year later. We demonstrated that both the acute and convalescent levels of α2,6 linked sialic acid on the endogenous IgG in IVIG-resistant KD patients were significantly lower than responders, suggesting a possible genetic predisposition for a pro-inflammatory state in IVIG-resistant KD patients.

We evaluated the transcript abundance patterns in whole blood of β-galactosamide α2,6-sialyltranferase (ST6GAL1), the enzyme that binds sialic acid to N-glycans on IgG in B cells, from two previous microarray experiments in 152 KD patients. Transcript levels of ST6GAL1were increased in IVIG-responsive vs. –resistant KD patients. Furthermore, in 19 of the 20 KD patients in whom endogenous IgG sialylation was assessed, whole blood transcript levels of ST6GAL1 were higher in IVIG-responsive compared to IVIG-resistant patients.

We postulate that higher ST6GAL1 levels correlate with a higher percentage of anti-inflammatory α2,6 sialylated IgG molecules. Using previously established EBV transformed B cell lines from IVIG-responsive and -resistant KD patients, we will quantify the expression levels of ST6GAL1 isoform 2, which is responsible for sialylation of IgG in B cells, and compare enzyme levels by Western blotting. Transcript levels of ST6GAL1 will be evaluated in CD19+/IgG+ B cells sorted by flow cytometry from 10 IVIG-resistant and 10 IVIG-responsive KD patients. These data will allow us to pursue NIH and AHA funding to genotype haplotype-tagged SNPs across the large ST6GAL1 gene and perform RNA seq to determine the genetic basis of this difference in sialylation patterns. These experiments will contribute to our understanding of the role of IgG sialylation in IVIG-resistant KD patients and may reveal a new paradigm for understanding host susceptibility to inflammatory and autoimmune diseases. Furthermore, uncovering a genetic basis for IVIG resistance may allow us to identify KD patients who require alternative therapy such as cyclosporine in place of IVIG. If sialylation of IVIG is critical for its anti-inflammatory properties, then recombinant sialylated Fc fragments, already in development as a therapeutic agent, may be a more effective, cheaper, and safer alternative to native IVIG for the treatment of KD and other inflammatory disorders.

 


 

Translational Research Pilot Projects

 

Dean Acheson, PhD                                    UCSD Department of Psychiatry                      $25,000*
Title: Intranasal oxytocin as a putative facilitator of virtual reality-based exposure therapy for arachnophobia

Project Description: In the past decade, a paradigm shift in pharmacotherapy for mental health has occurred. Drugs that enhance cognitive behavioral therapy compliance and long term efficacy are now being developed for a number of disorders. This shift originated from the positive effects of d-cycloserine (DCS) to enhance the efficacy of extinction-based therapies across a number of different anxiety disorders. We have recently found that oxytocin (OT), a mammalian peptide that modulates anxiety and cognition, may also facilitate fear extinction in humans. In healthy controls, OT treatment prior to extinction training significantly increased recall of extinction memory relative to placebo (Acheson et al., under review). This facilitative effect on fear extinction memory supports the hypothesis that OT may be a useful adjunct to extinction-based treatments, similar to DCS (Olff et al., 2010). In addition, the established pro-social effects of OT may encourage development of a therapeutic alliance between the patient and the therapist, reducing patient drop out as well as enhancing therapeutic efficacy. This application proposes to translate these initial findings into a treatment analogue study using a sample of patients with acrophobia (fear of heights). Exposure therapy in acrophobic patients is a relatively straightforward and brief protocol that utilizes fear extinction mechanisms. This same population and therapy was used in the proof of concept studies of DCS to enhance exposure therapy (Ressler et al., 2004), and was predictive for subsequent positive DCS effects on extinction-based therapies in more complex disorders (e.g. OCD, PTSD, social phobia).

The proposed project will enroll 24 subjects with acrophobia (diagnosed via Structured Clinical Interview for DSM-IV) in a randomized, double-blind, placebo controlled trial to assess the utility of OT in facilitating response to a virtual-reality-based exposure treatment for acrophobia (Rothbaum et al., 1995). Treatment will be delivered by a trained therapist with experience delivering exposure therapy and will utilize the virtual reality therapy system located at the San Diego Veterans Affairs hospital. Following Ressler et al. (2004), subjects will attend an initial assessment visit, two treatment visits separated by 1 week, and one-week and 3-month follow up assessments. During treatment visits, subjects receive 24 IU of OT or placebo via intranasal spray 45 min prior to each exposure session. Dependent measures include objective psychophysiological measures obtained during treatment sessions and during pre and post treatment behavioral assessments, well-validated self-report measures of acrophobia and general anxiety, as well as measures of attitude toward treatment and therapist given during the treatment sessions.

We hypothesize that the OT treated group will display greater reductions in measures of acrophobia and physiological responding post treatment and at follow up. Further, we expect that the OT group will display more positive attitudes toward the treatment and therapist. If our hypotheses are supported, these results will point to an additional avenue for improving treatment outcome in anxiety disorders. In addition, the results of this study will support funding applications to conduct larger trials investigating OT facilitation of treatment in a variety of anxiety disordered populations.

 

Beatrix Bartok, MD                             UCSD Department of Medicine (Rheumatology)                     $25,000*
Title: The novel role of YAP in regulating synoviocytes behavior in rheumatoid arthritis

Project Description: Rheumatoid arthritis (RA) is an immune mediated disorder that affects the synovial lining of the joints leading to chronic inflammation and joint destruction. Although treatment of RA improved with the introduction of biologic agents that target cytokines or lymphocytes, a significant proportion of patients are partial responders with continued disease activity. Fibroblast-like synoviocytes (FLS), the resident cells of the synovium, play a central role in the disease pathogenesis in concert with leukocytes by contributing to the synovial inflammation, intimal lining hyperplasia and by formation of the cartilage invasive pannus tissue. RA FLS, unlike normal or derived from osteoarthritic joints (OA) exhibit a persistently activated phenotype that is maintained in vitro. Increased proliferation, resistance to apoptosis, loss of contact inhibition and especially cartilage invasiveness are unique features of RA synoviocytes.

Therapeutic interventions that modulate FLS behavior have the potential to halt disease progression and even be curative. The first step towards this goal is to define key molecular pathways that regulate pathogenic behavior of RA FLS. Transcription factors are of particular interest, because they are able to regulate the expression of multiple genes and therefore regulate different pathways. The transcriptional coactivator Yes-associated protein (YAP) is emerging as a major regulator of proliferation, organ size and tumor genesis. In addition, it is also implicated in contact inhibition, cell migration and invasion. Our preliminary studies shows that 1) YAP translocates to the nucleus in RA FLS, which occurs with YAP activation, while it is cytoplasmic in OA FLS; 2) mRNA level for CTGF, which is directly regulated by YAP, is 2.5 fold higher in RA FLS compared with OA cells; and 3) stimulation of FLS with TNF further increases CTGF expression, suggesting that YAP activity is regulated by TNF. We hypothesize that YAP is dysregulated in RA and is responsible for persistent activation and pathogenic behavior of FLS. In addition, reprogramming RA FLS by modulating the YAP pathway represents a novel therapeutic approach for RA.

Specific Aim 1. . To determine the functional role of increased YAP activity in RA FLS proliferation, survival, migration and invasion. We will test the effect of YAP siRNA knock down and expression of a constitutively active YAP mutant on FLS phenotype and function.
Specific Aim 2. To determine how YAP activity is regulated by TNF in RA FLS. We will then determine whether the Hippo pathway, the major negative regulator of YAP, is responsible for TNF-mediated regulation.
Specific Aim 3. To determine the expression YAP in RA synovial tissue. We will determine if YAP expression is increased in RA tissue compared with OA and normal tissue. Immunohistochemistry will determine if the cells expressing activated YAP are FLS or other lineages.

We believe that these studies will establish the functional role of YAP signaling in RA and will demonstrate that modulating YAP pathway could potentially be a novel therapy for RA. If successful, these studies will lead to an RO1 submission that will determine the role of YAP in animal models of arthritis and define the mechanisms of FLS aggressive behavior.

 

Karen Christman, PhD                              UCSD Department of Bioengineering                                    $25,000*
Title: Novel biomaterial for preventing cardiac adhesions

Project Description: Postsurgical cardiac adhesions are a common complication following cardiac surgery. Adhesions increase the length of secondary cardiac surgeries, and the risk of heart injury and severe hemorrhaging. Severe adhesions can also restrict cardiac function. These complications are especially true for the thousands of children with congenital heart defects who will require multiple surgeries over the course of their lifetimes. A common approach to reduce or prevent adhesions is to apply a physical barrier between the heart and the chest cavity with a membrane. Due to the dynamic function of the heart versus other organs, utilization of protective barriers has proven difficult. A variety of different materials have been studied for this purpose; however, to date none have been capable of preventing the formation of severe adhesions, and therefore new approaches are needed, particularly for pediatric cardiac patients.

We propose a new approach to prevent postsurgical cardiac adhesions using rapidly forming poly(ethylene glycol) (PEG) hydrogels that are cross-linked by oxime bonds. Oxime bond formation is the reaction between hydroxyl amines and ketones/aldehydes, and will be used to both crosslink the material in place. These functional groups can easily be introduced into synthetic and biological materials; PEG was chosen due its known resistance to protein and cell adhesion. Our approach is a two component polymeric system that can be easily sprayed directly onto the heart forming an anti-adhesion layer within minutes. With this system we can control the degree of swelling as well as degradation time to prevent adhesions yet not interfere with cardiac function. Since the oxime bond is dynamic, the material can also be easily removed if necessary by addition of free hydroxyl amines (hydroxyl amine functionalized amino acids) or aldehyde/ketones (levulinic acid).

As part of this design oriented, translational proposal, we will accomplish the following two specific aims: 1) To tune the gelation rate, swelling ratio, and degradation rate of the PEG oxime system and demonstrate its anti-adhesion properties in vitro, and 2) To demonstrate anti-adhesion activity with the PEG oxime system in a rodent cardiac surgery model.

Aim 1 will allow us to finely tune our PEG oxime system to match the parameters necessary for clinical application and confirm resistance to protein and cell adhesion in vitro, while Aim 2 will allow us to assess the in vivo ability of the material to attach to the heart and prevent adhesions without interfering with cardiac function, as well as confirm biocompatibility. We will compare this to two clinically used materials: REPEL-CV and Co-SEAL. The proposed project will be accomplished through an interdisciplinary, translational research team of investigators that already has a successful track record of working together, is uniquely positioned to carry out this project, and possesses the necessary expertise and tools in biomaterials and cardiac physiology, pathology, and surgery. The objective of this project is the development of a new material for preventing adhesions following cardiac surgery, with the next steps being a pivotal large animal study and clinical translation.

 

Todd Costantini, MD                                   UCSD Department of Surgery                                             $25,000*
Title: Taxonomically restricted gene expression in injured patients

Project Description: Evolutionary biologists have long recognized that taxonomically-restricted genes (TRGs) differentiate phyla. The Trauma-Burn Research Laboratories recently discovered an epigenetically-induced TRG that can be directly linked to inflammation in primates. We propose a translational pilot project to investigate whether this primate TRG, called “SF-ORF, is up-regulated in the human inflammatory response to trauma and burn injury. If so, its expression will (1) challenge pre-clinical dogma that presumes the existence of universal mechanisms of injury and inflammation in tissue repair and regeneration, (2) add an important tool to the clinical diagnosis of infectious complications after injury and (3) provide statistically solid clinical translational data to seek NIH grant funding.

Background: Preclinical models of highly complex biological responses, like those found after trauma and burn injury, repeatedly fail to predict the efficacy of drugs in man. In human sepsis alone, over $3BN (US) in investments by the private and public sectors have failed to identify clinically effective drugs to improve mortality. Ironically, this is in spite of convincing biological efficacy of these same drugs in preclinical animal models and even insight into their molecular mechanisms. While mice are an ideal experimental tool for inflammation research, 65 million years of evolution has introduced significant differences and discrepancies in both the innate and adaptive immunity responses that govern the response to injury. Humans have evolved to respond to injury and infection in ways that must be fundamentally different from animals that are both a prey species and live less than 1cm from the ground. This project hypothesizes that primates differentially evolved a taxonomically unique response to trauma and burn injury that is more suitable to their specific physiology

Approach: Our ongoing work has established that SF-ORF is expressed in human leukocytes (unpublished). This allows use of FACS, immunoblotting, targeted PCR, and bisulfite sequencing to study its expression, regulation, and biological significance in different sub-types of circulating cells. Using an approved IRB protocol, blood is collected and leukocytes purified from severely injured burn and trauma patients. Changes in SF-ORF gene expression during the hospital course will be monitored and correlated with patient demographic data, multi-organ dysfunction scores (MODS, Denver), and outcome measures. In some cases, cells are placed in culture and challenged with agents to promote and inhibit function.

Innovation: Because SF-ORF is a primate TRG, this proposal is inherently translational and focuses on human tissues and human cells. Results generated by this translational research project will allow the PI to submit an R01 grant to the NIH on TRG expression in injured patients. To meet this overall objective, CTRI pilot research support will be used to (1) validate TRG-studies as a new and under-investigated area of translational research particularly in trauma and burn, (2) underscore the strengths of animal models in informative research and weaknesses in translational research, and (3) generate a clinically relevant molecular data-set describing an epigenetically regulated TRG loci in leukocyte homeostasis for the discovery and development of novel therapeutic strategies aimed at modulating the inflammatory response in injured patients.

 

Yo Suzuki, PhD               J. Craig Venter Institute, Department of Synthetic Biology          $15,000
Title: Using Yeast to Understand Malaria Drug Mechanisms of Action

Project Description: Widespread resistance to former first-line antimalarials, as well as emerging resistance to artemisinin derivatives used in current combination therapies, creates a pressing need for new classes of antimalarial drugs. Although large-scale screening efforts have led to the identification of over 20,000 compounds with activity against blood-stage parasites, further drug development has been hindered by the shortage of information on the mechanisms of action for these compounds. Finding targets for small molecule drugs has been described using malaria parasites, but the pace of these studies is not adequate for characterizing the great number of promising compounds. To develop a rapid approach for experimentally assigning biological information to these compounds, we formed a consortium involving UCSD (Elizabeth Winzeler), Genomics Institute of the Novartis Research Foundation (Case McNamara), and J. Craig Venter Institute (Yo Suzuki and Hamilton Smith) and recruited a student enrolled in the MSTP program. When we applied for an R01 grant, reviewers told us that the approach was innovative, but felt that the feasibility needed to be supported with more preliminary data.

Approach: The long-term goal of our T1 translational research is to accelerate the process of prioritizing compounds using a yeast surrogate system so that more anti-malarial compounds progress into pharmaceutical optimization and clinical trials. Our approach is to (1) evolve resistance to antimalarial compounds in a drug-hypersensitive yeast strain, (2) identify genetic changes in this strain using whole-genome sequencing, and (3) evaluate the identified changes defining targets and resistance mechanisms using facile genetic engineering in yeast.

Hypothesis: The mechanisms of action for some drugs are identical in yeast and Plasmodium falciparum so that yeast can quickly reveal biology of drug targets.

Specific Aim: To examine the similarity of the genetic causes for resistance in yeast and P. falciparum for a spiroindolone compound currently in a Phase II clinical trial, by exhaustively characterizing all mutations detected using whole-genome sequencing. We have already screened 3,835 antimalarial compounds available at GNF and identified over 300 drugs with effects on yeast. We have also evolved drug-resistant yeast lines for three ‘yeast-compatible’ antimalarial compounds including the spiroindolone compound.

Significance: The obtained data will be used for demonstrating the feasibility of our approach. The established methods will be applied to examining a larger number of compounds in our revised R01 proposal. Once target evaluation in the yeast system becomes a standard practice, we will be able to more readily develop candidates for clinical trials to keep pace with emerging resistance in malaria. In addition, the rapid chemical genomics approach in yeast is applicable to identifying the mechanisms of action for compounds for other diseases including cancer, with a potential to motivate the development of numerous additional drugs.

 


 

Innovative Technology Pilot Projects

 

Albert Remacle, PhD                 Sanford Burnham Research Institute – Cancer Center          $50,000
Title: Imaging of metastatic breast cancer cells via the active cell-surface proteinase

Project Description: Our application represents a translational continuation of our previous novel biochemical and chemical discoveries. To quantitatively assess and then to eradicate the membrane type-1 matrix metalloproteinase (MT1-MMP)-positive circulating carcinoma cells in both the blood and micrometastasis in the post-surgery patients, we will use an integrated approach involving imaging, drug design, nanotechnology and tumor biology studies. For these purposes, we will use the cell-surface active MT1-MMP enzyme as a target for imaging and as a vehicle for drug delivery of pharmaceuticals inside breast carcinoma cells.

Following trafficking through the cell compartment, the de novo synthesized MT1-MMP is proteolytically activated and presented at the plasma membrane. The resulting pro-invasive mature proteinase performs pericellular proteolysis and then, in 15-30 min, is internalized and next either degraded or recycled back to the plasma membrane. We have already developed the highly efficient and selective imaging/drug delivery reagent (IDDR) prototype. IDDR represents a 60-70 nm liposome loaded with a fluorochrome and functionalized with a PEG spacer. The latter is linked to a selective inhibitory warhead. The warhead is highly selective for MT1-MMP and, as a result, off-target interactions do not affect the IDDR’s ability to specifically target cell-surface MT1-MMP. For drug delivery, the drug cargo (doxorubicin, cyclophosphamide, docetaxel and/or paclitaxel) will be loaded into the liposomes instead of a fluorochome. IDDR binds the active site zinc atom of the catalytically active MT1-MMP enzyme alone, rather than the latent MT1-MMP zymogen or the inactive MT1-MMP●TIMP-2 stoichiometric complex in which the active site zinc atom is shielded by either the prodomain in the proenzyme orTIMP-2 in the enzyme●TIMP-2 complex. Following internalization via MT1-MMP, the liposomes would be hydrolyzed with the drug released inside the cell compartment.

As a result of our long-term studies of the MT1-MMP biology and as we reported in our multiple publications, we have already acquired all of the purified MT1-MMP constructs and MT1-MMP-transfected breast cancer cell lines, which are required for accomplishing, successfully and on-time, our current research goals.

Our Specific Aims are: (1) Determine the precise level of off-target activity of IDDR using a panel of purified MMPs, including MMP-2, MMP-8, MMP-9 and multiple membrane type MMPs, (2) Develop the methodology for the quantitative assessment of the cell-bound IDDR in breast carcinoma cells, which express the catalytically inactive (control) and the wild-type, active MT1-MMP constructs, and (3) Determine the efficiency of IDDR in the imaging of orthotopic xenografts, circulating cancer cells and micrometastasis in the lung, the liver and the brain using breast carcinoma models in immunodeficient mice. As a result of these in vitro, ex vivo and in vivo studies, we will propose the development of promising imaging/drug delivery tests applicable to breast cancer patients. We are confident that the knowledge we will gain through the proposed studies will help us to point the way to the development of safe imaging and therapeutic regimens for the post-surgery cancer patient cohort and, as a result, to reduce metastatic burden in survivors. Dr. Veronica Shubayev, MD – a co-investigator (UCSD) will help coordinate our studies.

 


 

Personalized Medicine Pilot Projects

 

Douglas Conrad, MD                       UCSD Department of Medicine (Pulmonary)        $50,000
Title: Metabolites as biomarkers in Cystic Fibrosis

Project Description: Mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) result in chronic polymicrobial airway infections. The immune responses to the microbial communities result in extensive airway remodeling and eventually significant gas exchange abnormalities. The disease is characterized by infectious exacerbations that result in permanent airway scarring. A primary goal of the therapy is to minimize the severity and frequency of these exacerbations.

The primary focus of these proposed studies is to use new technologies i.e. next generation sequencing, high throughput sputum metabolic product assessments and exhaled gas analysis to identify and begin to validate new biomarkers for CF pulmonary exacerbations. Two specific aims are proposed. SPECIFIC AIM 1: Identify metabolites unique to CF patients in routinely collected sputum samples. SPECIFIC AIM 2: Monitor for the presence of members of the acetoin metabolism pathway and to further explore our finding of 2,3-butanedione in the breath of CF patients.

Innovation: Existing assessments for disease flares include clinical symptoms, pulmonary function testing, radiographic studies and standard microbiological laboratory assessments. All of these have significant limitations in detecting pulmonary exacerbations before they are clinically significant and cause irrevocable airway damage. These pilot studies are likely to identify metabolic product biomarkers that are specific to microbial communities associated with disease flares. The technologies will identify metabolic pathways that are critical to the microbial communities to survive in the CF microenvironment and likely identify pathways that are expressed prior to disease exacerbations.

Approach: Patients followed by the UCSD Adult CF Clinic will be identified during exacerbation, treatment and clinically stable states. Exhaled gas and induced sputum samples will be obtained for metabolic and metagenomic analysis. Bioinformatic analysis will focus on identifying the metabolic pathways (metagenomic analysis) and metabolic products (exhaled gas and induced sputum analysis) that are specific to the exacerbation state.

Our work and others have identified, the unique nature of each CF patient’s microbial community and how it changes over time. The focus of our work is on the functional pathways that allow each member to survive and thrive in this airway environment. We believe that this work has a high probability for future funding from external agencies because of innovative nature of applying the technologies in a new context to answer important translational questions, in addition to our commitment and enthusiasm and track history in the area. To the extent that many other pulmonary diseases (asthma, COPD, bronchiectasis, fibrosis) are associated with altered airway microbial communities and disease flares, these studies are likely to provide new insights to identify exacerbations at an early point to minimize short and long term complications.

 

Daniel O’Connor, MD                     UCSD Department of Medicine (Nephrology)                       $50,000
Title: Natural human genetic variation and the metabolism: Studies in twin pairs

Project Description: The relative roles of heredity and environment in predisposition to human disease are of extreme interest, since once discovered they may guide diagnostic, prognostic, and therapeutic approaches to disease. However, precise estimates of the contribution of heredity (as the parameter “heritability”, or h^2) versus environment to trait variation are typically not available. Twin pairs are the best characterized study design in human genetics, wherein variance components are estimated from MZ versus DZ twin pair correlations: VP = VG + VE (where VP = total phenotypic variance, VG = genetic variance, and VE = environmental variance). Such partitioning readily enables estimation of h^2 = VG/VP, for any trait (biochemical, physiological, or disease). Coupling extensive twin phenotyping with genome-wide (genomic) approaches (linkage scan and genome-wide SNP scan/GWAS, already accomplished in our twins), then allows us to determine the influence of particular genetic loci on any trait. Here we propose a novel goal: coupling state-of-the-art phenotyping of global metabolic pathway activity (metabolomics), with genomics, in order to achieve new insights into how loci across the genome control not just single analytes but flux through entire biochemical and physiological pathways.

We begin with metabolomic characterization of urine, as an integrated output of systemic metabolic activity. Aliquots of freshly voided urine (n=750 subjects) were promptly frozen at -70C. Samples are processed on the Pegasus III GC-TOF-MS system , on which “non-targeted” primary metabolites (small molecules of 20-600 Da; organic acids, amines, and carbohydrates) are robotically derivatized (as trimethylsilyl ethers), separated, identified, and quantified analyzed by gas chromatography / time-of-flight mass spectrometry, with acquisition of up to 500 spectra/second. Typically after automatic peak deconvolution and BinBase data processing, ~150 such higher-abundance molecules are completely identified and quantified in biological samples; final structures of ~415 different target molecules have been defined in this system .

Pathways. We propose a novel goal: Coupling state-of-the-art phenotyping of global metabolic pathway activity (metabolomics), with genomics, in order to achieve new insights into how loci across the genome control not just single analytes but flux through entire biochemical and physiological pathways.

Disease: Novel biomarkers of disease susceptibility and contributory pathways. We have already carefully profiled each twin for multiple phenotypes (both biochemical and physiological) that constitute risk traits for disease: autonomic, metabolic, inflammatory, cardiovascular, and renal. Thus we are poised to develop new metabolomic biomarkers for disease risk, to quantify (as twin parameters h^2, rG and rE) the relative roles of heredity and environment in disease risk, allowing us to propose new strategies (diagnostic, prognostic, and therapeutic) to intervene in at-risk subjects. Coupling GWAS to metabolomics is anticipated to provide new genetic markers of disease risk, thereby providing evidence of novel, perhaps “druggable” pathways for intervention in disease. Extending these genetic studies in twin pairs, we will validate novel SNP marker-on-trait associations in independent case/control population samples, e.g., for hypertension, obesity, diabetes (type-2), and Chronic Kidney Disease (CKD).

 


 

Community Pilot Projects

 

Gregory Light, PhD                               UCSD Department of Psychiatry                                     $15,000
Title: Predicting Cognitive Enhancement Following Intensive Neuroplasticity-Based Training Intervention in Schizophrenia

Project Description: Neurocognitive impairments affect the majority of schizophrenia patients (SZ) and are correlated with functional disability. Given that current antipsychotic treatment does little to improve cognition, identifying the neural mechanisms and predictive biomarkers that underlie cognitive enhancing treatment response is needed to optimize therapeutic gains. Cognitive remediation approaches generally involve intensive behavioral training intending to improve the cognitive processes of attention, memory, executive function, and social cognition. This CTRI proposal aims to develop a collaborative partnership between UCSD and the Alpine Special Treatment Center, Inc. (ASTCI), a San Diego County-funded community mental health rehabilitation and transitional care facility. The funds will be used to generate feasibility data on the effectiveness of implementing an empirically-supported cognitive remediation intervention in the community that will support future NIH grant submissions and extend community relationships.

 

Monica Ulibarri, PhD                           UCSD Department of Psychiatry                                   $15,000
Title: Assessment of risk factors for commercial sexual exploitation of high risk adolescent girls in San Diego County

Project Description: The goals of this pilot study are to: (1) collect, analyze, and interpret descriptive qualitative data to better understand and characterize risk factors associated with commercial sexual exploitation of children (CSEC) among high risk adolescent girls; (2) develop an in-depth interview guide for use in a subsequent R01 mixed-methods study further examining risk factors for CSEC among high risk adolescent girls; and (3) establish an on-going collaborative research relationship between the PI/Co-Is and the San Diego County Office of Education (SDCOE). A collaborative approach between the UCSD investigators and SDCOE will be used to enhance the quality and validity of this research, equitably involving our community partner and its designated representative in all phases of the research process.
Specific Aim 1: Identify risk factors for CSEC utilizing in-depth interviews with key informants (i.e., previously sexually exploited adolescent girls attending school in the SDCOE system).
Specific Aim 2: Develop an interview protocol for a subsequent mixed-methods study of CSEC among high-risk adolescent girls in San Diego County.
Specific Aim 3: Provide data and recommendations specific to San Diego County to supplement SDCOE’s existing training materials related to CSEC prevention and education.


Pilot Project funded collaboratively with the UCSD Stein Institute for Research on Aging

 

Hemal H. Patel, PhD                 UCSD Department of Anesthesiology                         $20,000*  

Title: Membranes as a Regulator of Healthy Aging

With the aging of the United States population, it is estimated that the elderly (>65 years of age) will increase from 13-14% to 25% by 2035.1 If this trend continues, >50% of the United States population and >2 billion people worldwide will be “aged” in the next 50 years. Aged individuals face formidable challenges to their health, as aging is associated with a myriad of diseases. Cardiovascular disease is the leading cause of morbidity and mortality in the United States with >50% of mortality attributed to coronary artery disease and >80% of these deaths occurring in those age 65 and older. The driver for many of these statistics is poor lifestyle choices namely a poor diet and limited exercise. According to the American Diabetes Association, in the United States there are nearly 26 million individuals including adults and children that have diabetes. In addition, there may be as many as 79 million individuals that are pre-diabetic. In 2007 diabetes was listed as the underlying cause of >70,000 deaths and a contributing factor of an additional 160,000 deaths. Those aged 65 years or older represent an ever-growing population facing the sequelae of diabetes. In 2004, the most recent year for which statistics are available, heart disease was noted in nearly 70% of diabetes-related deaths among people 65 years or older, and adults with diabetes have heart disease mortality rates that are 2 to 4 times higher than adults without diabetes. Further, cardiovascular hospitalizations accounted for nearly 50% of all diabetic hospitilizations. Diabetes has also been described as “accelerate aging” and much of the pathology is reflective of an aging phenotype. Such statistics underscore the unusual link between healthy aging and associated secondary complication such as diabetes and heart disease.

A number of theories have been proposed to account for this aging deficit. These theories either invoke a genetic (i.e., there is a programmed regulation of gene expression that declines or accelerates with age in post-mitotic cells), a biochemical (i.e., altered metabolism leading to a mismatch of energy utilization/generation, diminished mitochondrial function leading to generation of toxic species such as free radicals and protein modification to reduce function), a catabolic (i.e., an inability to recycle cellular material at the cellular and organelle level), or a physiologic (i.e., declining heart function, neurologic deficits, and loss of vital organ function) component. Though the mechanisms that underlie an age-related deficit are not clear, they likely involve abnormalities at the cellular and subcellular level. Membranes are dynamic entities. They are not only barriers that separate two environments but are also structures composed of proteins and lipids that control and are controlled by numerous biological processes. The cell utilizes such structures at many different levels the most identifiable being the plasma membrane. However, intracellular structures such as the endoplasmic reticulum, mitochondria, nucleus, Golgi complex, and many others, create specified function through well-defined and characteristic membrane structures.

We hypothesize organismal aging is regulated by loss of membrane integrity in multiple cellular sites and this process is accelerated in disease conditions such as diabetes that are known to decrease lifespan. To test this hypothesis we will undertake to determine if membrane structures are compromised in aged organ and if this is accelerated by metabolic syndrome.


Pilot Project funded collaboratively with UCSD/UCLA Diabetes Research Center (DRC)

 

Neal K. Devaraj, PhD             UCSD Department of Chemistry & Biochemistry                      $42,500*
Title: Amplifying PET Imaging Signals for In Vivo Detection of Pancreatic Beta-cells

The objective of this proposal is to use redox amplified radionuclide deposition to enable the non-invasive imaging of pancreatic beta-cells in diabetic patients using positron emission tomography (PET). According to the World Health Organization, there are currently 347 million people worldwide who have diabetes, with the expectation that nearly double the number of people will be affected worldwide by 2030. Molecular imaging tools are needed to monitor the progression of disease, the effectiveness of novel therapeutics, and to track the viability of transplanted cells. However, despite this need, the development of a whole-body molecular imaging technique to monitor beta-cells has remained elusive primarily due to inadequate signal-to-background, which prevents the resolution of target rare beta-cells (~1-2% of tissue) against background pancreatic cells. We propose to utilize a fundamentally new approach to PET signal amplification through enzyme catalyzed radionuclide reporter deposition. Such methods could dramatically increase signal output from beta-cell specific reporters while simultaneously suppressing background. The development of generic methods to amplify PET signals could lead to valuable imaging tools for monitoring beta-cell mass and have an enormous impact on the clinical diagnosis, treatment, and understanding of diabetes.

 

Award amounts listed reflect the total award for the year.  Projects with asterisks are co-funded.