Clinical and Translational Research Pilot Program

Application of Optical Coherence Tomography to Airway Structure in Sheep and in Human Asthma

Principal Investigator: William Calhoun, MD

Asthma, a chronic inflammatory disease of the human airway, is associated with peribronchial fibrosis and other structural changes collectively called remodeling. These structural changes have physiologic correlates of airway hyperresponsiveness, and, perhaps, presage progressive loss of lung function. The current state of the art for assessing airway remodeling requires bronchial biopsy, using forceps that sample a portion of the airway mucosa approximating 1mm3. A variety of biologic and technical sources of variability have hampered progress in the field. We propose to use optical coherence tomography [OCT] a novel methodology for assessing the histologic structure of mucosa, to assess airway mucosal structure in an animal (sheep) model, validate the methodology against formal histology, and refine the methods for application to human airways. The purpose of these experiments is to develop preliminary data sufficient to support a multidisciplinary collaborative (Calhoun and Motamedi) application for federal funding via the R21 mechanism. 

Of note, there is no approved therapy at present that has been demonstrated to prevent airway remodeling, or to reduce its magnitude once established. This situation is in part due to the considerable difficulty in measuring airway remodeling in an objective, reproducible manner. Current ‘gold standard’ methods for measuring remodeling in human tissues include autopsy material, and bronchial biopsy. It would be a major advance in the assessment of the human airway if a technique could be applied repeatedly over time, which could be replicated many times during a bronchoscopy to provide better statistical inferences, and which was not associated with increased risk over that of bronchoscopy alone. This promise of Optical Coherence Tomography, a novel methodology for study of the airway structure, may offer a completely new strategy to assess long term consequences of asthma and its therapy.

Effects of Bisphenol A (BPA) on Asthma Pathogenesis

Principal Investigator: Terumi Midoro-Horiuti, MD, PhD

Co-investigators: Randal Goldblum, Cheryl Watson, Bhupendra Kaphalia, George Saade

This project will focus on the important topic of environmental contributions to childhood asthma.This project focuses on the important topic of a potential new environmental contributor to the epidemic of childhood asthma. The long-term goal of our research is to elucidate the role of environmental estrogens, such as bisphenol A (BPA), in the development of asthma and other allergic diseases.

The prevalence of asthma, particularly in children in industrialized countries has increased dramatically over the last 2-3 decades and is now the most common chronic disease of children in the U.S. Given the magnitude of the problem of childhood asthma and the evidence that asthma at any age usually has its genesis in childhood, the impact of our current studies on this public health issue is likely to be very large. We have recently found in an animal model that perinatal exposure to BPA enhances the development of asthma.

Our subsequent studies (submitted for publication) indicate that prenatal, but not early postnatal exposure to BPA was required to produce this effect in the mouse model. We also found that the developmental expression of a hepatic UDP-glucuronyl transferase, that is thought to be responsible for metabolism of BPA, is extremely low in the mouse fetus, but increased rapidly after birth. This is likely a factor in the sensitivity of the fetus to BPA exposures.

The next phase of this project will rapidly expand ongoing efforts on two campuses, to test the hypothesis that exposure to BPA in utero induces epigenetic changes that alter the early immunologic development that underlies asthma and other common allergic diseases. We plan to use the funding from this pilot project to continue translating our findings from the mouse model to humans, by investigating the effects of prenatal BPA exposure on epigenetic alterations and the function of cord blood mononuclear cells. During the previous cycle, we have recruited 59 mother-newborn pairs. Funds from this project are required to maintain periodic clinical assessments of these subjects and to test the stored sera and mononuclear cell samples for biomarkers of BPA exposure and immunologic precursors of asthma.

Given the short duration of the pilot project, our single specific aim will be to answer the question: Which of the biomarkers of fetal exposure of BPA relate to the propensity to develop asthma? Our results will form the basis for future studies to examine the relationship between selected biomarkers and the prevalence of asthma in an expanded cohort study. These studies will be a strong basis for extramural funding to continue these multidisciplinary and collaborative efforts. These studies should help to inform public health policies for preventing future toxic exposures of BPA.

A Pilot Study of Biomarkers Predicting Clinical Expression of Acute Porphyrias

Principal Investigator: Karl E Anderson, MD

Co-investigators: Sahil Mittal, Csilla Halberg, GA Shakeel Ansari, Heidi Spratt

The 3 acute porphyrias are autosomal dominant genetic disorders that present with identical attacks of neurological symptoms. Penetrance is low, and only 10-20% of heterozygotes develop symptoms, usually consisting of intermittent neurovisceral attacks that may be life threatening. Although some exacerbating factors are known and can be avoided to prevent attacks, the marked variation in disease penetrance remains poorly understood.

The recently developed Porphyrias Consortium (PC) provides an opportunity to study samples collected from patients with various degrees of disease expression from 6 centers, and to look for biomarkers of disease severity using ITS Key Resources at UTMB. We will use the methodology available in ITS resources, including the Core Laboratory, Translational Technologies and Biomedical Informatics.

Plasma and urine samples from at least 60 patients with well-documented acute porphyrias from the Longitudinal Study (LS) of the PC will be studied. Patients will be staged according to their history of disease manifestations into 2 groups of 30 each. Group 1 will comprise patients with history of 1-3 attacks but none for at least one year and Group 2 will be patients with repeated attacks that are ongoing or severe disease resistant to standard interventions including hemin.

Approaches to identifying biomarkers will include comparisons of the following between the 2 groups. 1) Patterns of comprehensive clinical test results collected as part of the LS. 2) Panels of proteins and inflammatory markers measured by the Millipore bioplex bead-based ELISA assay, including human metabolic and inflammatory panels in the ITS Core Laboratory. 3) Metabolomic/metabonomic analyses using methods available in the Translational Technologies Key Resource, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, and the application of statistical and multivariate methods for information extraction and data interpretation.

Analytic identification will include steroid metabolites and lipid profiles, some of which were previously noted to be associated with more active porphyria. 4) An exploration of differences in profiles of >27 free amino acids measured at physiological concentrations. This pilot study will address the variability in clinical expression (i.e. variable penetrance) of 3 human porphyrias that can present with the life-threatening neurological symptoms, and will likely lead to new methods of treatment and prevention and to generate new hypotheses regarding the pathogenesis of tissue damage in these inherited diseases.

Development and Validation of a Rapid and Inexpensive Sequence-Based Colorimetric Assay for Dengue Diagnosis and Serotype Determination

Principal Investigator: Kate McElroy Horne, PhD

Dengue is the most important arthropod-borne disease today, with a wide circulation that puts approximately 2.5 billion people at risk of infection with one of four related viruses (DENV-1 to 4). More than one serotype often co-circulates in a given area, and this hyperendemicity increases the risk of development of dengue hemorrhagic fever or dengue shock syndrome during secondary infection due to antibody-dependent enhancement. In addition, some serotypes and some genotypes of each serotype may be more likely to produce severe disease than others.

Because there is no vaccine against or treatment for dengue and vector control programs have been largely ineffective, rapid diagnosis of a DENV infection followed by effective patient management remains the only way to manage dengue disease.

Current DENV diagnostic tests are either inexpensive but slow and non-specific (antibody-based tests such as the ELISA) or relatively fast but expensive and complex (molecular tests such as multiplex, quantitative RT-PCR). Experiments outlined in this proposal will adapt a previously described genotyping assay, the oligonucleotide ligation assay, to identify and type DENV and validate this assay using contemporary DENV of all four serotypes collected in the Americas.

The long-term goal of this work is to improve DENV diagnostics through the development and implementation of a rapid and inexpensive DENV diagnostic assay. Use of such a test would alert clinicians to the possibility that a patient may progress to severe disease even when he or she presents with relatively non-specific symptoms. Improved early diagnosis and effective patient management would substantially reduce the burden of dengue disease.

Novel Method For High-Resolution Detection of Double-Strand DNA Breaks

Principal Investigator: Malgorzata Rowicka-Kudlicka, PhD

DNA double-strand breaks (DSBs) are caused by physical and chemical agents or replication fork collapse, and physiologically occur during apoptosis, meiotic crossing-over, and gene rearrangements. Despite the extensive knowledge on their sensing and repair, the precise distribution of DSBs in different cells and conditions remains obscure. No method has been able so far to map DSBs with high resolution (~100b). Megabase-long cytogenetic bands that break upon partial inhibition of DNA synthesis – common fragile sites (CFSs) – have been identified on multiple chromosomes by G-staining of metaphase spreads, but the exact breakpoints are not known due to poor resolution of the method.

ChIP-on-chip with antibodies against the phosphorylated histone H2A.X, a well defined DSB marker, has been used to map fragile sites in S.cerevisiae. Despite the great resolution improvement over G-banding, the applicability of this method is limited by the fact that phosphorylation of H2A.X can spread many kilobases away from a DSB, and mark structures other than breaks, such as sex and Barr bodies. Therefore, more resolved and specific genome-wide methods are needed to gain insights in the biology of DNA double-strand breaks in different cell types and conditions.

Recently, we developed the BLESS method to map DSBs. Our method was comprehensively validated and is suitable for genome-wide mapping of DSBs in various cells and experimental conditions with resolution at least three orders of magnitude better than cytogenetical methods. However, in initial experiments, due to low coverage of sequencing and lack of model-based, sensitive methods for detection of DSBs, we detected only 60 medium-resolution (several kb) and 191 low-resolution (several Mb) breaking hotspots. Here, we propose to improve sensitivity of the experimental method and develop data analysis approach and tools that would allow us to detect thousands of ultra-high-resolution (less than 100b) DSBs using affordable sequencing capacity of no more than one sequencing lane per sample.

A Pilot Study to Translate the Diabetes Prevention Program to Low Income Women

Principal Investigator: Abbey B. Berenson, MD

Co-Investigators: Karen Williams, PhD, Mahbubur Rahman, PhD, Dan Freeman, PhD, Elizabeth Reifsnider, PhD, Karen Williams, PhD, Helen Wu, PhD

The Diabetes Prevention Program (DPP) funded by NIDDK was implemented to compare the efficacy and safety of three interventions, 1) an intensive lifestyle intervention, 2) standard lifestyle recommendations combined with metformin, and 3) standard lifestyle recommendations with placebo. Differences were assessed between the three groups in the development of cardiovascular disease and its risk factors, changes in glycemia, ß-cell function, insulin sensitivity, obesity, physical activity, nutrient intake, health-related quality of life, and the occurrence of adverse events. 

Participants in the DPP were encouraged to follow the food pyramid guidelines and to consume the equivalent of the National Cholesterol Education Program step 1 diet which advocates goals to 1) lose 5% to 10% of their initial weight through diet and exercise, 2) increase activity gradually with a goal of at least 30 minutes of an activity such as walking 5 days each week, and 3) avoid excessive alcohol intake. Smoking participants are encouraged to stop. Additionally, those randomized to the lifestyle intervention group were given goals and provided with ongoing support to help them achieve and maintain 1) a weight reduction of at least 7% of initial body weight through healthy eating, and 2) a level of physical activity of at least 150 minutes per week through moderate intensity activity, such as walking or bicycling. Among the three groups in the DPP study, the 10-year cumulative incidence of diabetes was lowest in the intensive lifestyle group. As a result the 2002 DM Interagency Coordinating Committee4, which met to identify priority areas for future research, recognized the critical need to translate the clinically significant findings from the DPP into real-world clinical settings. 
Women are at high risk of Type 2 DM as over one-half of reproductive-age women in the US currently have a BMI >25 kg/m2. The DPP may provide an effective way to address this problem, but is too costly to use on a widespread basis. Thus, effective programs which can be incorporated into public clinics are critically needed. To fill this need, our multidisciplinary team of investigators will conduct a pilot study comparing two DPP-based interventions for weight loss and behavior change among young, low-income women receiving care in publicly funded clinics. UTMB’s network of maternal and child health clinics, which provide care to a large multi-ethnic low-income population, offers a unique opportunity to refine and test weight loss interventions in a population at high risk of Type 2 DM. Before initiating the pilot study, potential patients will help refine the interventions, thereby increasing our chance of obtaining extramural funding and the assuring the success of the project.

Imaging of CNS Invasion by Venezuelan Equine Encephalitis Virus

Principal Investigator: Slobodan Paessler, DVM/PhD

With dozens of emerging and reemerging viruses currently recognized around the globe, the need for vaccines or therapeutics has never been more important for both developing and developed countries. Advancing technologies such as In Vivo Imaging Systems (IVIS) are key tools for a new methodology of studying these pathogens. The development of an IVIS murine encephalitic model will assist not only in studying disease progression but also in identifying novel antivirals and vaccines. The capability to collect multiple data points from a single animal without the need for sacrifice insures IVIS modeling reduces research time, animal numbers, and animal morbidity while increasing accuracy of detecting virus progression and safety for researchers.

The study of Venezuelan Equine Encephalitis virus (VEEV) invasion of the CNS is an ideal system to utilize IVIS and develop a standardized model. VEEV is a reemerging zoonotic virus about which very little is known of the progression of the disease leading to encephalitis and neurological sequelae. VEEV is an ideal virus to develop an IVIS modeling system both due to the ease of inserting a transgenetic bioluminescent gene into the viral genome and how much is already known of the mechanisms of viral pathogenesis. There is no FDA approved human vaccine or therapeutic providing the opportunity to utilize an IVIS model to identify efficacy of untested drugs.

Our first aim is to develop a standardized modeling system for tracking CNS infection following intranasal challenge. The second aim is to utilize this model to track protection against VEEV with CNS invasion being the endpoint of the study. Protection regiments will include the IND vaccine strain TC83 and the TLR3 agonist Ampligen. As an innate immune stimulator and interferon inducer, Ampligen is a potential non-specific therapeutic for many viral infections. Standardization of this modeling system for VEEV can be translated to other viruses found worldwide known to cause neural sequelae and death, decreasing costs and decreasing research time by assisting with screening of new anti-virals and vaccines.

Novel Methods: Intrauterine Assessment of Fetal Growth and Development with High Resolution Ultrasound in a Mouse Model

Principal Investigator: Kathleen Vincent, MD

Although several observational studies have implicated uterine environment and genetic imprinting in the etiology of several disease processes, there is a lack of direct studies. Nitric oxide generated by NOS3 (endothelial NOS, eNOS) in the vasculature is believed to be one of the most important factors in modulating vascular tone. NO's role in regulating blood pressure is even more evident during pregnancy, a physiological state that requires plasma volume expansion and a decrease in vascular resistance in order to maintain adequate placental perfusion for fetal growth and development. Disturbances in the cardiovascular adaptations to pregnancy are believed to be responsible for some of the most common obstetrical complications, such as fetal growth restriction and preeclampsia. In humans, an association between polymorphisms in some of the genes controlling cardiovascular function and adverse pregnancy outcomes, including fetal growth restriction, have been described (Savvidou et al., 2001; Hefler et al., 2002; Zhang et al., 2003).

The relationship between the inheritance of these polymorphisms and the adverse outcome has not been extensively studied in humans. Transgenic mice offer an opportunity for the study of the genetic and uterine environment effects on fetal programming. Programming has permanent consequences that alter organ responses in later life and can modify susceptibility to disease. These effects differ between females and males. Studies in eNOS deficient mice show abnormal postnatal development and abnormal vascular function in later life depending on the parental source of the abnormal gene.

These mice represent a unique model to evaluate the role of the genetic background and uterine environment in fetal development especially the cardiovascular system. Studying the offspring of those mice provide a way to determine if the fetal programming is transmitted through generations. We have shown that lack of NOS3 expression is associated with abnormal fetal growth and vascular responses in mice. In preliminary investigations, we also found that the phenotype of the pups and adult offspring depends on the number and parental source of the allelic mutations. In the proposed study, we will measure parameters associated with placental perfusion, fetal size, and cardiac function in utero during fetal development with high resolution ultrasound. These findings will be correlated with postnatal parameters of weight and vascular response.

ABCB1 Gene Variants and P-Glycoprotein-Mediated Efflux of Methodone in Human Placenta

Principal Investigator: Sherif Abdel-Rahman, PhD

Co-investigator: T.Nanovskaya

Substance abuse during pregnancy is a major public health problem. More than 2.5 million women are pregnant in the US each year, and the prevalence of opiate abuse in this group ranges from 2% to as high as 21%. For the pregnant opiate addict, methadone maintenance therapy has been the standard of care for many decades. However, development of neonatal abstinence syndrome (NAS) is a major problem affecting newborns of mothers in methadone maintenance programs. To date, prescribing physicians are faced with the challenge of balancing benefits of methadone therapy (prevention of repeated intoxication/withdrawal and drug seeking behavior) with risks to the fetus (NAS). There is a lack of correlation between maternal methadone dose and incidence and severity of NAS, which strongly suggests that NAS correlates with methadone concentration in the fetal—and not the maternal—circulation.

Recently, our group demonstrated that the placental efflux transporter P-glycoprotein (P-gp), encoded by the multidrug resistance gene ABCB1(MDR1), actively regulates the extent of methadone transfer across the placenta and consequently affects its concentration in the fetal circulation. The ABCB1 gene is highly polymorphic, with more than 250 single nucleotide polymorphisms (SNPs) within its genomic region. These SNPs (commonly inherited genomic sequences variations) are arrayed as combinations (of many SNPs) in the genome forming specific haplotypes. These haplotypes could alter the level, structure, and function of P-gp, thus ultimately modifying the efflux activity in the placenta and its permeability to methadone. The relationship between ABCB1 haplotypes and P-gp function in human placenta is currently unknown.

The goal of this investigation is to generate preliminary data that will allow us to comprehensively characterize through an R01 project the relationship between ABCB1 haplotypes and P-gp function in the efflux of methadone from the human placenta. To achieve this goal, we will test the hypothesis that specific ABCB1 haplotypes (specific SNPs combinations) alter the efflux of methadone from the human placenta by affecting ABCB1 transcriptional processes and/or its encoded P-gp protein expression levels and activity.

To test our hypothesis, in Aim 1, we will (a) determine whole-gene, high-resolution haplotype structures encompassing the reported common SNPs in the ABCB1 gene and (b) determine the ABCB1 haplotypes of placentas obtained from uncomplicated pregnancies.

In Aim 2, we will characterize in human placentas the relationship between ABCB1 haplotypes and (a) ABCB1 transcription (ie, ABCB1 transcript levels and nature of splice variants) and (b) P-gp protein expression levels. In Aim 3, we will characterize the relationship between ABCB1 haplotypes and P-gp activity, determined by ATP-dependent uptake of [3H]-methadone by inside-out vesicles of placental apical membranes.

The data generated from our study will be used as preliminary data to respond to the critique of a scored R01 proposal that we are currently revising for resubmission. In addition, the data will be germane to larger translational studies already proposed in another submitted R01 project that focuses on elucidating the relationship between placental biodisposition of methadone and incidence and intensity of NAS in newborns of methadone-treated pregnant opiate addicts.

The information generated from our research program will bridge an important gap in knowledge by leading to a better understanding of the genetic factors regulating placental P-gp expression and activity, which, in turn, regulate the concentration of methadone (and other P-gp substrates) in the fetal circulation. The knowledge gained should also enhance our understanding of the genetic factors involved in incidence and intensity of NAS in newborn of women treated with methadone during pregnancy. In the near future, this could lead to the development of individualized treatment approaches for pregnant patients that minimize fetal and neonatal complications.

Novel Methods: Development of a Split Luciferase Complementation Assay for Rapid Screening of Pharmacological Inhibitors of Protein: Protein Interactions

Principal Investigators: Kathryn Cunningham, PhD and Fernanda Laezza, MD, PhD

Development of small molecule inhibitors targeting the protein: protein interface will enable the disruption of large protein:protein complexes and, theoretically, provide a rich source of vastly overlooked targets for drug discovery. A key component in the development of novel disrupters of protein:protein interactions is the capability to identify, detect and characterize these interactions. Here, we propose to optimize a novel cellular assay, the split luciferase complementation assay (LCA), to explore the biology of neuronal protein:protein interactions, in particular, those that occur between membrane-localized receptors and intracellular signaling molecules that control neuronal function, as a means to discover new pharmacological disruptors of these interactions. The LCA assay is a powerful tool to provide quantitative, real-time readout of protein:protein interactions in vitro and in vivo.

We propose to create and optimize the LCA assay to explore the biology of the protein:protein interactions of the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) macromolecular complex. Recently, disruption of coupling between the 5-HT2CR and its protein partner PTEN (phosphatase and tensin homologue deleted on chromosome 10) was shown to result in agonist-like effects in vivo, suggesting that a pathway-specific inhibition of the 5-HT2CR:PTEN interface provides a new way to enhance the efficiency of 5-HT2CR signaling and may prove therapeutically useful for a variety of physiological and psychological conditions. We will focus on optimizing the conditions for detecting the 5-HT2CR:PTEN assembly and assessing functional activity of new cellular models. We will then build on these results to develop a robust detection method suitable for screening of small molecules that interfere with the 5-HT2CR:PTEN interaction. We will engineer a new stable cell line for the 5-HT2CR:PTEN interaction for use in screening for peptides/small molecule inhibitors of the 5-HT2CR:PTEN interaction using the LCA assay.

The proposed studies will result in the development of the LCA technology and the creation of a stable cell line needed for screening assays geared to discover small molecule inhibitors of the 5-HT2CR:PTEN interaction. Further, this assay model will provide the foundation for subsequent adaptation of this novel approach to probe the involvement of protein:protein interactions in multiple arenas.

Development and Validation of an Inexpensive, High Throughput System for Detection and Quantification of Respiratory Viruses in Clinical Research Specimens

Principal Investigator: Tasnee Chonmaitree, MD

Respiratory viruses are the most common cause of symptomatic human infections. They are the major burden to human health and make significant impact on healthcare costs. In children, upper respiratory tract infections (URIs) are exceedingly common and often complicated by acute otitis media (AOM) and sinusitis, and may lead to bronchiolitis and pneumonia. Respiratory viruses are of wide variety and new viruses continue to be discovered. Research that will lead to control of respiratory illnesses overall requires knowledge and understanding of the contribution of each specific respiratory virus. Identification of the specific virus(es) in clinical research setting has been difficult due to a lack of uniform and sensitive assays that are cost-effective and able to identify broad spectrum of viruses.

Researchers of the CTSA-Multidisciplinary Research Team (MTT) on Pediatric Respiratory Infections have longstanding interests in studying: 1) the contribution of viruses to AOM complicating URI (Chonmaitree, et al.), and 2) pathogenesis of RSV and hMPV infections (Garofalo et al.), with the long-term goals to identify possible strategies for more effective prevention and/or treatment of these infections. One of the short-term goals is to increase collaboration between the two research groups within the MTT and secure a viral identification system that may serve as the ‘virology core’ for future research center grant/ program project grant applications.

We propose to develop and validate an onsite, inexpensive, high throughput system for identification and quantification of respiratory viruses in clinical research specimens. We will test more than 1000 archived specimens and specimens being collected as part of an ongoing research for the following viruses: adenovirus, influenza, respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and bocavirus. Validation will be performed by comparison with previously available results for adenovirus, influenza and RSV; testing for hMPV and bocavirus will be validated with results obtained from a study comparing two commercial molecular assays.

Success of this proposed project will generate new knowledge on the role of hMPV and bocavirus in AOM complicating URI as well as create an onsite, cost-effective system for virus identification in clinical research samples. This will build a strong foundation for future clinical research applications related to Pediatric Respiratory Infections.

Role of Fungi in Asthma and Allergies

Principal Investigator: Sanjiv Sur, MD

Allergic asthma is a major cause of morbidity and mortality, but the mechanism by which some humans become sensitized to allergens is unknown. We have shown that intrinsic pollen NADPH oxidases vigorously promote allergic inflammation in sensitized mice by inducing oxidative stress in the airway epithelium. However, ongoing work indicates that these oxidases are relatively weak inducers of allergic sensitization. The airway epithelium is the first line of defense against inhaled allergens, and it acts as a powerful barrier against allergic sensitization.

The factors that alter this epithelial barrier so that it promotes sensitization are an intense area of research. Epidemiologic studies have demonstrated an association between mold exposure and allergic sensitization and asthma, suggesting that exposure to molds could be the missing factor that initiates allergic sensitization to pollens. We propose to test the hypothesis that mold-exposure injures the airway epithelium and activates ROS pathways that mediate the release of “danger cytokines” IL-18 and High mobility group box 1 (HMGB1). The latter is chromatin protein that acts as a pro-inflammatory cytokine “alarmin” when released extracellularly. These cytokines drive Th2 differentiation and promote allergic sensitization to concomitantly inhaled bystander allergens.

We will perform bronchscopy, BAL and bronchial brushing on normal subjects and patients with mild allergic asthma. We will examine differences in levels of danger cytokine released from primary airway epithelial cells derived from subjects with mild allergic asthma and those from healthy control subjects when cultured with Alternaria extract, and test the ability of these epithelial supernatants to induce Th2 responses to bystander antigens. Achieving this aim will provide preliminary data for a later fully- developed proposal. Eventually, the information from this aim is likely to be useful for developing unique strategies and inhibitors that may prevent development of allergies throughout the world, thereby reducing morbidity from allergic rhinitis and asthma.

Testing PCP-Consensus Proteins for Multivalent Dengue Vaccine Design 2nd Stage

Principal Investigator: Catherine Schein, PhD

Objective: To produce a physicochemical properly (PCP)-consensus sequence of domain III of the envelope (EIII) protein of DENVs that will stimulate a neutralizing antibody response against all four Dengue virus (DENV) types.

Background: Flaviviruses include many dangerous pathogens. Mosquito control programs essentially eliminated yellow fever virus (YFV) and dengue viruses (DENV) in the US, but recent outbreaks due to West Nile virus (WNV) in the US, and DENV in Puerto Rico, show the need for antivirals and vaccines. Development of a vaccine for dengue has been complicated by the necessity of simultaneously inducing protective immunity against four related but distinct virus types. In the first stage of our project, we used annotated flavivirus genome and polyprotein sequences archived in our Flavitrack database to design physicochemical property (PCP)-consensus sequences of the EIII of DENVs (conEIII). A first conEIII based on 600 sequences was expressed from a synthetic gene, purified from E. coli, and shown to bind antibodies specific for 3 of the 4 types. The next stage of the project is to isolate a 2nd generation conEIII that accounts better for the distinct properties of the fourth DENV type and will generate neutralizing (i.e. protective) antibody response against all DENV.

Methods: Our PCP-consensus program automatically determines the amino acid that, at each column of an alignment of related protein sequences, most closely matches the properties of all others at that position. Synthetic genes have been obtained for PCP-consensus sequences for domain III of the Envelope proteins (E-con domIII), recloned and the proteins expressed in E. Coli. As with the first conEIII, the ability of our 2nd generation PCP-consensus protein to bind anti-DENV antibodies to all four virus types (monoclonal and polyclonal mouse sera, and characterized DENV patient sera) will be assayed, and the best protein further characterized structurally and for its ability to generate a neutralizing antibody response in mice and to protect cultured cells from DENV infection. The fold of the purified recombinant protein will be characterized with circular dichroism (CD), and preliminary NMR (15N-HSQC).

Anticipated results: As our first consensus protein bound antibodies in a fashion similar to that of three of the four individual serotypes of the proteins used to design it, we anticipate that the proteins designed to be closer to DENV4 in sequence will be tetravalent, and able to induce a neutralizing antibody response. Demonstrating that one of our PCP-consensus proteins can act as a multivalent antigen, and fold correctly will make the PCP-consensus protein an important candidate for novel vaccines.

We have already been able to add some of our initial data to other NIH applications in progress. Subsequent studies would undertake further sequence optimization and assessment of platform technologies (i.e. adjuvants, immunostimulatory fusion partners) to enhance vaccine performance.

In Vivo Imaging of Transplanted Stem Cells In The Brain

Principal Investigator: Maria Micci, PhD

Co-investigators: Douglas Dewitt, Massoud Motamedi

Stem cells represent a promising therapeutic approach for several neurological disorders of the central nervous system (i.e. Parkinson’s disease), as well as for traumatic brain injury (TBI). The successful development of stem cell therapy, and its translation to the clinical setting, is currently hampered by the lack of a reliable and safe method to accurately monitor the location, migration and phenotypical differentiation of transplanted cells. The long term-goal of our work is to develop a non-invasive imaging technique to facilitate the development of cell-replacement therapy for clinical use. In this application, will use the human sodium-iodide symporter (hNIS) as a reporter gene system for in vivo imaging of neural stem cells (NSC) in the brain. Our preliminary data show that the hNIS, in combination with SPECT/CT imaging, allows for the repeated visualization of NSC in vivo. The NIS is normally expressed in the thyroid, stomach, choroids plexus and salivary gland, but not in the brain. Previous studies have shown that the hNIS can be used as a reporter gene to track grafted cells in vivo in the heart. Currently, no studies have been performed to explore the use of the hNIS to track grafted NSC in the brain.

In this proposal we will pursue the following specific aims.

To characterize human-derived neural stem cells (hNSC) expressing the hNIS in vitro
To track the location and survival of hNSC-hNIS after transplantation in a rat model of TBI.
The completion of the proposed aims will validate the use of the hNIS as a safe, effective reporter gene for non-invasive imaging of stem cells after transplantation in the brain. The repeated, non-invasive tracking of implanted stem cells will contribute to a better understanding of the mechanisms of stem cell therapy and, therefore, will accelerate the development of effective stem cell therapies for TBI and other types of central nervous system injury.

 

Novel Therapeutic Treatment of Neuropathic Pain after Spinal Cord Injury

Principal Investigator: Olivera Nesic, PhD

Neuropathic pain sets in after an injury has healed, in contrast to acute pain, which happens before healing has occurred. Many different kinds of chronic pain are associated with spinal cord injury (SCI): burning, prickling pain; pain on touch; or pain caused by stimuli that normally do not produce pain (allodynia).The majority of people with SCI report chronic, unpleasant sensations or pain. About a third of SCI patients describe their chronic pain as severe and debilitating.

Chronic pain after SCI is a devastating condition primarily because there is no cure, or even adequate treatment. Pharmaceutical interventions are only partially and transiently effective; at best they reduce chronic pain by 20-30%. Unfortunately, even the results of clinical trials with new drugs for treating pain after SCI have been negative. Furthermore, there is a significant reinforcement of chronic pain with stress, depression, anxiety and posttraumatic stress disorder.

As a result, untreated SCI pain becomes worse with time, so effective treatment of SCI pain is greatly needed. We are proposing a new mechanism for the development of chronic pain after SCI. Exaggerated, pathological signaling in pain-processing pathways in almost all neuropathic pain conditions, including SCI pain, involves amplified glutamatergic signaling. However, inhibitors of glutamate (Glu) signaling, although effectively reducing neuropathic pain in animal models, are toxic in humans because they also block Glu transmission which is necessary for normal functioning of the brain and spinal cord.

Therefore, we propose to target glutamate release pathway that is activated only after SCI, and thus can be safely inhibited. We hypothesize that SCI-induced swelling of astrocytes in chronically injured spinal cords produces excessive release of Glu via volume-regulated anion channels (VRAC), overactivation of Glu receptors in nociceptive pathways and chronic pain. We also hypothesize that inhibition of VRACs with FDA approved drug tamoxifen will reduce chronic pain in a rat model of contusion SCI.