The John B. Graham Student Research Society
43rd Annual Student Research Day
January 20, 2011
The Harold C. Pillsbury, M.D. Student Research Awards
Best Basic Science Poster
Christina Lee, “Notch signaling functions as a cell-fate switch between the endothelial and hematopoietic lineages.”
Senyene Hunter, “DNA Double Strand Break Repair in Human Mitochondrial Extracts”
Best Clinical Science Poster
Kimberly Niles Newton, “The effect of infant feeding on maternal affect, cardiovascular and autonomic regulation in post-partum women”
William Kesler, “Decreasing complications in external fixation pins via time-released nitric oxide coatings”
Best Basic Science Talk
Klara Klein, “Determining the role of CXCR7 in Adrenomedullin Signaling”
Best Clinical/Public Health Talk
Samuel Ross, “Trauma Patient Mortality at a Level 1 Trauma Center: Is Inter-hospital Transfer Associated with Adverse Patient Outcomes?”
The Michiko Kuno Research Award
Stephen Vance – “Health Care Accessibility Barriers for the North Carolina Hispanic Population”
The Scott Neil Schwirck Fellowship
James Byrne, “Overcoming Barriers: Electric Field-Assisted Delivery Devices for the Treatment of Pancreatic Cancer”
Basic Sciences – Poster Presentations
Viral Protein Mimics and Their Characterization
Amarpreet Kaur, William Graham, Paul F. Agris
The HIV nucleocapsid protein NCP7 aids in the recruitment of the human tRNALys3 species as the primer of reverse transcription during viral replication. Reverse transcription is initiated by a rearrangement of the tRNA’s 3’-terminal sequence and its anticodon stem and loop (ASLLys3) into conformations that promote annealing to the Primer Binding Site (PBS) and the A-rich Loop I of the HIV RNA genome, respectively. NCP7 has a demonstrated specificity for the ASLLys3 with its posttranscriptional modifications and an ability to relax the RNA structure. Our goal is to develop and characterize phage display selected peptides as mimics to NCP7 in their binding to ASLLys3 and in their abilities to change the ASL’s conformation. Using fluorescence quenching as an indication of binding, a library of fluorescein-conjugated peptides were screened to determine their affinity to bind the ASLLys3. Peptides numbered 6 and 17 demonstrated a high affinity for binding to the modified ASLLys3, but not the unmodified ASLLys3, and an ASL of tRNAVal. The abilities of these peptides to alter the conformation of ASLLys3 were compared to that of NCP7 using circular dichroism and fluorescent competition assays. Peptide 6 was found to mimic NCP7 in its ability to relax the ASL structure, whereas in contrast peptide 17 was found to enhance base stacking within the structure. The preliminary results suggest that the peptides are able to bind to the ASLLys3 in such a way that prevents the rearrangement of the ASL by NCP7. Thus, selected peptides can mimic HIV’s NCP7 in its binding of the ASL of the human tRNALys3, and in its ability to alter the conformation of the ASL, allowing for an understanding of protein/RNA interaction at the atomic level.
The Role of Alternative Splicing in Lymphoma
Cassandra L Jacobs, Amee Patel, Dereje Jima, Qingquan Liu, Adrienne Greenough, Jenny Zhang, Sandeep Dave
Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma and these tumors demonstrate a striking molecular and clinical heterogeneity. Gene expression profiling has demonstrated that these tumors comprise at least 2 molecularly distinct subsets. These 2 subsets, termed activated B-like DLBCL (ABC DLBCL) and germinal center B-like DLBCL (GCB DLBCL) have major differences in response to chemotherapy. Further, the expression profiles of a number of different pathways including germinal center differentiation, proliferation, and stromal response have been shown to be associated with outcome in these lymphomas.
Alternative splicing has been shown to be a ubiquitous mechanism of gene regulation in eukaryotes. The role of alternative splicing in DLBCL is unknown. We hypothesize that alternative splicing might play an important role in the regulation of cellular processes known to be important in the biology of DLBCL.
We applied microarrays to measure genome-wide expression levels of exons in 106 primary DLBCL tumors. Our initial studies using exon-specific microarrays indicate that over a thousand genes are alternatively spliced when comparing ABC and GCB DLBCL.
In this paper, we aim to study the global changes in alternative splicing that occur in DLBCL tumors through the examination of exon-specific microarray data from over 100 patients diagnosed with DLBCL. Further, we will study the role of alternative splicing in key oncogenic pathways that are known to be associated with survival rate after treatment. Our analysis indicates that alternative splicing plays a key role in the activation and repression of these important oncogenic pathways through differential expression of various isoforms of a gene.
Through a comprehensive analysis of alternative splicing in DLBCL, we hope to add another tool in classifying lymphoma subtypes to provide more accurate prognoses. Further, our findings set the stage for investigating targeted therapies through isoform specific drugs.
Generating a library of HIV-1 env mutants to map determinants of coreceptor usage
Andrew P Morgan, Kate T Arrildt, William I Ince, Sarah Joseph, Ronald I Swanstrom
The HIV-1 retrovirus, causative agent of AIDS, targets human T-cells for infection. Entry of the virus into host cells is mediated by its envelope glycoprotein (gp120 subunit of Env polyprotein, encoded by env) via binding to two cell-surface molecules: a primary receptor CD4, ubiquitous on white blood cells; and a coreceptor, either CCR5 or CXCR4, both seven-transmembrane chemokine receptors. Most infections of new hosts involve a CCR5-tropic (“R5”) isolate, but CXCR4-tropic (“X4”) isolates emerge during disease progression in about half of patients. Presence of X4 isolates is correlated with increased disease severity and more rapid disease progression in vivo, and with altered CD4 affinity and sensitivity to neutralizing antibodies in vitro. Characterizing the selective forces shaping Env during evolution of X4 viruses, and consequences for pathogenicity and susceptibility to antiretroviral therapy, is thus of both basic and clinical interest.
To that end we undertook the creation of a library of env pseudotypes with single-amino-acid substitutions in the second variable loop (V2) region of Env. Determinants of coreceptor usage in the third variable loop (V3) region, which makes physical contact with the coreceptor, are well-known. But data from evolution experiments in a mouse model with humanized immune cells implicated previously unappreciated determinants in V2. Starting with an R5-tropic env and using site-directed mutagenesis PCR and a high-throughput screen for desired mutants, we have generated ~110 of a target 399 clones. These clones will be assayed for viral fitness, CD4 affinity, and coreceptor preference in order to map residues in V2 potentiating the transition to the more pathogenic X4 phenotype.
In adition we used a database of patient-derived HIV sequences and an in silico coreceptor-usage prediction algorithm to generate a list of positions statistically associated with coreceptor phenotype in humans. Several of these predictions corroborate results obtained from the mouse model.
Role of the hippocampus in remembering the past and imagining the future
L.R. Squire, Anna van der Horst, Susan McDuff, Jennifer Frascino, Ramona Hopkins, and Kristin Mauldin
It has been proposed that a core network of brain regions, including the hippocampus, supports both past remembering and future imagining. We investigated the importance of the hippocampus for these functions. Five patients with bilateral hippocampal damage and one patient with large medial temporal lobe lesions were tested for their ability to recount autobiographical episodes from the remote past, the recent past, and to imagine plausible episodes in the near future. The patients with hippocampal damage had intact remote autobiographical memory, modestly impaired recent memory, and an intact ability to imagine the future. The patient with large medial temporal lobe lesions had intact remote memory, markedly impaired recent memory, and also had an intact ability to imagine the future. The findings suggest that the capacity for imagining the future, like the capacity for remembering the remote past, is independent of the hippocampus.
Genetic dissection of MEK1/2 function in the developing brain
Ayumi Nakamura, Jason Newbern, Xiaoyan Li, Allison McKell, William Snider
The mitogen-activated protein kinase (MAPK) pathway is an elaborate signal transduction cascade that regulates cellular proliferation, differentiation and survival in response to extracellular signals. The MEK/ERK signaling cascade, one of the MAPK pathways, has recently been shown to contribute to synaptic plasticity in neurons. Mutations in the MEK/ERK signaling cascade are responsible for neuro-cardio-facial-cutaneous (NCFC) syndromes, which are developmental disorders characterized by craniofacial abnormalities, heart defects and cognitive impairment; however, the underlying mechanism of how mutations in the MEK/ERK signaling cascade lead to problems in cognition remains unknown. Dendritic spines, the protrusions on a neuron that form excitatory synapses with other neurons, have been implicated in learning and memory. Here, we sought to determine whether MEK1/2-deficient neurons have abnormal dendritic morphology in vitro and in vivo. Unexpectedly, a two-month post-natal inactivation of MEK1/2 using the Cre recombinase system in mice resulted in a marked reduction in the number of neurons in the dentate gyrus of the hippocampus. A shorter three-week post-natal inactivation of MEK1/2 displayed a similar neuronal loss phenomenon. Although we have yet to characterize the dendritic morphology and dendritic spine formation in MEK1/2-deficient mice, our results suggest that MEK1/2 has a crucial role in maintaining the survival of neurons in the nervous system during development.
MicroRNA Turnover in Cell Culture
Catherine Fahey, Scott Hammond
MicroRNAs are 21-23 nt noncoding RNAs that regulate translation of specific mRNA targets by interacting with the 3’ UTR of the target in a sequence specific manner. miRNAs can regulate translation by degradation of the target, or by direct inhibition of translation. How miRNA itself is regulated is an area that is still largely unexplored. Previous work by the Hammond lab has shown that miRNA is post-transcriptionally regulated at multiple processing steps, in addition to regulation at the transcriptional level. One additional step at which regulation can occur is in miRNA turnover. Previous half-life studies have shown that as a group miRNAs are very stable, with half-lives that exceed 8 and 12 hours. Because of this, it is impossible to study general miRNA half-life using traditional, transcriptional blocking methods, as the cells will die before the majority of the miRNAs have decayed. The goal of this study was to determine if miRNA microarrays are a viable method by which to measure miRNA half-life. To test this method, we used a siRNA knockdown of Dicer, a key protein involved in miRNA generation, in two different cell lines. The cells lines used were p19 and MEF, which express very different complements of miRNA due to variation in the state of cellular differentiation. The Dicer knockdown prevents generation of new miRNA, and level changes should correspond to turnover of the cells' miRNA. Using 24 hour time increments, total RNA was arrayed on an miRNA microarray to determine relative levels of miRNA over a time course of 8 days. Ultimately, the microarrays showed a decline in overall levels of miRNA, suggesting that miRNA turnover can be examined using miRNA microarrays. This study provides support for the use of microarrays to determine miRNA half-life.
Notch signaling functions as a cell-fate switch between the endothelial and hematopoietic lineages
Christina Lee, Kevin Vogeli, Didier Stainier, and Suk-Won Jin
Endothelial cells are a major component of the vascular system, the function of which is essential to normal development and survival; however, the molecular mechanisms regulating the differentiation of the endothelial lineage remain largely unknown. In this study, we tested whether Notch signaling is involved in the segregation of endothelial cells from mesodermal progenitors by modulating signaling levels in zebrafish embryos. Embryos with decreased levels of Notch signaling, achieved pharmacologically or by using the zebrafish mind bomb mutant, showed a significantly increased number of endothelial cells. Conversely, using the GAL4-UAS system to genetically induce Notch activity in the embryos resulted in a decreased number of endothelial cells, confirming that Notch signaling negatively regulates the endothelial lineage. This early requirement for Notch activity can be separated from its previously described function in promoting arterial endothelial fate. Furthermore, when Notch signaling is perturbed, cell proliferation rates are unaffected, but there is a dramatic reduction in the number of hematopoietic cells. Lineage-tracing analyses indicate that the ectopic endothelial cells in embryos with decreased Notch activity originate from mesodermal cells that normally produce erythrocyte progenitors. Taken together, our data indicates that Notch signaling negatively regulates endothelial specification and potentially functions as a cell-fate switch between the endothelial and hematopoietic lineages. These studies provide further understanding of the regulatory mechanisms involved in the specification of different progenitor populations within the nascent mesoderm.
Altered gene silencing in leukocytes of lupus nephritis patients
Elizabeth R. Blyth, Jia Jin Yang, Dominic Ciavatta, Susan Hogan, Yichun Hu, Gloria Preston, Charles Jennette, Ronald Falk and Keisha Gibson
ANCA disease patients aberrantly express neutrophil granule genes PR3 and MPO due to depleted histone H3K27me3 modifications and loss of normal gene silencing. A subset of patients with lupus nephritis also exhibit aberrant leukocyte PR3 and MPO gene expression. The objective here is to determine the underlying factors attributable to changes in gene expression in lupus; specifically, can expression be linked to positive ANCA titers? Is necrosis in the renal biopsy a factor? Is aberrant transcription in lupus nephritis initiated by epigenetic changes similar to those observed in ANCA disease?
Increased PR3 and/or MPO mRNA expression (2 SD above mean of healthy controls) was present in patients with lupus nephritis (27/68), but not RA (n=46) or IBD (n=40). All lupus patients were negative for ANCA titers. There was no difference in PR3 or MPO expression between lupus patients with histological evidence of necrosis (n=44) versus those with no necrosis (p=0.56). Expression did not differ based on race, age or SLEDAI score.
Patients with lupus nephritis had significantly lower levels of PR3 compared to patients with ANCA disease (p=0.009) but comparable levels of MPO (p=0.95). Statistical analysis of MPO to PR3 ratios comparing all disease groups was significant (p<0.0001, F-test), with a higher ratio in lupus nephritis than ANCA.
Epigenetic studies (ChIP) of four lupus patients determined that H3K27me3 was depleted at the MPO gene and reduced at PR3 compared to MYO-D, which is transcriptionally silent in neutrophils; however, there was slight enrichment at the PR3 gene compared to mock immunoprecipitated samples. These data suggest that MPO is not epigenetically silenced in lupus, while PR3 may be silenced in a fraction of neutrophils. Thus, regulation of MPO and PR3 is altered in individuals with lupus nephritis, but the stimuli for this modulation of gene silencing deviates from ANCA disease.
Investigating Reward Perception in Two Mouse Models of Autism
J. Elliott Robinson, Eric W. Fish, Thorfinn Riday, Megan M. McGuigan, and C. J. Malanga
Background: Treatments for Autism Spectrum Disorders (ASD) involving positive or negative reinforcement rely on intact limbic reward pathways to enhance or diminish motivation to engage in specific behaviors. These reward systems may also be involved in stereotyped behavior and perseverant focus on ideas and interests, and can be studied in rodents using the operant behavioral paradigm, intracranial self-stimulation. This study examined the effects of a reward potentiating agent, cocaine, on animal models of two heritable autism spectrum disorders: Fragile X Syndrome (FXS) and Rett Syndrome (RS).
Methods: Mice lacking Fmr1 (FXS) or MeCP2 (RS) protein products were implanted with unipolar stimulating electrodes in the medial forebrain bundle and taught to self-administer rewarding electrical stimuli (brain stimulation rewards or BSR). Using a curve-shift method, the BSR threshold (θ0) was determined before and after administration of intraperitoneal cocaine (1.0, 3.0, 10.0, 30.0 mg/kg) or saline.
Results: When compared to wild type (WT) controls, fmr1-null mice displayed greater BSR threshold reductions at 1.0 mg/kg and 3.0 mg/kg doses. In the MeCP2-null mice, BSR threshold change was blunted at 10.0 mg/kg when compared to WT littermates. Cocaine dose-dependently decreased BSR threshold in all animals.
Conclusions: The Fmr1-null mice displayed a left-shift in the rate frequency curve when compared to WT controls, which suggests that they are more sensitive to the reward-potentiating effects of cocaine. Though the MeCP2-null mice displayed decreased sensitivity to cocaine at the 10.0 mg/kg dose, BSR threshold differences were not robust when compared to WT littermates. Future studies will be necessary to investigate limbic reward system differences in these animals.
Monitoring the Status of Residual Hearing During Cochlear Implantation in an Animal Model: Use of a Clinically Relevant Electrode Design
Jacob Wang, BA; Douglas C. Fitzpatrick, PhD; Oliver F. Adunka, MD
Objective: The goal was to use a flexible electrode, similar to those used clinically in cochlear implantation, to detect damage to cochlear structures caused by electrode insertion in a normal-hearing gerbil model. The hypothesis is that the reversible and irreversible interactions of the electrode with cochlear structures can be detected by reductions in intracochlear potentials.
Methods: The flexible electrode was provided by a cochlear implant manufacturer (Med-El Corp), and had one or two iridium contacts embedded in a silastic carrier. It is similar in design to their clinical devices but with fewer contacts and of a size appropriate for the gerbil cochlea (about 200 um diameter). It was implanted in normal-hearing gerbils through the round window, which was accessed via an opening made in the bulla. Recordings of the cochlear microphonic (CM) and compound action potential (CAP) in response to stimuli of 1 kHz, 2 kHz, 4 kHz, 8 kHz, and 16 kHz were made as the electrode was advanced. Anatomical damage was assessed histologically from dissections.
Results: Reversible and irreversible physiologic damage was observed with the flexible electrode. However, even in cases of irreversible physiologic damage, major anatomic damage was rare. Many cases demonstrated a tonotopic CM response. Three cases also demonstrated a pattern in the 16 kHz CM response that may have been an early indication of electrode interaction with cochlear structures.
Conclusions: We were able to detect decreases in CM and CAP with a flexible electrode, although the CM seems to be a more reliable indicator of electrode-cochlea interaction. Anatomical damage was difficult to investigate with the flexible electrode, and modifications to the experiment may be warranted in the future.
Strain Modulates Tenomodulin Isoform 1 Expression and Nuclear Localization in Porcine Tenocytes
Dmochowski, J.M.; Qi, J.; Banes, A.N.; Bynum, D.; Patterson, M.; Gomez, A.; Banes, A.J.
Introduction: Tenomodulin (Tnmd), a putative tendon marker gene exists in three isoforms. We hypothesized that physiologic levels of strain to tenocytes cultured in vitro with and without applied strain, would regulate Tnmd expression to maintain tenocyte phenotype.
Methods: Porcine Achilles tendons (PAT) of pigs were collected, minced and treated with collagenase to release cells. Cells were cultured in DMEM-H with 10% fetal bovine serum. Cells (P1-4) were seeded on silicone bottomed culture plates (Bioflex, Flexcell Corp) then either grown statically for 48h or grown 48h then subjected to 3% strain at 1Hz for 1,4 or 7 h or 1Hz, 3% strain for 1h/day for 3 days in vitro (n=3/group; three cell isolations). Total RNA and quantitative PCR (Q-PCR) performed for three Tnmd isoforms (I1, full-length; I2, truncated; I3 secreted). Cells were immunochemically stained for Tnmd using C-and N-terminal recognizing antibodies.
Results: Expression of Tnmd I1 rapidly increased after just 1 h of strain stimulation, then decreased at 4 h and returned to control levels after 7 h of continual strain. The second stretching regimen significantly decreased expression of I1 and I2 recognized (Fig. 1). Immunochemical analysis of the strained cells showed localization of Tnmd in the nucleus with the C-terminal, but not the N-terminal recognizing antibodies (Fig. 1). Given the identification of Tnmd with only the C-terminal antibody, we conclude that either isoform 3 or a cleavage product of isoform 1 enters the nucleus since I2 does not contain the C-terminal epitope sequence. This is the first report of tendon Tnmd regulation by strain and nuclear localization.
Overcoming Barriers: Electric Field-Assisted Delivery Devices for the Treatment of Pancreatic Cancer
James D. Byrne, Adrian O'Neill, Colleen Stack N'diaye, Suzan Hanna, Bill Zamboni, Xianxi Wang, Jen Jen Yeh, Joel Tepper, Matt Mauro, Richard Stack, Mary Napier, Joseph M. DeSimone
Pancreatic cancer is the fourth leading cause of cancer death in the world, with an incidence rate approximately equivalent to the death rate. The very poor prognosis for pancreatic cancer can be attributed to late diagnosis and the ineffectiveness of drug delivery to the primary tumor. Poor tissue perfusion plays a substantial role in preventing adequate drug exposure to primary pancreatic tumors. The systemic administration of gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer, has shown limited efficacy for the treatment of pancreatic cancer, with only 5 to 10% of patients demonstrating an objective radiographic response at the primary tumor site. Furthermore, the toxicity associated with systemic chemotherapy has been found to reduce the quality of life of the patient and in extreme cases can be fatal. In an attempt to address the lack of effectiveness of systemically administered chemotherapy and associated toxicity, we have developed modalities for the localized delivery of chemotherapies to pancreatic tumors. These devices rely upon an applied electric potential difference between electrodes to drive chemotherapy into the tumor. This use of an electric potential for local delivery of chemotherapies offers the capability of overcoming considerable flow and pressure gradients. Significant work using an applied electric potential difference for drug transport has been performed in transdermal drug delivery. In the area of oncology, electric field-assisted delivery techniques have been proposed for skin and ocular cancers and have been clinically translated to the treatment of bladder cancers in the electromotive delivery of mitomycin C. This technique, combined with novel device approaches, is particularly well suited for the local treatment of the primary pancreatic tumor. Here, we report the development of electric field-assisted delivery (EFAD) devices and the testing of these devices in a canine animal model. The results show that our devices provide a significant increase in the local delivery of gemcitabine while limiting systemic exposure.
Proteomic Analysis of Laser Capture Microdissected Mouse Myocardium
Joel L. "Jock" Moore, Jr., Jitka I. Virag, Barbara J. Muller-Borer
Background: Heterogeneous cellular micro-environments exist across tissues. Traditional protein assays analyze whole tissue homogenates, which may dilute minute regional expression differences. Laser capture microdissection (LCM) allows excision of distinct cellular regions, even single cells, from sectioned tissues in order to assay these regions more precisely. Proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) can simultaneously identify a broad spectrum of proteins. Here, LC-MS/MS was used to analyze LCM samples of peptide-treated vs. non-treated ischemic mouse myocardium.
Methods: Transverse sections of mouse myocardium were obtained from three experimental groups: 1) naïve mouse myocardium, 2) peptide-treated ischemic myocardium, 3) saline-treated ischemic myocardium. Ligation of the left anterior descending artery created an ischemic insult to the left ventricular (LV) wall. Cells were microdissected from the ischemic borderzone and remote regions of the interventricular septum according to the following groups: A) LV of naive mouse, B) septum of treated mouse, C) ischemic borderzone of treated mouse, D) septum of saline mouse, E) ischemic borderzone of saline mouse. Proteomic analysis was conducted at the David H. Murdock Research Institute. Cells were lysed with two freeze/thaw cycles and collected via centrifugation. Protein content was reduced, alkylated, and digested with trypsin. Following protein content normalization, LC-MS/MS analysis was conducted using a ThermoScientific LTQ Orbitrap XL instrument. A database search of these spectra was performed using the SwissProt mouse/human peptide database and MASCOT search engine.
Results: Over 50 proteins from 300-500 peptides were identified from a few hundred cells. Differential expression of mitochondrial proteins suggested increased mitochondrial activity in peptide-treated vs. untreated animals.
Conclusions: Proteomic analysis, specifically LC-MS/MS, is a viable analysis tool for LCM samples. Based on these results, further studies will analyze mitochondrial activity in live tissue samples. Western blot, repeated LC-MS/MS experiments, and other protein analysis techniques can also be used to verify these expression differences.
A “Computational Sieve” for Selecting Candidate Disease-causing Mutations from Whole Genome Sequence Data in Patients with Likely Hereditary Cancer Susceptibility
Jonathan P. Mathew, Jonathan S. Berg
Background: Members of the UNC Cancer Genetics Clinic have evaluated over 100 families in which a family history was strongly suggestive of hereditary cancer susceptibility but genetic testing was either unavailable or yielded a negative result. We hypothesize that these families are segregating novel cancer-predisposing alleles.
Aims: We sought to develop a computational algorithm for selecting candidate mutations from whole genome data and to apply it in two individuals.
Methods: Whole genome sequence for two unrelated probands was generated by Complete Genomics. Our major prior assumptions included that candidate mutations would: 1) occur within a protein-coding gene, 2) cause premature truncation or significantly alter structure, and 3) be absent in polymorphism databases. 222 variants were manually annotated with a “high/medium/low” ranking by one of the investigators (JSB), from which we identified likely “true positive” and “false positive” variant characteristics. A Perl script was composed, scoring variants according to: variant type, exon count, homozygosity, gene ontology, common polymorphisms, proximity to repeat sequences, and other factors. Fifteen variants were selected and PCR samples were sent for sequencing to confirm each variant in patient 1. Subsequently, manual and automated annotations were performed for patient 2.
Results: In patient 1 89.6% of variants were filtered from consideration after an initial selection by mutation subtype, and 307 of 4,067,143 variants met the above criteria. By comparison to hand annotation, an 18.6% “false inclusion” rate and a 14% “false exclusion” rate were observed. Results from patient 2 demonstrated an 82.7% filtration rate with a 38.1% “false inclusion” rate and a 6.3% “false exclusion” rate.
Discussion: Our results are promising, and the effort required to select candidate mutations from whole genome data has been lessened. Further work will allow us to reduce erroneous classification and increase power to differentiate variants.
Development of a CXCL12 deficient Claudin-low tumor cell line
Julia Gambone, Jon Serody
Despite recent advances in therapy, Breast Cancer remains the number one cause of cancer death in women between the ages of 20 and 60. The Claudin-low subtype of breast cancer is characterized by decreased expression of cell-cell adhesion and tight junction proteins. In general, Claudin-low tumors are poor prognosis triple negative (ER-/PR-/HER2-) invasive ductal carcinomas. In order to further elucidate the immunologic background for carcinogenesis and tumor progression in this recently identified breast cancer subtype we have examined the involvement of lymphocyte recruitment in the growth and development of tumors formed from a Claudin-low tumor cell line in FVB mice. Utilizing flow cytometry, ELISA, and Real-Time PCR this study has shown increased lymphocyte recruitment into tumors formed from a Claudin-low tumor cell line that is likely dependent on lymphocyte chemotactic factor CXCL12. Further, a stable CXCL12 deficient Claudin-low tumor cell line was developed by Lentiviral CXCL12 ShRNA transduction in order to further evaluate the involvement of this chemotactic factor in the development of Claudin-low breast tumors.
Isoform-specific palladin upregulation in epithelial to mesenchymal transition: a role in Rac1 activation
M.E. Lambert, S. Glaubiger, S. Goicoechea, H.J. Kim, and C. Otey.
Epithelial-to-mesenchymal transition (EMT) is a form of cellular plasticity in which epithelial cells acquire a motile myofibroblast-like morphology, and it plays a critical role in embryogenesis, organ fibrosis and cancer metastasis . Recent results have elucidated that EMT is accompanied by increased activity of the small GTPase Rac1. The actin-associated protein palladin is known to be upregulated in myofibroblasts of skin wounds, so we asked if palladin is upregulated in EMT, and if this upregulation is necessary for increased Rac activity. To model EMT in cultured cells, we treated the kidney epithelial cell line LLC-PK and the pancreatic tumor cell line Panc-1 with TGF-Beta1. To monitor levels of palladin expression during EMT, we used isoform-specific palladin antibodies in immunoblot analysis. Three days of treatment with TGF-Beta1 resulted in the dramatic upregulation of palladin isoform #4 (85-90 kDa) in both cell lines, and isoform #3 (140 kD) in Panc-1 cells. These results suggest that upregulation of specific palladin isoforms may be a hallmark of EMT, such that palladin may contribute to the changes in cell morphology and behavior that accompany the acquisition of the mesenchymal phenotype. To explore the relationship between palladin upregulation and Rac activity, we used knockdown and overexpression approaches. Importantly, we have previously shown that baseline levels of palladin isoform #4 in epithelial and mesenchymal cells are low and high, respectively. In Panc-1 cells, Rac activity increased when palladin isoform #4 was overexpressed. Knocking down palladin isoform #4 in a mesenchymal cell line resulted in a concomitant decrease in Rac activity in knockdown cells but not control cells. Taken together, these results strongly suggest that palladin plays an integral role in upregulating the activity of Rac in EMT. Future efforts will explore the possible pathway linking upregulation of palladin isoform #4 and increased Rac activity in EMT.
The Serine Protease Tmprss6 Regulates Hepcidin Expression, But Its Loss Does Not Cause Systemic Iron Deficiency in the Fetal and Neonatal Periods
Ramsey M. Wehbe, Rebecca L. Whittlesey, Nancy C. Andrews, Karin E. Finberg
Mutations in TMPRSS6 (matriptase-2), a transmembrane serine protease expressed by the liver, result in the clinical phenotype of iron refractory iron deficiency anemia (IRIDA). TMPRSS6 increases iron absorption by reducing expression of the hepatic hormone, hepcidin. Hepcidin promotes the internalization and degradation of the duodenal iron transporter, ferroportin, thereby inhibiting iron absorption. Genetic loss of Tmprss6 was shown to result in significant elevation of hepatic hepcidin expression in mice at birth; however, whether this hepcidin elevation was associated with abnormalities in iron homeostasis was not reported. We therefore intercrossed Tmprss6+/- mice to generate Tmprss6+/+, Tmprss6+/-, and Tmprss6-/- progeny for phenotypic characterization at either gestational day 17.5 (E17.5) or postnatal day 0 (P0). At E17.5, Tmprss6-/- fetuses showed a 50-fold increase in hepatic hepcidin mRNA compared to Tmprss6+/+ littermates (p=.005). However, Tmprss6-/- fetuses were not pale, and they showed no significant difference in body mass compared to Tmprss6+/+ and Tmprss6+/- littermates. Surprisingly, hepatic non-heme iron concentration at E17.5 was significantly higher in Tmprss6-/- fetuses than in Tmprss6+/+ fetuses (p=.003). Consistent with prior observations, Tmprss6-/- pups at P0 showed a 4.6-fold increase in hepatic hepcidin mRNA compared to Tmprss6+/+ littermates (p=.006). However, Tmprss6-/- pups also were not pale, and they showed no significant differences in body mass or hepatic and splenic non-heme iron concentration. To determine if the increased hepcidin expression of Tmprss6-/- fetuses might affect iron homeostasis in their pregnant mothers, we measured iron parameters in Tmprss6+/- females gestating E17.5 litters that were enriched for either Tmprss6+/+ or Tmprss6-/- fetuses. No significant effects of fetal genotype on maternal iron parameters were observed. In summary, our results demonstrate that Tmprss6 regulates hepcidin expression in the fetal and neonatal periods in mice, but Tmprss6 deficiency does not appear to be associated with systemic iron deficiency at these stages of development.
Spike-Timing Dependent Plasticity in Layer IV to II/III of the Primary Auditory Cortex
Russell Coletti, Deepti Rao, Paul Manis
Spike-timing dependent plasticity (STDP) has been implicated as a possible mechanism by which the brain can code and store information, and generally states that synaptic strength can be enhanced by way of long-term potentiation or weakened by way of long-term depression, depending upon the timing of activation of presynaptic and postsynaptic neurons. Little work has been done with STDP in the primary auditory cortex, especially with layer IV to II/III synapses, which is the first ascending corticocortical connection in the auditory system. Earlier research in our lab has suggested that at this synapse, the general rules of STDP are upheld, though the amount of potentiation (induced in positive pairings) and depression (induced in negative pairings) is very small. This experiment’s objective was to determine whether an increase in stimulation frequency of the presynaptic (layer IV) cells and postsynaptic (layer II/III) cells during a conditioning protocol leads to a greater amount of potentiation and depression in the context of STDP. Experiments were performed in vitro on brain slices from rat pups ages p11-p15 by way of making extracellular field stimulations in layer IV and recording from individual cells in layer II/III. The results suggest that at a frequency stimulus of 20Hz, depression is induced in positive pairing as well as in negative pairing. However the difference in depression between the two pairing scenarios did not appear to be significant at this time, and data from more cells will needed to be gathered to establish whether there is true significant difference.
Immune-Induced Epithelial to Mesenchymal Transition in the Generation of Breast Cancer Stem Cells
Sabrina Heman-Ackah, Jennifer Reiman, Keith Knutson
OBJECTIVES: A significant portion of the mortality associated with breast cancer is due to recurrence and metastasis. Therefore it is of interest to examine the mechanisms by which primary tumors produce secondary tumors in recurrence, and how these cells spread to novel sites in metastasis, even following seemingly effective treatment with conventional therapies. Breast cancer stem cells have been proposed as a potential source for the observed recurrence and metastasis experienced by breast cancer patients. Initiation of metastasis involves invasion, which is phenotypically similar to epithelial to mesenchymal transition (EMT), both of which are characterized by loss of cell to cell adhesion, e-cadherin repression, and increased cell mobility. The project described here is based on the hypothesis of immunoediting, particularly the notion that immune pressure may result in selection or induction of a tumor phenotype with reduced immunogenicity, such as that seen in the breast cancer stem cell. HYPOTHESIS: After seeding with immune cells, tumors will express increased mesenchymal cell markers and decreased epithelial cell markers. METHODS: The experiments described utilize direct versus transwell co-culture of mouse mammary carcinoma cells and CD8+ T cells to determine if these cells, a component of the immune system, have the capacity to release cytokines that induce EMT and to produce cells which are phenotypically similar to breast cancer stem cells. RESULTS: Cells were analyzed at variable intervals and found to have the expected increase in mesenchymal cell markers, but an anomalous increase in epithelial cell markers. CONCLUSIONS: Although our hypothesis stated that tumor expression of mesenchymal cell markers would increase and expression of epithelial cell markers would decrease, results showed an unexpected increase in both. Experiments are currently underway to examine the source and significance of this phenomenon.
DNA Double Strand Break Repair in Human Mitochondrial Extracts
Senyene Hunter, Joel Meyer, Bennett VanHouten
Double strand break (DSB) repair is essential for the maintenance of mitochondrial DNA (mtDNA) integrity in yeast and is expected to play a similar role in animal mtDNA. We are investigating human mitochondrial DSB repair. MtDNA is prone to DSBs; however the nature of its repair in human cells is not well understood. To analyze the presence and potential repair of DSBs in mammalian mtDNA we have developed a highly sensitive, quantitative PCR-based assay for the detection of DSB repair. Using this assay, we demonstrated that highly purified mitoplast protein extracts execute DSB repair. DNA containing 5’ or 3’ overhangs is repaired more efficiently than blunt-ended DNA (13%, 8% and 3% repaired, respectively). Further investigation of the rejoining of PstI-generated DSBs showed appreciable DNA processing, resulting in the loss of ~50 bases surrounding the PstI site. Sequence analysis of over 100 ligation products revealed several patterns of repaired DNA, most with deletions spanning 4-7 bp direct repeats. These DNA deletions are strikingly similar to those observed in patients with mitochondrial disorders and certain cancers; suggesting that these clinical syndromes may be associated with DSB repair. We have created a model of DSB repair in which broken DNA is resected to reveal regions of microhomology, allowing annealing and ligation. We show that the 3’ to 5’ exonuclease activity of DNA polymerase gamma is necessary for the efficient repair of DSBs by mitochondrial protein extracts. Additionally, we propose that hSNM1B is the exonuclease responsible for the 5’ to 3’ resection. hSNM1B has a predicted mitochondrial targeting sequence (MTS) with a putative cleavage site of 70 amino acids. Using immunofluorescence, we have shown that hSNM1B-GFP is targeted to mitochondria. Furthermore, the hSNM1B MTS is both necessary and sufficient for mitochondrial localization. Investigating mammalian mitochondrial DSBs and their potential repair may help to develop an understanding of the diseases involving mtDNA damage and their possible treatments.
Searching for the Y. enterocolitica DygSR Signal
T. Jordan Walter, Kim Walker, Markus Obrist, Virginia Miller
Yersinia enterocolitica is a pathogenic Gram-negative bacteria that causes gastroenteritis in humans. Y. enterocolitica possesses three Type Three Secretion Systems (T3SSs), which are needle-like protein appendages located on the surface of the bacteria that inject virulence factors into host cells. One of these T3SSs, the Ysa system, is not well understood. One means of elucidating the function and significance of the Ysa system is to investigate its regulation. It is known that the “two-component” regulatory system YsrRST is able to activate expression of the Ysa proteins in response to high NaCl and 26°C conditions. Interestingly, duplicates of the YsrSR genes, called the “DygSR” genes, exist in the Y. enterocolitica genome. Since YsrSR is involved in regulating the Ysa system, and DygSR is highly homologous to YsrSR, it is possible that the DygSR genes also regulate the Ysa system. However, virtually nothing is known about the DygSR genes other than the fact that they are autoregulatory. The goal of this project was to investigate the DygSR system by determining its activating signal. In order to do this, two strains of mutant Y. enterocolitica were engineered. Both strains had a lacZ fusion at the dyg promoter. One strain lacked the dygSR genes, while the other stain contained the dygSR genes. Both strains were exposed to various test conditions and then assayed for β-galactosidase activity in order to determine whether the dyg promoter had been activated. None of the conditions tested activated the Dyg system (osmotic shock, low Ca2+, low O2, pH, temperature or low Fe). Further searching for the activating signal will be undertaken by testing more conditions, such as redox state. Alternatively, the DygSR system will be studied from a different perspective by determining which genes it activates. This will be done by overexpressing DygSR and determining differential gene expression using a microarray. These experiments might elucidate a role for DygSR in the expression of the Ysa T3SS, which would provide further insight into the pathogenesis of Y. enterocolitica.
Volumetric Growth Model of Human Medulloblastoma in the Nude Mouse Cerebellum
Thomas S. Gavigan, M.S.
Medulloblastoma is the most common brain tumor in children, accounting for 10-20% of primary central nervous system (CNS) neoplasms and approximately 40% of all posterior fossa tumors. It is a highly invasive embryonal neuroepithelial tumor that typically arises in the cerebellar vermis and has a tendency to disseminate throughout the CNS early in its course. The molecular mechanisms of the disease largely remain uncharacterized, as the clinical treatment is still associated with mortality and severe side effects. The development of a clinically relevant in vivo model is important not only to further understand the disease but also a method with which to test novel therapeutics. This study attempts to quantify the volumetric growth of a human medulloblastoma (VC312) in the athymic nude mouse cerebellum using Gd-enhanced T1-weighed MRI scans. Additionally, a medulloblastoma flank tumor model was used to explore the in vivo effect of the oral anti-cancer agent that inhibits Akt activation in the phosphoinositide 3-kinase (PI3K) pathway. Intracerebellarly, perifosine increased the survival of treated mice while qualitatively reducing leptomeningeal dissemination. In the flank model, perifosine effectively suppressed the volumetric growth, active AKT pathway and cellular proliferation in treated mice.
Clinical Sciences – Poster Presentations
The Clinical Utility of Endoscopic Ultrasound Before Ablation in Patients with Barrett's Esophagus and High-grade Dysplasia or Intramucosal Carcinoma
William J. Bulsiewicz, Albert J. Rogers, Evan S. Dellon, Sarina Pasricha, Ryan D. Madanick, Ian Grimm, Nicholas J. Shaheen
BACKGROUND: Endoscopic ultrasound (EUS) is often used to stage patients with Barrett?s esophagus (BE) and high-grade dysplasia (HGD) or intramucosal carcinoma (IMC) prior to ablation, since tumor invasion or lymphadenopathy might preclude endoscopic therapy in favor of esophagectomy. The clinical utility of EUS in this role is unclear.
AIM: To assess the utility of EUS prior to ablation in BE patients with HGD or IMC.
METHODS: We performed a retrospective analysis of endoscopy records to identify all BE patients with HGD or IMC, as confirmed by histology, who underwent upper EUS in anticipation of endoscopic ablative therapy at our institution between June 2006 and November 2010. Pertinent data were extracted, including demographics, disease-specific parameters (histology before EUS, time with BE/dysplasia prior to EUS), endoscopic findings (Prague C/M classification, presence of esophagitis or nodule), EUS findings (tumor invasion, submucosal nodule, wall thickening, lymph nodes) and fine needle aspiration results. Contraindications to ablation were defined as tumor invasion beyond the superficial submucosa on EUS, regional lymph node involvement, invasive adenocarcinoma on endoscopic mucosal resection specimen, or distant metastasis.
RESULTS: Among 273 patients identified by endoscopy records, 7 did not have BE, 37 did not have an EUS, and 94 had an EUS for an alternative indication. The remaining 135 patients with BE and an EUS for pre-ablation staging were analyzed. Forty four subjects underwent EMR. Ablation was contraindicated in 14 patients (8 with invasion beyond superficial submucosa on EUS, 6 with invasive adenocarcinoma detected by EMR). Ablation contraindications were more likely among those with IMC (78.6% vs. 14.9%, p<0.01), mucosal nodularity (100%vs. 34.7%, p<0.001), wall thickening on EUS (100% vs. 32.2%, p<0.001), and suspicious lymph nodes on EUS (35.7% and 3.3%, p=0.001). Stratified by histology and nodularity, ablation was contraindicated in 11/21 with nodular IMC (52%) and 3/35 with nodular HGD (9%). EUS identified invasion beyond the superficial submucosa in 5/21 with nodular IMC (24%) and 3/35 with nodular HGD (9%). EMR upstaged histology in 10/18 with nodular HGD (56%) and 6/10 with nodular IMC (60%), all 6 of which were invasive adenocarcinoma. All 79 patients with non-nodular disease (HGD or IMC) lacked contraindication to ablation. There were no complications attributed to EUS.
CONCLUSIONS: EUS identified a contraindication to ablation in 9% with nodular HGD and 24% with nodular IMC but did not alter management in patients with non-nodular disease. The diagnostic utility of EUS in such subjects is low.
Using cardiothoracic surgery simulators to improve technical aptitude in PGY-1 cardiothoracic surgery residents
Aaron Webel, Melissa Readio, Philip Pepple, Staci Beamer, Thurston Bauer, Richard Feins
The premise behind simulator-based training is that surgical techniques can be effectively taught without putting patients at risk while allowing for complete control over the operating environment and opportunities to attain proficiency through repetition. Our hypothesis was that using an open lobectomy simulator would improve residents’ operative performance between their first and last dissections of the case. The open lobectomy model consisted of a porcine heart and lung block mounted in a left-sided torso model, perfused by a blood substitute, and ‘beating’ with a pneumatic device. First year residents in cardiothoracic surgery at the 2008 and 2009 resident boot camps held at UNC were instructed and graded by attending surgeons with global rating sheets. We asked each surgeon to grade each resident that they instructed on the operation’s component tasks, both for the first and last dissections of the case. These were termed their first and last Objective Structured Assessment of Technical Skills (OSATS) scores, respectively. We evaluated whether using the simulator was associated with an intraoperative performance improvement. Our data indicated that residents’ OSATS scores did improve between first and last dissections (matched pairs T-test: t = -8.59, t Critical two-tail=2.02, p=1.25*10^-10). Furthermore, residents operated in pairs, with one resident operating and the other assisting, and then switched roles so that each would get a chance to operate. We asked whether residents who operated second had higher OSATS scores or whether they had greater OSATS score improvement. There were no significant differences in OSATS scores for first dissection, last dissection, or OSATS improvement between residents operating first and those operating second. These data suggest that cardiothoracic surgery simulators may indeed be useful in helping residents improve their operative technical skills. Moreover, the data support the notion that it is hands-on practice, not observation, which produces this improvement.
Pain and Psychological Symptom Differences in African Americans and European Americans Experiencing Motor Vehicle Collision
Brandon Roane, April Soward, Danielle Orrey, Robert Swor, Isreal Liberzon, Daniel Clauw, Samuel Mclean
Each year, approximately 4 million people are evaluated in United States Emergency Departments (ED) after experiencing motor vehicle collision (MVC) and discharged to home. North American studies indicate that 10%-20% of these individuals develop persistent post-MVC neck pain. Evidence from a variety of pain conditions suggests that African Americans (AA) may have increased vulnerability to pain compared to European Americans (EA). However, rates of post-MVC neck pain in AAs and EAs have not previously been compared. In this investigation we assessed rates of acute and persistent post-MVC neck pain in EA vs. AA individuals presenting to the ED after MVC. ED assessment included an assessment of demographic characteristics, neck pain severity (0-10 NRS), and peritraumatic distress (Peritraumatic Distress Inventory). One month follow-up assessment included an evaluation of neck pain severity (0-10 NRS) and posttraumatic stress disorder symptoms (Impact of Event Scale-Revised). To date, 129 patients have been enrolled in this ongoing study (22 AA and 107 EA). In the ED, AAs had more intense neck pain than EAs (5.3(3.4) vs. 3.2(3.1), p = .011), and had greater peritraumatic distress (25.5 (9.9) vs. 19.9 (9.6), p = .022). One month after MVC, AAs had more PTSD symptoms than EAs (37.4 (18.5) vs. 18.8 (17.4), p <.0001), and a trend toward greater neck pain (3.0 (3.8) vs. 1.7 (2.8), p = .146). These preliminary data suggest that AA may experience increased rates of pain and psychological sequelae after MVC. Whether these differences are due to lower socioeconomic status, greater life stress, challenges associated with racism and discrimination, and/or genetic differences is not known. Further studies are needed comparing pain and psychological symptom outcomes after MVC in EA vs. AA individuals.
Use of Human Simulation to Evaluate Out-of-hospital Management of Acute Respiratory Distress in the Pediatric Patient and a Training Concept for Improvement
Brian Lanier BS EMT, Valerie J. De Maio MD MSc, Paul Hinchey MD MBA, Amar Patel MS NREMT-P, J. Brent Myers MD MPH, Robert Lee MS MA, Joseph Zalkin BSBH, Ryan Lewis MS EMT-P, José Cabañas MD, Ashley Elkins RN EMT for the CanAm Study Group
Objectives: For children, prompt management of respiratory distress may prevent deterioration and even death. We identified barriers to quality care for pediatric respiratory distress using human simulation and developed a focused intervention to address those barriers.
Methods: This observational cohort included randomly selected paramedics from a large EMS system participating in validated respiratory distress scenarios using a human simulator before (Phase 1, October 2008) and after (Phase 2, June 2009) an educational intervention. The intervention was developed from barriers identified during Phase 1 and offered to system paramedics in April 2009 online. Clinical end-points were collected from simulator logs. Experts reviewed videos and scored subjects on an inventory for dichotomous measures of performance, timing, and success of assessment and intervention items. Subjective ratings were made on a five-point scale for assessment, airway control, oxygen administration and team work and analyzed using cumulative logistic regression. All other analyses were descriptive using univariate methods.
Results: Fifty-nine Phase 1 and 26 Phase 2 subjects had complete simulation video/logs and were analyzed. Major deficiencies identified in Phase 1 were: incorrect use of length-based pediatric tape (76% of 25 subjects that used the tape); failure to remove patient from carrier prior to performing BVM ventilations (43.5% of 46 subjects which used BVM; 25 (42%) subjects initiated an IV during Phase 1 with 8 (32%) doing so prior to managing the airway vs. no Phase 2 subjects used IV. All subjective ratings for Phase 2 were significantly higher (p < 0.05). No significant differences were identified for clinical endpoints with our sample sizes but positive trends were observed.
Conclusion: Human simulation was useful in identifying issues with patient management that could lead to poor outcomes. Focused training proved to reduce deficiencies and resulted in streamlined care that adhered more closely to system protocols and best practices.
Over-commitment of EMS personnel in North Carolina
Cameron Watkins, Fran Shofer, Greg Mears, Ted Delbridge, Jeff Robertson, Jane Brice
Background: While large-scale disasters are uncommon, our society relies on emergency personnel to be available to respond and act. Faith in their availability may lead to a false sense of security. Many emergency personnel obligate themselves to more than one agency and so may be torn between two (or more) duties leaving agencies with unfilled positions in a disaster. We sought to describe the frequency of over-commitment of emergency medical services (EMS) personnel in North Carolina. Methods: We conducted a cross-sectional study utilizing the Credentialing Information System (CIS) of the North Carolina Office of EMS. The CIS database manages demographic and certification information for all EMS personnel in North Carolina. The state is divided into 100 EMS systems based on county boundaries. Utilizing de-identified provider data from the CIS, we collected system(s) affiliation(s), and level of certification. To calculate a system over-commitment rate we divided the number of personnel with more than one system affiliation by total number of system roster personnel. To compare urbanicity and certification level with over-commitment, analysis of variance and the chi-square test were used, respectively. Results: North Carolina credentials 14,717 EMS providers (8346 EMT, 1709 EMT-Intermediate (EMT-I), 4662 EMT-Paramedic (EMT-P)). 10,928 (74%) are affiliated with a single system. Of the 3789 committed to more than one system, 3020 (21%) were committed to two systems, 571 (4%) to three, 138 (1%) to four, and 60 (<1%) to five or more. EMT-Is and EMT-Ps were more likely to be overcommitted when compared to EMTs (37%, 32%, 20% respectively, p<.0001). Statewide, median system over-commitment rate was 24% (IQR 16-37%). Personnel working in systems servicing less densely populated areas were more likely to be overcommitted: 33% wilderness, 29% rural, 20% suburban and 11% urban (p<.0001). Utilizing the 75th IQ percentile, 40% wilderness, 23% rural, 4% suburban, and 0% urban systems had >37% of their personnel engaged in 9-1-1 response in more than one system. Conclusion: The number of EMS personnel with multiple jobs is significant. Disaster planners and emergency managers should consider over-commitment of emergency responders when calculating the work force on which they can rely.
Renal Perfusion Pressure Predicts Plasma Renin Activity Better than Angiographic Stenosis or Translesional Pressure Gradient in Patients with Unilateral Renal Artery Stenosis
Claud Grigg, Jingjing Yin, George A Stouffer
BACKGROUND: Angiography and translesional pressure gradients (TPG) are commonly used to determine the need for revascularization in renal artery stenosis (RAS), but some studies have suggested that renal perfusion pressure may be a more appropriate physiologic determinant of plasma renin and aldosterone levels. METHODS: In a single institution, prospective study, 63 patients with hypertension undergoing evaluation for atherosclerotic RAS had selective renal angiography, measurement of TPG with a 0.014” pressure-sensing wire and simultaneous measurement of plasma renin activity (PRA) and plasma aldosterone concentration (PAC). Multiple regression models accounting for non-normally distributed outcomes were developed using age, sex, race, ejection fraction, diabetes, anti-hypertensive medications and transformed PRA and PAC variables. RESULTS: The patient sample included 36 males (57%), 48 Caucasians (76%), mean ± SD age of 64 ± 12 years and blood pressure of 151 ± 30 / 71 ± 15 mm Hg. Comorbidities included diabetes (52%, 33 of 63), hyperlipidemia (91%, 57 of 63) and coronary artery disease (87%, 55 of 63). Patients with angiographic stenosis by QCA of >60% (n=24) had a trend toward higher PRA (median=1.2 vs 0.5 ng/ml/hr, p=0.07) but not aldosterone (median=124.6 vs 130.2 pg/ml, p=0.58) compared to patients with stenosis <60% (n=37). Among all patients, the strongest correlates of PRA were systolic (r=-0.392, p=0.002) and mean perfusion pressure (r=-0.451, p<0.001). There was a weaker correlation between PRA and angiographic stenosis (r=0.29, p=0.03) and between PRA and TPG (r=0.27, p=0.03). In regression models controlling for confounders, mean and systolic perfusion pressures were significant predictors of PRA. For every 10mm Hg decrease in mean perfusion pressure, PRA increased by 1.1 ng/ml/hr (95% CI 0.3-2.9, p=0.009) and by 0.25 log units (95% CI 0.11-0.39, p=0.001). CONCLUSIONS: In patients with unilateral RAS, renal perfusion pressure is a better predictor of PRA than either angiography or TPG. Aldosterone is not elevated in these patients.
Behavioral changes may contribute to lower blood pressure and glucose in Juventino Rosas
Health disparities have a large impact on the minority community and in particular the Hispanic community, which experiences high rates of obesity, diabetes, alcoholism, hypertension, and overall cardiovascular risk. PPS ( Proyecto Puentes de Salud) is a medical school research and service project that was founded in 2005 with the goal of better understanding and working to improve the health of the Latino community in Juventino Rosas, a region of approximately 100,000 persons in central Mexico, from which immigrants to central North Carolina have come. PPS students, with support from the local parish priest and physicians, established a program of health screening and educational forums in the rural communities surrounding Juventino Rosas. This study sought to determine the program’s impact on individuals who received screening and health education, by seeking one year later to identify and re-evaluate persons who had screened positive for either high blood pressure or glucose from 2009. In addition to re-testing their glucose, blood pressure, weight, and body mass index (BMI), we sought to determine if they had made life style changes after receiving their results in 2009.
In 2009, 367 adult residents of eleven communities in the state of Guanajuato, Mexico underwent cardiovascular screening. Participants with an abnormal result had a consultation with a physician, who discussed the results and made treatment recommendations. We sought to re-screen all 106 participants from six communities who had had a systolic pressure greater than 140 and diastolic pressure greater than 89, or a capillary glucose measurement greater than 100. As in 2009, blood pressure evaluations were conducted using an anaeroid blood pressure cuff and stethoscope, and glucose measured using an Accu check Aviva glucometer. Of the 106 eligible participants, 73 participants were screened and 33 were not screened due to moving out of the state, refusing an interview, or inability to be contacted. The rescreened patients were given the same nutritional survey as the previous year and this again consisted of a 15-minute one-on-one interview. Extra questions pertained to their follow-up care after the screening, asking if they remembered their results from the previous year, went to see a physician about the results, and made changes in their lifestyle.
There was no significant change in mean BMI or weight, which showed a slight increase in 2010. There was a significant decrease in both systolic blood pressure (mean ∆= - 6.59, p=0.002) and diastolic blood pressure (mean ∆= 7.26, p=<0.001).
It was encouraging to note that blood pressure had decreased. Additional analysis will look at the relationship between behavior change and blood pressure, and will evaluate changes in blood glucose.
Understanding risk factors for cystic fibrosis-associated liver disease
Emily Ray, Adam Shapiro, Elisabeth Dellon
Background: Although lung disease continues to be the main cause of morbidity and mortality in patients with cystic fibrosis (CF), CF-associated liver disease (CFALD) is common and has an impact on quality of life and survival. Regrettably, diagnosis of CFALD is difficult and often inaccurate due to the general absence of symptoms of developing fibrotic liver lesions. Elevations in liver function tests (LFTs) offer some suggestion of underlying liver disease, but these elevations are not always consistent with histological and radiographic findings and are often dismissed by providers as artifacts associated with antibiotics used to treat pulmonary exacerbations. The limited understanding of CFALD beckons further study of the disease itself and of risk factors for its development.
Methods: Using structured chart review, we have developed a comprehensive data set to examine characteristics of 147 children with CF with (n=44) and without (n=58) liver disease as well as an indeterminate group (n=45) with periodic elevations in LFTs.
Results: The mean age of the patients within each group is similar: 11.7 years for controls, 12.2 years for intermediates, and 14.1 years for those with liver disease. Our preliminary data reveal that 56% of patients have signs of underlying liver disease even in the absence of recent antibiotic use, a finding that could alter the approach to evaluation of CFALD.
Conclusions: Delineating risk factors for CFALD will provide the basis for future clinical investigations intended to improve screening, diagnosis, and treatment of this condition. Given the complexity of subsequent analyses needed to more appropriately evaluate our outcomes of interest, we have enlisted the help of a biostatistician. We are in the process of preparing our data for analysis and plan to have more results in the next six months.
Feasibility and efficacy of a home spirometer quality improvement program in a cohort of adult cystic fibrosis patients
E.J. Formeister, L.B. Bills, A.W. Brown, C. Jasper, K. Amann, K.W. Hohneker, S.H. Donaldson
Pulmonary exacerbations in cystic fibrosis (CF) patients (pts) contribute to accelerated lung function decline. Earlier detection of exacerbations through enhanced patient self-monitoring could improve clinical outcomes and slow the rate of lung function deterioration by prompting earlier therapeutic interventions. The objective of this study was to administer a combined home spirometry-based quality improvement (QI) program in a population of adult CF patients. We hypothesized that increased surveillance of lung function and symptoms would facilitate earlier reporting of pulmonary exacerbations. One-hundred nine adult CF pts (65 females, 44 males) from the UNC Adult CF Clinic chose to participate following physician referral. Patients received a portable spirometer and logbook used to record FEV1, weight, and CFQ-R Respiratory symptom scores weekly. FEV1 data were downloaded from spirometers at each clinic visit. One year into the program, surveys were mailed to 88 patients to assess patient perceptions of the program. To date, 91 pts (83%) have returned to clinic after receiving a spirometer. Of those returning at least once, 30 (33%) have never returned with their spirometer after an average of 2.2 clinic visits. The average proportion of clinic visits in which the 91 patients brought their spirometer (the “return rate”) was 47% (median, 50%). The average return rate of the 61 patients who have returned with their spirometer at least once was 70%. Patients returning at least once used the spirometer on average 0.67 times per week (median, 0.49/week; SD, 0.91; range, 0-5.95/week). Based on returned surveys, the most frequently reported barrier to home spirometer/logbook use was forgetfulness (67%); 13% also reported that they did not want to see their lung function numbers. Overall, administration of this program was feasible. Additionally, respondents to the survey overwhelmingly supported the value of the program for helping them detect exacerbations earlier and preserve their lung function.
Pain and Physical Function in Elderly Adults Following Motor Vehicle Collision: A Pilot Study
Young M. Lee; Robert A. Swor, DO; Erin Z. Zaleski, BA; Daniel J. Clauw, MD; Samuel A. McLean, MD, MPH
Musculoskeletal pain after motor vehicle collision (MVC) is a substantial public health problem. The number of elderly individuals experiencing MVC is increasing. We performed a pilot longitudinal study of pain outcomes among elderly vs. non-elderly individuals experiencing MVC. Adults presenting to the Emergency Department following MVC without severe or life-threatening injury were recruited. Outcomes were assessed via one month follow-up telephone call. The frequencies of persistent MVC-related moderate or severe pain were similar among elderly (age > 60, n=22) and non-elderly participants (age < 60, n=134), both in the neck region (32% vs. 27%, p=.61) and in any region (59% vs. 52%, p=.65). Persistent pain was associated with high levels of interference with physical activity and mood. These results indicate that persistent pain is common in elderly individuals experiencing MVC and is associated with substantial morbidity. Further studies of this rapidly increasing injury population are needed.
Treatment of Endometrial Hyperplasias and Cancers with systemic and local hormone therapies: The UNC-Chapel Hill Experience
Jessica Hubbs, Reagan Saig, Paula Gehrig
Background: With the evolution of modern lifestyles, increasing obesity epidemics and desire for fertility preservation, physicians are being asked to consider alternatives to tradition treatments, surgery and radiotherapy (RT), for patients with endometrial hyperplasia and cancers. While the efficacy of these traditional modalities in patients with early-stage disease have been well documented in randomized control trials, the efficacy of hormonal therapy as primary treatment for non-surgical candidates patients is less well-defined. We herein analyze treatment rationale and efficacy of patients treated with LVG-IUDs for endometrial hyperplasia/cancer.
Methods: The records of all patients who received LVG-IUDs for endometrial hyperplasia/cancer at UNC between 1999 and 2010 were reviewed. The cumulative incidence of disease progression/recurrence was estimated using the Kaplan-Meier method. A multivariate analysis assessed factors associated with development of disease recurrence. Outcome differences between patients who received tradition systemic progestins vs. LNG-IUD, were assessed for statistical significance using Student’s t-test, and chi-squared. Outcomes for patients who received LNG-IUD, were compared to traditional therapies based on literature recorded values.
Results: 169 patients were identified from the UNC tumor board and Gynelogic Oncology Disposition Board. The average age at diagnosis was 49.6 years (range: 21-92) with the majority of patients presenting with abnormal premenopausal bleeding 56% (69/166). Of consecutive patients reviewed, 157 patients were treated with hormonal therapy with adequate follow-up and did not have surgery as part of their primary treatment. When known, comobidity was the major reason for treatment with hormones, document in 44% (69/157) of patients, with desire for fertility preservation being the second most common reason 18% (28/157).
Conclusions: In this large series of patients, we consider outcomes and treatment rationales in patients treated with primary hormone therapy for complex hyperplasias and stage I endometrial cancers. While IUD hormonal treatment offers essentially 100% compliance, more work is needed to better assess the outcomes as compared to systemic therapy and conservative surgical treatment. Further, risk factors for recurrence will be considered on univariate and multivariate analysis. We will determine if for those patients who underwent hormonal therapy for fertility preservation if those patients went on to achieve pregnancy.
HIV/AIDS and Intimate Partner Violence: Barriers to Antiretroviral Adherence
Julia Brant, Shruti Gupta, Amy Weil, and Katherine Schafer
Non-adherence to HIV medications is known to contribute to the severity of the disease, and is associated with increased hospitalizations, poor immunological outcomes, and death. Barriers to adherence can consist of major life stressors including, but not limited to, intimate partner violence (IPV). A study was developed to look at the prevalence of IPV in a rural clinic in the South with a population of about 630 patients. Participants complete a 30 minute interview consisting of 8 questionnaires which focuses on their experience with IPV as well as with other life stressors like traumatic events, depression, substance abuse and neglect. Those who completed all 8 questionnaires will receive a $5 Walmart gift card. Not participating in the study does not change patient perception in clinic or quality of care. This is an ambidirectional cohort study with no randomization. Controls consist of those who have not experienced IPV. 131 patients of an expected 400 patients have been enrolled in this pilot study. Out of this population, 40% have experienced IPV at some point in their lives. Additionally 37% of participants have experienced childhood sexual abuse. The goal of this study is to identify to prevalence of patients exposed to IPV and other stressors, so that IPV and other identified covariates can be addressed early among clinic patients. If these barriers are dealt with at each patient’s initial clinic appointment, we hypothesize that we can reduce the rate of non-adherence and improve a patient’s overall life expectancy.
Incidence of Comorbidities in UNC's Turner Syndrome Population
Marsha Davenport, Katerina Constantacos, Kari Tomlinson
Individuals diagnosed with Turner Syndrome have a plethora of conditions associated with the loss of genes due to their missing X chromosome. Commonly known issues are growth retardation due to the loss of the SHOX gene, infertility due to gonadal streaking, and cardiac and musculoskeletal abnormalities. However little is known about other categories of diseases which may be of significant clinical importance. For instance, the prevalence and severity of autoimmune conditions in TS patients has not been fully explored. This study aims to examine the records of active Turner syndrome patients at UNC Hospital for relevant trends in their diagnosed autoimmune diseases. The records of eighty six patients regularly seen at the Turner Syndrome Clinic were examined and organized into a database which charted their diagnosed autoimmune disease and the date of their onsets if available. A smaller than expected incidence of celiac disease and hypothyroidism, conditions associated with Turner Syndrome was observed. The incidence of eczema was below that of the population at large and the incidence of food allergies was about the same as the general population. These results may have been skewed by the very small number (83) of patient records that were used in the study.
The effect of infant feeding on maternal affect, cardiovascular and autonomic regulation in post-partum women
Newton KN, Soh A , Stuebe A, Grewen K
Background: Breastfeeding causes release of the neuropeptide Oxytocin (OT). OT has been implicated in mother-infant bonding and autonomic cardiovascular regulation.
Objective: To measure the effect of infant feeding on endocrine and autonomic cardiovascular regulation.
Methods: We enrolled 22 women in the postpartum period who were categorized by current infant feeding practice (12 breastfeeding and 10 formula-feeding). Women underwent an experimental laboratory protocol in which we measured endocrine and cardiovascular markers during infant interaction, feeding, stress (speech and cold pressor), and rest. From these data, respiratory sinus arrhythmia (RSA), a measure of vagal tone, was derived from the R-R interval. Plasma OT and Urine OT were also collected. Data were analyzed using SAS 9.2 and significance was set at p≤0.05.
Results: Breastfeeding women had significantly higher plasma oxytocin (pg/mL) at baseline rest when compared with formula feeding women (4.92 ± 2.3 vs 2.62 ± 1.2; p=0.01). Among breastfeeding women, these higher plasma oxytocin levels were associated with lower Edinburgh Postnatal Depression Score (r=-0.82, p=0.002). Heart rate (HR) and RSA were negatively correlated in all mothers at baseline (r=-0.70, p=0.003) and during feeding (r=-0.71, p=0.001). This effect was driven primarily by breastfeeding mothers, among whom HR and RSA were more negatively correlated during feeding (r=-0.77, p=0.009), indicating greater vagal cardiac control.
Conclusion: Breastfeeding is associated with higher tonic plasma oxytocin and greater vagal cardiac control. These differences may improve cardiovascular and affective regulation among breastfeeding mothers, compared with formula-feeding mothers.
Ultra-Late Recurrence of Cutaneous Melanoma to the Tonsil
Kristopher Swiger, Darrell Klotz, Asim Amin
The incidence of melanoma has risen dramatically in recent years. Late recurrence of melanoma is now increasingly recognized and is seen in 1.1 percent patients with a follow-up of 10 years or more. Two cases have reported recurrences as far out as 24 and 41 years. The usual patterns of spread in Melanoma include involvement of regional lymph-nodes, in-transit metastases, or distant metastases. The most common locations include the liver, lungs, and brain, but melanoma is noteworthy for metastasizing to nearly any organ. While most commonly melanoma arises from the skin, mucosal membranes of the sino-nasal, gastrointestinal and genital tracts as well as the choroidal layer of the eye can serve as sources. Henderson et al. reported only 54 cases of metastases to the mucosal surfaces of the head and neck out of 8,823 patients with cutaneous melanoma. Metastatic spread to the palatine tonsils, however, is exceptionally rare with fewer than 30 cases available in literature since 1912. Furthermore, case reports have shown a disease-free interval ranging from four to 84 months for spread to the palatine tonsils. We present the case of a patient with cutaneous melanoma of the back who presented with metastases to the palatine tonsil, duodenum, and brain 216 months after wide local excision.
Social Support for adolescents with chronic illness promotes self-esteem and quality of life
Laura Robinson1, Gary R Maslow, MD1, Kristin Wolbert2, Peter Sim, MD2 Kristi Bickford1, Edwin Fisher, PhD1, and Maria Ferris, MD,PhD,MPH1. 1University of North Carolina, Chapel Hill, NC, United States and 2 Victory Junction, Randleman, NC, United States.
Background: Adolescents with chronic illnesses are at risk of lower self-esteem and quality of life than their healthy peers. Various forms of social support contribute not only to disease management, but may improve self-esteem and quality of life (QoL).
Objective: To examine the relationship between social support from parents and from friends with self-esteem and QoL for adolescents with chronic illness.
Methods: A web-based survey was conducted among 13-16 year old adolescents with several childhood-onset chronic medical conditions before arriving at Victory Junction Camp. Measures included the PedsQL and the Rosenberg Self-esteem scale. Participants also completed a survey about the social support they receive from parents and from friends. Support was divided into instrumental (concrete action) vs. emotional support and into directive (friends tell me what to do) vs. nondirective (friends let me decide what to do) support. Correlations between different forms of support and self-esteem and QoL were examined, followed by partial correlations examining each type of support controlling for the others.
Results: Overall response rate was 55% (N=135). The mean self-esteem score was high at 3.4 out of 4. Generally, correlations indicated significant positive relationships of social support with self-esteem and QoL. Nondirective emotional support from both parents and friends were significantly associated (p<0.01) with both self-esteem and QoL. Nondirective instrumental support from friends was significantly associated with QoL.
Conclusions: Greater social support from friends and parents, particularly nondirective emotional support, is correlated with higher self-esteem and quality of life for adolescents with chronic illness.
The Impact of a Multidisciplinary Tumor Board on the Management of Complex Benign Head and Neck Tumors
Lauriane Auvergne BBA, Kibwei A. McKinney MD, Stephen A. Wheless BS and Adam M. Zanation MD
The complex nature of head and neck neoplasms has warranted the use of multidisciplinary tumor board meetings with medical, dental, and surgical subspecialists. In the absence of an established evidence-based best treatment approach, it is important to reach a consensus regarding the management of these patients. While such conferences have been assumed important in the management of complex cancers, no studies have evaluated the impact of such tumor boards on benign tumors. The objective of this study is to describe and analyze changes in the management of benign neoplams made by the multidisciplinary head and neck tumor board. A prospective observational study was conducted from December 2009 and May 2010, during which weekly head and neck tumor board meetings were held at the North Carolina Cancer Hospital in Chapel Hill, NC. Patients with benign head and neck neoplasms reviewed by the tumor board during this period were enrolled in this study. Descriptive statistics and an unpaired two-tailed Student’s t-test (p<0.05) were conducted. The population of benign head and neck tumors consisted of 57 patients, among which 31.6% were salivary gland and 21.1% were skull base tumors. The most frequently diagnosed pathology was adenoma (31.6%), with 77.8% encountered in the salivary gland, 11.1% in the pituitary, 5.6% in the thyroid, and 5.6% in the parathyroid gland. Ten patients (17.5%) received a change in treatment plan or diagnosis, and 10.5% required additional diagnostic testing or consultation. In the study cohort, no significant differences were noted with comparisons of tumor location. In conclusion, the tumor board plays an important role in the management of patients with complex benign head and neck tumors with 1 of 6 patients having treatment plan changes as a result of discussion.
Endoscopic and Open Skull Base Surgery Outcomes by a Single Team: Analysis of Initial 100 Cases
Oluwaseun Omofoye, M.S., Anand Germanwala, M.D., Adam Zanation, M.D.
Introduction: Comprehensive skull base surgery often requires a neurosurgery and otolaryngology partnership. While endoscopic skull base surgery becomes increasingly popular, familiarity with open approaches continue to be paramount. As the variety and complexity of different skull base procedures grow, the different skill sets of the team become increasingly challenged. We report initial outcomes of a single skull base team with both endoscopic and open skull base surgeries.
Methods: A retrospective review of the initial 100 cases performed by one neurosurgeon together with one otolaryngologist at the University of North Carolina was performed. Attention was paid towards skull base procedure, pathology, complication rate, and patient outcome.
Results: Over a period of 2 years, 100 cases were performed for a variety of pathologies by a single skull base team. Analysis reveals 76 endoscopic endonasal cases, including transcribriform, transplanar, transsellar, transclival, transodontoid, and infrapetrous approaches, intermixed with 24 open skull base surgeries, including anterior fossa, middle fossa, and transfacial approaches. Dural reconstruction included pedicled nasoseptal, pericranial, and temperoparietal flaps. Complications included CSF leak (n=2), permanent pituitary insufficiency (n=2), infection (n=1), cerebral infarcts with no clinical sequela (n=1), vascular injury with no clinical sequela (n=1), and temporary cranial nerve palsy (n=1). Symptomatic improvement and gross total resection were achieved in the majority of cases.
Conclusion: A comprehensive skull base surgery team must have experience with endoscopic and open approaches. Acceptable outcomes with initial endoscopic and open approaches can be achieved by a single skull base surgery team.
Toward Uniformity in the Diagnosis of Eosinophilic Esophagitis (EoE): The Effect of Guidelines on Variability of Diagnostic Criteria for EoE
Sarah L. W. Sperry BA, Nicholas J. Shaheen MD MPH, Evan S. Dellon MD MPH
Objectives: Recent consensus guidelines for diagnosis of eosinophilic esophagitis (EoE) have been published. Whether these guidelines have standardized diagnostic criteria for EoE is unknown. We aimed to determine if the EoE guidelines had an impact on the diagnostic criteria reported in the EoE literature, and whether the previously observed variability in diagnostic criteria has become more uniform.
Methods: Two investigators independently conducted a MEDLINE search from January 1, 2007 through June 30, 2010 for all publications reporting EoE in human subjects, and also searched the proceedings of the 2007-2010 American College of Gastroenterology and American Gastroenterological Association meetings, using a pre-defined search strategy. Data were extracted from all relevant publications.
Results: Of the 799 publications identified, 149 original reports, 99 reviews and 165 abstracts were included. Thirty two original reports (21%) used diagnostic criteria consistent with each of the three components of the consensus guidelines. There was a significant increase when comparing original articles published after the release of the guidelines with those published earlier (31% vs. 6%, p< 0.001). The proportion of original articles using 15 eos/hpf as a histologic cut-point increased significantly (p=0.001). There was still substantial variability in biopsy protocols and eosinophil count methodology. The majority of original articles did not report microscope high-power field area.
Conclusions: The proportion of original reports with diagnostic criteria consistent with the consensus guidelines has increased significantly. However, the majority of articles still did not conform to all three of the criteria in the guidelines, and biopsy and eosinophil count protocols continue to demonstrate significant variability. Standardization of biopsy and eosinophil count protocols is needed.
Evaluations of patterns of care for RCC
Angie Smith, Sophia Delpe, Raj Pruthi, Matthew E. Nielsen, Eric M. Wallen
Purpose: The trend of national rates of RCC have been increasing steadily concomitant with an increasing rate of small tumors. With this changing composition, the patterns of RCC treatment over time have been increasingly scrutinized, focusing on the importance of renal-sparing techniques especially for patients at higher risk for renal insufficiency. Based on this, the purpose of our study is to evaluate the diffusion of surgical treatment for RCC at our own institution within the past 5 years, focusing on radical and partial, open and laparoscopic techniques as well as evaluating their correlation with comorbidities.
Methods: A retrospective analysis was initiated, evaluating patterns of care for RCC from 2005-2010. Patients with RCC were included in the study, excluding those undergoing nephrectomy for non-oncologic disease. Univariate analysis was performed using chi-squared test and multivariate analysis using logistic regression.
Results: As shown in the table below, the proportion of open partial nephrectomies increased from 5% to 14% while laparoscopic radical and partial nephrectomies remained relatively stable. Interestingly, the number of small tumors referred to our institution decreased over time from 58% in 2005 to 38% in 2009. Including only small tumors, we found that 43% of those with diabetes & 50% with HTN underwent partial nephrectomy in 2005 whereas in 2009, those numbers increased to 62% and 61%, respectively. Multivariate analysis, controlling for demographic variables as well as stage, size, and comorbidities, found that significant predictors of partial nephrectomy included year (p=0.04), size (p<0.001), and smoking status (p=0.0039). Comorbidities such as HTN & diabetes mellitus were nonsignificant. These findings remained the same when evaluating small renal tumors.
Conclusions: In an academic center, we found that the proportion of renal-sparing procedures increased slightly over time. However, diabetes mellitus and HTN were non-significant predictors. Future quality of care research will be necessary to ensure that those at highest risk of renal insufficiency are offered renal sparing procedures.
Year Open Radical Open Partial Lap Radical Lap Partial
2005 7 (16%) 2 (5%) 26 (60%) 8 (19%)
2006 8 (14%) 2 (3%) 39 (67%) 9 (15%)
2007 15 (22%) 0 (0%) 41 (60%) 12 (18%)
2008 8 (16%) 2 (4%) 31 (61%) 10 (20%)
2009 15 (15%) 14 (14%) 54 (56%) 14 (14%)
Analyzing the Key Components of a Pediatric IBD Quality Improvement Collaborative
Thomas Runge, Erica Peterson, Sue Tolleson-Rinehart, Wallace Crandall, Richard Colletti, Michael D. Kappelman
Background: In 2007, nine pediatric gastroenterology practices formed a QI network for inflammatory bowel diseases [Improve Care Now (ICN)] and used established QI methods to improve the reliability of care delivery to children with IBD. Prior work suggests that the proportion of children in remission has increased over the course of this intervention. An in-depth exploration of participant perceptions can be used to further evaluate this intervention.
Objectives: To explore self-reported perceptions of ICN participation to better understand 1) specific QI changes that sites implemented as a result of collaborative participation, 2) how much of the reported improvement in remission rates participants believe is attributed to the collaborative intervention, and 3) possible co-interventions which might have accounted for the reported improvement.
Methods: We first conducted in-depth telephone interviews with 16 key physicians, nurses, and other members of 10 participating QI teams. Interviews gathered information on current QI activities underway at each practice, whether participants believe the observed improvement remission rates is a direct result of the collaborative itself, and non-ICN related changes that may have contributed to this observed improvement, and additional perceptions of participation. Interviews were recorded, transcribed, and coded, and a thematic analysis was performed. We next distributed a web-based survey to a larger group of physicians, nurses, and other QI team members at all current collaborative sites, totaling 69 respondents (of 90 possible) at 24 institutions. The survey assessed the relative importance of a number of factors identified in the in-depth interviews.
Results: Most interview respondents reported that their practices had implemented “previsit planning sessions” during which providers review scheduled patients prior to their clinic appointments to identify necessary actions, and found these sessions valuable. Nearly all interviewees reported that a standardized algorithm for management of nutrition and growth status was another valuable approach utilized in their practices. Although the majority of interview respondents stated that “population management reports”, interactive, excel-based tools created by ICN on a monthly basis to summarize the disease status of all registered patients at each site (disease severity, current weight-based dose, etc) were an innovative tool, fewer than 50% of sites had been using this on a regular basis. Two-thirds of interview informants thought that collaborative activities were responsible for improved remission rates; the remaining stated that the collaborative was partially responsible. When asked about whether any co-interventions may have significantly changed remission rates, 4 of 12 (33%) said no; 2 of 12 (17%) said yes; and 6 of 12 (50%) were uncertain. From the surveys (scale 0-10), participants identified the use of standardized IBD patient encounter templates (8.53, SD 1.8), meetings to review the population management reports described above (8.53, SD 1.5), and reorganizing work-flow to support dedicated IBD clinics (8.3, SD 2.4) as the most important collaborative activities. Less valuable collaborative activities were programs to support patient self-management(7.42, SD 2.8), daily auditing of process (6.80, 3.1), and completion of narrative reports describing QI activities (5.11, 2.8). When asked to rate the relative effect of ICN and other practice changes on improved remission rates, respondents indicated that advances in medical therapy or management (8.40, SD 1.56), continuing education about IBD, independent of ICN (7.90, SD 1.92), and the collaborative intervention itself (7.85, SD 2.19) were rated similarly.
Conclusions: Our study indicates that in the first-ever pediatric IBD collaborative, participants made meaningful changes in their practices based on collaborative learning. However, respondents also indicated some promising tools remain underutilized. Participants indicated that ICN was partially responsible for improved outcomes, but continuing education and medication advances were also felt to be important contributors. As ICN moves forward, analytical methods which account for potential co-interventions may increase the robustness of the collaborative’s outcomes data.
Decreasing complications in external fixation pins via time-released nitric oxide coatings
Kesler, W W; Addison, K J; Watts, A M; Weinhold, P S; Holt, J B; Storm, W L; Schoenfisch, M H; Dahners, L E
Bacterial pin tract infections represent the primary source of complications limiting the utility of external fixators. The initial adherence of bacteria to titanium pins represents a critical stage in pin tract infections because the polysaccharide matrices of biofilms formed by bacterial colonies are resistant to normal immune system responses and conventional antibiotics. Holt et al. demonstrated that acid-etched titanium pins coated with a diazeniumdiolate nitric oxide (NO) donating xerogel had significantly smaller bacterial counts forty-eight days following implantation in a rat model. Our objectives were to use NO-releasing donors in a similar xerogel with a longer time-release mechanism and multiple treatments of a topical gel containing NO-releasing nanoparticles to reduce infection rates and increase osseointegration of the bone-pin interface. Three 2 mm diameter acid-etched titanium pins belonging to either the xerogel-coated group, the group to which the topical gel was applied, or the control group were implanted dorsally into the third, fourth, and fifth tail vertebrae. Implant sites were allowed to heal for forty-two days before the rats were sacrificed, cultured, and subjected to torsional testing. We found the rats on which the topical gel was applied had significantly fewer bacteria than the control. We noted no significant differences in the torques required to initiate loosening between the experimental groups and the control. Because the NO-releasing topical gel significantly improved infection results without compromising the osseointegration of the pins, we believe such treatments could potentially be optimized to reduce infection rates in external fixators and other orthopaedic implants in humans.
Public Health – Poster Presentations
Urinary Metabolites of Polycyclic Aromatic Hydrocarbons and Markers of Inflammation
Emily Cohn and Kirstin Huiber
Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals which result from the incomplete combustion of organic matter. Both human autopsy and animal studies have suggested that PAH exposure plays a role in the development or progression of cardiovascular disease (CVD) through atherogenesis. A recent population study of the urinary metabolites of eight PAHs found an increase in the odds of self-reported CVD when comparing the highest to the lowest tertiles of 2-napthalene, 2-phrenanthrene, and 3-phrenanthrene, after adjusting for age, sex, and race. An association only persisted for 2-phrenanthrene after further adjusting for serum cotinine, alcohol consumption, BMI, and hypertension. This is currently the only study of human subjects that directly links PAH exposure to CVD. The 2003-2004 NHANES data was used to investigate the relationship between exposure to PAHs and biomarkers indicative of increased CVD risk. Exposure to PAHs was measured through the urinary metabolites 2-napthol and 1-pyrene. C - reactive protein (CRP) and white blood cell count (WBC) were used as biomarkers of increased CVD risk. After adjusting for current smoking, a 25th percentile of 2-napthalene (1.22 ug/L) to the 75th percentile (7.82), predicted a 1.34 mg/dL increase in CRP (95% CI: 1.10, 1.63). Likewise, going from the 25th to the 75th percentile of 2-napthalene was associated with an increase of 250 white blood cells per uL, after adjusting for current smoking (beta = 0.25, 95% CI: 0.03, 0.48). However, after further adjustment for age, sex, race, SES, systolic and diastolic blood pressure, total and HDL cholesterol, and BMI, only the effect for continuous CRP remained statistically significant. After adjusting for smoking, age, sex, SES, BMI, blood pressure, and cholesterol, moving from the 25th percentile (0.03 ug/L) of 1-pyrene to the 75th percentile (0.17 ug/L) predicted an increase of 1.30 mg/dL CRP (95% CI: 1.08, 1.57).
Maternal Perceptions of Child Weight in Rural Mexico
Jamie Carter, Katie Turek, Will Martin, Renee Johnson, Philip Sloane
In the past, Mexicans mothers tended to prefer children to be heavy because they believed that larger figures looked healthier. This was likely a compensatory preference that resulted from the prevalence of under-nutrition and household food insecurities. Now that Mexico is undergoing a nutritional transition with childhood obesity on the rise, it is important to reevaluate maternal perceptions and preferences of child weight. This study evaluated 175 children ages 5 to 11 from rural communities around San Miguel de Allende, Guanajuato, Mexico for BMI as an indicator of weight status, and surveyed the children’s mothers regarding how they perceived their children’s weights and how they wanted their children’s weights to be. Perceptions and preferences were evaluated using a pictorial scale with values that were converted to z-scores and compared with actual BMI z-scores using paired t-tests. Mexican mothers underestimated the weights of those children whose weights were normal, but accurately perceived the weights of underweight and overweight children. Mothers of underweight children preferred their children to be larger, and mothers of overweight children preferred their children to be smaller, which reflect appropriate health changes. However, mothers of normal weight children preferred their children to be heavier. These results indicate interesting patterns where mothers of children whose weights are unhealthy are able to recognize the problem and form their preferences in line with a making a healthy change. In contrast, mothers of normal-weight children still have a tendency to prefer their children to gain weight, which perhaps stems from past and present problems with malnutrition. Ideally, this information could be used to develop educational resources for mothers regarding what healthy weight is and how to maintain it, in order to prevent maternal perceptions and preferences from supporting the rise in childhood obesity.
Level of Disaster Preparedness in Patients Visiting the Emergency Department: Results of the Civilian Assessment of Readiness for Disaster (CARD) Survey
Nicholas True, Juliana Adedoyin, Frances Shofer, Ph.D., Eddie Hasty, Jane Brice, MD, MPH
Background: Disaster preparedness of the general public is thought to be poor. Patients seeking care in public hospitals are often resource-limited populations who have in past disasters become the most vulnerable. We sought to determine the personal disaster preparedness of emergency department (ED) patients and to identify predictors of low levels of preparedness. We hypothesized that vulnerable populations would be better prepared for disasters.
Methods: We conducted a prospective cross sectional survey of patients in a public university hospital ED (census 65,000) over an 8 month period. The survey was developed utilizing the 15 items from the Federal Emergency Management Agency’s disaster preparedness checklist as well as demographics characteristics. Excluded subjects were mentally impaired, institutionalized, or non-English speaking. Summary statistics were used to describe general preparedness. Chi-square tests were used to compare preparedness by demographics.
Results: During the study period, 943 patients completed the survey. Participants were predominantly male (57%), Caucasian (64%), middle-aged (mean 45 years), and high school graduates (83%). 56% had an annual income below $40,000. 17% reported having special needs, and 17% were single parents. Most participants were not prepared: 393 (42%) had food and water for 3 days, 451 (53%) had >75% of checklist items, and 318 (34%) had food, water, and >75% of items. Level of preparedness was not associated with age, gender, education, race, income, parenting, or special needs (p>.05 for all).
Conclusions: The majority of participants in this survey were unprepared for a disaster. Only a third of the surveyed population had food and water for 3 days and 75% of the recommended preparedness items. Though single parents and those with special needs are traditionally vulnerable populations, no association with higher preparedness was found. Current disaster preparedness education has not been effective in reaching this survey population.
A Unique and Previously Untapped American Health Care Resource: The Results of a US Army Special Forces Medical Sergeant Survey
Nicholas True, Aaron Conway, Bruce A. Cairns, MD, FACS
Background: US Army Special Forces Medical Sergeants (SFMSs) provide exceptional medical service, including primary health care for many host countries, in some of the most dangerous and resource limited places in the world. Although well trained and very experienced, SFMS qualification does not directly translate into civilian health care certification. Given this structural limitation, we sought to determine the interest of SFMSs in pursuing careers in other areas of health care.
Methods: We conducted a survey of 1230 people on the SFMS list serve via the Army Knowledge Online accounts during November 2010. The survey included questions about completion of an undergraduate degree, the importance of SFMS skills compared to other Green Beret skills, and interest in further pursuing a career in health care.
Results: During the study period, 243 graduates of the Special Forces Medical Sergeant Course completed the survey. 70% of participants reported that being a medic is very important to their professional identity. 91.8% reported being interested in pursuing health care as a career. 83.5% have pursued an undergraduate degree and 42.6% have completed an undergraduate degree. 95% agreed that rotating through academic medical centers would be helpful. 36.6% were interested in attending medical school and 48.5% reported interest in obtaining a Physician Assistant degree.
Conclusions: The majority of SFMS participants in this survey expressed an interest in pursuing a career in health care beyond the role of a SFMS. Given the unique skill set and interest of this highly capable and experienced group of individuals, SFMSs represent a previously untapped resource for training and service in other health care fields. We believe additional career support would be valuable for SFMSs and in particular, that an academically based health care career program would facilitate a better transition from a SFMS to a career in medicine.
Presumptive and definitive virologic HIV diagnosis in hospitalized Malawian infants
Eric D. McCollum, Irving Hoffman, Mina Hosseinipour, Charles van der Horst, Peter Kazembe, Charles Mwansambo, Geoffrey Preidis, Portia Kamthuzi, Agness Moses, William Miller, Susan Fiscus, Dan Olson, William Buck, Carrie Golitko
BACKGROUND: There are over 91,000 HIV-positive (HIV+) children in Malawi. In order to manage the pediatric HIV/AIDS epidemic in the country, Malawi has instituted policies of early infant diagnosis (EID) and universal ART therapy for children under 12 months. However, the mean time from diagnosis to ART initiation at the Baylor Pediatric AIDS Initiative clinic in Lilongwe, Malawi is 2.4 months and children are often lost to follow up before therapy can be initiated. The mortality of infected infants at 12 months without treatment is 50%. OBJECTIVE: To improve on these diagnostic and delayed treatment issues this investigation seeks to validate presumptive diagnosis (PD) and definitive inpatient virologic testing (“expedited PCR”) as two alternatives to the current diagnostic algorithm of PD and dried-blood spot (DBS) PCR. METHODS: To validate the accuracy of PD performed by clinical officers (CO) versus pediatricians, hospitalized children younger than 1 year without definitive PCR test result were evaluated by both independently. In order to investigate the impact of expedited PCR, enrolled patients were randomized to the control group (standard of care) or the expedited PCR group and followed prospectively from time of clinical evaluation until twelve months after initiating outpatient care at the Baylor HIV clinic. RESULTS: Results are pending, preliminary data is expected in January, 2011. DISCUSSION: The current systems to process the DBS-PCR are inefficient and it often takes more than six weeks for results to return to the clinician. This extensive waiting period compromises the ability of the clinicians to provide the best care for their patient and can worsen the prognosis for pediatric HIV patients who are not initiated on antiretroviral therapy in a timely manner. In this setting, a reasonably sensitive and specific PD of HIV and the ability to receive PCR results in a timely manner are essential for infant survival.
School Feeding Programs in Rural Mexico--Successful but not Comprehensive?
Proyecto Puentes De Salud. Renee Johnson & Will Martin
School feeding programs are the cornerstone of improving the nutritional status of children in isolated and marginalized communities throughout the world, although doubts exist about their effectiveness. In many, but not all, of the poor rural communities within the municipality of San Miguel de Allende, Guanjuato, Mexico, the government agency DIF( Desarrollo Integral de la Familia or Integrated Development of the Family) provides one meal a day to elementary school children. We went to eleven of these communities, eight that receive DIF support and three that do not, to conduct health fairs that assessed the nutritional status of 174 primary school students by Body Mass Index, intake of fruits, vegetables, and meats. We found that children in communities receiving DIF support were much less likely to be underweight by the CDC’s age-adjusted BMI scale (LR of 4.5) demonstrating that this school feeding program is effective in preventing under-nourishment. However, this program is not comprehensive in addressing nutritional needs-- according to the health indicators of consumption of fruits, vegetables and red meat, there was a great range of nutritional food being consumed within the communities receiving the DIF program. We suspect that while the feeding program is effective in preventing gross under-nourishment, other factors including isolation from larger communities, selling “junk food” within the school, and access to local community gardens have a larger effect on nutrition. Future studies will aim to more clearly define these and other factors to make these feeding programs more comprehensive.
Health Care Accessibility Barriers for the North Carolina Hispanic Population
Stephen Vance, Aida Lugo-Somolinos M.D.
Background: The Hispanic population is rapidly growing in the state of North Carolina. Despite this population growth, numerous Hispanics still struggle to access adequate health care. Many barriers to Hispanic health care have been cited in the literature. However, the magnitude of these barriers and strategies to reduce them remain relatively unknown.
Methods and Findings: This study utilized an IRB approved questionnaire disseminated to Hispanics both within and outside of the health care setting. The questionnaire asked patients to agree or disagree that proposed barriers impeded their access to care. Overall, The data indicate that lack of insurance (2.330 (95% CI 2.242-2.418)), the belief that other sources of health information are equally sufficient as a physician encounter (1.979 (95% CI 1.890-2.068)), wait time (1.964 (95% CI 1.876-2.052)), cost (1.881 (95 % CI 1.791-1.971)), language (1.812 (95% CI 1.722-1.902)), and transportation (1.706 (95% CI 1.624-1.788)) are the largest barriers believed by Hispanics in North Carolina to be impeding their health care access. Both subsets of patients in the study identified the same six barriers as the most significant.
Conclusions: Of the six most significant barriers identified in this study, three have the potential for strategies to be implemented at the local level by individual hospital and clinical settings. Improved patient education about the importance of physician contact, adequate availability of Spanish for documentation and signage, Spanish education for health care providers, and rural transportation services are all feasible and cost-effective strategies which could reduce the health care access barrier. Ultimately, this research serves as a foundation for further discussion on strategies to improve Hispanics’ access to health care.
Prevalence Rates of Newborn Screening Disorders by Race/Ethnicity in California
Sunaina Dowray MPH; Lisa Feuchtbaum DrPH, MPH; Robert J Currier PhD; Fred W Lorey PhD
The California Genetic Disease Screening Program screens for 76 disorders as part of mandatory newborn screening (NBS), including endocrine and hemoglobin disorders, cystic fibrosis (CF), and 46 MS/MS disorders. The state’s large, diverse population provides an opportunity to study the prevalence of these disorders among 18 races/ethnicities.
Between 7/7/2005 and 7/6/2009, 3,237 disorders were identified among 2,202,460 NBS births, or 16.3 disorders/10,000 births. Except for 47 cases, all had initial positive test results. The disorders with the highest rates (per 10,000 births) were Primary Congenital Hypothyroidism (CH) (5.1), CF (1.4), Hb S/S disease (0.9), Homozygous EE disease (0.80), and Hb H disease (0.8). For MS/MS disorders, the highest rates were for MCADD (0.5), SCADD (0.3), 3MCC (0.2) and PKU (0.2).
For a majority of subpopulations the most common disorder was CH.The overall disorder rate was highest for Laotians (200.6) and Cambodians (194.2).These rates are approximately 16 times those observed in Whites or Hispanics. Homozygous EE, though benign, was the most common disorder in these groups. For Blacks the most common disorders were Hb S/S (14.7) and Hb S/C (8.8). For CF, Whites (3.0) had 3 times the rate of Hispanics (1.0). For MCADD, Whites (0.8) had twice the rate of Hispanics (0.4), although the highest rate was observed in Native Americans (1.5). The Other Southeast Asian (1.1) and Middle Eastern groups (1.1) had the highest rates of SCADD followed by Asian Indians (0.8); with a lower rate for Whites (0.36) and Hispanics (0.23). 3MCC was highest in Native Americans (1.5) and the Middle Eastern group (1.1); Hispanics (0.3) had twice the rate of Whites (0.2). For MSUD the highest rates were in the Middle Eastern (1.7) and Filipino (0.3) groups; Whites (0.02) had 5 times the rate of Hispanics (0.1). This data suggests that in California, specific smaller ethnic/race groups share the highest burden of a variety of disorders detected through NBS.
Basic Sciences – Oral Presentations
Computational analyses of alternative splicing from RNA sequencing of tumors
Christian Parobek, Derek Chiang
It is estimated that 15 - 50% of disease causing mutations affect pre-mRNA splice sites. Splice-site mutations are relevant in cancer progression, because cancers accumulate mutations that cause splicing variants (isoforms) and aberrant protein products. Identifying and understanding these isoforms is key to understanding the molecular mechanisms of cancers. In order to identify these isoforms, we have assembled a software workflow that uses RNA sequence data. Here, we demonstrate that our workflow can identify alternate isoforms that are expressed differently across tissues.
Modulating the enteric microbiota to treat radiation enteritis
Christopher D. Packey, Ian M. Carroll, Tope Keku, Christian Jobin, R. Balfour Sartor
Background: Radiation is utilized to treat abdominal/pelvic malignancies, and catastrophic civilian exposure to radiation is a concern. The rapidly-dividing intestinal epithelium is particularly susceptible to radiation-induced damage and is in contact with trillions of bacteria. Objectives: Characterize effects of radiation on the intestinal microbiota and epithelial cells and identify means to modulate the microbiota to impact host responses to radiation. Methods: 1) Intestinal epithelial cell (IEC) apoptosis/proliferation were quantified before/after irradiation in specific pathogen free and germ-free mice (n=10-11/group)by morphological H & E/BrdU labeling. 2) 16S rRNA terminal-restriction fragment length polymorphism (T-RFLP) and qPCR assays were performed with fecal DNA from pre-/post-irradiated mice (n=6-24/group). 3) Six clinically-relevant antibiotics or water were administered to mice (n=8/group) one hour after exposure to 15 Gy total body irradiation, and tissue and blood were harvested. Results: 1) Mice lacking detectable microorganisms had increased radiation-induced IEC apoptosis in the mid-jejunum at six (p=0.0001) and 24 hours (0.004) and cecum at six hours (p=0.001) after exposure, as well as decreased IEC proliferative responses in the mid-jejunum (p=0.0001) and cecum (p=0.02) at six hours. 2) The composition of the murine microbiota shifted following radiation (p=0.001), with diminished diversity and increased concentrations of Bacteroides fragilis, (>300-fold; p=0.009), Enterococcus faecalis (>100-fold; p<0.009), Escherichia coli (>100-fold; p<0.0001) and Pseudomonas fluorescens (>5-fold; <0.0001), and decreased segmented filamentous bacteria (four log) and (initially extremely abundant) Lactobacillus reuteri (four log; p=0.02). This shift began by 24 hours. 3) Ciprofloxacin reduced clinical scores in irradiated mice(p<0.0001), delayed mortality (p<0.0001), prevented dysbiosis, delayed sepsis, decreased serum pro-inflammatory cytokines IL-2, IL-6, MCP-1 and KC, reduced gross colonic inflammation (p=0.05), and preserved intestinal crypts (p<0.0001). Conclusions: Modulating the enteric microbiota may be useful to prevent and/or treat radiation enteritis in a low-toxicity, targeted, patient-specific manner. L. reuteri and ciprofloxacin are candidates for hypothesis-driven trials.
Co-Culture of Osteoblast and Osteoclast Precursors for Tissue Engineered Bone
Justin Woodlief; Montserrat Caballero, PhD; John van Aalst, MD
The ultimate goal of bone tissue engineering is to produce a material in the lab with the same physiological and sustainable characteristics as human bone in vivo. Bone forming osteoblasts have traditionally been the focus of bone engineering research, but mature bone formation cannot occur without the help of bone resorbing osteoclasts. We investigated the co-culture of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), the precursors to osteoblasts and osteoclasts, as a novel approach to bone tissue engineering research. Our study focused on examining the sequence and timing of cell introduction to co-culture and the effects on cell differentiation and bone matrix deposition. We hypothesized that osteoblastogenesis and osteoclatogenesis would occur in co-culture, and that more robust bone matrix formation would result from MSCs induced to osteoblasts prior to the addition of HSCs. MSCs and HSCs were harvested from human umbilical cord and umbilical cord blood, respectively. The precursors were grown to early passage in a standard growth media. Osteoblastogenesis and osteoclastogenesis was then induced in both single culture and co-culture. Evidence of osteoblastogenesis, osteoclastogenesis, and bone matrix deposition was verified by standard staining methods and RT-PCR. This preliminary study showed that MSCs and HSCs could produce osteoblasts and osteoclasts in co-culture, and increased cell activity was found with cells induced simultaneously in co-culture. With osteoclasts serving pivotal roles in remodeling and mature bone formation, introducing osteoclasts and their precursors into co-culture could push bone engineering strategy forward by more closely mimicking cell interactions in vivo.
Determining the role of CXCR7 in Adrenomedullin Signaling
Adrenomedullin (AM), a 52 amino acid peptide, has a dynamic role throughout the body. In particular, AM has been shown to be critical to heart development. In the Caron lab, AM knockout and AM overexpression has been shown to lead to hypo- and hyper-plastic hearts respectively. Adrenomedullin signaling has been widely studied and determined to be through a G-portein coupled receptor, CLR and its binding proteins, RAMP1, RAMP2 and RAMP3. Recent data, however, showing enlarged hearts in CXCR7 knock-out mice have suggested that CXCR7 may also play a role in AM signaling. In the Caron lab, our working hypothesis that CXCR7 acts as a AM decoy receptor was studied. BRET was employed to measure the levels cAMP in AM treated cells transfected with AM receptors and CXCR7. Though preliminary, our data shows that transfection with CXCR7 does decrease the production of cAMP in HEK cells. These data suggest that indeed CXCR7 could act as an adrenomedullin decoy.
Subcutaneous grafting of isolated murine CD24+CD45- intestinal stem cells, with the goal of showing growth and differentiation of these cells
Isolated CD24+CD45- intestinal stem cells (ISCs) from mice have been shown in vitro to produce enterospheres in the absence of a mesenchymal niche. Others have demonstrated the in vivo growth and differentiation of organoids or intestinal tissue into crypt-like or crypt-villus-like structures when grafted subcutaneously. However, no one has ever demonstrated the in vivo growth of isolated ISCs. This study hypothesized that CD24+CD45- ISCs are capable of proliferation and differentiation into the four lineages of intestinal epithelium when grafted subcutaneously. Following EDTA and dispase epithelial cell isolation from GFP donor jejunum, ISC were collected by FACS. Cells were suspended in Matrigel+ DMEM with multiple growth factors and supplements added. The study aimed to assess the necessity of a mesenchymal niche for the ISCs; half of the hosts received mesenchyme in their cell suspensions, the other half did not. In both host groups, the suspension was injected subcutaneously into a WT host over the right shoulder, and also injected into a natural scaffold (denuded trachea) which was subsequently implanted over the left shoulder of the same host. Subcutaneous injections and tracheal implants were retrieved after five weeks. The retrieved samples were analyzed for the presence of GFP cells in the graft (indicating donor ISC survival), however no crypt-like structures could be identified in the grafts of any of the twenty hosts. Though this experiment failed to show growth and differentiation from isolated ISCs, future efforts to demonstrate such growth are worthwhile, as intestinal stem cells are a promising source of future medical therapies. Overall, better understanding is needed in terms of the conditions necessary for ISCs' survival in the context of isolation from their niche, and subsequent transplant.
Identifying Race-Associated Genetic Risk Factors for Breast Cancer via a Semi-Supervised Clustering Method
Michael Iglesia, Chuck Perou
Breast cancer is a very heterogeneous disease both in its cellular biology and its pathogenesis. Previously, six different subtypes of breast cancer have been identified based on patterns of overall gene expression. These subtypes are reliable indicators of prognosis in a patient. It has long been known that breast cancer prognosis is much poorer in African American women than in Caucasians despite a higher incidence among Caucasian women. This disparity can be partially explained by the uneven distribution of breast cancer subtypes between the two groups. African American women have a higher incidence of basal-like breast cancer, a poor-prognosis subtype, at the expense of a lower prevalence of luminal-A breast cancer, the subtype with the best prognosis. Using gene expression microarray data from 344 breast cancer patients of known race and subtype, a least squares linear regression model was used to identify genes that correlate with race independent of subtype, clinical grade, and other possible confounders. The resulting 94 genes were verified using a semi-supervised clustering algorithm to predict race in both the original 344-patient data set and a validation data set of 233 patient samples. Self-reported race was predicted correctly in 81.69% and 62.22% of training and validation set samples respectively, indicating good generalizability for the gene signature. Additionally, the genes identified in both the original and validation data sets as highly correlated with race were selected. These genes, PSPH, CRYBB2, AMFR, and c17orf81, have individually been previously implicated in mechanisms associated with cancer pathogenesis, and will each be further pursued in future studies. Overall, our results point to specific genetic differences between African American and Caucasian women that predispose African American women to different subtypes of breast cancer and to an overall poorer prognosis.
Implication of BBS5 in resistance to chemotherapy
Current therapies for advanced non-small cell lung cancer are inadequate with response rates usually less than 30%. One of the reasons for such low response rates is the high degree of resistance among many cell lines to the chemotherapies used. In order to address this problem a genome wide siRNA knockdown screen was conducted within a cell line (HCC366) highly resistant to the first-line agent paclitaxel, to determine which genes are necessary for this resistance. Using oligo deconvolution, western blotting and qPCR this project validates a hit from that screen, BBS5. BBS5 is a member of a multimeric protein complex known as the BBSome due to its role in Bardt-Biedl syndrome, a cluster of symptoms due to malformation of the primary cilia in different organs of the body. Currently the BBSome’s only known functions involve the primary cilia, however results of this project argue against the effects of BBS5 knockdown being due to a loss of primary cilia function, hinting at a novel role for either BBS5 or the entire BBSome.
NVP-BEZ235: Future Therapy for Kaposi's Sarcoma?
Introduction: Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a gammaherpesvirus present in Kaposi’s sarcoma (KS) and linked to primary effusion lymphomas (PEL) and multicentric Castleman’s disease. KS is a highly angiogenic tumor driven by KSHV-infected endothelial cells present in the tumor. The KS lesion shows hyperactivation of Akt and mTOR kinases and the Damania lab has previous reported that activation of the PI3K/Akt/mTOR signaling pathway is critical for the survival of KSHV-infected endothelial cells and for KSHV-mediated angiogenesis. Two KSHV proteins (K1 and vGPCR) have been implicated in positively regulating this pathway in the infected cell. Recently, the Damania lab has reported that a dual inhibitor of the PI3K/AKT/mTOR pathway, NVP-BEZ235, (currently in clinical trials for treatment of solid tumors) inhibits the growth of KSHV-positive primary effusion lymphomas by down-regulating protein synthesis and cell proliferation, and up-regulating apoptosis in target cells. NVP-BEZ235 is an imidazo(4,5-c) quinoline derivative compound that acts as a competitive inhibitor of both PI3K and mTOR kinases.
METHODS: For this project I measured survival rates endothelial cells infected with KSHV with the MTS assay in the presence or absence of NVP-BEZ235. Levels of activated kinases in the PI3K/Akt/mTOR pathway were evaluated by Western blot analysis of cell lysates from mock or NVP-BEZ235-treated KSHV-infected endothelial cells. Mouse xenograft models of KS were used to determine efficacy of NVP-BEZ235 in vivo.
RESULTS. NVP-BEZ235 treatment decreased survival of KSHV-infected endothelial cells (IC50 ~7.0 nM). Additionally 36 hr NVP-BEZ235 treatment of KSHV-infected TIVE cells reduced phospho-protein levels in the mTOR pathway (at 200 nM). Slight growth delay of KSHV-infected TIVE tumors in NOD-SKID mice was observed with daily drug treatment (50 mg/kg).
CONCLUSION: Low IC50 & inhibition of cell growth & reproduction suggests a role for NVP-BEZ235 in treatment of KS.
Clinical Sciences/Public Health Oral Presentations
A Biomechanical comparison of short segment long bone fracture fixation techniques: Single large fragment plate versus two small fragment plates.
Adriel Watts, Paul Weinhold, Weaver Kesler, Laurence Dahners
To determine whether using two small fragment plates (3.5 mm screw size) side by side is biomechanically superior to the use of one large fragment plate (4.5 mm screw size), in the fixation of “short segments” in long bone fractures.
Fiber filled epoxy bone surrogate were plated across 1 cm gaps with three different constructs. Six surrogates were fixed using two side by side 3.5 mm waisted compression plates and six 3.5 mm screws, six surrogates were fixed using one 4.5 mm waisted compression plate and two 4.5 mm screws and six surrogates were fixed using one 3.5 mm waisted compression plate and three 3.5 mm screws. These constructs then underwent cyclic axial compression in 100 N increments until 500 N was reached. Then they underwent cyclic cantilever bending at 2 Hz and 175 N until fatigue failure occurred. Also a single load to failure test was performed in cantilever bending.
The cumulative strain after 500 cycles of loading up to 500 N was 3.4 +/- 0.4% for the 3.5 mm double-plated construct, 9.5 +/- 1.4 % for the 4.5 mm single-plated construct and 14.4 +/- 0.9% for the 3.5 mm single-plated construct. In cantilever bending the 3.5 mm double-plated construct failed after 15,345+/- 2493 cycles, the 4.5 mm single-plated construct failed after 2713 +/- 1811 cycles and the 3.5 mm single-plated construct failed in its first cycle. In single load to failure testing the load at offset yield was higher in the 3.5 mm double-plated construct than the 4.5 mm single-plated construct.
Conclusions: This study suggests that in situations where anatomy or other limitations limit the length of bone segments available for fixation, it may be preferable to use two small plates with more screws rather than one large plate with fewer screws.
Genetic Modifiers of Cystic Fibrosis Lung Disease
Clayton W. Commander*, Ph.D. and Michael R. Knowles, M.D. *Reporting for the North American CF Gene Modifier Consortium
Cystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The primary cause of morbidity and mortality for patients with CF is progressive bronchiectasis, which results from defective chloride ion secretion in the airway. There is great heterogeneity in lung disease severity, even between patients with identical CFTR mutations. Many factors account for the variation in lung disease severity including environmental factors such as second-hand smoke; however, twin and sibling studies have shown that lung function differences between CF patients are highly heritable (h2 > 0.5). This suggests that non-CFTR modifier genes are accountable for much of the variation in lung function. Until now, few candidate genes (TGFB1, IFRD1, and MBL2) have been shown to significantly modify lung disease severity in CF, and these only account for a small proportion of the overall variation. In this talk, we present the results of a genome-wide association study (GWAS) of n=1978 patients all homozygous for the F508del mutation in which a significant association (P=3.34 x 10-8) was identified near EHF and APIP (chr11p13). Our results are promising and will increase our understanding of CF lung disease progression and suggest novel therapeutic targets for this debilitating disease.
Surgical Resection of Low Grade Spetzler-Martin Arteriovenous Malformations Without Preoperative Embolization
Oluwaseun Omofoye, M.S., Anand Germanwala, M.D.
There are currently three main therapeutic modalities for treating cerebral arteriovenous malformations (AVMs): microsurgical resection, endovascular embolization, and stereotactic radiosurgery. Microsurgical resection is generally regarded as the most definitive form of therapy. Preoperative endovascular embolization is commonly used to reduce the volume of the nidus and obliterate deep arterial feeders with the goal of minimizing operative blood loss; however, this step does add additional risk to the patient. We present our experience with the microsurgical resection of Spetzler-Martin grade I and II intracranial AVMs without preoperative embolization. Between 2007 and 2010, a total of 12 patients with intracranial AVMs were treated by microsurgical resection alone at the University of North Carolina. AVMs were located in noneloquent frontal (n=10) and temporal (n=2) regions. All patients had Spetzler-Martin
grade I (n=8) or grade II (n=4) AVMs. Patient age ranged from 15 to 72 years, and only 1 patient presented with a prior history of hemorrhage. All patients had routine preoperative diagnostic imaging and intraoperative angiography immediately after microsurgical resection. Intraoperative post-resection angiography revealed no residual AVM in all cases. Average estimated blood loss was 304ml. There were no immediate surgical complications. Follow up ranged from 6-30 months. No long term morbidity was identified. Our results suggest that endovascular embolization should be used judiciously, particularly as an adjunct to surgery in cases with large AVM volumes and deep arterial feeders. Microsurgical resection alone can be a safe and effective form of therapy for low Spetzler-Martin grade AVMs.
Trauma Patient Mortality at a Level I Trauma Center: Is Inter-hospital Transfer Associated with Adverse Patient Outcomes?
Samuel Ross, Anthony Charles
Background: The goal of the trauma system is to minimize patient mortality by facilitating rapid transport, stabilization, and early definitive care through higher level inter-hospital transfer of the severely injured. However, evidence is currently mixed on whether inter-hospital transfer is associated with worse patient outcomes. We sought to evaluate whether patients transferred to a Level I trauma center had worse outcomes compared to patients directly transferred from the scene of injury.
Methods: This was a retrospective cohort study utilizing the University of North Carolina (UNC) Trauma Registry database. The study period included patients from January 1, 1999 to December 31, 2008 admitted to trauma services at UNC. The primary outcome of interest was in-hospital mortality, with intensive care unit (ICU) stay and hospital stay as secondary outcomes. Mortality was compared using logistic regression and lengths of stay were compared using analysis of covariance adjusting for injury severity, time to care, and other key physiologic indices.
Results: 11,315 patients were included in the analysis and there were 610 deaths. There was no difference in mortality or ICU stay among the entire sample or within strata. Mean hospital stay was longer within the lowest injury severity strata for transferred patients (3.38 days vs. 3.95, p <0.001), but the difference was not clinically relevant.
Conclusion: When adjusting for key patient characteristics, inter-hospital transfer is not associated with adverse outcomes in trauma patients. Additionally, it may be that EMS personnel are already directly transporting the most severely injured patients to Level I trauma centers.
Differences in consumption of Obesity Promoting Foods in the South vs. Non Southern Regions of the US for Children and Adults
Oren J Mechanic, MPH; Kiyah J Duffey, PhD; Barry M Popkin, PhD
Background: The Southern population in the United States (US) has the highest rates of obesity and related chronic diseases, such as stroke and congestive heart failure. Excessive consumption of obesity promoting foods may explain the rise in obesity, yet there is little regional data to support this hypothesis. Our objective is to examine differences in the dietary intake in populations from the South and Non South regions of the U.S.
Methods: We used data from the Continuing Survey of Food Intakes by Individuals (CSFII 1994-1996) to determine percent consuming and per-capita consumption of ten food groups for children (2-18y) and adults (≥ 18y). Regional populations were stratified by income and education.
Results: Adults in the South obtained significantly more energy from obesity promoting food groups such as Soda and Fruit Drinks, Sweet Tea, and French Fries (p<0.05). Conversely, Non Southerners obtained a higher amount of calories from potentially protective foods such as Low Fat/High Fiber Breakfast Cereal, Fruits, and Alcohol (p<0.05). At lower education and high income levels, persons in the South had higher consumption of Obesity Promoting Foods as compared with Non Southerners with the same levels. Generally, less drastic trends were observed for children.
Conclusion: Differences in dietary patterns between Southern and Non Southern regions may partially explain the higher prevalence of obesity and chronic diseases observed in the South.