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Round 2: 2013-2014 Pilot Projects

Assessing and Reducing Risk of Violence and Violent Injury Among Victims of Urban Violence

Victims of urban violence face a perilous trek even after their physical injuries heal. For victims,violent trauma is a recurrent event and many later even die from violence. Victims of urban violence often have histories of violence perpetration and are at a heightened risk for violence in the wake of traumatic injury. Multiple, interrelated risk factors predict later violence and violent injury. Risk behaviors are often exacerbated following traumatic injury, potentially leading to a self-perpetuating cycle of violence and victimization. Interventions to promote behavioral changes that reduce risk may thus be critical in preventing further violence and violent injury. The hospitalization immediately following violent trauma may be a unique window for such care, yet few hospital-based programs exist and empirical evaluations of these programs have yielded mixed results. Our lab has developed an initial draft protocol of a hospital-based intervention for victims of urban violence that is rooted in Transtheoretical Model of Change and Motivational Interviewing.

We propose to refine, implement, and pilot test this intervention among patients hospitalized following a violent injury at an urban Level I Trauma Center. The proposed study will be conducted in two phases: (Year 1) a pre-randomized development phase to systematically refine and standardize our intervention and (2) a pilot randomized clinical trial of efficacy. The RCT will randomly assign violently injured patients to (a) treatment as usual (brief mental health screening and referral (n = 50); (b) a hospital-based Ml protocol (n = 50). Phase 1 outcome measures include both quantitative and qualitative assessments of acceptability and feasibility Phase II primary proximal outcome variables are motivation to change and actions taken to mitigate risk. Phase II distal outcome variables are violent behavior, arrests for violence crime, violent injury, and hospital service utilization due to violent trauma. Risk behaviors (substance abuse, weapon use, anger/irritability, belief in the utility of violence, post-traumatic stress symptoms, unemployment, lack of education, and unstable housing) are included as medial outcomes to examine possible mechanisms of influence between proximal and distal outcome variables. 

Investigation of novel genetic factors predisposing to multiple epiphyseal dysplasia and osteoarthritis

Multiple epiphyseal dysplasia (MED) is a relatively common chondrodysplasia characterized by delayed and irregular ossification of epiphyses and early-onset osteoarthritis (OA). This clinically and genetically highly heterogeneous disorder is caused by mutations in six genes. However, mutations in these genes only explain approximately 50% of all cases, which indicates that defects in other genes are involved. Currently, the overlapping MED/OA phenotype is being reinvestigated since it may provide new insights towards understanding the genetic basis for common forms of osteoarthritis. Our central hypothesis is that there are new candidate genes for MED and mutations in these genes may also contribute to the development of common forms of OA. We plan to identify a new gene(s) for MED using exome-sequencing in four families for whom we have previously excluded all currently known candidate genes and identified a high lod score (5.45) on chromosome 11 indicating a new locus for this disease. We will then determine if changes in the new MED candidate gene predispose to primary OA, by sequencing of the new candidate gene in Louisiana patients with primary knee OA. With respect to expected outcomes, this project has a high chance to result in discovery of a new gene that mutations cause MED and OA. Identification of this gene may also help to develop a new treatment for these disorders; however, this strongly depends on the function of a coded protein. We are planning to analyze this gene in patients with OA from Louisiana and to estimate the genetic predisposition in this group in collaboration with Dr. Vinod Dasa of LSU.

HPV and EBV Serum Antibodies as Prognostic Markers for Cervical Cancer

Human papilloma virus (HPV) is necessary but not sufficient for development of cervical cancer, an epithelial cell malignancy. We have evidence supporting Epstein-Barr Virus (EBV) as a co-factor for HPV related cervical disease in HIV+ women. EBV is the causative agent of the epithelial cell malignancy nasopharyngeal carcinoma (NPC). Serum antibodies against EBV antigens are the best predictor of NPC in high risk populations. This proposal will assess whether serological assays for HPV and EBV antibodies can serve as prognostic indicators of cervical disease. HIV+ human serum samples will be evaluated for 5 antibody markers against EBV and 6 antibody markers against HPV. This data will be combined with my preliminary data to determine the best combination of serum antibodies for predicting cervical disease. The advantages of serum testing will likely increase patient compliance for screening and follow up of cervical disease and therefore improve outcomes for cancer patients.

Evaluation of peg-Arginase I as a Therapeutic for Virus-Mediated Ocular Diseases

Pathogen-associated ocular diseases are a complex combination of pathogen-mediated trauma and hostmediated inflammation-associated pathologies. We have developed an ophthalmic formulation of pegylated-ArginaseI(peg-ArgI) that has broad anti-pathogen, anti-inflammatory, and antineovascularization activities, while also promoting healing of traumatic corneal wounds (Patents:US13/828,669;PCT/US13/31623). No current drug has all these combined activities. Targeting host metabolic pathways by peg-ArgI to simultaneously ameliorate pathogen- and host-mediated disease sequelae is a novel therapeutic approach with wide-ranging clinical potential. The rabbit eye is an FDA recognized model for establishing an ophthalmic drug's pharmacological parameters and evaluating clinical efficacy prior to initiating human trials. We will utilize this model to: 1) Establish a dosing regimen for peg-ArgI that prevents both HSV-1 replication and presentation of ocular pathologies and 2)Evaluate peg-ArgI's ocular safety and toxicity according to FDA recognized protocols. Establishment of these pre-requisite parameters will provide critical support for future human trials of peg-ArgI as a broadly applicable ophthalmic therapeutic.

Analysis of the Pathogenesis of HIV/EBV associated DLBCL

Currently, the state of Louisiana has the 4th highest annual AIDS diagnosis rate and the 10th highest HIV/AIDS population in the United States. Nearly 70% of AIDS patients in Louisiana are African American (Kaiser family foundation / HIV/AIDS is considered one of the major health problems in Louisiana, especially in the medically underserved population. AIDS associated malignancies such as Diffuse Large B-cell lymphomas (DLBCLs) are one of the major factors for AIDS-related morbidity and mortality. The objective of this project is to determine the precise role of Epstein-Barr virus (EBV) in the pathogenesis of the AIDS/EBV associated DLBCLs using the most clinically relevant model system available - clinical specimens. EBV is a primary driver of AIDS associated DLBCLs with a penetrance of nearly 100%. We will use expression and genetic signatures of clinical lymphoma biopsies to determine EBV signaling mechanisms driving lymphomagenesis in AIDS patients.

Targeted microRNA analysis in Multiple Sclerosis associated cognitive decline

Two-hundred-thousand Americans with Multiple Sclerosis (MS) will develop cognitive impairment (CI) resulting in unemployment and social isolation. Innate immunity plays a key role in widespread injury to the brain underlying CI in MS. Currently we cannot measure innate immunity activation in the brain non-invasively and no treatments are available to arrest it. MicroRNAs (miRs) are small RNA molecules that regulate genes in their cell of origin and surrounding cells. MiRs regulating innate immunity activation or linking it with neurodegeneration are over-expressed in CSF of people with Alzheimer disease. Our hypothesis is that these same miRs play an important role in CI in MS; to test our hypothesis we will compare CSF miR in people with MS with and without CI and correlate CSF miRNA with MRI measures of tissue injury. This is a first step in testing this hypothesis, opening novel diagnostic and treatment possibilities for CI in MS.

Development of a PACAP Analog as a Therapeutic for Contrast-Induced Nephropathy

The incidence of contrast-induced nephropathy in the general population is low, but can exceed 50% in some subpopulations. No drugs have been approved by the FDA specifically for the prevention of contrast-induced nephropathy. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional peptide with potent anti-inflammatory and cytoprotective properties. We have shown that PACAP is efficacious in a novel model of contrast-induced nephropathy. We have synthesized and characterized two series of proprietary PACAP analogs. We have submitted a proposal to NIH to acquire the funds needed to move a lead compound from the preclinical stage of development past the Investigational New Drug application stage of development. The proposal was scored but not funded. There were three major criticisms. The receptor binding data has now been determined by our collaborators. The purpose of this proposal is to obviate the other two major criticisms and then resubmit the scored NIH proposal.

Screening for glycosylation disorders and study galactose intake in PGM1-CDG

Hypoglycemia is the most common metabolic condition in newborns and infants. We defined a novel inborn error of metabolism due to Phosphoglucomutase (PGM1) deficiency. The novel condition presents with severe neonatal, and recurrent infantile hypoglycemia associated with abnormal growth and endocrine abnormalities of variable degree (Mohamed et al, BBA, 2011, Timal et al., HMG, 2012). Since 2012 more than 20 patients have been diagnosed with this condition. PGM1 deficiency leads to progressive increase of glycogen concentration in muscle and liver and abnormal protein glycosylation (Congenital disorder of glycoslation: CDG) in blood. In an adolescent patient we observed variable disease severity, depending on the daily milk intake (galactose intake), over a one-year period. One year of therapy with extra oral galactose intake resulted in improved N-linked glycosylation, normalization of liver function tests, prevention of hypoglycemic episodes, and normalization of N-glycosylated hormones and IGF1/IGF3. These observations led us to the hypothesis that PGM1 deficiency alters the balance of glucose-1- and galactose-1-phosphate concentrations leading to abnormal N-glycosylation, and that this phenomenon is most likely influenced by dietary galactose availability.

Aims of our study:

Hypoglycemia is a common symptom of the Congenital Disorders of Glycosylation (CDG). None of the types of the CDGs have been screened in Louisiana, and no metabolic diagnostic test is available in our state for the more than 50 different types of this condition. We will start a collaborative effort with LSU Health Sciences Center and Children's Hospital, to set up selective CDG screening, using10 ul plasma or dried Blood spot on filter paper, by transferrin isoelectric focusing (TIEF) in hypoglycemic newborns and infants.
We clinically follow five patients with the recently discovered inborn error; PGM1 deficiency. The patients are of different age and have a natural, variable galactose intake and remain under regular metabolic and dietary control. In order to study the effects of oral galactose intake on the clinical and biochemical features of affected patients, we will evaluate the correlation between galactose intake, growth parameters, clinical symptoms, diet, and follow the N-glycosylation pattern by TIEF and glucose regulaton related endocrine parameters. These evaluations will be performed on a monthly basis over a one-year period.
Background: The principle investigator has been active as a clinician and scientist in Europe and joined the faculty at Tulane University in 2012. She is a clinical and biochemical geneticist. She has significant expertise in congenital disorders of glycosylation including the description of the PGM1-related glycosylation defect and other new gene discoveries. Deliverables: This study will allow (1) establishment of a novel research line on protein glycosylation at the Hayward Genetics Center (2) collaboration between the Departments of Pediatrics and Genetics at Tulane University and LSUHSC (3) initiation of selective screening for protein glycosylation defects in Louisiana, (4) development of more pathophysiological insight of a novel metabolic disease.

Boron-based 4-hydroxytamoxifen and endoxifen prodrugs for treatment of breast cancer

Poor initial response to tamoxifen therapy and persistent side effects remain major clinical challenges in the treatment of breast cancer patients. The boron-based 4-hydroxytamoxifen and endoxifen prodrugs are designed to address both problems by guaranteed delivery of a more potent metabolite of tamoxifen without requiring a functional enzyme (CYP2D6) to convert tamoxifen to 4-hydroxytamoxifen and endoxifen, and by lowering the dose requirement to reduce side effects such as hot flash. The proposed animal study is expected to confirm that the superior activities of the prodrugs in breast cancer cells can be extended to in vivo models. These results will lay the ground work for more extensive pre-clinical studies leading to clinical trials.

Plaque Destabilization through Shear Stress Mediated Decreases in miR-221/222

Our limited understanding of the mechanisms behind plaque rupture is a critical barrier to developing methodologies for prevention of myocardial infarction and stroke. Our lab has found the expression of two microRNAs and a related circular RNA are altered in plaques postrupture and in relation to shear stress. We hypothesize that increased shear stress alters noncoding RNA (ncRNA) expression to promote plaque destabilization. We will test this via two aims. Aim 1 will identify ncRNAs associated with plaque rupture using next generation sequencing and relate their expression to shear stress. Aim 2 will use a custom-designed bioreactor to demonstrate in vitro that increases in laminar shear stress reduce proliferation and increase apoptosis in vascular smooth muscle cells through changes in ncRNA expression. This project thus combines an innovative approach to studying plaque rupture with cutting edge methodology to determine the role of flow mediated changes in ncRNA in plaque destabilization.

Selective targeting of the metastatic-invasive phenotype of triple-negative breast carcinoma

Introduction: Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.

Methods: TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.

Results: Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat upregulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.

Conclusions: This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.

Keywords: Panobinostat, LBH589, triple-negative breast cancer, xenograft, histone deacetylase inhibitor, E-cadherin, CDH1, epithelial-to-mesenchymal transition