Graduate Students Alphabetical by Graduate Student last name

1. Development of a mouse model to study SMN splicing and replacement therapy Presenting author: Bebee, Thomas

Proximal (SMA) is a neuromuscular disease caused by low levels of SMN protein due to loss of the Survival Motor Neuron-1 (SMN1) . Humans have a duplicate gene, SMN2, that generates low levels of SMN protein due to a C>T point mutation that skips exon 7. We generated a mouse model that recapitulates SMN2 altered splicing by engineering the exon 7 C>T mutation into the homologous mouse Smn gene. The C>T mutation in Smn induces skipping of exon 7 in multiple tissues in the mouse, reduces SMN protein levels, and leads to a mild form of SMA exhibited by reduced hind limb grip strength, rearing, and activity. Increasing SMN expression by correcting splicing or increasing transcription of SMN2 are attractive therapeutic options in SMA patients. However, the timing of SMN replacement will be crucial in the treatment of SMA. Studies in zebrafish predict SMN function during embryogenesis may be important for axonal pathfinding, while mouse models and SMA disease progression in humans suggest that post-natal treatment may be sufficient to protect motor neurons. AAV9 encoding SMN has shown that early postnatal SMN expression in spinal cord motor neurons is sufficient to rescue survival though complete rescue of normal development was not observed, indicating that earlier treatment may be required for full restoration of SMN function and disease correction. To address the question of optimal therapeutic timing for SMA we have developed a temporally inducible transgenic mouse in which the expression of human SMN cDNA is under the control of tamoxifen inducible Cre-recombinase. In our inducible mouse, induced transgene recombination following tamoxifen treatment was validated in 4 week old weanling mice, PND1.5 treated neonatal mice, and E13.5 treated embryos. The temporal induction of transgene recombination and SMN expression was further validated in these mice in various tissues including the brain and spinal cord. When crossed to SMA mouse models our inducible SMN mouse model will allow for the temporal induction of SMN at varying time-points during development, both in utero and postnatally, to evaluate the time-point in which SMN replacement is required for SMA correction. The therapeutic window determined from these experiments can then be used in our Smn C>T mouse model to validate the use of drug therapies targeting splicing correction in the treatment of SMA mice, and in turn SMA patients.

2. Muscle enhancement in SMA Presenting author: Bevan, Adam K.

Purpose: Spinal Muscular Atrophy (SMA) is a devastating neurological disease marked by lower motor neuron death and the subsequent atrophy of skeletal muscle. SMA patients can present with various severity of disease; SMA type 1 is very severe, usually leading to respiratory failure by 2 years, while SMA type 3 is non-fatal but patients are weak and are often wheelchair-dependent by their teenage years. While this disease primarily affects only spinal motor neurons, enhancement of skeletal muscle may be a good approach to treat patients with less-severe disease to increase their quality of life. We have therefore tested the muscle enhancing effects of follistatin gene therapy in severe (“Δ7”) and mild (“C/C”) mouse models of SMA. Methods: Newborn Δ7 (SMN2+/+, Δ7SMN+/+, Smn-/-) and C/C mice (SMN2+/+, hybSmn+/+, Smn-/-) were injected with 1e+11 particles of AAV9 carrying the follistatin transgene (AAV9.CBA.FS344) via the facial vein. Behavioral tests were performed starting at 6 days of age and continued until either 1) death in Δ7 mice or 2) at least 60 days of age in C/C mice. Muscle-related physiological measures were also performed in C/C mice at 200 days of age including muscle mass and in situ and ex vivo force generation. Transgenic follistatin expression was also confirmed by body mass, serum follistatin concentration and muscle fiber diameter measurements. Results: Both Δ7 and C/C mice showed early, positive effects of muscle enhancement, though no significant extension of survival was observed in the Δ7 group. In C/C mice, follistatin treatment had a positive effect on righting ability and, as predicted, significantly increased lean body mass and grip strength. Importantly, the ex vivo force generated by stimulation of the nerve was greater in follistatin-treated C/C mice despite having a pathological neuromuscular junction. Conclusions: While follistatin had little effect in the rapidly progressing Δ7 model, our data suggests that muscle enhancement may be a very useful therapy for patients with more mild forms of SMA.

3. Gene Therapy and Ovarian Cancer Presenting author: Bolyard, Chelsea M.

Ovarian cancer is the leading cause of death due to gynecological malignancy, and a leading cause of death due to cancer in developed countries. Because patients often present in late stages of disease, aggressive multi-modal therapies may provide the most efficacious results. Within our study, we investigate how Adeno-associated virus (AAV) gene therapy can be combined with chemotherapy to treat ovarian carcinoma. Adeno- associated virus can be modified to incorporate targeting ligands into the viral capsid, facilitating AAV transduction into tumor-associated vasculature when administered systemically. These ligands bind receptors up-regulated on the angiogenic vasculature within the tumor microenvironment. AAV transduction can be increased by treatment with certain types of anti-cancer drugs, such as DNA-damaging agents (doxorubicin), histone deacetylase inhibitors (bortezomib) and proteasome inhibitors (depsipeptide). We examine the ability of TNFα-Related Apoptosis-Inducing Ligand (TRAIL) gene therapy to augment chemotherapy-mediated killing of ovarian cancer cells. Chemotherapeutics can also increase tumor cell sensitivity to TRAIL-mediated apoptosis. In this study, we will deliver TRAIL using the vasculature-targeted self-complementary AAV vector, after treatment with chemotherapeutics. Because our in vivo model of gene therapy targets the tumor-associated vasculature, our in vitro work focuses on the paracrine effects of TRAIL. When ovarian cancer cell lines were pre-treated with drugs, their sensitivity to the paracrine effects of TRAIL increased markedly. With this study, we have determined that TRAIL can be delivered within the context of the AAV genome, and that the TRAIL secreted from transduced cells maintains paracrine activity. Furthermore, the effects of chemotherapeutics can be potentiated by subsequent TRAIL treatment. This implies that with a multi-modal therapy, chemotherapeutic doses could be reduced and systemic toxicity minimized.

4. Aspirin Therapy in Neonatal Hyperoxia Presenting author: Britt, Rodney

Purpose: Preterm infants are born with immature lungs, often requiring oxygen therapy to maintain homeostatic blood oxygen levels. Infants with prolonged oxygen exposure are at highest risk of developing chronic lung diseases such as Bronchopulmonary Dysplasia (BPD) and pulmonary complications later in life. Oxygen toxicity induces a pro- inflammatory response which is associated with lung injury and impairment of lung development which may persist through life. Aspirin is a nonselective cyclooxygenase (COX) inhibitor which is known to disrupt pro-inflammatory processes through multiple mechanisms. We tested the hypothesis that aspirin therapy will improve lung function and minimize airway hyperresponsiveness in lungs of adult mice following newborn hyperoxia exposure. Methods: Immediately after birth, newborn mice were exposed to room air (RA) or hyperoxia (>95% O2) and were injected daily with vehicle (PBS), 5, or 10 mg/kg aspirin (ASA). On day 7, hyperoxia exposed mice were returned to room air. Pulmonary function tests (PFT) were performed using the SCIREQ Flexivent system on day 28 and day 70. Results: Airway reactivity was assessed by measuring total resistance following intratracheal treatment with 0, 5, 10, 15, 25, and 50 mg/mL acetylmethacholine. Pressure- Volume loops (PV loops) indicated an increase in compliance in PBS/>95% O2 mice which was ameliorated in the hyperoxia exposed pups treated with aspirin. Total resistance was significantly higher in PBS/>95% O2 mice in response toacetylmethacholine challenge compared to room air controls, however both ASA/>95% O2 treatment groups exhibited only mild increase in resistance in response to 50 mg/mL acetylmethacholine (6.18±0.59 vs 2.93±0.35). Conclusions:Aspirin treatment attenuated detrimental effects of hyperoxia on lung function and airway reactivity. Aspirin may be a beneficial therapy for preterm infants who are at risk of developing BPD.

5. rAAV recombination Presenting author: Cataldi, Marcela P.

The palindromic terminal repeats (TR) of adeno-associated virus (AAV) form DNA hairpins (HP) that are essential for replication and for priming the conversion of single- stranded virion DNA to double-strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. Recombination with chromosomal DNA poses a risk for genotoxicity, raising the question as to whether the hairpin structures formed by the TRs are more likely to integrate than other forms of DNA ends. Because we cannot generate rAAV viral vectors without HP ends, we investigated the contribution of AAV TR to chromosomal integration by comparing DNA molecules with the TR sequences constrained in the hairpin conformation to molecules with linear duplex ends, with or without TR sequences. In order to measure integration efficiency, 293 cells were transfected with a high dose of each of these AAV genome-like molecules carrying a GFP-expression cassette, and continuously passaged until a stable percentage of GFP-expressing cells was reached, which represented the percentage of cells with DNA molecules integrated into the host genome after loss of episomal DNA by dilution. The results show no significant difference in terms of integration efficiency between the three DNA molecules, suggesting that AAV TR in a hairpin conformation is not more or less likely to integrate than open blunt ends with or without AAV TR sequences. In addition, we found that a previously reported decrease in gene expression mediated by silencing of HP-containing molecules is not observed when molecules are transfected at high doses, suggesting that the pathway that specifically recognizes the hairpin structure and leads to DNA silencing is saturated under these experimental conditions.

6. FIND: Software for enhanced flow cytometry analysis Presenting author: Dabdoub, Shareef M.

Flow Cytometry is a process by which cells, and other microscopic particles, can be identified, counted, and sorted mechanically through the use of hydrodynamic pressure and laser-activated fluorescence labeling. Unfortunately, widely adopted analysis software limits users to manual separation of events based on viewing two or three simultaneous dimensions. While this may be adequate for experiments using four or fewer colors, advances have lead to laser flow cytometers capable of 24 colors, and mass- spectrometry based machines capable of recording 100 channels are being developed. Analysis of such high-dimensional data by visual exploration alone can be error-prone and susceptible to unnecessay bias. The field of Data Mining provides tools for automated group classification of multi-dimensional data, and many algorithms have been applied to flow cytometry. However, the majority of this research has not been made easily available to users, and are not in wide use. Here we present a new software application for analysis of multi-color flow cytometry data: FIND (Flow Investigation using N-Dimensions). The main goals of this effort are to provide a user-friendly tool for automated gating of multi-color data, and a platform for development and dissemination of new analysis tools. Users can easily load single or multiple data sets, perform automated event classification, and graphically compare results between experiments. We also make available a powerful plugin system that allows researchers to implement and share their data analysis, visualization, and classification algorithms. This will greatly reduce development time as well as provide a common platform for distribution of new techniques to flow cytometry users around the world.

7. Genetic modification of AAV9 Presenting author: Davis, Adam S.

Purpose Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. To date, the FDA has approved a single treatment showing modest benefit; however, several therapeutic neuroprotective factors (IGF-1, NGF) have demonstrated significant potential in animal models (e.g., SOD1 mouse). Due to the short half lives of these neuroprotective agents using invasive delivery methods, we are interested in developing a neuroprotective gene transfer strategy capable of targeted and prolonged neuroprotective factor expression following simple intravenous (IV) infusion.Methods Adeno-associated virus (AAV) has been widely used as a human gene transfer vector. It is non-pathogenic and can exhibit prolonged gene expression. Previous studies have shown that AAV tolerates short linear peptide inserts into its capsid proteins. We have previously identified a short peptide, tet1, which binds the motor neuron axon membrane specific receptor Gt1b. We have successfully incorporated tet1 into the capsid proteins of AAV serotype 1 (AAV1) increasing its specificity for motor neurons while simultaneously decreasing its specificity for non-Gt1b expressing cells in vitro. We are now interested in extending this work using AAV serotype 9 which is capable of crossing the blood brain barrier (BBB) after IV administration. Using computer modeling of the AAV9 capsid structure, we have identified the AAV1 homologous tet1 insertion site. We will incorporate tet1 into AAV9 via PCR mutagenesis and assess its efficacy for increasing motor neuron specificity in the context of AAV9, following rAAV9tet1 vector production.Results Modification of AAV1 to include tet1 following D590 significantly increases the vector’s specificity for cells expressing the motor neuron axon membrane receptor Gt1b while simultaneously decreasing the vector’s specificity for cell types not expressing this receptor. Using PCR mutagenesis, the tet1 peptide coding sequence has been inserted into the AAV9 cap gene and we are evaluating the impact that the insertion has on vector yields.Conclusions Our results indicate that tet1 modified AAV exhibits the potential for therapeutic delivery of neuroprotective factors in the context of the ALS SOD1 mouse model. Based on recent studies that demonstrate AAV9’s ability to cross the BBB, we hypothesize that inclusion of tet1 into the capsid proteins of AAV9 will increase its transduction of motor neurons by a non-invasive delivery route resulting in long-term expression of several neuroprotective agents at therapeutic levels.

8. Diesel exhaust particle exposure exacerbates contractile dysfunction in diabetic cardiomyocytes Presenting author: Gorr, Matthew W.

Diesel particulate matter (DEP) pollution and diabetes mellitus (DM) are two key contributors to heart disease in modern cities. The purpose of the present study was to evaluate contractile function, calcium signaling and reactive oxygen species (ROS) formation in ventricular myocytes from diabetic adult rats exposed to DEP (10 µM). Ventricular myocytes were isolated from both control and streptozotocin-induced diabetic (55 mg/kg, i.p.) adult male Sprague-Dawley rats and sarcomeric contractile properties and calcium flux were evaluated using an IonOptix Field-Stimulator system. Intracellular ROS formation was assessed using a 2′,7′- dichlorodihydrofluorescein (DCF) assay. We found that several measurements of contractile function and calcium flux were reduced with DEP treatment in cells from both the control and diabetic rats, but with a greater degree in the diabetic rats. Further studies indicated that antioxidant co-culture (0.25 mM Tiron and 0.5 mM N-Acetyl-L-cysteine) completely restored contractile function in DEP-treated myocytes. Our final data confirmed that the effects of DEP on the function of the myocyte are exacerbated in cells from the diabetic heart.

9. GM-CSF for Unresolving Infection Presenting author: Greathouse BSN, MS, Kristin

Purpose: Persistent infection is a leading cause of morbidity and mortality in critically ill children. The innate immunostimulant GM-CSF (LEUKINE®[sargramostim]) has been shown to speed clearance of infection in critically ill adults but pediatric data are limited. We present our institutional experience with the use of GM-CSF as adjuvant therapy (in addition to antibacterials and source control) for the treatment of persistent infection in critically ill children. Methods: Retrospective chart review. Data are presented as median (IQR). Results: From 2003 to 2010, nine critically ill children underwent therapy with GM-CSF for the purpose of augmenting immune function in the setting of persistent culture positivity (age: 0.9 [0.3-9] years, 55% female). Sites of infection included lung (n=4) and bloodstream (n=5). Pathogens included Candida species (n=4), Pseudomonas species (n=4), and MRSA (n=1). Duration of culture positivity prior to initiation of GM-CSF therapy was 9 (8-15) days. Duration of GM-CSF therapy was 7 (5-7) days. Dosing was by IV infusion in all patients with doses ranging from 125-150 μg/m2/day. Two children were receiving extracorporeal membrane oxygenation support at the time of GM-CSF initiation. Clearance of infection was achieved in 7/9 children (78%), with a median time to clearance (among the 7 responders) of 3 (2-5) days from the time of GM-CSF initiation. There was one relapse of infection among responders. 28-day mortality (from the time of GM-CSF initiation) was 100% in the non-responders (n=2) and 43% in the responders. Oxygenation index, modified inotrope score, pediatric logistic organ dysfunction score, and white blood cell count values were similar pre- and post-GM-CSF therapy (p=1.0, 0.24, 0.71, and 1.0 respectively). Conclusions: Our case series suggests that GM-CSF therapy may have a role in facilitating clearance of unresolving infection in critically ill children. Its use was not associated with worsening of organ failure. Prospective randomized, controlled trials are needed to evaluate the efficacy of GM-CSF in this setting.

10. MACROPHAGES ARE REQUIRED FOR CONTROL OF UTI IN MICE Presenting author: Horvath, Jr., Dennis J.

The innate immune system is critical for the protection of the urinary tract from bacterial infections of the bladder (cystitis) and kidney (pyelonephritis). Phagocytes constitute the predominant innate effector cells of the urinary tract, and these cells are essential for clearance of bacteria from the bladder during the first 24-48 hrs following infection. Although it is known that neutrophil recruitment is critical during the first 24 h upon infection, macrophages are also found within the bladder during a urinary tract infection but how these phagocytic cells contribute towards bacterial clearance is unknown. The aim of this study was to determine the functional role of resident and recruited inflammatory macrophages in a murine cystitis model. Toward this end, we injected diptheria toxin (DT) intraperitoneally (i.p.) into CD11b-DTR transgenic mice to selectively deplete CD11b+ macrophages prior to transurethral inoculation of uropathogenic Escherichia coli (UPEC) into the murine bladder. Flow cytometric quantitation of the inflammatory responses of the bladder and kidney obtained from mice infected with UPEC and depleted of CD11b+ cells revealed the absence of a subpopulation of macrophages expressing CD11b, CD43, and CD62L. This subpopulation of macrophages correspond to resident rather than inflammatory macrophages and were found in the infected but non-depleted control mice. In contrast, we found that the bacteria spread from to the bladder to the kidneys in mice that received the DT treatment. The higher bacterial burden within the kidneys compared to non- depleted controls occurred despite the presence of greater numbers of neutrophils. Interestingly, the bladder colonization levels were not significantly different between the control and experimental groups up to 48 hours post infection. These studies indicate that neutrophils appear to be responsible for bacterial clearance in the bladder while resident macrophages appear to be responsible for preventing bacterial spreading and the clearance of bacteria in the kidney.

11. Treatment of disseminated medulloblastoma with measles virus Presenting author: Hutzen, Brian

Purpose Medulloblastoma is the most common malignant brain tumor of childhood. Although the survival rate of afflicted children has improved considerably over the past several years, a small subset of these patients will present with a disseminated variant of the disease and face a much bleaker prognosis. We have previously demonstrated the efficacy of a recombinant oncolytic measles virus (MV) to treat localized medulloblastoma in a mouse xenograft model. In the present study, we sought to extend our findings to the treatment of disseminated disease. To this end, we developed and characterized a mouse xenograft model of disseminated medulloblastoma using serial bioluminescent imaging techniques in combination with histopathological examination. Methods D283med-Luc medulloblastoma cells were injected into the lateral ventricles of athymic nude mice. These mice were randomly divided into groups receiving treatment with an oncolytic measles virus expressing GFP (MV-GFP) or an inactivated form of the virus as a control. Tumor establishment and growth were subsequently monitored via bioluminescent imaging techniques. Mice were sacrificed if they developed neurological deficits or at the experiment endpoint, and their brains and spinal cords were subjected to histopathological examination. Results Mice injected with D283med-Luc cells showed tumor growth in their ventricles and both intracranial and spinal subarachnoid spaces, closely recapitulating the human disease. Subsequent intraventricular administration of MV resulted in stabilization and shrinkage of the tumor, significantly prolonging the survival of the treated animals compared to those treated with an inactivated virus. These data demonstrate that oncolytic MV may be of use in treating disseminated medulloblastoma. Conclusions While advances in treatment modalities have increased the survival of children diagnosed with medulloblastoma, approximately 20% of these patients will present with disseminated disease, and face a much worse prognosis. Because of the limitations and significant drawbacks associated with conventional therapy, alternative means for treating all variants of medulloblastoma are sorely needed. The results presented here demonstrate that modified measles virus has therapeutic potential in the treatment of disseminated medulloblastoma and provide initial data to be pursued with the goal of using the virus in a clinical trial.

12. Damage Induced Alternative Splicing of is Controlled by a Balance of Positive and Negative Splicing Regulators Presenting author: Jacob, Aishwarya G. Introduction:MDM2 is an oncogene, whose primary function is to negatively regulate the levels of tumor suppressor . Numerous alternatively spliced forms of MDM2 have been identified and have been associated with various cancer types. The MDM2 ALT1 form, which consists of exons 3 and 12 spliced together, lacks the p53-binding domain but can bind full length MDM2. MDM4 is a close family member, which along with MDM2 suppresses p53 transcriptional activity. MDM4 is also alternatively spliced. We have observed the occurrence of the MDM2 ALT1 form in >85% of Rhabdomyosarcoma tumors along with MDM4 ALT2, an alternatively spliced form of MDM4, suggesting a coordinated regulation of their splicing. MDM2 ALT1 and MDM4 ALT2 are also generated in response to genotoxic stress like UV and cisplatin treatment. We have shown interaction of MDM2 ALT1 and MDM4 ALT2 with full length MDM2 and MDM4 and consequent upregulation of p53 and its targets. The mechanism of this alternative-splicing phenomenon is poorly understood. Hypothesis: We hypothesize that MDM2 ALT1 and MDM4 ALT2 are induced in response to genotoxic stress by coordinated action of cis elements and modified splicing factors and bind the full length proteins thus sequestering them in the cytoplasm and preventing their function. This may initially confer protection to the cell due to p53 upregulation but on accumulation of mutations leads to cell survival and oncogenesis. Purpose: To identify the cis elements and trans factors regulating the splicing of MDM2 transcripts under normal and stress conditions. Methods: We used an MDM2 minigene system that is responsive to stress induced splicing in an in vitro, cell free splicing assay. We then used RNA affinity chromatography to identify the trans factors that bind to the minigene and immunointerference assays to understand their roles in MDM2 splicing. Results: We have shown that intron 11 of MDM2 contains important cis elements that positively regulate splicing under normal conditions and confer stress responsive negative regulation. We have also identified novel trans factors FUBP1 and PTB that bind the minigene differentially in response to stress and possibly play a positive role in splicing regulation. Significance: These studies enable an understanding of MDM2 splicing regulation, its biological significance and help identify potential therapeutic targets for cancer.

13. Proteomic analysis of the dystrophin-associated protein complex reveals differences between cardiac and skeletal muscle Presenting author: Johnson, Eric

Dystrophin is a large cytoskeletal protein that localizes to the inner surface of the membrane of skeletal muscle fibers and cardiomyocytes. It is the central organizer of the dystrophin-associated protein complex (DAPC) that functions as a signaling complex and as a structural stabilizer of the striated muscle membrane. Mutations in dystrophin result in skeletal muscle degeneration and cardiomyopathy leading to early mortality due to respiratory or heart failure. However, there is no correlation in the severity, age of onset, or progression of disease between skeletal and cardiac muscles. This suggests that the DAPC may differ in composition and therefore function between these two striated muscles. This is further supported by gene therapy studies utilizing micro and mini dystrophin constructs that fully rescue skeletal muscle function yet appear to lack domains required to fully restore cardiac function. Therefore, the identification of differences in DAPC composition between cardiac and skeletal muscle is highly relevant to our understanding of the molecular mechanisms that underlie heart failure and the development of effective therapeutics for muscular dystrophies. To this end, we preformed a comprehensive comparison of all known members of the DAPC between cardiac and skeletal muscle. Because of its complexity, we chose to purify the DAPC from tissues by immunoprecipitation and subsequently identify all DAPC members simultaneously by Shotgun Proteomics. This approach revealed novel differences between mouse cardiac and skeletal muscle that primarily involve DAPC proteins known to mediate intracellular signaling. DAPC members unique to the mouse heart were confirmed in human cardiac biopsy samples, indicating conservation of cardiac-specific functions of dystrophin in the human. These differences could underlie the reported discrepancies in clinical presentation and response to treatment between cardiac and skeletal muscle.

14. Import and degradation of AMPs by NTHI Presenting author: Johnson, Sara M.

Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative inhabitant of the human nasopharynx yet can cause opportunistic diseases of the upper and lower respiratory tract. To colonize and establish infection, NTHI must acquire nutrients and resist insult by the host innate immune response. Host antimicrobial peptides (AMPs) are innate immune molecules that promote sterility of environments such as the middle ear. Previously, we demonstrated that the Sap ABC-transporter binding protein, SapA, binds AMPs and equips NTHI to resist AMP lethality. A SapA mutant is attenuated for survival in the middle ear, demonstrating that Sap transporter function is essential for pathogenesis. Thus, we hypothesize that Sap-dependent AMP transport and subsequentcytoplasmic degradation is vital for removal of AMP accumulation within the bacterial periplasm, providing a mechanism of bactericidal defense concordant with nutrient acquisition. Toward this end, we incubated whole bacteria with sublethal concentrations of AMPs and monitored AMP localization by immunogold transmission electron microscopy, bacterial cell fractionation and western blot analysis. We demonstrated that AMPs localize to the periplasm and cytoplasm of whole bacterial cells. Transport of AMPs to the bacterial cytoplasm was dependent upon a functional SapBC permease complex since AMPs accumulated in the periplasm of the SapBC permease-deficient strain. In order to monitor the fate of AMPs transported to the bacterial cytoplasm, whole cells were incubated with a sublethal concentration of AMP, washed and then incubated for increasing time to monitor AMP trafficking and degradation. We demonstrated that AMPs transported to the bacterial cytoplasm were susceptible to proteolytic degradation. We hypothesize that intracytoplasmic transport and degradation of AMPs would nutritionally benefit NTHI. In support of this, we observed increased growth of NTHI when exposed to sublethal concentrations of AMPs. Collectively, this novel mechanism of innate immune evasion via AMP transport and degradation benefits NTHI survival. Blockade of the functional Sap transporter could hinder this mechanism thus rendering NTHI more susceptible to the host innate immune response and promoting clearance of the bacteria

15. Effects of extracellular DNA within a nontypeable Haemophilus influenzae- induced biofilm Presenting author: Jones, Eric A.

Biofilms formed by the commensal bacterium nontypeable Haemophilus influenzae (NTHI) are frequently associated with chronic infections of the upper airway such as otitis media. The resistant nature of bacterial biofilms to mucosal immune effectors and antibiotic therapy plays an important role in NTHI persistence and disease chronicity. Extracellular DNA (eDNA) contributes to the structure of NTHI biofilms, and has been reported to co-localize with host defense molecules such as antimicrobial peptides (APs) in vivo. We hypothesized that eDNA would interact with cationic APs and that this association would have important consequences for NTHI biofilm formation.

Herein, we demonstrated that the mucosal immune effector human beta defensin- 3 (hBD-3) bound eDNA in a dose dependent manner in vitro. Furthermore, this binding altered the biochemical migration of eDNA in an agarose gel, which indicated that hBD-3 altered the biochemical properties of eDNA and suggested that NTHI biofilms would be impacted as well. As such, we incubated NTHI with sub-lethal concentrations of hBD-3 and showed that NTHI biofilms demonstrated an altered architecture with bacteria arranged along unique structures, compared to medium alone. The concentrations of eDNA isolated form hBD-3 treated and untreated biofilms were equivalent, which indicated that the altered morphology was not due to increased eDNA content. Experiments to determine the functional impact of hBD-3 on biofilms formed in an experimental model of NTHI-induced OM are underway.

Together, our data demonstrated that eDNA bound hBD-3 and that this association influenced the development of NTHI biofilms. These results further suggested that the presence of eDNA in NTHI biofilms could provide a niche in which host defense peptides are sequestered and their activity diminished, which in turn contributes to the chronicity of NTHI-induced diseases.

16. A shared ATPase for carbohydrate transport in S. pneumoniae Presenting author: Marion, Carolyn

The opportunistic pathogen Streptococcus pneumoniae is responsible globally for over five million deaths each year. Acquisition of carbohydrates for nutrition is essential to pneumococcal fitness in the human host, and ATP binding cassette (ABC) transporters are one of the main mechanisms for import of carbohydrates. ABC importers require two permeases which create a pore for transport, two ATPases which hydrolyze ATP, and in Gram-positive bacteria, a substrate binding domain which confers specificity. S. pneumoniae is predicted to encode six carbohydrate-specific ABC transporters; three have been characterized experimentally. Interestingly, the operons encoding each of the six transporters lack for the necessary ATPases. We sought to identify the gene(s) encoding the ATPases required for these transporters. Through bioinformatics, the predicted gene msmK was selected for its similarity to the carbohydrate-specific ATPase from S. mutans. An msmK mutant was unable to utilize substrates of the characterized ABC transporters, indicating that MsmK likely energizes multiple transporters. The complete elimination of growth on two of the tested substrates further implies that MsmK is the only ATPase interacting with these transporters. Together, these data suggest that regulation of msmK may affect transport of multiple sugars. Ongoing work is focused on expressing recombinant MsmK and confirming its ATPase activity as well as determining the regulation of msmK in the presence of different carbohydrates. As similar genetic arrangements have been noted in many Gram positive organisms, S. pneumoniae may provide a model for a novel mechanism of regulation of carbohydrate uptake.

17. Social Information Processing in Pediatric Epilepsy Presenting author: Moran, Lisa

Previous research suggests children with epilepsy are less socially competent than healthy peers, although the specific reasons for this deficit are undetermined. This study seeks to evaluate social information processing (i.e., attributional styles, coping strategies, and emotional responses) in children with epilepsy in response to hypothetical stressful social scenarios.At present, the sample includes 17 of 28 recruited children with epilepsy and 6 matched healthy controls. For each of five scenarios, children selected the reason the situation occurred, how they would handle the situation, and how they would feel. The five scenarios were completed twice, with the antagonist being an unknown peer in one instance and the child’s reported best-friend in the other. Preliminary findings from a series of repeated-measures ANOVA revealed few differences between children with epilepsy and healthy controls. All children reported more externalizing attributions (e.g., “He did it on purpose”) toward unknown peers than friends. No differences were found in coping strategies at the group level or for antagonist type. Overall, children reported feeling less anger, embarrassment, and sadness in situations involving friends versus unknown peers. However, group differences were nearly significant (p = 0.07) for sadness, such that healthy controls experienced more sadness in response to stressful social situations than children with epilepsy. The results are preliminary, and may change as we continue to recruit participants, but suggest that the social difficulties experienced by children with epilepsy are not necessarily linked to deficits in social information processing on hypothetical social scenarios.

18. Transcutaneous immunization against experimental NTHI-induced otitis media Presenting author: Novotny, Laura A.

Transcutaneous immunization (TCI) is a noninvasive immunization strategy that utilizes the immunocompetence of the skin to induce systemic and mucosal immune protection. As otitis media (OM) is a disease of the respiratory mucosa, we examined TCI as a means to prevent and treat nontypeable Haemophilus influenzae (NTHI)-induced OM. Immunogens that target NTHI OMP P5 and the Type IV pilin adhesins admixed with the potent adjuvant LT(R192G-L211A) were applied to the pinnae of chinchillas followed by NTHI challenge. Immunogen-specific IgG and IgA were detected in serum and nasopharyngeal lavage fluids, which demonstrated the induction of both systemic and mucosal immune responses. Moreover, receipt of the immunogens resulted in 2.5-4 log fewer NTHI within the nasopharynges and middle ears (p< 0.001) compared to adjuvant only. The mechanism for this response was attributed to the migration of DC-SIGN+ dermal dendritic cells from the pinnae to local lymphoid tissues. Therefore, TCI with the NTHI adhesin-directed immunogens was efficacious against experimental NTHI-induced OM. As the chronic nature of OM is attributed to the formation of biofilms within the middle ear, we next evaluated the therapeutic potential of TCI. Chinchillas were first challenged transbullarly to induce biofilm formation, then immunized by TCI. Within 1 week after receipt of adhesin-directed immunogens, pre-established biofilms were markedly reduced (p< 0.05) compared to adjuvant alone. Immunogen-specific IgG and IgA were detected in middle ear fluids, in addition to 2-5-times more of the inflammatory mediators IL-4, IFN-g and IL-17A. These data advocate TCI as a noninvasive and inexpensive immunization regimen. Moreover, TCI could expand the use of vaccines to both protect against as well as treat OM, in addition to other diseases of the respiratory tract due to NTHI.

19. Muscle Side Population Cells: Shifting the Balance in Muscular Dystrophy Presenting author: Penton, Christopher

Skeletal muscle regeneration is a complex process that requires the coordinated action of muscle stem cells to repair damaged muscle fibers and mesenchymal cells to secrete pro- myogenic factors. In acute muscle injury models where regeneration fails, mesenchymal precursor cells are deregulated and produce large numbers of fibroblasts and adipose cells. This hinders regeneration by muscle stem cells. It is currently believed that muscle stem cells and mesenchymal progenitors do not share a common stem cell origin within skeletal muscle. We show here that muscle-resident interstitial stem cells (known as CD31-/CD45- side population (SP) cells) can give rise to both Pax7+ muscle stem cells and PDGFRα+ mesenchymal precursor cells. Furthermore, we found that muscle damage alters the lineage choices of CD31-/CD45- SP cells. CD31-/CD45- SP cells isolated from cardiotoxin-injured mice or from mice with muscular dystrophy do not form muscle but instead give rise to PDGFRα mesenchymal precursors that differentiate into fibroblasts and adipocytes. These results show that a damaged muscle environment has the potential to alter CD31-/CD45- SP stem cell fate choices thus shifting the balance away from muscle regeneration towards fibrosis and fat deposition. CD31-/CD45- SP cells could represent an important therapeutic target in muscular dystrophy and other muscle pathologies where the balance between myogenesis and fibrosis is disrupted

20. The Haemophilus Sap transporter is required for commensal establishment and pathogenesis Presenting author: Raffel, Forrest K.

Commensal bacterial colonize host environments, and in turn provide nutrients and limit pathogen colonization at these sites. Mechanisms used to establish commensalism can likewise equip these bacteria in the transition to an opportunistic pathogen. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx, yet mediates diseases of the airway such as otitis media. Commensalism requires adaptation to host nutrition and innate immune mechanisms, therefore coordination of adherence, metabolism and bactericidal resistance mechanisms are necessary. We previously demonstrated that the NTHI Sap transporter, an inner membrane multifunctional complex, mediates resistance to host antimicrobial peptides and is required for heme homeostasis. We hypothesized, therefore, that NTHI commensal behavior required these Sap transporter functions. Thus, wild-type and SapA-deficient NTHI were cultured on the surface of respiratory epithelial cells to examine NTHI-host cell membrane interactions. SapA-deficient NTHI were less adherent to bronchial, middle ear and nasopharyngeal epithelial cells yet demonstrated dramatic alterations in NTHI-epithelial cell homeostasis as characterized by epithelial cell membrane ruffling, increased actin polymerization and enhanced invasion of epithelial cell layers. Additionally, cytokine profiles indicated altered inflammation induced by SapA-deficient NTHI. Our preliminary data support the hypothesis that the Sap transporter is required for commensalism; however we seek to better understand the outer membrane changes in SapA deficient NTHI and the epithelial host cell response to these changes. This study supports the essential role for the Sap transporter in the commensal and pathogenic behavior of NTHI. Thus, targeted therapies to block Sap transporter function could significantly reduce the clinical and economic burden of NTHI mediated diseases.

21. Greyhound structural variation Presenting author: Rowell, Jennie

Purpose: DNA segments that are 1kb or larger and present at a variable copy number are known as DNA copy-number variation (CNV). Many CNV’s span genes and likely affect genetic networks, suggesting significant effects on phenotypes. In the last 200 years, hundreds of pure dog breeds were created by selection of morphological and behavioral traits. Because of these extreme population bottlenecks, dogs are a powerful resource for studying CNV genetics. Furthermore, dogs are an excellent model to study many human diseases, as they share 650 Mb of additional genetic material with humans beyond that of the mouse. Many genes identified in dogs have a human homolog, making them an ideal model study CNV and disease for comparative genomics. We are particularly interested in Greyhound specific variation, as they have a high rate of many diseases, including cancers, that are similar to humans. Another advantage of Greyhounds is a recent divide in breeding into racing and show dogs. This has resulted in two highly related populations with different disease predispositions. We are conducting studies to identify biologically relevant genetic variation that predisposes racing and show Greyhounds to certain diseases. Here we report the first ultra high resolution CNV map in Greyhounds. Methods: Our lab conducted CNV discovery in a small panel of pure bred Greyhound racing and show dogs. We quantified CNV by Comparative Genome Hybridization on a custom designed ultra high resolution whole genome microarray including regions of segmental duplication (1 M oligonucleotides probes <2 kb Genome-wide mean spacing; <1.2 kb segmental duplication mean spacing; Agilent). Selected regions were validated by Southern blot and PCR-based methods. Results: We identified 288 CNV regions affecting >150 known genes, with 16 CNVs unique to racing Greyhounds and 33 unique to show Greyhounds (as compared to the reference breed). In addition, many CNV regions overlapped both racing and show Greyhounds, suggesting CNVs may contribute to show and racer similarities. This analysis reveals CNV regions that overlap genes of biological importance in many human relevant diseases. Conclusion:We suspect that CNV contributes the increased prevalence of specific diseases among racing and show Greyhounds. We are currently conducting validation studies to determine if the observed variation contributes to specific diseases.

22. Cardiac Teratogenicity in a Murine Model of MPKU. Presenting author: Seagraves, Nikki J.

Phenylketonuria (PKU) is an inborn error of metabolism due to phenylalanine hydroxylase (PAH) deficiency which leads to elevated phenylalanine (Phe). Elevated Phe levels during pregnancy cause a syndrome of congenital anomalies known as Maternal PKU (MPKU). The mechanism of Phe teratogenicity upon the heart remains elusive. The BTBR Pahenu2 mouse provides a model to characterize the MPKU associated CVMs. We hypothesize there is a maternal serum Phe level and developmental period of exposure required to induce CVMs in offspring of mouse PKU females and the genetic background will influence the manifestations of CVMs.

We established four treatment groups to investigate the critical maternal serum level. We screened exposed BTBR fetuses and observed gross defects of the outflow tract (OFT) and vessel morphology. CVMs occurred in dose-dependent manner as maternal serum Phe levels increase (Nonparametric test for trends, p=0.03).

Preliminary data to investigate the period of exposure required to induce CVMs implicates Phe exposure during E8.5-E12.5 as the critical period.

We have generated congenic mice to investigate the effects of genetic background, and have observed CVM differences on each strain.

Defects of OFT and vessel morphology implicate the cardiac neural crest cells (cNCCs). We hypothesize migration or cell signaling of cNCCs is perturbed by high Phe. We began experiments using chick neural tube explant, and have not observed Phe exposure to affect migration or growth of the explant. Wnt-1 Cre X Pah mutant females will be utilized to observe the in vivo effect of Phe exposure on the neural crest cell population. Also, we have chosen genes important in early cardiovascular development and in many pathways to characterize the effect of Phe exposure on gene expression. In situ hybridization experiments are ongoing to determine gene expression changes between wild-type and exposed fetuses. Our studies provide strong evidence that model of MPKU is an excellent model to investigate the mechanism of Phe teratogenicity on heart development, which can aid in the understanding of the development of CVMs.

23. The transcriptome of NTHi Presenting author: Santana, Estevan

Next generation sequencing technology, is rapidly gaining use and popularity in the biomedical field. One aspect of this technique involves massive parallel sequencing of cDNA generated from RNA transcripts (RNA-seq). The RNA-seq approach gives whole transcriptome information. Transcriptome information includes both the relative abundance of each transcript and the identity of novel transcripts. We want to understand how the transcriptome of nontypeable Haemophilus influenzae (NTHI) changes in response to stress stimuli. Moreover, we want to identify small non-coding RNAs (sRNAs) that, in other bacteria, are known to have a regulatory role. To test the feasibility of our sequencing approach we carried out a preliminary RNA-seq experiment. NTHI were grown in supplemented Brain Heart Infusion medium to mid-log phase and total RNA isolated. Approximately 95% of all RNAs in a bacterial cell are either ribosomal RNA (rRNA) or transfer RNA (tRNA). To enrich our samples for mRNA we used a Ribo-Zero kit to physically remove rRNA. We then used an Illumina TruSeq mRNA kit to generate cDNA libraries of untreated total and Ribo-Zero treated RNA samples. These libraries were sequenced using the HiSeq2000 instrument housed in the Biomedical Genomics Core. The Integrative Genome Viewer (Broad Institute) was used to visualize sequencing reads aligned to the NTHI reference genome. The total RNA sample yielded 87 million reads of which 5.6% aligned to non-rRNA or tRNA regions. The Ribo-Zero treated sample yielded 100 million reads with 94.4% of reads aligning to non-rRNA or tRNA regions. This indicated that RiboZero treatment was an effective method to remove rRNA from total RNA. We were also able to determine transcriptional start sites for many genes based on read alignments. Finally, we identified 251 intergenic transcripts. In other species of bacteria, sRNA-encoding genes have a predominantly intergenic localization. The intergenic transcripts thus most likely indicate the presence of multiple sRNAs not previously reported in NTHI. NTHI is normally a commensal of the upper respiratory tract. However, NTHI has roles in multiple upper respiratory tract diseases, which includes otitis media. During infection NTHI is faced with numerous stresses from the host environment. Sensing this stress and quickly responding is essential to NTHI survival in the host. Using an RNA- seq approach to investigate gene expression changes under various stresses on the transcriptome level will allow us to elucidate exactly how the bacteria colonize and survive in the host.

24. Minicircle plasmid DMD vectors for gene transfer studies in mdx Presenting author: Simmons. Tabatha

Duchenne Muscular Dystrophy (DMD) is a lethal X-linked disorder that is the result of mutations in the DMD gene that lead to an absence of the dystrophin protein. DMD mutations causing the allelic Becker Muscular Dystrophy (BMD) preserve an open mRNA reading frame, typically via an internal in-frame deletion of some number of the gene’s 79 exons, and results in the expression of some residual dystrophin. Adeno- associated viruses have emerged as the front-runner for clinical gene transfer in DMD, but because AAV vectors are limited as to the size of the transgene, only internally truncated BMD-like genes (mini- or microdystrophins) may be encapsulated by the virus. Although these have shown benefit in studies in the mdx mouse model of DMD, the ultimate clinical effect in humans may be less pronounced. Transfer of the full-length gene would be predicted to be of the greatest clinical benefit, but full-length gene transfer using lentiviral or adenoviral techniques increases the risk for a greater degree of toxicity and lacks the muscle tropism of AAV. Additionally, IM injection of full-length naked DNA plasmid in humans showed minimal expression in a sole clinical trial.We seek to explore the feasibility of full-length gene transfer via a biopolymer-mediated delivery. In order to minimize the size of the plasmid, we will use minicircle vectors; these have not previously been used as a potential vector in DMD gene transfer studies but have been shown to allow for sustained transgene expression in quiescent cells and tissues. In preparation for studies of biopolymer delivery, we are creating minicircle vectors of DMD and control constructs, each driven by the MHCK7 promoter. These include MHCK7 only (negative control); MHCK7 plus hrGFP; MHCK7 plus a microdystrophin construct with exons 17-58 deleted (5.0 kb); and MHCK7 plus full length dystrophin cDNA (12.2 kb). Our initial ongoing studies will test delivery to mouse skeletal muscle of the GFP reporter construct using poly(cystaminebisacrylamide-diaminohexane) [poly(CBA-DAH)], a bioreducible cationic polymer. We intend to then test whether attachment of a published muscle targeting hepta-peptide to the polymer-transgene complex allows for increased muscle delivery efficiency. Finally, we will deliver both full-length and micro-dystrophin minicircle DNA vectors that should theoretically increase and prolong expression in muscle tissues. We will present details of our strategy and the ongoing results of our experiments, directed at developing a novel therapeutic approach to Duchenne Muscular Dystrophy.

25. The role of Galgt1, a glycosyltransferase, in muscle regeneration. Presenting author: Singhal, Neha

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disease that results in progressive muscle weakness and muscle wasting. It is the most common form of muscular dystrophy, affecting 1 in every 3500 male births. DMD is a very debilitating disease wherein the patient is usually wheelchair bound by the age of 12 and dies in early 20s due to cardiac and/or respiratory failure. A sub-sarcolemmal muscle membrane protein dystrophin is absent in DMD patients. Dystrophin anchors the dystrophin- associated glycoprotein complex in the muscle membrane, which helps to connect the extracellular matrix (ECM) that surrounds every muscle fiber to the F-actin cytoskeleton. Disruption of this linkage causes mechanical damage in DMD muscle, which in turn leads to a chronic cycle of degeneration followed by regeneration. The regeneration in DMD isn’t robust enough to maintain the muscle mass and eventually the regenerative capacity of the muscle decreases significantly. MDX, the mouse model for DMD, has a homologous (to humans) mutation in dystrophin gene. Regeneration in MDX, however, is more robust than in humans and prevents significant loss of muscle mass. In MDX muscle, there is a massive upregulation of a glycosyltransferase; Galgt1. Galgt1 is also the most up regulated protein in the normal regenerating muscle. Galgt1 synthesizes the building block for major complex gangliosides, which are involved in many signaling processes. In this study, we wanted to investigate the role played by Galgt1 and gangliosides in regeneration of muscle. Our hypothesis is that the lack of Galgt1 gene in mdx muscle should significantly reduce the regenerative process of the skeletal muscle. The following mouse models were used for the study; WT (wild type), Galgt1 knockout (KO, WT mice with non-functional Galgt1 gene), MDX, MDX/Galgt1 knockout (KO, MDX mouse with non-functional Galgt1 gene). We induced regeneration in muscles by injecting the gastrocnemius and tibialis anterior with 10µM of cardiotoxin (CTX, Cardiotoxin destroys the muscle thus inducing regeneration). Muscle sections were immunostained with antibodies against Galgt1 and gangliosides, to localize the expression of Galgt1/gangliosides in normal and regenerating muscle fibers. RNA was isolated from regenerating and normal muscles of mice of different genotypes to understand changes in Galgt1, and regenerative marker’s gene expression. By comparing the RNA from all these different conditions we can estimate how the gene expression of regenerative markers change with (WT, MDX) and without (Galgt1 KO, MDX/Galgt1 KO) the presence of Galgt1. Further, we want to investigate the role played by these complex gangliosides in disease severity. Disease severity can be measured by various disease parameters like serum creatine kinase as a measure of muscle damage (creatine kinase is an muscular enzyme, that increases in serum with muscle damage), central nuclei as a measure of regeneration (in regenerated muscle fibers the nucleus remains in the center of the muscle fiber as compared to normal muscle, in which the nucleus moves to the periphery), fiber diameter (various factors can lead to a change in diseased state). These studies have shown that the dramatic elevation of Galgt1, and the ganglioside it synthesizes, in regenerating and dystrophic muscles. Elevated Galgt1 and ganglioside expression occurs primarily in satellite cells, the stem cell population of regenerating muscles. Further, absence of these results in MDX/Galgt1 ko muscles suggests a role for Galgt1 in the regenerative process. The process of regeneration is vital to the maintenance of muscle mass and the failure of this process has lethal consequences for the DMD patients. Our results suggest that Galgt1 and complex gangliosides play a role in regeneration of muscle. Galgt1 has potential to be used as a therapeutic for DMD that will prevent or reduce the course of disease by inducing enhanced regeneration in the dystrophic muscle.

26. MKP-1 and acetaminophen toxicity Presenting author: Wancket, Lyn M.

Purpose: MAPK phosphatase (MKP)-1 is a critical negative regulator of p38 and JNK MAP kinases. Mkp-1-/- mice exposed to bacteria and bacterial ligands have prolonged p38 and JNK activation, leading to enhanced cytokine production, multi-organ failure, and high mortality rates. While the function of MKP-1 is well characterized for microbial models, little is known about the role of Mkp-1 in other diseases where JNK activation plays a key role in the pathogenesis. JNK activation is linked to hepatocyte death in acetaminophen overdose, one of the most common causes of drug-induced liver failure in the United States. In this study, we evaluated the role of Mkp-1 in the host response to acute acetaminophen toxicity. Methods: Fasted Mkp-1+/+ and Mkp-1-/- mice received a single intraperitoneal injection of PBS or 300 mg/kg acetaminophen. Mice were then euthanized and tissues collected 1-6 hours post dosing to assess glutathione levels, organ damage, and MAPK activation. Separately, fasted Mkp-1+/+ and Mkp-1-/- mice received a single intraperitoneal injection of 400 mg/kg acetaminophen, re-fed after 6 hours, and were monitored up to 120 hours post treatment to evaluate survival. Results: Serum ALT levels, a marker of hepatocyte injury, were significantly higher in Mkp-1-/- mice at 4 hours post dosing and continued to trend higher than wildtype mice at 6 hours. Histologically, Mkp-1-/- mice had more rapid and extensive hepatocyte degeneration and necrosis than did wild type mice at 2, 4, and 6 hours post dosing. While both Mkp-1+/+ and Mkp-1-/- livers had marked phosphorylation of JNK at 2 hours post dosing, JNK activation was more sustained through 6 hours in Mkp-1-/- livers than in Mkp-1+/+ livers. Although hepatic Glutathione (GSH) and Glutathione disulfide (GSSG) levels were higher in PBS-treated Mkp-1-/- compared to Mkp-1+/+ mice, both mouse strains had marked GSH and GSSG suppression at 2-6 hours, indicating that higher initial glutathione stores were not protective in Mkp-1-/- mice. Finally, Mkp-1+/+ animals had a significantly higher total survival than Mkp-1-/- mice (43% vs. 32%) and Mkp-1+/+ mice had a longer median survival time than knockout animals (71 vs. 23.5 hours). Conclusions: Together, these data support the model that Mkp-1 plays an important protective role in mediating host survival and tissue damage during acute acetaminophen toxicity, likely through regulation of JNK.

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27. Persistent antigen presentation after gene therapy Presenting author: Xu, Dan

Foreign proteins persistently expressed from the genome of recombinant adeno- associated virus (rAAV) gene therapy vectors elicit a sustained but ineffective CD8+ T cell response. To define the requirements for CD8+ T cell priming, a rAAV vector encoding the b-galactosidase model protein was delivered intramuscularly to mice 3 weeks before lethal irradiation and immune reconstitution with semi-allogeneic bone marrow. CD8+ T cells were detected after reconstitution, indicating continuous priming by persistent b-galactosidase under conditions that ruled out vector transduction and antigen expression in dendritic cells. Dendritic cells instead acquired antigen from transduced myocytes for cross-presentation to CD8+ T cells. These results indicate that restricting transgene expression to myocytes or hepatocytes using cell-type specific promoters will not prevent antigen acquisition by dendritic cells and CD8+ T cell priming. This experimental model may also be generally useful to define defects in antigen presentation that cause ineffective CD8+ T cell priming by rAAV vectors.

28. CANINE BETA-GLOBIN GENE CLUSTER Presenting author: Zaldivar, Sara

We reported that hemoglobin in retired racing Greyhounds (RRG) has higher oxygen carrying properties and affinity than other breeds. Surprisingly, very little is known about canine hemoglobin genetics. The purpose of this study was to characterize genetics of canine beta globins. Using computational BLAST analysis of the dog genome, we identified five beta globin genes in a single : two human HBE-like followed by three HBB-like genes. We isolated DNA and RNA from blood of RRGs, AKC registered Greyhounds (AKCG), and German Shepherd Dog (GSD). All beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom 1M Agilent array). Additionally, we determined the number of common haplotypes that span this locus in RRGs and AKCGs using high density SNP array (180k Illumina HD). Expression and sequence analysis of cDNA showed all five beta globin genes are actively expressed in adults. CanHBB1 and 2 were created by relatively recent segmental duplication and have identical protein sequence. CanHBB1/2 are abundantly expressed in adults; CanHBB3 is expressed at greatly reduced levels. Sequencing results revealed one rare non-synonymous single nucleotide polymorphism (SNP) in HBE1 of RRGs, but no variation that could explain their abnormal hemoglobin. We did not detect structural variation overlapping or near the beta globin locus. Notably, RRG and AKCG do not share haplotypes spanning the beta globin locus. This is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults.

29. RECIPROCAL REGULATION OF SYNDECAN-2 AND NOTCH IN MURAL CELLS Presenting author: Zhao, Ning

Purpose: Precise interactions between endothelial and mural cells are required for proper blood vessel formation. In a quest to investigate the mechanisms that govern this interaction, we focused on Syndecan-2 and Notch3, which were shown to be regulated by the interaction of endothelial cells and mural cells using a coculture model of angiogenesis.

Methods: Endothelial and mural cells were cultured alone or together and the regulation of Syndecan-2 expression and Notch signaling were assessed by quantitative PCR, Western blotting, and luciferase assays. Expression of relevant genes was modulated by siRNA knockdown and over expression using lentiviral expression vectors.Results: We identified Syndecan-2 as one gene that is induced in mural cells by cocultured endothelial cells. Syndecan-2 is a heparan sulfate proteoglycan that serves as a receptor for extracellular matrix proteins and growth factors, and has been implicated in regulating angiogenesis. Investigations into the mechanism that governs endothelial-dependent induction of Syndecan-2, revealed a role for Notch signaling. Treatment with Notch signaling inhibitor DAPT and dominant-negative Mastermind blocked Syndecan-2 upregulation. In addition, silencing of Notch2 and Notch3 by siRNA prevented Syndecan-2 induction, and overexpression of the intracellular domain of either Notch2 and Notch3 drove the expression of Syndecan-2. Our lab previously showed induction of Notch3 by heterotypic cell interaction regulates vessel formation. The activation of Notch signaling is initiated by the binding of ligand to receptor, and this event is a potential target of Syndecan-2, as a transmembrane coreceptor. To test this hypothesis, we silenced Syndecan-2 and found the induction of Notch signaling was attenuated.

Conclusion: These finding indicate Syndecan-2 and Notch signaling regulate each other reciprocally, and partially explain the mechanism of autoregulation of Notch3 in mural cells.