40th Annual John B. Graham Research Society Abstracts

A Novel Methodology for Endothelial Progenitor Cell Enumeration in Clinical Trials Based on Mononuclear Cell Cryopreservation

 

Stacie Adams BS, Sunil V. Rao MD, Robert A. Harrington MD, Thomas J. Povsic MD PhD

 

Background:  Increasing evidence of the role played by circulating endothelial progenitor cells (EPCs) in vascular repair has led to interest in the clinical use of EPC enumeration.  EPC mobilization is a key secondary endpoint of the REVEAL study, a multi-site clinical trial assessing the effect of acute erythropoietin administration on infarct size.  Current EPC enumeration techniques require specialized laboratory techniques and immediate analysis, which poses significant barriers to assessing EPCs in the clinical setting where personnel may have limited laboratory experience and access to only basic equipment.  To overcome these barriers, the development of a simplified protocol for isolating and storing mononuclear cell specimens is essential.        Methods:  We compared the numbers of EPCs enumerated in samples processed immediately after acquisition with EPCs enumerated in samples stored for 24 hours or cryopreserved mononuclear cell samples using two EPC identification strategies: cell surface marker expression (CD133/CD34) and aldehyde dehydrogenase activity (ALDH^br cells).  EPCs identified in duplicate time points were plotted, and correlation co-efficients determined.  We then implemented and assessed the feasibility of the mononuclear cell cryopreservation strategy in the REVEAL trial.        Results:  The correlation coefficients for duplicate samples using the CD133/CD34 analysis (r=0.59 for cryopreserved samples, r=0.841 for overnight storage) and the ALDH^br analysis (r=0.894 for cryopreservation, r=0.880 for overnight storage) were high, however, correlation was higher with the ALDH^br assay (p<0.002 for comparison of correlation coefficients).  Of the first 58 REVEAL study participants drawn from 9 clinical sites, samples from 32 participants from 7 sites provided sufficient cells for EPC analysis at multiple time points.        Conclusion:  EPCs can be reliably determined in cryopreserved mononuclear cell samples based on ALDH activity.  Despite barriers, this strategy combined with central core lab EPC analysis is feasible in clinical trials.

 
 

Evaluating Knee and Total Body Burden of Osteoarthritis with Synovial Fluid and Serum Cartilage Oligomeric Matrix Protein (COMP)

 

Shelby Addison, R Edward Coleman, Sheng Feng, Gary McDaniel, So Yeon Kong, Virginia Byers Kraus

Objective. To evaluate the association of cartilage oligomeric matrix protein (COMP) levels, both in the serum and in the synovial fluid, with local and systemic measures of osteoarthritis (OA) severity.   Methods. Knee joint synovial fluid (SF) and serum were obtained from 159 human study participants (total 275 knees) with symptomatic OA of at least one knee.  Knee OA severity was evaluated by early (EP) and late phase (LP) technetium-99m-methylene disphosphonate bone scintigraphy, and radiograph scored semi-quantitatively. Late phase whole body bone scintigraphy was scored semi-quantitatively for assessment of total body OA load.  Correlations of biomarker levels and disease assessments utilized generalized estimating equations where appropriate.  Principal components analysis was used to explore the contribution of each joint site to the variance in serum COMP.    Results. The overall correlation of synovial fluid COMP and serum COMP was significant but modest (r value of 0.206, p value of 0.006).  Synovial fluid COMP correlated most strongly with EP knee bone scans (p value of 0.0003) even accounting for OA severity by late phase bone scan (p value of 0.015), and SF volume (p value of less than 0.0001).  Serum COMP correlated with total body bone scan score (r value of 0.188, p value of 0.018) and best reflected bone turnover in the shoulder, spine, lateral knee and sacroiliac joints.   Conclusion.  Synovial fluid COMP correlated strongly with two indicators of knee joint inflammation, early phase bone scintigraphy and synovial fluid volume. This same biomarker measured in the serum correlated with total body measures of joint disease severity by late phase bone scintigraphy. In considering the ease with which bone scintigraphy is conducted, the adoption of this method for clinical use in the quantification of OA burden can be envisioned.

 
 
Redox Regulation of RhoA
 

Amir Aghajanian, Erika Wittchen, Sharon Campbell and Keith Burridge

 

The endothelial lining of the vasculature is a tightly regulated, selectively permeable barrier between blood and tissues. Junctions between endothelial cells are critical in the regulation of this permeability, modulating the passage of fluid, macromolecules, and leukocytes. Excessive vascular leakage is pathological as seen during inflammation, shock, and ischemia-reperfusion injury. Reactive oxygen species (ROS) increase endothelial permeability, which can be inhibited with free radical scavengers and antioxidants. We have shown that H2O2 increases permeability across a monolayer of human umbilical vein endothelial cells (HUVECs) using real-time electrical impedance measurements. RhoA and other members of the Rho family of small GTPases are critical factors in the regulation of endothelial permeability because of their role in cytoskeletal and junctional reorganization; intriguingly, RhoA has recently been shown to contain two redox reactive cysteines within the phosphoryl-binding loop (C16 and C20). We hypothesize that ROS may influence endothelial permeability by directly affecting the activity of RhoA via modification of these cysteine residues. In agreement with this, exogenous treatment with H2O2 induces a concentration-dependent biphasic activation/inactivation of RhoA in both HUVECs and HeLa cells. We show that mutant RhoA containing cysteine to alanine substitutions at C16 and C20 (C16A/C20A) is not susceptible to the effects of H2O2 treatment when expressed in HeLa cells. We tested the significance of this by analyzing cell spreading on fibronectin, which normally requires a transient decrease in RhoA activity. Efficient cell spreading on fibronectin is limited when HeLa cells are treated with N-acetylcysteine, a radical scavenger. Similarly, cells expressing C16A/C20A RhoA spread to a lesser extent than cells expressing wildtype-RhoA, presumably due to the mutant’s resistance to modification by redox agents. These results suggest a novel, redox-based mechanism of regulating RhoA activity, which may have a role in the modulation of vascular permeability.

 
 

Endothelian antagonists lead to alterations in both breast cancer and bone metastases

 

Didier Dreau, Heather Wyan, Lauren D. Allen, Mark G. Clemens

 

We investigated the effect of various endothelian-1 antagonists on the angiogenesis associated with breast cancer growth and development of bone metastases. Murine mammary carcinoma 4T1 cells were injected in a skin-fold chamber implanted with bone explants on immunocompetent CB6 mice. Mice were treated with or without various antagonists of either ETRA, ETRB, or both ET-1 receptors. The progression of the vascularization within the chamber was monitored over time by intravital microscopy (IVM). The development of bone metastases was assessed by histological studies and the presence of cytokeratin-19 in bone explants. In addition, mRNA and protein expressions of endothelians, VEGF, and their receptors were evaluated in both tumor and bone metastases. Results indicate that only some of the ET-1 antagonist treatments tested were associated with significantly reduced tumor mass and tumor bone metastases. Decreased vessel numbers were observed in both tumor and bone explants following treatment with efficient ET-1 antagonists (p<0.05). mRNA and protein expressions for ETRA, ETRB, and VEGFR2 were decreased in the tumor mass of treated animals compared to control mice (p<0.05). These changes were correlated with smaller tumor masses and reduced bone invasion (p<0.05). Our data support the hypothesis that ET-1 through its receptors may play a key role in the angiogenesis associated with the development of breast metastases to bone.

 
 

Cardiac Risk and Knowledge of Risk Factors in Patients Presenting to the Cardiac Catheterization or Cardiac CT Labs

 

James Barnwell, Larry Klein MD, Mike Gillespie MD, Eric Yang MD

 

Background: The dilemma of cardiovascular risk assessment has resulted in screening tools such as the Framingham Risk Calculator and the NCEP guidelines for risk appraisal. However, the effectiveness of these evaluations hinges on the patient’s perception of self risk in comparison to what is being quoted to them. The purpose of this study is to explore patient knowledge and understanding of cardiac risk analysis and to evaluate the possible disconnect between a patient’s views of self risk with that of their physician.   Methods and Results: Three hundred and twenty two patients undergoing Cardiac Catheterization or CT cardiac imaging were divided into recurrent cardiac event and initial cardiac presentation. All patients’ with an obtainable lipid panel received a 10 year risk calculation using the NCEP’s guidelines. The outcomes of this calculation stratified our patients into low, medium, and high risk. Of the 322 patients screened, 147 were presenting with their first cardiac event, 110 agreed to answer a questionnaire, and 93 of those questioned had an obtainable lipid panel and a diagnostic cardiac study. The results were: 27% recalled a physician estimating their cardiac risk, 55% had an incorrect self risk assessment in comparison to Framingham, 65% stated that they would have made lifestyle changes following a physician statement of cardiac risk, 85% stated they would have made lifestyle changes following an imaging study, and 68% commented that they would put more effort into life style changes following a positive imaging technique as opposed to physician verbal warning.   Conclusion: Our study demonstrated that a significant portion of our patient population could not recall receiving a cardiac risk assessment from a physician. Of those that did receive this evaluation, there was a significant discord between patient self risk assessment and that of their physician. Patients with this disparity retrospectively believed they may have altered their risk patterns with further imaging to correct this disbelief.   

 
 

Parental Literacy and Pediatric Peritoneal Dialysis Outcomes

 

Michael J Belsante, BSE, Otis Burnett, BS, Darren DeWalt, MD, MPH, Phil Icard, MS, Debbie Gipson, MD, MSPH, William Primack, MD, David Tauer, RN, Hyunsook Chin, MPH, Paul Fields, BS, BA, Kristi Bickford, BA and Maria Ferris, MD, MPH, PhD

 

Purpose: To correlate parental literacy scores with health outcomes amongst pediatric peritoneal dialysis (PD) patients.   Methods: Literacy scores of the parents (n=34) of children previously or currently receiving PD were assessed using the Word Reading subtest of the Wechsler Individual Achievement Test (WIAT) II, an individually administered test, which provides standardized subsets and composite scores (M=100; SD =15). A parental survey and chart review for the children ascertained health outcomes including peritonitis rates, adherence to treatment and emergency department (ED) visits. Results: Parental mean age was 40 years (range = 21-77). Mean parental literacy score was 85.3. The median score for the cohort was 90. White (n=10) parents had higher mean literacy scores than Black (n=20) parents (p<.01). Children of parents who had standard literacy scores less than 90 had more episodes of peritonitis per patient month than those whose parent had a higher score. Low literacy is associated with low adherence to treatment whereas ED visits did not correlate with literacy scores. Conclusion: PD patients whose parents have lower literacy scores have significantly more peritonitis episodes and poorer adherence to treatment than patients whose parents have higher literacy scores.

 
 

Parasite Burden and IBS: A Case Control Study

 

Matthew Benshoff, Loreto Cortes, MD, Douglas Morgan, MD

 

Objective: To determine if intestinal parasite presence is correlated to increased incidence of irritable bowel syndrome (IBS) in a non-sterile environment using a case-control study.      Methods: The study was conducted using the existing nested case-control component (N=379) of a cross-sectional survey (N=1,642) of the inhabitants of Leon, Nicaragua. The cases were selected using the Rome II Modular Questionnaire followed by physician confirmation. The controls were selected at random from FGID-negative subjects. Stool samples from the case-control group were analyzed for parasite presence and type. Data were analyzed using SPSS statistical software and chi-squared tests were employed to determine statistical significance of relationships.         Results: A nested case-control population (N=357) had an (incidence) prevalence of intestinal parasite carriage of 16% (N=57) with 17% of cases and 15% of controls testing positive for intestinal parasites. There was no correlation found between parasite carriage and IBS incidence among the sample population (OR=1.09, 95% CI=0.62-1.91). Additionally, no individual parasite was found to increase the risk of IBS.      Conclusion: While parasite infection has been associated with post infectious IBS in sterile environments, there is no statistically significant relationship between parasite carriage and IBS incidence in the developing nation environment of Leon, Nicaragua.

 
 

Effectiveness of Manipulating Arachidonic Acid Metabolism in Healing Fracture Nonunions

 

Berry, John, Daniel Cavanaugh, Paul Weinhold, PhD, Laurence E. Dahners, MD

 

The American Academy of Orthopaedic Surgeons defines a fracture nonunion as one which has not healed for three months and shows no promise of healing.   The two of the most effective treatment options are surgical repair of the fracture and treatment with Levodopa (L-Dopa). Surgical repair of a fracture nonunion is both invasive and expensive. However, surgery presents a high success rate (80-90%) of uniting and repairing a fracture. Merck and Company have stopped producing L-Dopa in favor of Sinemet, a combination of L-Dopa and Carbidopa.   Carbidopa is useful in the treatment of Parkinson’s because it reduces the side effects of L-Dopa.   In the case of fracture nonunions, Costa et al found Sinemet was not as effective healing fractures as L-Dopa.       Genetic knockout experiments have provided evidence that bone growth can be stimulated through manipulation of arachidonic acid metabolism. Additionally, Lovastatin has been shown to be effective in stimulating bone growth when the local concentration is kept high enough on the fracture site. We evaluated three drugs, zileuton, montelukast sodium, and lovastatin in healing nonunion fractures. After obtaining IACUC approval, the rats underwent an operation under 2.0-3.0% isoflurane anesthesia. The operation exposed the right femur with then stripped the bone of its periosteum then filed a notch until the bone may be easily broken transversely through the the diaphysis. Omega pins were inserted into the two ends of the fracture. The loop created a fracture nonunion of 1-2 mm. The wound was closed using a subdermal stitch.     Rats were given zileuton and montelukast sodium in chow while lovastatin was injected at the fracture site. The rats were X-Rayed at 3, 4, and 5 weeks then sacrificed at 5 weeks. Stress testing shows that montelukast sodium improved over control, lovastatin did not make a difference and zileuton actually impaired healing. 

 
 

Regulation of Cathepsin L by the Serpin Protein C Inhibitor

 
Ryan Bialas, Yolanda Fortenberry
 

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation and fibrinolysis including thrombin and activated protein C (APC). The cysteine protease, cathepsin L, has been shown to play a role in many physiological processes including cardiovascular disease and tumor cell invasion. Recently, several serpins have been described to inhibit serine and cysteine proteases, termed cross-class inhibitors. The goal of this project was to determine if PCI inhibits cathepsin L activity and if so, does this inhibition process mimic the mechanism of serine proteases. Studies have shown that the prototypical serpin, antithrombin (AT), inhibits the cysteine protease cathepsin L. We found that PCI is a more efficient inhibitor of cathepsin L than AT with an inhibition rate (k2) of 1.5 x 106 M-1min-1 compared to 5.2 x 104 M-1min-1 for AT. Also, PCI is a more efficient inhibitor of cathepsin L than either thrombin or APC whose inhibition rates are 5.7 x 105 M-1min-1 and 3.4 x 104 M-1min-1, respectively. Interestingly, a reactive site P1 mutant (R354A) of PCI does not inhibit thrombin but does inhibit cathepsin L at rates comparable to wild-type PCI. This implies that the P1 residue of PCI does not determine specificity for inhibition of cathepsin L unlike for thrombin and APC. We believe that the specificity is primarily determined by the hydrophobic Phe residue located at the P2 position since other serpins that inhibit cathepsin L contain either a Phe or Val at the P2 position. The wild-type PCI-cathepsin L interaction has a stoichiometry of inhibition (SI) value of 1.6. This indicates that PCI is an effective and possibly a physiologically relevant inhibitor of cathepsin L. Regulating cathepsin L by serpins like PCI may be a novel and new pathway of regulation of hemostasis-thrombosis and metastatic diseases.

 

Antimicrobial treatment in the postoperative care of neonates with severe necrotizing enterocolitis

 
Jason Blatt, J. Duncan Phillips MD
 

Background " "Necrotizing Enterocolitis is a common disease of prematurity, which causes severe inflammation of the GI tract. Severe cases require resection of necrotic bowel or peritoneal drainage. The post-operative care these patients is poorly described; management is based on the subjective judgments of the responsible physicians.   Current care for surgical NEC includes heavy antimicrobial therapy for extended periods, exposing patients to superinfection. Studies of wound infection comparing neonates to older children have found no increased incidence of infection, suggesting that extended prophylaxis may be unnecessary.    Aims " We will determine whether patient outcomes are significantly influenced by antimicrobial choice and duration in postoperative NEC neonates. It may be possible to elucidate certain subgroups of patients that benefit from more intensive therapy.     Methods   "Data was collected on the last 109 patients who underwent surgery for NEC or perforation at UNC. Numerous variables were recorded from the medical charts of these infants. Statistical tests will be used to examine how antimicrobial treatment times affected survival rates and morbidity at 30, 60, and 90 days.    Results " "Much data remains to be collected in this study. However, we have made a number of interesting observations with the data we do have: 1)" 90% of neonates with peritoneal drains at UNC require subsequent laparotomy. This is a much higher rate of secondary laparotomy than at other institutions.    2) Surgical NEC at UNC has a 38% mortality. Despite our best efforts this remains a dire illness. 3) "57% of babies who die from surgical NEC have pan-necrosis of the bowel on exploration. Since NEC is poorly understood, we must consider whether operating sooner may arrest the necrosis before it reaches a fatal extent.    Discussion " "The data collected thus far has armed our pediatric surgery team with new knowledge about NEC and its consequences. They are better able to council families now that they know the mortality rate of surgical NEC. We are identifying factors allow surgeons to identify better surgical candidates, and are reconsidering peritoneal drain placement strategies. 

 
 

Comparison of Catheter-Based Radiofrequency Ablation and Photodynamic Therapy for Barrett’s Esophagus

 

Joseph M. Bumgarner, Millie D. Long M.D., Nicholas J. Shaheen M.D., M.P.H.

 

Background: Two novel modalities for the treatment of Barrett’s esophagus (BE) are currently commercially available. These include catheter-based radiofrequency ablation (RFA) and photodynamic therapy (PDT).    Objectives: We aimed to evaluate outcomes of % remaining BE after initial treatment, cure of dysplasia over treatment course, and stricture formation in patients with BE undergoing either RFA or PDT.      Methods: A chart review of patients with BE was performed at two tertiary referral centers. Patient characteristics and procedure specific data were abstracted. Bivariate analyses comparing ablation type to each outcome were performed; Wilcoxon rank-sum or chi square test statistics were used as appropriate. Multivariable logistic regression models were used to assess each outcome controlling for potential covariates. Variables were dropped from the models based on change in beta coefficients using a backwards elimination approach.       Results: 225 patients were assessed (103 RFA, 122 PDT). The majority of the patients were male and in their 60’s. The median % remaining BE after the initial procedure was 20%. On bivariate analysis, the median % remaining was 15% vs. 30% for RFA vs. PDT (p=0.40). There were a total of 18 strictures (12%) in 145 patients undergoing repeat endoscopy. Of patients with RFA as the initial modality, significantly fewer had a stricture complication compared to patients undergoing PDT (p=0.05). On multivariate analysis, the odds of stricture was lower with RFA as compared to PDT (OR 0.29, 95% CI 0.09-0.95). On multivariate analysis the odds of cure of dysplasia for RFA was not different when compared to PDT (OR 0.69, 95% CI 0.26-1.85).    Conclusions: RFA therapy is associated with significantly fewer stricture complications as compared to PDT. There is no difference between the modalities for either % remaining Barrett’s after initial endoscopy or cure of dysplasia. These results may impact choice of ablation technique in patients with BE.

 
 

A Comparison of Systemically and/or Locally Applied Antibiotics for Surgical Infection Prophylaxis: An in vivo study

 

Daniel L. Cavanaugh, John Berry, Seth R. Yarboro, and Laurence E. Dahners

 

Background    Prophylactic systemic antibiotics significantly lower the risk of postoperative infection. In a previous study from this laboratory injection of antibiotics directly into the wound cavity was even more effective. In this study, we investigated the efficacy of direct antibiotic injection into a wound cavity after wound closure, alone and in combination with systemic antibiotics. We hypothesized that a combination of preoperative systemic administration and postoperative local injection would be the most effective.   
Methods    Rats were divided into six treatment groups: no treatment, local gentamicin, systemic cefazolin, local cefazolin, local gentamicin   systemic cefazolin, and local cefazolin   systemic cefazolin. A wound cavity was opened along the femur, a cerclage was placed and 2.5 x 108 CFUs of S. aureus was inoculated. Systemic antibiotics were injected subcutaneously 30 minutes before initial incision. Local antibiotics were injected percutaneously into the wound cavity after closure. Rats were sacrificed at 48 hours postoperatively and quantitative cultures obtained.  
Results: All groups receiving antibiotics showed significantly lower bacterial counts than the no treatment control (p < 0.001). Local gentamicin treatment decreased CFU isolates by approximately two orders of magnitude over systemic cefazolin (p = 0.00005) and five orders of magnitude over the control (p = 0.00003). The combination of local gentamicin and systemic cefazolin decreased bacterial counts by approximately seven orders of magnitude over the no treatment control group and provided a statistically significant improvement over local gentamicin alone (p = 0.00006).
Conclusions: In support of our hypothesis, the combination of systemic cefazolin and local gentamicin proved the most effective regimen. Local injection of gentamicin again proved more effective than systemic administration of cefazolin but did not perform as well as the combination treatment of both antibiotics. The initially high concentrations of locally applied antibiotic and the utilization of two different antibiotic classes may have contributed to the observed efficacy.
Clinical Relevance: If this work is supported by clinical trials such a treatment could prove valuable as a regimen for prophylaxis of surgical wound infection.

 
 

Does the murine formyl peptide receptor play a role in LPS signaling?

 

Rebecca Chasnovitz, Ji-Liang Gao, Phil Murphy

 

Background: Lipopolysaccharide (LPS), or bacterial endotoxin, is a major mediator of septic shock, a rare life-threatening complication of bacterial infection. Determinants of fatal outcome in septic shock are poorly understood.  Since neutrophils normally control bacterial infection, and since the formyl peptide receptor (Fpr) is a major neutrophil chemoattractant receptor, we asked whether this receptor was important in septic shock. Hypothesis: Fpr regulates pathogenesis of septic shock. Design: Fpr-/- and WT mice were injected with 200 mg LPS iv. We observed the mice clinically for 7 days and measured serum levels of lps and the inflammatory cytokines TNFα, IL-6, IL-1α, and IL-1β at 1, 2, 4.5, 8, 13.5, and 23.5 h after lps challenge. Neutrophils elicited by thioglycollate installation ip were harvested from Fpr-/- and WT mice and Ca++ flux was measured in response to lps. Results: After lps injection, Fpr-/- mice experienced markedly increased mortality compared to WT mice (50% v 7%, p<0.005). This was associated with increased serum concentration of lps 13.5 h after injection (p=0.0105), and higher concentration of all four inflammatory cytokines tested 13.5 and 23.5 h after injection. LPS induced a rapid and transient flux of intracellular Ca++ in neutrophils from WT mice ex vivo that was reduced by 69% in neutrophils from Fpr-/- mice. Conclusion: Fpr protects against fatal outcome after lps injection of mice. The mechanism may involve accelerated lps clearance and reduced production and/or accelerated clearance of inflammatory cytokines.  Our preliminary data indicate that lps may be a direct agonist at neutrophil Fpr.  We speculate that lps-Fpr interaction may induce internalization and clearance of lps by neutrophils.

 
 

Use of three-dimensional chest wall imaging in the evaluation and management of complex pediatric chest wall disorders

 

Ali Chhotani and J. Duncan Phillips, MD

 

Chest wall deformities are often identified in infancy, but patients normally do not become symptomatic until adolescence. Symptoms can include dyspnea with mild exertion, pain in the anterior chest, and palpitations. Stroke volume, cardiac output, and lung expansion may also be reduced. Repair of the defect is typically recommended for patients who are symptomatic or have a relatively severe defect. Modern surgical repair of pediatric chest wall deformities began in the early 1950’s with the description of the so-called “Ravitch procedure.” Currently numerous surgical procedures exist that correct the complications of older procedures or avoid the complications altogether to improve patient outcomes. The procedure used depends largely upon the characteristics of the deformity. To help characterize the deformity, surgeons are using three-dimensional imaging. It remains unclear, however, which subgroups of chest wall deformity patients benefit from three-dimensional imaging and how exactly that information may alter surgical planning.   Data was collected on patients in which a CT scan performed and interpreted at UNC between 2001-2006 to characterize their chest deformity. A total of 67 patients met all the inclusion criteria. Preliminary results indicate that the control arm of the study (CT imaging without 3D reconstruciton) is too small for reliable statistical analysis. The experimental arm of the study (CT imaging with 3D reconstruction) shows that while the use of 3D imaging has increased since 2001, there was a period in the intervening years when the technology appears to have been used less by the UNC surgeons.

 
 

Antimicrobial Susceptibility Surveillance and Development of Algorithms for Clinical Management at Kamuzu Central Hospital in Lilongwe, Malawi

 

Rushina Cholera, Gabriel Malunga, Debbie Kamwendo, Mina Hosseinipour, Bill Miller, Mwai Makoka, Peter Gilligan, Irving Hoffman

 

Infectious diseases are a major part of clinical practice in the developing world. Various antimicrobial agents are used alone or in various combinations to treat these infections. However, over time, many microorganisms develop resistance to these antimicrobial drugs. In the developed world, real-time determination of antimicrobial susceptibility is used to guide therapeutic decisions. In contrast, most resource-poor countries cannot provide such real-time support and instead broad spectrum antimicrobial therapy is initiated empirically, based on clinical observation and patient histories. This is undesirable, as empiric and broad spectrum antibiotic use is a major factor in the emergence of antibiotic resistance and as a result, clinical care is algorithm driven. The UNC Microbiology Survey at the UNC Project in Lilongwe, Malawi has two major goals: to characterize the antibiotic drug resistance patterns at Kamuzu Central Hospital (KCH) and to use this information along with clinical data to design updated algorithms for patient care. This type of information is collected and used regularly in the United States but has not been collected in Malawi for years. This project is currently in its final stages and should be completed by December 2007.

 
 

Cardiovascular Risk Factors in Rural Mexico: Results from the Proyecto Puentes de Salud

 

CR Clover, KA Olson, LA Temming, MG Cohen

 

Background   Hispanics are the largest and fastest growing minority in the United States (U.S.), with Mexicans being the largest Hispanic subgroup. However, little is known about their antecedent health risks and how these risks contribute to their health when they immigrate to the U.S. The primary purpose of this study was to begin evaluating the cardiovascular (CV) risk in Mexicans who live in high migration areas of Mexico in order to better understand the health of immigrant Mexicans. Furthermore, because our studies took place in high migration areas, we looked to see if the migration of relatives to the US leads to a change in CV risk.    Methods Participants were recruited from 15 rural communities in Guanajuato, Mexico. Participants were screened for hypercholesterolemia, impaired glucose tolerance, elevated blood pressure, abdominal obesity, and low HDL cholesterol. Participants were also interviewed regarding their past medical history, lifestyle behaviors, and social factors.    Results   A total of 699 participants were screened. Prevalence of elevated blood pressure was 35.6%, and 22.5% of the sample had impaired glucose tolerance. Frequency of hypercholesterolemia was 18.3% and frequency of low HDL was 82.5%. Abdominal obesity was present in 78.6% of subjects. Of note, 39.6% met modified ATP III criteria for metabolic syndrome. Although we found high rates of CV risk, we found no statistically significant relationship between having a relative in the U.S. and increased CV risk.     Conclusions   Our study shows a high prevalence of cardiovascular risk in rural Mexicans. The prevalence of metabolic syndrome and of low HDL cholesterol levels was particularly noteworthy in this young cohort. Future research may focus on the role of lack of health-literacy in perpetuating poor diet and limited exercise. Improving the health of Mexicans in their place of origin may improve the health of Latin American immigrants to other nations. 

 
 

Corpus Callosum and Lateralized or Asymmetric Brain Hemisphere Function in Subjects at High Risk for Schizophrenia

 

Asa L. Cordle, Ayseil Belger, PhD, Martin Styner, PhD, Robert K. McClure, MD

 

The corpus callosum is an anatomical substrate for inter-hemispheric communication. Both first-episode and chronic schizophrenic patients have exhibited structural changes in the corpus callosum. Disturbance in normally lateralized or asymmetric functions across brain hemispheres are currently being investigated in schizophrenia. If such a disturbance is the result of developmental defects beginning in adolescence or earlier, then differences should be evident in subjects at high risk of developing the disease. DTI and MRI from 26 at risk subjects are being analyzed with a probabilistic tractography model to identify corpus callosum subdivisions connecting to individual cerebral lobes.   Previous approaches use arbitrary geometric subdivisions. Design fluency and verbal fluency tasks were completed in the high-risk subjects as functional testing. Correlation between functional testing performance, corpus callosum subdivision size, and cortical white matter changes are being investigated. The statistical analysis of this data is in progress at the time this abstract was submitted.

 
 

CHOLESTEROL SCREENING DOES NOT MOTIVATE OBESE ADOLESCENTS TO DECREASE THEIR BMI

 

Nipa Doshi BSPH, Eliana M. Perrin MD, MPH, Denise Esserman PhD, Michael J. Steiner MD

 

Purpose: The utility of routine cholesterol screening in obese adolescents is controversial. Proponents argue that in addition to detecting potential lipid abnormalities, routine screening may help motivate obese patients to decrease their BMI by reinforcing high physician concern regarding patients’ weight. We sought to determine the effect of cholesterol screening on subsequent BMI change in obese adolescents. Methods: We performed a retrospective cohort study of overweight youth (BMI > 85% on CDC standardized growth charts) aged 10-18 who attended a well child visit in a general pediatric clinic at an academic medical center.   Patients who had a cholesterol screening performed between June 2004- June 2005 were individually matched based on age, gender, ethnicity, and BMI at baseline to patients who did not have the “exposure” of cholesterol screening. Subjects in both groups were followed for a minimum of 3 months and up to 2.5 years for change in BMI from baseline; our primary outcome variable. In addition to the BMI change difference, we also decided a priori to compare BMI trajectory between groups. We used t-test analysis for comparison of the mean changes between groups and a linear mixed model to compare BMI trajectories after exposure. Results: Twenty-four matched pairs met inclusion criteria for enrollment in the study (n=48). Subject characteristics of matched pairs were as follows: 50% were female; 66% were African American and 8% were Latino; baseline BMI ranged from 26 to 46. Comparing the means of best individual BMI change within each group over time, the screened group did not decrease BMI more than unscreened subjects (mean BMI change, -0.04, sd=1.8 and -0.99, sd=3.3 respectively, p=0.24). Using a linear mixed model, screened children actually had an increase in BMI of 0.81 units per year of follow-up, while the matched controls decreased their BMI by an average of 0.44 units per year (p=0.053). In exploratory subgroup analysis, there was no difference in BMI trajectory between those with normal cholesterol screening results and those with abnormal results (p=0.91). Conclusion: Cholesterol screening of obese adolescents does not appear to positively impact short term BMI change. The trend of increased BMI in the screened group raises the possibility that screening may somehow “demotivate” adolescents from making important weight changes. While further confirmatory research is necessary, the results of this study suggest that cholesterol screening should not be undertaken solely to motivate children to lose weight. 

 
 

LPS preconditioning and its effects on oligodendrocyte precursors and neonatal white matter disease

 
Courtney Failor, Dr. Wayne Price
 

Inflammation is one of the proposed major etiologic factors influencing the development of white matter damage (WMD) in premature infants. However, recent clinical studies have found that prenatally, low levels of inflammation are protective, while more significant chorioamnionitis predisposes the preterm infant to WMD. Inflammatory injury to precursor oligodendrocytes (preOLs) is thought to be a key event, as loss of these cells disrupts further OL development and results in impaired myelination; part of the pathophysiology of WMD in these patients. Mice at post natal days 1-5 were chosen for this study due to the correspondence of abundance preOLs during this time and their correspondence to the “high risk” period for WMD in preterm infants.      To investigate the protective nature of inflammation, a sensitizing dose of LPS was administered prior to induction of inflammatory injury via a second injection of LPS on PND 4. We evaluated the effect of varying the time interval (6 h vs. 24 h) between the first and second doses of LPS and the outcome was assessed by direct quantitation of MBP staining of OL cells in the cortex and caudate-putamen regions at PND 10. Based on these results, we found that LPS enhanced brain injury when administered 6 h before the second injection; a so called sensitizing effect. Conversely, brain injury was significantly decreased at the 24 h interval. Elucidating the mechanisms behind this possible neuro-protective effect holds significant implications for understanding the development and possible prevention of WMD. 

 
 

Conformational Restraints on Reactivity of Human PR3-Specific Autoantibodies Facilitated through Protein Folding Manipulations

 
Lila Farrag, Ronald J. Falk
 

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) recognize conformational epitopes on PR3. This study evaluates PR3-ANCA target epitopes utilizing a novel recombinant PR3 (rPR3) produced to accommodate manipulations of the N-terminal domain. The rPR3 molecule contains an N-terminus six histidine tag, which can be removed by enterokinase (EK) cleavage of an adjacent EK cleavage site. Once cleaved the remaining amino acids correspond to the mature N-terminus of PR3. This rPR3 can be manipulated to produce three variant forms: tagged rPR3+his, EK-cleaved (his-tag removed) rPR3-his, and EK-cleaved, denatured/refolded rPR3-his/dr (the proteolytically active form). Patients with clinically positive PR3-ANCA titers (n = 40) were confirmed for reactivity against purchased native PR3 in our system. Controls included 29 healthy volunteers and 34 MPO-ANCA patients. All PR3-ANCA sera samples tested were reactive with one or more forms of the recombinant protein (greater than mean ELISA OD 405 + 2 SDs of controls). Of significance, three sera were reactive with non-active forms only and three others were more reactive with rPR3-his/dr than with native PR3. The results of our evaluation of PR3-ANCA sera for reactivity against the three forms of our rPR3 protein uniquely exemplify the diverse array of epitopes within the PR3-ANCA population. This new recombinant form of PR3 should provide a suitable approach to mapping ANCA epitopes using site-directed mutagenesis.

 

Does Recreational Drug Use in Elderly Drivers Impact Mortality?

 

Justin Fender, Anthony Charles, Sharon Schiro, Annabelle Fonseca, Howard Chen

 

Background: The correlation between substance use in the elderly trauma patient and increased morbidity and mortality is as of yet undetermined. Aims: The primary goal of my research was to examine the relationship between recreational drug use and outcome in the elderly driver. Methods: The North Carolina Trauma Registry (NCTR) was queried to identify elderly trauma patients, over 65 years old, presenting to a trauma center over a 6-year period from January 2000 to December 2005. Using SAS statistical software, patients with detectable levels of ethanol or illicit drugs were compared to those with no evidence of substance use. Logistic regression analysis was used to model outcomes as predicted by several independent variables. Co-morbidities were controlled for using the Charleson comorbidity index. Results: The data showed that out of 3481 trauma patients involved in a motor vehicle crash, 2987 patients were not tested for illicit drugs. After controlling for injury severity score, age, gender, race, age, and Charleson comorbidity score, a positive screening for drugs, ethanol, or both was found to not predict mortality in drivers. Conclusion: The elderly trauma patient may also be predisposed to substance use and abuse, including prescription drugs that may result in cognitive impairment and diminished neuromotor response. Our study does not show worse outcome (mortality) in drug positive elderly drivers. However, screening rates in this subgroup of the population is low.

 
 

Malaria Care-Seeking Behaviors in Rural Ghana

 
Kristine Folkerts
 

Malaria is the leading cause of morbidity and mortality in Ghana. Significant barriers to the use of official sector health facilities exist, and thus many people rely on alternative treatments. An understanding of these activities at the local level is needed to inform policies aimed at reducing malaria’s burden. There have been few studies on malaria care-seeking behaviors in Ghana’s Central Region. Most studies in Ghana have not highlighted women’s voices about the difficulties they face when caring for their children with malaria, nor have they presented mothers’ suggestions for improvements. This study was designed to address these gaps in the existing literature. In-depth interviews were conducted with key informants and with twenty-four female caretakers of children under age five in four rural communities. The interviews addressed the women’s perceived causes of malaria, the treatment activities they employed for the last illness episode in each of their children under age five, as well as their preferences, difficulties, and suggestions. Although two-thirds of the women implicated mosquitoes in the transmission of malaria, most cited additional erroneous causes. As expected, the women used a wide range of treatment activities, including home-based treatments, over-the-counter medications, consultations with herbal practitioners, and formal hospital/clinic care. In almost half of cases, care-seeking behaviors included multiple treatments. Nearly a third of children received no antimalarial medications at their last episode. Of those children who were brought to the hospital/clinic, their mothers waited an average of about five days before bringing them. The women identified poverty, poor education, and lack of transportation as significant barriers to the timely use of hospitals/clinics. The results point to the continued need for education about the transmission and prevention of malaria, for campaigns to increase awareness in rural communities about Ghana’s new malaria treatment guidelines, and for innovative initiatives to financially empower women.

 
 

Identifying Nerve Fibers in Kidneys Using Laser Confocal Microscopy

 

Nadezhda Gavrilova, Dr. Richard Bukoski

 

It has been shown that renal transplant patients develop hypertension over time. The mechanism may be connected with the loss of sensory neural innervation and especially with the loss of calcium-sensing receptors located on sensory nerves in transplanted kidney. They were shown causing reduction in blood pressure. However, little is known of the sensory neural innervation within the kidney. The performed study was designed to determine the nerve fiber distribution within the kidney. To determine the nerve fibers immunostaining of kidney slices with anti-CGRP (calcitonin gene related peptide), anti-NCAM (neural cell adhesion molecule) and anti-TH (tyrosine hydroxylase) followed by confocal microscopy visualization was performed. We detected red fluorescence, indicating nerve fibers, inside the kidney glomerulus, in Bowman’s capsule, between and inside kidney tubuli. Our results confirm the possible role of sensory innervation in development of hypertension. 

 
 

Referral Patterns and Patient Demand for Acupuncture Services at Kaiser Permanente Northwest

 

Jason R Goldsmith, Charles Elder, Richard Hammerschlag, Remy Coeytaux

 

Referral patterns and patient demand for acupuncture services at Kaiser Permanente Northwest (KPNW) for a one- year period were collected and analyzed. A combination of medical records and compiled data from KPNW and its outsourced self-referral network were filtered for all uses of the keyword acupuncture. Chief complaint, frequency of use, and (in the case of the self referrals) range of clinical conditions was documented when available. For the KPNW referrals, the data showed that the majority of referrals were for pain (78%) or neurological symptoms (11%). For self referrals, the chief complaint was not obtainable, but the range of diagnoses treated was much larger than for doctor referrals. The usage and demand patterns also differed between self- referrals and doctor referrals. Self-referred patients averaged 6.5 visits/year, compared to 7.4 among KPNW patients. This difference is even greater when looking at only those KPNW patients in Washington state whose referrals were authorized, with a mean of 12.9 visits per year. A mean of only 6 visits per year was observed among KPNW patients who were identified in their charts as requesting the referral. These findings suggest that patients who manage their own care of acupuncture use it for a wider range of conditions, and that they seek fewer treatments compared to patients referred for acupuncture by physicians.  

 
 

Clinical Manifestations of Lactic Acidosis in Malawi

 

Danica Gomes, Cecilia Kanyama, Mina Hosseinipour, George Joaki, Ralf Weigel, Nasinuku Saukila

 

Background/Study Objectives In Malawi, the first-line treatment for HIV/AIDS is D4T/3TC/NVP. It is a well-known fact that lactic acidosis is a major side effect of the Stavudine (D4T) drug and can be fatal if not addressed. In resource poor Malawi, lactate laboratories are not readily available, thus it is imperative that we identify the features of lactic acidosis so that a clinical diagnosis can be confidently made based on presentation. Our goal is to formulate a clinical criterion for lactic acidosis presentation in Malawi.      Methods Reviewed charts came from Lighthouse clinic (HIV clinic), Tidziwe center, and Kamuzu Central Hospital. Tidziwe center, where UNC Project is based, represents the only location in Malawi that has the resources to do lactate labs. We did a retrospective chart review of confirmed and suspected lactic acidosis cases. Information such as demographics, antiretroviral history, signs and symptoms leading to suspicion of lactic acidosis, and lactate levels were recorded. Confirmed lactic acidosis cases were dependent on symptoms and their resolution upon stopping ARVs or lactate levels greater than 3.5mmol/liter. Frequencies, means, medians and tests of associations were calculated.     Results 235 charts were reviewed. 120 were confirmed lactic acidosis cases. 115 were found not to have this diagnosis. Among confirmed cases, median age was 39 years, median duration of ART was 439 days and the median increase in CD4 on ART was 183 cells. 97% patients had gastrointestinal symptoms. 93% had neuropathy, 72% had weight loss. The mean lactate level was 6.5mmol/L. 7 patients were known to have died. Among survivors maintained in care at the Lighthouse (69), 66% were switched to AZT/3TC/NVP. Patients resumed ART after a mean of 79 days.      Conclusion Among confirmed cases, gastrointestinal symptoms accompanied by rapid weight loss and neuropathy or rapid progression of neuropathy supports a clinical diagnosis of lactic acidosis. 

 
 

The Designer GPCR: A new tool for selective modulation of signal-transduction pathways in vivo

 

John Hartmann, Atheir Abbas, Bryan Roth

 

     A novel approach to the study of G-protein coupled receptors (GPCRs) involves the development of “designer” receptors, or more specifically designer receptors exclusively activated by a designer drug (DREADD). For proof of concept, we worked with a human M3 muscarinic receptor (hM3), which had undergone directed evolution in yeast to be activated solely by the pharmacologically inert compound, clozapine-N-oxide (CNO). The DREADD is potently activated by CNO, but not by its endogenous ligand, acetylcholine (ACh), allowing selective activation of G-protein signaling in vivo. We created two lines of mice, an M3 DREADD transgenic with a tet response element (TRE) driving expression of an HA-tagged M3 DREADD, and another with the CamkIIalpha promoter driving tetracycline transactivator (tTA) expression. Thus, when the two lines are crossed, the HA-tagged M3 DREADD is expressed in CamkIIalpha-expressing neurons, which are primarily located in the hippocampus and cortex. Immunofluorescence using a mouse monoclonal anti-HA antibody provided initial verification of M3 DREADD expression in these regions. Furthermore, i.p. injection of CNO caused dramatic seizures in CamkIIalpha-tTA( /-)/M3 DREADD( /-) mice, but causes no apparent behavioral effects in CamkIIalpha-tTA(-/-)/M3 DREADD( /-) mice. Lastly, competition binding assays have confirmed the presence of two acetylcholine binding sites in double transgenic mice, owing to the expression of the normal M3 receptor and the M3 DREADD, each with a different affinity for ACh. This is in contrast to the wildtype mouse, which has only one binding site. Thus, we have demonstrated that modified GPCRs are suitable for in vivo studies. DREADD technology will prove to be a powerful tool for selective modulation of signal-transduction pathways, elucidating receptor-specific functions, and providing insight into disease states resulting from overstimulation of certain signaling pathways. 

 
 

The Effects of Leg Dominence and Strength Imbalance in the Kinematics of a Stop-Jump Task

 

Daniel C. Herman, Andrea L. Moore, Anna R. Cruz, William E. Garrett, Bing Yu, Darin A. Padua

 

INTRODUCTION Lower extremity strength imbalance has been shown to be possible risk factor for lower extremity athletic injury. The purpose of this study is to examine the effects of limb dominance and strength imbalances in the kinematics of female recreational athletes during a task often associated with anterior cruciate ligament injury.    METHODS Three cohorts were created, consisting of 1) subjects with at least 15% less strength in their gluteus medius (GMED) of their non-dominant (NDOM) limb compared to dominant (DOM); 2) subjects with at least 15% less strength in their quadriceps (QUAD) of their NDOM limb compared to DOM; and 3) subjects with less than a 10% strength imbalance between NDOM and DOM (NONE). Strength was assessed by hand-held dynamometry. The first 10 qualifying subjects for each group were selected for this study for 30 total subjects. GMED (age 22.5yrs, ht 1.67m, wt 62.2kg, ave. diff. 20%), QUAD (age 22.6yrs, ht 1.68m, wt 63.3kg, ave. diff. 23%), and NONE (age 21.4yrs, ht 1.65m, wt 58.4kg, ave. GMED diff. 3%, ave. QUAD diff. 3%).    Lower extremity kinematics were collected during 5 stop-jump trials using three-dimensional videography. Dependent variables included knee valgus and flexion angles, and hip abduction and flexion angles. Statistical analyses were performed using a 3 [group] X 2 [limb] ANOVA (alpha<.05). Dependent samples t-tests were performed to investigate significant interactions.     RESULTS   No significant main or interaction effects were observed in any of the dependent variables.    SUMMARY/CONCLUSIONS The results indicate that lower extremity limb dominance and moderate lower extremity strength imbalances may not significantly alter athletic task performance in female recreational athletes, and thus may not be a significant risk factor for injury. Further study into larger strength imbalances and multi-muscle strength imbalances using prospective study designs, alternative athletic tasks, and larger sample sizes may be warranted. 

 
 

Exploring the Relationship between Neutrophils and Cystic Fibrosis Biomarkers

 
Chris Horvat and Charles Esther
 

BACKGROUND: Adenyl purines are promising biomarkers of lung inflammation. These compounds could be especially useful in studying and treating cystic fibrosis (CF), a disease characterized by repeated episodes of neutrophil-dominated inflammation that result in progressive lung failure.    AIMS: A biomarker must have a strong link to the relevant physiology. This project begins to look at adenyl purine production by neutrophils, the hypothesized source of these biomarkers.    METHODS: Neutrophils were isolated from bronchoalveolar lavage (BAL) fluid and peripheral blood from patients with CF and healthy controls. Purine production was subsequently measured at different incubation time points using etheno-derivitization and high proficiency liquid chromatography (HPLC).      RESULTS: Measurements of purine production by neutrophils revealed slight increases in adenosine and ADP, decreases in ATP, and significant increases in AMP over time.    DISCUSSION: An observed increase in AMP over time among isolated neutrophils is consistent with accounts of neutrophil purine metabolism in the literature. High initial values of both AMP and ATP, as well as a sharp initial decline in ATP levels, suggest that premature activation of neutrophils occurred during the isolation protocol. Our results are consistent with an initial burst of ATP, followed by ecto-ATPase metabolism to the associated metabolites (predominantly AMP). Future goals of this work include enrolling more patients and collecting more BAL fluid and blood samples, as well as refining the neutrophil isolation protocol to prevent premature activation. 

 
 

Prenatal Cocaine Exposure and Oxytocin: Social Behavior in Young Adult Male Rats

 

Jarrett, T.M., McMurray, M.S., Walker, C.H., and Johns, J.M.

 

Background: Many rodent models of prenatal cocaine exposure document heightened levels of adult-social-aggressive behavior and differences in brain-oxytocin levels. None of these models assess the impact of prenatal-cocaine-exposure combined with being reared by a cocaine-treated rat mother. Hypothesis: Male prenatal-cocaine exposed offspring reared by a cocaine-treated mother will exhibit more social aggression and have lower brain-oxytocin levels. Methods: Prenatal-cocaine-exposed offspring were cross-fostered at birth and reared by either cocaine-treated or control rat mothers. At 60 days of age male offspring were tested for unprovoked aggression and success in obtaining water in a social behavior task. Results: Prenatal exposure to cocaine combined with rearing by cocaine treated dams, significantly increased aggressive behaviors such as threats and attacks and reduced success of attaining water. Males reared by cocaine treated dams regardless of their prenatal exposure condition, had significantly reduced levels of amygdaloidal oxytocin following completion of the water competition task. Conclusions: Prenatal-cocaine-exposed male offspring raised by a cocaine-treated mother are more socially aggressive and have lower brain-oxytocin levels. Interestingly, they are also less successful in attaining water suggesting this model of prenatal-cocaine exposure and rearing by a cocaine-treated rat mother also induces maladaptive social behavior. These studies were supported by NIH R01 grants DA -13283 and DA-13362 awarded to Dr. Johns. These data were first presented at the 2006 Annual Meeting of the International Society on Behavioral Neuroscience, Whistler, Canada.

 
 

Changing Indications for Revision Total Knee Arthroplasty

 
Lachiewicz and Lachiewicz
 

Introduction: There is relatively little data on the indications for revision total knee arthroplasty (TKA). Two previous studies have suggested that infection and loosening are the most common reason for failure. The purpose of this study was to determine if the indications for revision have changed over the past decade.     Methods: This is a retrospective review of demographic data and the indications for revision TKA in two cohorts of patients performed 10 years apart by one surgeon at a university referral hospital. Patient gender, age, weight, time in-situ and reason for revision (8 categories) were recorded and analyzed by chi-square tests. The first cohort was 61 revisions performed between 1990 and 1997. The second cohort was 96 revisions performed between 2000 and May 2007.    Results: There were significantly more male patients in the more recent cohort. There was no significant difference in the mean age or weight (corrected for gender) between the cohorts. There were significantly more revisions for instability and wear in the recent cohort.  There were significantly fewer revisions related to the patella prosthesis. The leading cause for revision in both cohorts was still aseptic loosening of the components. There was no significant change in the number of revisions for infection between the two cohorts.    Discussion and conclusion: The indications for revision total knee arthroplasty have changed significantly over the past decade. More emphasis may be needed on proper balancing techniques to prevent instability and new bearing surfaces should be considered to decrease wear.

 
 

Reducing the Global Burden of Amebiasis: Investigations in Diagnosis, Treatment, and Prevention

 
Rita Marie Lahlou
 

The burden of morbidity and mortality associated with diarrheal diseases presents a considerable challenge to health care providers, researchers, and policy makers, world-wide. The infectious parasite, Entamoeba histolytica causes amebiasis, one form of diarrheal disease. This thesis attempts to provide a comprehensive review of some of the current trends in research and clinical practices relating to the diagnosis, treatment and prevention of amebiasis infection. Accurate and timely diagnosis of amebiasis is necessary for proper and effective treatment. Unfortunately the current methods of diagnosis are not appropriate for the low-resource settings where amebiasis is most prevalent. Consequently, a new, cost-effective, rapid diagnostic tool that detects antigen in stool specimens and antibody in serum samples was developed and verified through clinical trials in Dhaka, Bangladesh. The device proved to be equally sensitive and specific for the diagnosis of infection with E. histolytica as traditional methods like ELISA and RT-PCR. Histone deacetylase inhibitors are being explored as potential chemical therapies for persons suffering from amebiasis, as they cause cell cycle arrest and cell death. To determine the effects of HDAC inhibitors such as TSA and SAHA on E. histolytica, the growth rate, adherence capabilities, protein production, and HDAC activity of amoeba grown in the presence of inhibitors were measured using cell counting, CHO cell adherence assays, Western Blots and an HDAC activity assay kit. The experimental results suggest a real potential for the use of HDAC inhibitors in treating amebiasis infections. Finally, a brief overview of the promise and obstacles of preventative vaccine research for is presented. An argument for increased funding and support for neglected tropical diseases is made, with the ideals of cost-effectiveness and setting-appropriateness in mind.

 
 

Effects of Cytochrome P450 Epoxygenase Overexpression on Lipopolysaccharide-Induced Cell Adhesion Molecule Expression in Murine Lung Endothelial Cells

 

Garjae D. Lavien, Matthew L. Edin, Craig Lee, J. Alyce Bradbury, Laura M. DeGraff, Joan P. Graves and Darryl C. Zeldin

 

Background: Atherosclerosis represents the major cause of morbidity and mortality in developed countries. Lipopolysaccharide (LPS), a constituent of the outer membrane of gram-negative bacteria exhibits pro-inflammatory effects central to the etiology of atherosclerosis, such as upregulating endothelial selectin (E-Selectin), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) on the surface of endothelial cells. Epoxyeicosatrienoic acids (EETs), synthesized from arachidonic acid by cytochrome P450 epoxygenases CYP2J2 and CYP2C8, have anti-inflammatory properties. Exogenous administration of EETs reduces ICAM-1, VCAM-1 and E-selectin expression on endothelial cells.     Aim: Elucidate the role of overexpression of CYP2J2 and CYP2C8 in LPS-induced VCAM-1, ICAM-1 and E-selectin expression in murine lung endothelial cells (MLECs).    Methods: MLECs were harvested from female mice with endothelial Tie2 promoter driven overexpression of CYP2J2 (2T HET) or CYP2C8 (8T HET), as well as their respective wild type littermates (2T WT, 8T WT). MLECs were cultured, grown to confluence, and incubated with either E. coli LPS or phosphate buffered solution (PBS) as a control for four hours. Cell lysis was followed by RNA extraction and reverse transcription. Real-time RT-PCR quantitation of VCAM-1, E-selectin, ICAM-1, CYP2J2, CYP2C8 and GAPDH were conducted on cDNA samples.     Results: Data from the real time RT-PCR indicates that 2T HET and 8T HET MLECs express CYP2J2 and CYP2C8 mRNAs, respectively whereas 2T WT and 8T WT MLECs do not. LPS increases VCAM-1, ICAM 1, and E-selectin RNA levels in all four cell lines compared to PBS in a dose-dependent fashion, which is consistent with previous studies. In comparison to 2T WT MLECs, preliminary data suggests that 2T HET MLECs have lower baseline mRNA levels of ICAM-1, VCAM-1, and E-selectin, as well as lower mRNA levels after LPS administration. In contrast, 8T HET MLECs did not show any differences in mRNA levels at the baseline or after LPS dosing compared to 8T WT MLECs. 

 
 

Creating a Novel Nanofiber-based Epithelial Tissue Construct Using Human Umbilical Cord Blood Stem Cells

 
Gita Madan, John van Aalst
 

The goal of our project is to culture umbilical cord blood (UCB)-derived stem cells on nanofiber scaffolds made of biodegradable polycaprolactone (PCL) with the intention of differentiating these stem cells into keratinocytes. This stem cell based skin graft will not have the limitations seen in traditional cultured epithelial autografts (CEA): these grafts take a considerable time to culture and are not clinically durable. The goal for the nanofiber-stem cell constructed skin will be quicker clinical availability and a skin graft that will have greater mechanical integrity,   Nanofibers can be used to create an ideal structure that can mimic the extracellular matrix (ECM) by providing a path for exchange of nutrients and metabolic waste between the scaffold and the environment. Nanofiber scaffolds have a higher rate of gas-exchange making them an ideal substrate for tissue culture. Fibroblasts are initially seeded onto the nanofiber matrix, and the collagen secreted by the fibroblasts functions as a substrate for cell attachment and differentiation.    Recent in vitro studies suggest that UCB stem cells can be differentiated into keratinocytes. Our aim is to differentiate CD34+ hematopoietic stem cells derived from human umbilical cord blood into epidermal keratinocytes for in vivo studies on nanofiber mats. In order to simulate the multistratified 3-dimensional structure of the skin in vitro, a method known as an organotypic culture is employed. It is possible to get full differentiation of the keratinocytes in vitro into skin’s component layers by simply raising the cell-layer to an air-liquid interface. To date, our laboratory has successfully grown keratinocytes, fibroblasts and combinations of these cells on nanofiber scaffolds in preliminary steps of creating an organotypic model.   In conclusion, our protocol describes a 3-dimensional organotypic tissue culture model to study the differentiation of human UCB stem cells into keratinocytes, creating a more robust skin substitute.

 
 

Recreating breastfeeding as the normative feeding behavior: what are the risks associated with formula feeding?

 
Melinda E. McNiel, Miriam H. Labbok
 

 Context: The majority of studies published on the topic construct the data in a way that portrays formula-use as the standard behavior and exclusive breastfeeding merely as an ideal, associated with particular benefits. In this secondary analysis we have converted odds and risks ratios so that the data reflects the risks of formula-use and normalizes exclusive breastfeeding. The association between formula-use and increased risk for several adverse infant health outcomes is analyzed.   Objective: To normalize exclusive breastfeeding and establish formula-use as a risky behavior by converting data related to breastfeeding sensitive outcomes.       Study Selection: We referenced data primarily from the recently published, comprehensive AHRQ study on breastfeeding and maternal and infant health outcomes. By using only studies including exclusive breastfeeding as a comparator group we limited the conditions to otitis media, asthma, type 1 diabetes, type 2 diabetes, atopic dermatitis, and hospitalization secondary to lower respiratory tract diseases (all infant outcomes). We performed a Pubmed search or similar studies that were published after the AHRQ and included their data in the analysis. Data Extraction: From the studies found we identified odds ratios or risk ratios and their corresponding confidence intervals. We then converted these values and calculated the associated significance (p-values) of each.   Results: Data collected from this secondary analysis reflects the risk associated with formula-use and the six aforementioned infant health outcomes. Significant associations were found in all of the studies on otitis media, half of the studies on type 1 diabetes, and half of the studies on type 2 diabetes. For the remaining conditions, the majority of associations were found to be insignificant.    Conclusions: Providing the data in this format, we construct formula-use as the deviant behavior we can adjust the language and attitudes surrounding exclusive breastfeeding and impact the way researchers approach and care-takers relay study results.   

 
 

Perceptions and Attitudes Toward the New Health Care Reform—"Universal Coverage"—Among Health care Providers in Thailand

 

Patcharica Meteesatien and Sohini Sengupta

 

Perceptions and Attitudes Toward the New Health Care Reform— “Universal Coverage ”—Among Health care Providers in Thailand    Patcharica Meteesatien and Sohini Sengupta    ABSTRACT     Background: Thailand implements universal health coverage policy to their new health care reform in 2001. Under the plan, patients pay 30 Baht per each visit until 2006 when the co-pay was revoked and medical service is now free of charge.   Aims: The goal of this study is to qualitatively explore (a) Thai physicains’ knowledge and perceptions about the current policy, and (b) physicians’ satisfaction with their practices after the implementation of the policy. Methods: Qualitative in-depth in-person interviews of 17 physicians from three provinces (Lampang, Buri Rum, and Bangkok) of Thialand and observation. Notes are taken during the one hour interview.   Results and Discussion: Most of physicians interviewed express good understanding of the new health care reform and are able to incorporate the reform into their practices and make appropriate advices to patients regarding the policy and their rights. Physicians’ satisfaction in practice shows strong correlation to their personal attitude toward the program. Fifteen out of seventeen physicians express some sort of dissatisfactions with the reform. Sources of dissatisfaction include: 1) increase risk of patients filing complaints against physicians and the hospital causing physicians to lack motivation and wary of increase responsibility. 2) Physicians’ concern with patients’ complaints to a lesser extend lead to increase in referral rates and a decrease in-patient service in the primary hospital. 3) Patients’ abuse of their privilege, particularly free medications that is a part of the universal coverage program. 4) Moral hazard that association with decrease self care and promotion of health. 5) Lack of a formal referral system and referral centers. To improve the quality of care and increase physicians’ satisfaction under the universal coverage, the issues stated above must be addressed.  

 
 

Acute and Persistent Pain after Breast Surgery

 

Haley Meyer, Dr. Karamarie Fecho, Dr. Silvia Wilson

 

Acute pain after breast surgery is a relatively common problem. For most patients, postoperative pain is limited to the first few days after surgery. However, persistent postoperative pain after breast surgery is a problem that was not recognized until recently, and it is unclear how many patients suffer from persistent postoperative pain and how best to treat such pain. The present study determined the incidence of, and identified risk factors for, acute and persistent pain after breast surgery. Data sources were medical records of females undergoing partial mastectomy (n equals 79), complete mastectomy (n equals 92) or complete mastectomy with immediate reconstruction (n equals 71). Outcome variables were pain scores (0-10 VAS scale) measured in the post-anesthesia care unit (PACU) and 1-30 days and 6-12 months postoperatively. Evaluated risk factors were preoperative and PACU pain, age, race, insurance, prior radiation or chemotherapy, obesity, and hypertension. Preoperative pain scores averaged 0.79 plus or minus 0.17. PACU pain scores averaged 4.95 plus or minus 0.22, with 61% of patients experiencing severe pain (VAS>5). PACU pain increased with surgical complexity (p<0.0005). Pain scores within the first postoperative month averaged 2.22 plus or minue 0.29, with 22% of patients experiencing severe pain. Pain scores measured 6-12 months postoperatively averaged 0.75 plus or minus 0.20, with 8% of patients experiencing severe pain and 5% of patients reporting severe pain that arose after surgery. Severe preoperative and PACU pain, younger age and non-white race were identified as risk factors. Systolic hypertension was a protective factor. These findings indicate that acute pain is problematic for the majority of breast surgery patients, while persistent pain is problematic for a small subset.

 

Impact of Oxacillin-resistant Staphylococcus aureus in Cystic Fibrosis

 

Meredith E. Miller, Dr. Marianne Muhlebach, Dr. Jennifer Goodrich

 

Background: Infection with ORSA is an increasing problem in the healthcare system and has important implications for CF patients. Studies on the impact of ORSA on CF disease progression show conflicting results. It remains unclear how infection with CA versus HA strains affects the outcome and whether a universal eradication protocol is warranted.      Aims:   The aim of this study is to determine the prevalence of CA and HA-ORSA at UNC’s CF center and to assess the clinical impact of ORSA vs. OSSA on CF patients.    Methods: ORSA isolates were prospectively collected from respiratory cultures of CF patients for an 18 month period. A cohort of CF patients colonized with OSSA was selected based on age, sex, and Pseudomonas aeruginosa status. A retrospective review of clinical charts was then conducted to create a database of clinically relevant parameters associated with CF disease severity.      Results: Eighty-two each of ORSA and OSSA patients were included in the final analysis. There was a significant difference (P < 0.001) in age at first S. aureus infection (13.3 yrs. for ORSA vs. 7.8 for OSSA). There was no significant difference in FEV1% values before S. aureus infection but at 6 and 12 months post-infection, the mean FEV1% for ORSA patients was significantly lower (P < 0.05). ORSA patients also had significantly more pulmonary exacerbations requiring IV antibiotics in 2006 (1.0 vs. 0.6). Of the ORSA patients, 68 (83%) had HA and 14 (17%) had CA-ORSA; analysis of this aspect is ongoing.      Discussion: Analysis of local CF patients infected with ORSA vs. OSSA indicates a greater decline in FEV1 % during the year following infection but no significant long-term effect on pulmonary function. A multicenter study would be more effective for assessing the impact of ORSA, and CA vs. HA strains, on CF patients.   

 
 

Continental Variations in Pre-operative and Post-operative Management of Patients with Anterior Cruciate Ligament Lesions

 

Lam Nguyen BS, Chad Cook PT, PhD, MBA, Eric Hegedus PT, DPT, MHSc, Allyson Sandago ATC, Ricardo Pietrobon MD, PhD, MBA, Claude T Moorman III, MD

 

Background: Surgeon decision making for non-operative anterior cruciate ligament (ACL) treatment and post-operative rehabilitation is influenced by a myriad of factors. The purpose of this study was to investigate inter-continental differences in surgeon decision making for care of the ACL deficient patient.     Materials and Methods: A survey which met the CHERRIES guidelines was administered to surgeons in 15 countries. Questions related to non-operative care management and post-operative/rehabilitative management were provided to each respondent. Statistical analyses included multivariate comparisons among continents and regression findings for likelihood of targeting longer term non-operative treatment.     Results: Over six hundred (634) surgeons completed the survey, representing six continents. Continental variations were found in non-operative surgical decision making and post-operative/rehabilitative management. Activity level and age greater than 10 years was associated with the likelihood of longer non-operative bouts of care.     Conclusion: Variations do exist across continents in the non-operative and post-operative/rehabilitative management of patients with an ACL tear. Continental variations and disparate emphases such as activity level, age during injury, and bracing influenced treatment decision making

 
 

Preliminary study of the impact of the daily dosage of antiretroviral therapy on adherence among people living with HIV/AIDS

 
Musheni Nsa

Although the use of combination antiretroviral therapy (ART) in HIV management reveals to be effective and to dramatically decrease the morbidity and mortality, treatment failure can result from nonadherence. This study was designed in order to determine how adherence is affected by dosage. Methods: Two hundred and one patients were followed during one year at the UNC Infectious Diseases Clinic and the Wake County HIV Clinic for the SafeTalk study. Participants were interviewed at baseline, 4, 8, and 12 month follow-up sessions. Adherence level was assessed by pill count and the questionnaires. Data is expressed as mean percent of adherence and it was determined by using SAS program (Cary, NC). Results: The mean adherence was decreased by the dosage 93.59%, n=944 observations, vs. 89.70%, n=460, one- and two-doses respectively. The adherence was not affected by the number of pills per dosage ranging from 1 to 5 pills. Discussion & Conclusion: In this study we found that a single daily dose of pills was much easier to follow by the patients than multiple dosages. Furthermore, the number of pills per dosage seemed not to have affected the adherence. Taken together, these observations suggest that adherence to ART is affected by dosage per day rather than the number of pills per dosage.

 
 

Use of Color-Coded BMI Charts Improves Parent Understanding of BMI

 

Joanne Finkle, RN, JD, Steven Pattishall, Lisa Whitehead, MD

 

Background: Many parents underestimate their children’s weight status and many not make appropriate nutrition and physical activity changes as a result. While body mass index (BMI) is an effective and efficient obesity screening tool, pediatric health care providers underuse BMI, and little information exists about whether parents understand the concept of BMI    Aims: This study aims to determine how well parents of young children (ages 3-8) understand BMI using both standardized and augmented (color-coded) BMI charts. Furthermore, it seeks to determine the relationship between literacy and numeracy skills and the ability to correctly comprehend BMI.      Methods: Fifty parents were interviewed at the UNC Pediatric Continuity Clinic using a demographics survey, a math skills test (WRAT), a literacy test (STOFHLA), and the “Understanding BMI” survey, a survey we devised to determine parents’ understanding of both standard and color-coded BMI charting. Analysis with STATA 10.0 determined the relationships between demographic variables and BMI understanding, as well as correlations between the WRAT, the STOFHLA, and the Understanding BMI survey results.     Results: Race, number of children, education, insurance, WIC enrollment, primary language, and income all were related to understanding of BMI. Positive correlations were found between scores on both the WRAT and STOFHLA and the Understanding BMI survey (r equals 0.58, p less than 0.0001, and r equals 0.59, p less than 0.0001, respectively). Those of lower (as opposed to higher) literacy and numeracy were more likely to increase their understanding with augmented, color-coded BMI charts.     Discussion: Individuals with higher literacy or numeracy had greater understanding of both standard and augmented BMI charts. Color-coded BMI charts will likely improve overall understanding of children’s BMI by their caregivers, particularly those with lower math and literacy skills. Further research is needed to determine if improved understanding of their children’s BMI leads to greater parental motivation to adopt healthy lifestyle changes and improved childhood weight trajectories.  

 
 

Functional Analysis of Secreted frizzled related protein 2 (Sfrp2) in Mouse Endothelial Cells

 

Demore, Nancy M.D. Olsson, Erik Ketelsen, David

 
 

Background Secreted frizzled related protein 2 (Sfrp2) has been investigated for its role as a paracrine factor mediating myocardial cell survival and repair (1). The gene has also been identified as overexpressed in breast cancer tumor endothelial cells and confirmed at the phenotypic level using IHC (2). Sfrps have been implicated in a variety of cellular processes including cell proliferation and cell death (3). Methods 24 and 48 hour proliferation assays were performed in the presence and absence of serum and Sfrp2. After incubation in experimental conditions, cells were counted using a hemocytometer and compared to controls with and without serum. For the migration assay cells were serum-starved overnight and added to a 12-well migration plate with experimental conditions in the base and allowed to migrate for 6 hours before staining. Apoptosis was determined using the Caspase 3 Assay Kit (Sigma) after treatment with Sfrp2 for 24 hr under hypoxic conditions. Results After addition of 10 uL rmSfrp2 (R&D Systems Minneapolis, MN) and incubation at 37C for 24 hours the median cell count for the control group was 2.25x10^4 (95% confidence interval 2.22 to 2.28). The median cell count for the wells treated with Sfrp2 was 4.0x10^4 (95% CI 3.98 to 4.02). A T-test was performed on the data and the value was calculated to be 5.2x10^-6. We were unable to achieve these results in repeat experiments under the same conditions. Unfortunately Analysis of Migration assays was unsuccessful due to undetermined reasons.   Discussion Our data suggest that secreted frizzled related protein 2 (Sfrp2) is not involved in promoting the proliferation of MECs in vitro. Further investigation is warranted to determine the role of Sfrp2 in migration and apoptosis. Our lab plans to perform knockdown experiments with siRNA against Sfrp2 to provide further evidence for Sfrp2’s involvement in MEC proliferation

 
 

Shifting from mitigation to prevention using Geographic Information Systems to identify and reach vulnerable populations

 

Andy Hull, Jeff Davis, Dohyeong Kim, Marie Lynn Miranda

 

Geographic Information Systems (GIS), a tool for managing, visualizing, querying, and analyzing information linked to geographic locations, have been used to analyze environmental exposures and their relationships to health outcomes. Our environments are important determinants of health status and outcomes. Science and medicine are growing to understand that even the expression of our genes is heavily influenced by our environments. A tool to map populations within their environments can be used to better understand the interaction between vulnerable populations, their environments, and their health outcomes. We have developed a tool to help prevent childhood lead poisoning and exposure before it begins by linking several datasets within a GIS. Collaborating with the North Carolina Children’s Environmental Health Branch and health care providers, the Children’s Environmental Health Initiative has designed a GIS based tool to identify children as risk of lead exposure to try to move from mitigation to prevention. We combined tax assessor housing information at the tax parcel level, childhood blood lead level testing results, and census data to produce county wide lead exposure risk models at the individual tax parcel level. These models are used to identify children living in homes where they might be exposed to bioavailable lead. Once identified, efforts are made to screen older children as a method of secondary prevention. Primary prevention is done by educating parents of infants before they are exposed about lead exposure risks and exposure prevention strategies. This tool is now being converted to a web-based application that can be used by parents and providers to better understand the potential for exposure. GIS holds great potential for applications to other heath outcomes and can be used to explore and analyze the relationship between health outcomes and the social and physical environment.

 
 

Shifting from mitigation to prevention using Geographic Information Systems to identify and reach vulnerable populations

 

Andy Hull, Jeff Davis, Dohyeong Kim, Marie Lynn Miranda

 

Geographic Information Systems (GIS), a tool for managing, visualizing, querying, and analyzing information linked to geographic locations, have been used to analyze environmental exposures and their relationships to health outcomes. Our environments are important determinants of health status and outcomes. Science and medicine are growing to understand that even the expression of our genes is heavily influenced by our environments. A tool to map populations within their environments can be used to better understand the interaction between vulnerable populations, their environments, and their health outcomes. We have developed a tool to help prevent childhood lead poisoning and exposure before it begins by linking several datasets within a GIS. Collaborating with the North Carolina Children’s Environmental Health Branch and health care providers, the Children’s Environmental Health Initiative has designed a GIS based tool to identify children as risk of lead exposure to try to move from mitigation to prevention. We combined tax assessor housing information at the tax parcel level, childhood blood lead level testing results, and census data to produce county wide lead exposure risk models at the individual tax parcel level. These models are used to identify children living in homes where they might be exposed to bioavailable lead. Once identified, efforts are made to screen older children as a method of secondary prevention. Primary prevention is done by educating parents of infants before they are exposed about lead exposure risks and exposure prevention strategies. This tool is now being converted to a web-based application that can be used by parents and providers to better understand the potential for exposure. GIS holds great potential for applications to other heath outcomes and can be used to explore and analyze the relationship between health outcomes and the social and physical environment.

 
 

Pharmacological Observation of the Acute Reciprocal Relationship of Vagal Tone and Sympathetic Nerve Activity

 
(1) Palvia, Tanuj P. (2) Taylor, J. Andrew, Ph.D.
 

PURPOSE: In the human body, there are indications that the sympathetic and parasympathetic systems share a reciprocal relationship. We wish to identify that an acute reciprocal relationship exists and if this relationship can be maintained via continuous exercise training through the aging process. We observe if pharmacologically decreasing vagal tone can incur an acute increase in the resting level of sympathetic activity in young sedentary individuals and master’s athletes.   METHODS: ECG, arterial pressure, and mean sympathetic nerve activity (obtained via microneurogrpahy in the peroneal nerve) were recorded before and after administering vagolytic doses of atropine on 8 young sedentary and 7 master’s athletes. All waveforms were stored to computer for subsequent processing and analysis.    RESULTS/STATISTICS: In young sedentary individuals, mean heart rate increased from 64.67 to 70.02 beats/min (p=0.14), mean systolic BP increased from 111.7 to 117.39 mmHg (p = 0.053), mean diastolic BP increased from 60.43 to 62.63 mmHg (p=0.23). Mean MSNA decreased 40.68% from 17.66 to 10.47 bursts/heart rate (p=0.1369). In master’s athletes, mean heart rate increased from 52.35 to 64.22 beats/min (p=0.007), mean systolic BP increased from 117 to 123.32 mmHg (p=0.12), and mean diastolic BP increased from 72.47 to 78.52 mmHg (0.015). Mean MSNA decreased 21.28% from 61.7 to 48.5675 bursts/heart rate (p=0.5879). CONCLUSIONS: This study demonstrates that pharmacologically decreasing vagal tone does not acutely increase the resting level of sympathetic activity in young sedentary populations. Also, this relationship cannot be identified in master’s athletes, thus preventing us from identifying preserved reciprocal relationship of the autonomic limbs via continuous exercise training through the aging process. Limitations of the study include confounding by the baroreceptor reflex, small sample size, and noise in sympathetic nerve activity measurement.

 
 

The Effect of Literacy and Numeracy on How Parents Understand Instructions for Medical Care of Their New Baby

 

Steven Pattishall, Matthew Oettinger, Joanne Finkle, RN, JD, Russell Rothman, MD, MPP and Eliana Perrin, MD, MPH

 

Purpose: New parents receive many health-related recommendations and instructions from a variety of sources concerning the care of their baby, but this information may not always be correctly understood leading to potentially serious health outcomes. We sought to examine the connection between parents’ general literacy and numeracy skills and their ability to interpret basic, common instructions regarding the care of their babies. Methods: Parents or caregivers of children less than 1 year of age were approached during a visit to the pediatric continuity clinic at UNC Hospitals and asked to participate in the study. They completed demographic information, a standardized literacy test (STOFHLA), numeracy test (WRAT-3), and answered questions related to various common pediatric health-related activities. The health-related questions were part of the 20 question Pediatric Health Activities Test (PHAT), which included questions regarding interpretation of thermometer read-outs, directions for formula preparation, and dosing of medications, among others. Completing the entire study took approximately 45 minutes and the parents were reimbursed for their time. The data were analyzed to determine correlations between both STOFHLA and WRAT scores with PHAT scores using the statistical program STATA (version 10.0). Results: Fifty-two caregivers were recruited and successfully completed all parts of the study. The average age of the parents was 26, with an average of 13.3 years of education. We found the STOFHLA literacy test results to be correlated with the PHAT scores (r0.35, p0.01). We also discovered an even stronger correlation between an individual’s WRAT numeracy scores and their PHAT scores (r0.47, p0.001). Conclusion: Parents literacy and numeracy skills both appear to be correlated to their ability to successfully interpret information regarding health-related information concerning their infant. When providing instructions or anticipatory guidance to parents of infants, pediatric providers should be aware of their potential limitations in literacy and numeracy. 

 
 

Interrogating the Serine Hydrolase Proteome of Human Platelets with Carbamate Probes

 

Ryan Phillips, Stephen P. Holly, Zhengyan Wang, Weiwei Li, Ben Cravatt, Leslie V. Parise

 

CONTEXT: The American Heart Association estimates that coronary heart disease and stroke will account for approximately 600,000 deaths in the US this year. Platelet aggregation is a key factor in the formation of thromboemboli that occlude blood vessels and lead to these conditions.    OBJECTIVES: To screen a library of carbamate moiety containing small molecules for human platelet activity modulation in order to identify potential drug candidates and novel platelet signaling pathways.    METHODS: Washed human platelets were incubated in each of 120 library compounds, activated with thrombin receptor-activating peptide (TRAP) and then subjected to flow cytometry to measure fibrinogen binding and P-selectin exposure. The effect of selected library compounds on platelet aggregation was then probed through aggregometry. The toxicity, with respect to human platelets, of these compounds was determined using an LDH assay.    RESULTS: Flow cytometry analysis led to identification of 13 library compounds that decreased platelet fibrinogen binding and P-selectin exposure by greater than 50%. Aggregometry confirmed that platelets treated with these compounds showed decreased aggregation when activated with TRAP. LDH assays of platelets treated with these compounds suggest little or no lysis or permeablization.    DISCUSSION: The most potent library compound demonstrated the ability to decrease platelet aggregation to just 6.25% of maximum. Further testing is in order to confirm these results, as well as to probe the broader toxicities of this and other compounds of promise. In addition, investigation into the possible targets of these compounds is ongoing and will potentially elucidate novel signaling molecules and pathways. 

 
 

Quantitative Assessment of the p53-Mdm2 feedback loop using protein lysate microarrys

 

Sundhar Ramalingam1, Peter Honkanen2, Lynn Young3, Tsutomu Shimura4, John Austin2, Patricia S. Steeg1,4 and Satoshi Nishizuka1,5

 

Mathematical simulations of the p53–Mdm2 feedback loop suggest that both proteins will exhibit impulsive expression characteristics in response to high cellular stress levels. However, little quantitative experimental evaluation has been performed, particularly of the phosphorylated forms. To evaluate the mathematical models experimentally, we used lysate microarrays from an isogenic pair of gamma ray–irradiated cell lysates from HCT116 (p53 / and p53-/-). Both p53 and Mdm2 proteins showed expected pulses in the wild type, whereas no pulses were seen in the knockout. Based on experimental observations, we determined model parameters and generated an in silico 'knockout', reflecting the experimental data, including phosphorylated proteins.

 
 

Volumetric Analysis of Brain Metastases Following Stereotactic Radiosurgery

 
Suzanne Rhodes
 

Background: Stereotactic radiosurgery is frequently offered to patients with brain metastases as a minimally invasive treatment option. Following stereotactic radiosurgery, the volume of metastatic lesions measured with magnetic resonance imaging may not change until months after treatment. However, in other patients, the volume of treated lesions may appear to decrease or even increase during the weeks following treatment. We have performed an analysis of volume changes of metastatic brain lesions following radiosurgery treatment. Methods: Sixty adult patients having undergone Gamma Knife stereotactic radiosurgery (SRS) for metastatic brain disease between August 2001 and March 2007 at the University of North Carolina, Chapel Hill were enrolled in a retrospective study. Thirty-six patients (60%) had a single lesion, and twenty-four (40%) had multiple lesions (range 2-5) at initial treatment.   Ninety-six metastatic lesions were analyzed for lesion volume using RECIST criteria (uni-dimensional), WHO criteria (bi-dimensional), and manual image segmentation software (3-dimensional). Measurements of tumor volume were taken at baseline and available follow-up dates. Results: Thirty-four lesions (35%) increased in volume ≥ 20% when compared to baseline at any follow-up time point, whereas sixty-two lesions (65%) never increased in volume ≥ 20% from baseline on follow-up MRIs. Twenty-four patients (40%) had at least one treated metastasis to increase in volume ≥ 20% from baseline on follow-up MRI, whereas thirty-six patients (60%) never had a lesion to increase in volume ≥ 20% from baseline on follow-up.

 
 

Parathyroidectomy for Renal Hyperparathyroidism at UNC: A Retrospective Review

 
Christina Russell
 

Chronic kidney disease (CKD) requiring hemodialysis is common in the United States and currently affects about 100,000 Americans. End-stage renal failure often results in hyperparathyroidism as a result of calcium ion homeostasis imbalance. In 2004 cinacalcet HCl (Sensipar) was introduced to treat renal hyperparathyroidism by lowering the threshold at which parathyroid chief cells detect calcium ions. Still, even with this innovation in medicine, parathyroidectomy is sometimes necessary for renal hyperparathyroidism that does not respond to treatment. This study is a retrospective chart review of patients who have undergone parathyroidectomy for renal hyperparathyroidism at UNC Chapel Hill hospitals from 1997 to 2007. Patients who were taking cinacalcet immediately before their parathyroidectomy were found to have a significantly higher chance of developing hypocalcemia during their post-operative course, 61.5% compared to 40.6% of the control group. Hypocalcemia was defined in this study as requiring IV calcium supplementation. Cinacalcet’s mechanism of action lowers serum calcium; however more research is needed to determine if the drug is causing this hypocalcemia phenomenon directly or by selecting out the sickest patients for surgery.

 
 

Identification of new foci of artesunate-mefloquine resistant Plasmodium falciparum in Cambodia using molecular surveillance

 

Shah NK, Alker AP, Sem R, Ariey F, Duong S, Wongsrichanalai C, Rogers W, Meshnick SR

 

Emergence of multi-drug resistant /Plasmodium falciparum/ malaria along the Thai-Cambodia border led to the introduction of artesunate-mefloquine combination therapy in 2000. Reports of increasing treatment failures of artesunate-mefloquine and geographic variations in resistance suggest the need for expanded surveillance in Cambodia. Increased /pfmdr1/ gene copy numbers predict /in vivo /treatment failure to mefloquine alone as well as to artesunate-mefloquine. We evaluated the feasibility of a national molecular surveillance program for artesunate-mefloquine resistance by assessing /pfmdr1/ mutations. Clinical isolates of /P.falciparum/ were collected from sites in Western Cambodia (Pailin, Kampong Seila), Central Cambodia (Chumkiri), and Eastern Cambodia (Memut and Rattanakiri). Samples were genotyped for /pfmdr1/ copy number and codons 86, 184, 1034, and 1042 using real-time PCR. We assessed the relationship between clinical characteristics and /pfmdr1/ mutations with parametric and non-parametric analyses. From 2004-2006, 768 isolates were collected and 730 (95.1 %) were successfully genotyped of which 197 (26.9 %) possessed multiple copies of /pfmdr1/. Median copy number varied by site: Pailin (1.34, n=177), Kg. Seila (1.53, n=10), Chumkiri (2.11, n=112), Memut (1.07, n=165), and Rattanakiri (1.05, n=287). The proportion of isolates with multiple copies of /pfmdr1/ was also greater in the west and center of the country compared to the eastern regions (54.0% versus 6.1%, OR = 8.72, 95%CI: 6.73, 10.72). /pfmdr1/ SNPs were largely wild-type and did not vary by site with the exception of 184-Phe whose prevalence was higher in the West and Central regions (Chi-square = 341.7, p<0.001). In multivariate analysis site and parasite burden remained associated with /pfmdr1/ amplification (p<0.001).// The results reveal a high prevalence of geographically clustered artesunate-mefloquine resistance in Cambodia which cannot be explained by variations in clinical or demographic characteristics. Artesunate-mefloquine resistance was believed to be restricted to areas along the Thai border and its presence in Central Cambodia is alarming. Malaria control programs in endemic settings can conduct molecular surveillance and detect previously unknown foci of antimalarial resistance prior to reports of clinical failure.

 
 

The Role of iNOS in Hepatic Progenitor Cell Proliferation and Differentiation

 

Yiqing Sheng, David Gerber, Natasha Wright

 

The regenerative capacity of the liver has attracted the interest of many who continue to explore the cellular pathways within the hepatic microenvironment. They do so in hope of developing alternative therapies that may avoid the limitations in whole-organ transplant in treating end-stage liver disease. The Gerber lab at UNC-CH has isolated from the adult liver a somatic-derived hepatic progenitor cell (HPC) population that demonstrate bipotent capability to differentiate towards hepatocytic and biliary phenotypes. After partial hepatectomy, increased expression of a cytokine tumor necrosis factor (TNF)-alpha has been shown to indirectly increase the production of Interleukin-6 (IL-6). Together, TNF-alpha and IL-6 upregulate inducible Nitric Oxide Synthase (iNOS), which converts L-arginine to nitric oxide. iNOS seems to be involved in both proliferative and apoptotic processes in the mature hepatocyte. Recent studies have shown that TNF-alpha supplementation alone or in combination with IL-1B in cultures can induce iNOS expression within HPC’s, which subsequently expressed hepatic and biliary genes as shown by PCR analysis. Further investigation after incubation of HPC’s with TNF-alpha alone and in combination with IL-1B using Western Blot and MTT assay are needed to confirm their capabilities in differentiation and proliferation. In this study, preliminary Western Blot data showed that Sca-1 HPC’s consistently expressed albumin markers on day 0 and 20 hours after addition of cytokines. CK-19 markers were never observed. MTT assay indicated a drop in cell count at 20 hours after the addition of cytokines followed by a rise in cell numbers until day 7 in Sca-1 HPC’s incubated with both TNF-alpha and IL-1B and with TNF-alpha alone. However, proliferation was more apparent under the influence of TNF-alpha alone than with both TNF-alpha and IL-1B. Due to time constraints, the preliminary data is inconclusive. Repeats of the same work needs to be done for reproducibility and confirmation of current results.

 
 

Vascular Tissue and Thrombin Generation

 
Samar Sheth, Alok Pathak, Rick Stoffer MD
 

Background: Despite treatment with antiplatelet agents, thrombotic events occur in patients. Thus questioning the role of platelets in the clotting cascade and acute thrombosis. Previous data from this lab has shown that in the presence of healthy tissue (freshly harvested internal mammary artery) and in cell culture (human aortic smooth muscle cells (HASMC)) it is possible to generate significant amounts of thrombin in the absence of platelets.     Methods: HASMCs, cultured in 24 well plates using a DMEM:SMBM media mix, were grown to 80% confluence. They were then incubated with reptilase treated, ultracentrifuged- platelet poor plasma (PPP) for 1.5 hours, or incubated with specific factor deficient platelet poor plasmas (FIX, FVIII, FVII, etc) using the same preparation as normal PPP. Tissue factor and CaCl2 were then added at final concentrations of 0.6pM and .5mM respectively. At 5 min intervals for 120min, 15microliters of plasma was added to 3.8% sodium citrate in a 96 well plates. S-2238 was added and optical density was measured at 405 lamba and thrombin generation was quantified.     Results/Conclusions: Preliminary data using HASMCs has shown that PPP thrombin generation reaches a peak at approximately 20 mins. FVIII deficient PPP reaches a peak as well, but the peak is delayed, and doesn’t appear to reach the level of normal PPP. Interestingly data suggests FIX deficient PPP also reaches a peak similar to the peak of normal PPP, however at a delayed time point. These data suggest that the intrinsic pathway is necessary for the propagation of thrombin and speed at which thrombin generated. These findings are supported by various other models of thrombin generation. We hope to further elucidate the roles of the intrinsic and extrinsic pathways in thrombin generation in cultured HASMCs, as well as understand pathways responsible for thrombin generation in these platelet poor systems.  

 
 

Prenatal Mild Ventriculomegaly Predicts Abnormal Development of the Neonatal Brain

 

John H. Gilmore, MD; Lauren Smith, Honor Wolfe, MD; Barbara Hertzberg, MD; J. Keith Smith, MD; Nancy Chescheir, MD; Dianne Evans, MA, Chaeryon Kang; Robert M. Hamer, Weili Lin, PhD, Guido Gerig, PhD

 

Background: Although mild ventriculomegaly (MVM) is one of the most common fetal brain abnormalities, little is known the effects it has on neurodevelopment after birth.     Aims: The main aim of this study is to determine if children with prenatal isolated MVM have alterations in brain development after birth.     Methods: Patients were recruited to the study from UNC Hospitals and Duke University Medical Center after a diagnosis of MVM on routine ultrasound. Using magnetic resonance imaging (MRI) in the first weeks after birth, we assessed gray and white matter development with automated tissue segmentation and quantitative tractography of diffusion tensor images (DTI) in 34 children with MVM and 34 age and gender-matched controls.     Results: Neonates with prenatal MVM had significantly larger lateral ventricle volumes (286.4 percent; p less than 0.0001), intracranial volumes (7.1 percent, p equals 0.0063), and cortical gray matter volumes (10.9 percent, p equals 0.0004) than matched controls. MVM cases have overall larger gray matter volumes (p equals 0.0010) and smaller white matter volumes (p equals 0.0125) than controls when normalizing for intracranial volume. DTI tractography revealed a significant increase of mean diffusivity in most white matter tract regions, while fractional anisotropy was significantly decreased in several white matter tract regions.     Conclusions: Prenatal enlargement of the lateral ventricle is associated with significant enlargement of the lateral ventricles after birth, as well as increased gray matter volumes, reduced white matter volumes, and delayed or abnormal maturation of DTI properties in the corpus callosum and cortico-spinal tracts. This study suggests that prenatal ventricle volume may be an early structural marker of dysmaturation of the cerebral cortex after birth and a risk for neuropsychiatric disorders, such as schizophrenia, that are associated with ventricle enlargement.

 
 

DYSREGULATION OF MICRORNA IN THE PREFRONTAL CORTEX OF BIPOLAR PATIENTS

 

Benjamin N Stepp, Clark D Jeffries, Joel S Parker, Diana O Perkins

 

MicroRNA (miRNA) expression has become an area of intense research due to the critical role they have been found to have in post-transcriptional regulation. These regulatory mechanisms have been found to be dysfunctional in bipolar disorder and schizophrenia, specifically regarding neurodevelopment and synaptic plasticity, respectively. We investigated the miRNA expression profile in the prefrontal cortex (PFC) of subjects with bipolar disorder compared to that of pyschiatrically unaffected and schizophrenic subjects. PFC from 14 subjects with bipolar disorder, 15 with schizophrenia, and 21 unaffected were case matched for post-mortem interval (PMI), age, gender, and hemisphere. Oligonucleotide probes were synthesized in duplicate for 264 human miRNAs antisense to the mature sequence reported in the Sanger miRNA registry and hybridization was performed. Assuming a FDR of 5%, 16 miRNAs were differentiated in PFC of bipolar compared with unaffected subjects. Trends of these comparisons were mirrored in bipolar versus schizophrenia. We also compared 12 miRNAs previously shown to be differentially expressed in schizophrenia with bipolar subjects and found a pattern similar to that of schizophrenia versus unaffected, with the same 12 miRNAs differentially expressed in a similar direction. The results of this study show a set of miRNAs differentially expressed in the PFC of persons with bipolar disorder as compared to psychiatrically unaffected subjects and to subjects with schizophrenia. In addition, the schizophrenia subjects similarly have a characteristic set of miRNAs differentially expressed when compared to psychiatrically unaffected and bipolar subjects.

 
 

Direct Comparison of Noninvasive Physiologic Measurements in a Multicenter Cohort of Emergency Department Patients Evaluated for Suspected Pulmonary Embolism

 

Michael Steuerwald, Brian J. O'Neil, David C. Portelli, Michael C. Plewa and Jeffrey A. Kline

 

Objectives: We tested which of 15 physiologic parameters discriminate PE from PE–.     Methods: 437 emergency department (ED) patients with suspected PE were prospectively enrolled at 7 U.S. hospitals. We first obtained room-air vital signs, arterial blood gases, pulse oximetry, and breath samples. All patients then had pulmonary vascular imaging and 90-day follow-up. PE was diagnosed (PE ) and excluded (PE–) via rigorous peer-reviewed criteria.     Results: 97 (22%) patients were PE and 340 (78%) were PE–. We observed no significant difference in body mass or vital signs. PE patients were older (56 ± 18 years vs. 49 ± 16 years), with lower oxygen saturation (SaO2) (94% ± 6% vs. 96% ± 4%) but not partial arterial oxygen tension (PaO2) (72 ± 20 torr vs. 80 ± 20 torr). Alveolar minute ventilation was increased (8.3 ± 4.0 L vs. 6.5 ± 2.8 L) with trend of increased tidal volume (565 ± 288 mL vs. 503 ± 263 mL) and with shortened exhalation time (1.8 ± 0.8 sec vs. 2.1 ± 0.9 sec). Alveolar dead space (VD/VTalv) was increased (33% ± 20% vs. 19% ± 17%) with proportional decrease in mixed expired CO2 (PeCO2, 17 ± 6 mm Hg vs. 22 ± 5 mm Hg) and end-tidal CO2 (PetCO2, 29 ± 7 mm Hg vs. 36 ± 6 mm Hg). PeCO2 and PetCO2 had the highest areas under the ROC curve (AUCs) (0.71 and 0.74), with lower-limit 95% confidence intervals >0.6. No other parameter had an AUC >0.64.     Conclusions: In symptomatic ED patients, age, weight, vital signs, SaO2, and PaO2 poorly discriminated PE from PE–. PE markedly increased alveolar dead space, manifested as low exhaled CO2. Among 15 physiologic parameters, PeCO2 and PetCO2 had the highest, albeit marginal, discriminatory value. 

 
 

High Rates of Depression and Cardiovascular Risk Factors in Rural Mexico

 

Lorene A. Temming, Kristoff A. Olson, Charles R. Clover, Sandra C. Clark, Mauricio G. Cohen

 

Background:   Cardiovascular disease (CVD) and depression are estimated to be the first and second largest contributors to the global burden of disease by 2020. Depression is an independent risk factor for developing CVD. There is a paucity of data regarding CVD risk and depression among rural Mexicans, a population likely to emigrate to the US. We assessed the prevalence of CVD and depression among rural Mexicans.    Methods:   Participants were recruited from 9 rural communities in Guanajuato, Mexico. We screened for depression using the nine-question Patient Health Questionnaire (PHQ-9). Participants with scores of 10-14 were considered moderately depressed, and scores >15 were considered severely depressed. Participants were screened for elevated blood pressure, impaired glucose tolerance, hypercholesterolemia, waist-to-hip ratio, and for low HDL cholesterol.    Results:   A total of 432 participants were screened and interviewed. Mean age was 45.8 years and 79.4% were female. Prevalence of elevated blood pressure was 38.4% and 21.3% of participants had impaired glucose tolerance. Frequency of elevated total cholesterol and HDL cholesterol was 16.6% and 81.8%, respectively. Abdominal obesity rates were 82.2%. According to modified ATP III criteria, 49.8% of our sample had metabolic syndrome. Moderate or severe depression was present in 25.9% of subjects. Aside from age, depression showed no statistical correlation with CVD risk factors.     Conclusions:   We found a high prevalence of CVD risk factors and depression among rural Mexicans. However, there was no clear correlation between CVD risk factors and depression in our sample. Most noteworthy in our study were high rates of metabolic syndrome and low HDL in this relatively young cohort of mostly women. Further research is needed to gain insight into the reasons for the high prevalence of depression and CVD risk factors in the rural Mexican population. Strategies for early preventive therapies should be considered in this population.  

 
 

Three-Dimensional Images and Vessel Rendering Using Optical Coherence Tomography

 

Meghan W Thomas, James M. Grichnik MD PhD (DUMC), and Jospeh A. Izatt PhD (Duke University)

 

Purpose: To evaluate improvements in optical coherence tomography’s handheld technology and software on dermatological applications.     Method: An institutional review board approved a small clinical trial at Duke’s Dermatology clinic. Patients coming to the clinic for previously scheduled appointments were asked to participate. Once consented, their skin lesions were imaged with the OCT machine and photographed for comparison. The system had a 1310 nm laser with a lateral resolution of 20 microns and a penetration depth of approximately 1.8 mm. Vessel rendering software was then used to trace imaged vascular lesions.    Results: 3-dimensional images of hemangiomas, telangiectasias, and a psoriatic plaque were acquired. Videos of the scans were recorded and telangiectatic vessels were rendered.    Conclusion: The use of OCT in the clinical setting is increasing especially with improvements in technology and software. Optical coherence tomographic technology may play a significant role in future bedside dermatologic diagnostics.  

 
 

Integrin Associated Protein (IAP) Cleavage and Vascular Smooth Muscle Proliferation

 

Diabetic patients with elevated blood glucose levels have a greatly increased risk of developing atherosclerosis due to increased vascular smooth muscle proliferation. Vascular smooth muscle cells grown in high glucose (25 mM) are more responsive to IGF-1 than smooth muscle cells that are grown in physiologically normal concentrations of glucose (5 mM). IGF-1 activates a signaling cascade in vascular smooth muscle cells that are grown in high glucose, and this signaling cascade results in altered gene transcription and increased smooth muscle cell growth. In order for this signaling cascade to be activated, the extra-cellular domain of Integrin Associated Protein (IAP) must first associate with SHPS-1. In physiologically normal 5mM glucose, IAP is known to be cleaved by MMP-2, which prevents activation of the signaling cascade and results in no increased smooth muscle cell proliferation. In high glucose (25mM), IAP is not cleaved and is able to associate with SHPS-1. The aim of this experiment was to determine the IAP cleavage site and to study the effect that protecting the IAP cleavage site from cleavage would have on smooth muscle cells grown in both high and low glucose. Smooth muscle cells containing a mutated version of IAP were developed, and gel electrophoresis was used to determine that the mutation blocked the cleavage of IAP. A cell growth assay was used to examine what effect blocking IAP cleavage would have on smooth muscle cells grown in low glucose. Preliminary data suggests that protecting IAP from cleavage is not enough to trigger smooth muscle cell proliferation in low glucose, and it is hypothesized that other pathways must also be involved.

 
 

Surgical Treatment of Locally Recurrent and Locally Advanced Rectal Cancer with and without the Addition of Intraoperative Radiation Therapy

 

Matthew Triplette, Michael Meyers, MD, Benjamin Calvo, MD

Introduction: Intraoperative Radiation Therapy (IORT) has become increasingly utilized in a multimodal approach including surgical resection with curative intent of recurrent and locally advanced rectal cancer; however, many studies have been inconclusive as to its benefit on local control and recurrence rates.      Objectives: It is important to establish whether IORT has consistent tangible benefits to patient disease-free survival as its use is associated with logistical hurdles and significant perioperative morbidities. We hypothesize that the addition of IORT to a curative resection in the treatment of locally recurrent and locally advanced rectal cancer reduces further recurrence rates and improves survival in these patients without significantly affecting operative outcomes such as morbidity and hospital stay.    Methods: This is a single institution, retrospective review of all locally recurrent and locally advanced rectal cancer patients who underwent IORT in addition to curative surgical resection between the years of 2001-2006, and an adequate number patients to serve as a control, who underwent the same surgical procedure and adjuvant therapy without IORT in the proceeding years. Complete data sets were collected on all patients including tumor staging and surgical margins, as well as operative variables. Longitudinal information was also collected on all patients to determine post-operative status and further recurrence. The data was then analyzed.    Results: After preliminary analysis, it appears that there is significant difference between overall survival and local recurrence rates in the two populations. For patients with IORT, local recurrence rate and death rate was found to be 14.6% and 43.9% respectively, while these values were significantly higher at 23.7% and 60.5% in those patients without IORT.    Conclusions: While much of the data needs to be analyzed, there does appear to be significant benefits to the incorporation of IORT into a multimodal surgical treatment for patients with locally advanced and recurrent rectal cancer. 

 
 

SPINAL PATHWAYS ACTIVATED WITH THE STIMULATION OF THE HYPOGASTRIC NERVE IN FEMALE RATS

 
UGOEKE, J., YU, G-Z., AND MARSON, L
 

Recent studies in women and female rats suggest that activation of the sympathetic nervous system can facilitate sexual genital responses. In addition, women with injury of the spinal cord below the sympathetic region can still experience sexual arousal. To further understand the spinal pathways and mechanisms involved in relaying afferent and efferent information during sexual behavior; the present study examined the location of neurons in the spinal cord and lower brainstem that were activated with stimulation of the hypogastric nerve. " "Mapping of c-fos, an immediate early gene that is expressed when cells are activated, was used to localize the spinal segments and regions that were activated with stimulation of the hypogastric nerve. Animals were anesthetized and the left hypogastric nerve was exposed. Control rats received similar surgical manipulation, except that the nerve was not stimulated, 2 other groups received either 40uA or 100uA stimulation over a 30min period. Animals were subsequently perfused and the brain and spinal cord was cut and stained using immunocytochemistry to visualize the fos-immunoreactive nuclei using standard techniques. Comparisons were done with t-test to examine any difference between segmental activation between stimulated and control animals. " "Hypogastric nerve stimulation with 40uA resulted in a significant increase in cell activation in T12-L1 of the spinal cord, particularly in the dorsal horn, medial and lateral gray. Some increase in cell activation was also observed in the ventral horn of L3-L4. In the L5-S1, 40uA stimulation resulted in increased c-fos in the medial and lateral gray. The group stimulated with 100uA showed less cell activation compared to the 40uA stimulated group. C-fos expression was found in the nucleus of solitary tract and the dorsal motor nucleus of the vagus nerve, with more expression seen in stimulated animals, especially in the dorsal motor of the vagus. These results map the spinal segments and regions that relay afferent information from the hypogastric nerve and suggest that stimulation of the hypogastric nerve activates vagal afferents. This information will aid our understanding of sensations and ability for sexual arousal after spinal cord injury.        

 
 

Effects of Supplemental Oxygen Treatment on Oxygen-Induced Retinopathy in Neonatal Rats

 

A.Uppal, Y.Saito, D.K. Sutton, P.Geisen, G.Cui, L.J. Peterson, M.E. Hartnett.

 

Purpose: Retinopathy of prematurity is a leading cause of vision loss in children.  This study investigates the effects of supplemental oxygen treatment on retinopathy in neonatal rats using oxygen stresses similar to those experienced by pre-term infants.   Methods: We used a 50/10 oxygen-induced retinopathy (OIR) model that exposes Sprague-Dawley newborn rat pups to cycles of 24 h of 50% oxygen alternating with 24 h of 10% oxygen until postnatal 14 days (p14). Some of the litters were then exposed to 28% O2 (OIR+SO model) until p20 while others were moved to room air (OIR model). Pups were given IP injections of Pimonidazole (Hypoxyprobe: HP) 90 minutes before sacrifice and retinal dissection. Half of the retinas were fixed for flatmounts or cryosections and stained with Griffonia simplicifolia isolectin and HP antibody, while the rest were collected for western blot analysis of HP vs. actin levels. The flat mounts were scored for intravitreous neovascularization (IVNV) by counting clock hours and for avascular areas as a percent of total retinal area (%AVA) using Image J (NIH). Data was analyzed by Mann-Whitney U-test and ANOVA using SPSS software.   Results: HP was significantly increased at p18 vs. p13 and p17 vs p13 in the OIR model (one-way ANOVA p=0.002 & Bonferroni p<0.05), and was observed to localize within the ganglion cell layer to outer nuclear layer of peripheral avascular retina in flatmounts and cryosections. There was no significant difference between OIR+SO model and OIR model at p20 in clock hours IVNV (SO: 3.7±1.0 S.E., OIR: 4.2±0.6), %AVA (SO: 7.30%±1.24, OIR: 9.26%±1.63) or retinal HP levels.   Conclusion: Supplemental oxygen might not be useful for reducing retinal hypoxia, neovascularization or peripheral avascularity following oxygen-induced retinopathy.  Hypoxyprobe proved to be a useful tool for locating and quantitatively measuring hypoxic areas of the retina.

 
 

Inhibition of NAD(P)H Oxidase Activity Reduces Intravitreous Neovascularization after Oxygen Stress in a Rat Model of Retinopathy of Prematurity

 
Y.Saito, A.Uppal, G. Byfield, S. Budd, M.E. Hartnett
 

Purpose: Retinopathy of prematurity is a leading cause of vision loss in children. This study measures NAD(P)H oxidase-dependent outcomes after oxygen stresses, similar to those experienced by preterm infants today, using a rat model of retinopathy of prematurity.    Methods: Within 4 hours of birth, pups and their mothers were cycled between 50% and 10% oxygen daily for 14 days, and returned to room air (21% O2, 50/10OIR) or supplemental oxygen (28% O2, 50/10OIR SO) for four days. Pups received intraperitoneal injections of the specific NAD(P)H oxidase inhibitor, apocynin (10 mg/kg/day), or PBS from postnatal day (p)12-p17, and some received intraperitoneal injections of pimanidazole (Hypoxyprobe: HP) prior to sacrifice. Outcomes were intravitreous neovascularization (IVNV) and avascular/total retinal areas, VEGF, phosphorylated/total p47phox, and hypoxic retina (conjugated HP). Human retinal microvascular endothelial cells (RMVECs) treated with apocynin or PBS were exposed to 1% or 21% O2 and assayed for phosphorylated JNK and p47phox.    Results: Compared to 50/10OIR, 50/10OIR SO had increased NAD(P)H oxidase activation (phospho- p47phox), reduced VEGF, but not less hypoxic retina. 50/10OIR SO treated with apocynin had less hypoxic retina and IVNV area, but not reduced VEGF. RMVECs treated with 1% O2 had increased signaling through JNK than did cells exposed to room air.    Conclusions: Varying degrees of NAD(P)H oxidase activation by different oxygen stresses differentially trigger signaling pathways leading to angiogenesis (IVNV) or apoptosis (avascular retina), features important in severe ROP. NAD(P)H oxidase dependent IVNV appears to involve pathways besides VEGF.

 
 

Risk Factors for Dengue in Laredo, Trujillo, Peru

 
Anshu Verma
 

Dengue is a mosquito-borne viral illness whose incidence has increased continuously in South America over the last 25 years. Many people view this increase as a failure in the politics of public health in this region. Peru, in particular, has noticed a reemergence of dengue in the past two decades. The purpose of this project was to examine awareness of risk factors for dengue and risk behaviors in the community of Laredo, in Trujillo, La Libertad, Peru. We surveyed households in the community about their practices concerning water storage, raising animals, use of mosquito nets while sleeping during the summer. Overall, we found that the community was well aware of risk factors and was taking appropriate steps to avoid dengue outbreaks. 89% of the households surveyed reported storage of water in their homes. Of these households, 88% reported using something to cover the containers in which water was stored. 52% reported emptying and cleaning their water storage containers each day, 44% reported doing so every 2-3 days, and 4% reported doing so once weekly. 44% reported raising animals domestically in their homes. 12% reported use of mosquito nets while sleeping during the summer. 

 
 
Diversity of IGRP-specific CD8 T cells in NOD mice
 

Benjamin Vincent, Adam Buntzman, Ellen Young, Tom Kepler, Jeff Frelinger

 

Background / Objectives  The non-obese diabetic (NOD) mouse develops autoimmune-mediated beta-islet destruction and provides a model for the study of type I diabetes (T1D).  It has been shown in our group that the diversity of a cell population important in diabetegenesis (IGRP206-214-specific CD8+ T-cells) is exquisitely low in the pancreatic islets and peripheral blood of 20wk-old NOD mice.  It is important to know whether this restriction obtains in younger mice, whether tissue-specific differences in diversity exist, and to what degree there is overlap in IGRP206-214-specific T cell pools between mice, tissues, and ages.    Methods  IGRP206-214-specific CD8+ T cells were isolated from various tissues of NOD mice at ages 8-16 weeks then single-cell sorted.  RT/PCR was done on the single-cell lysates and product dna was sequenced.  TCR-beta gene usage was determined using the SoDA software tool.  Population diversity accounting for unseen species was calculated using a novel method developed by one author TK).  Results  The diversity of the IGRP206-214-specific CD8+ T cell population was significantly lower in islets & pancreatic lymph nodes (PLNs) and higher in the spleen and thymus at 8 weeks.  Diversity in the spleen and thymus remained high for all time points.  At 10 weeks, islet and PLN diversity had increased, but by twelve weeks had decreased to a midpoint between 8 week and 10 week levels.  Through 12 weeks, diversities in the islets and PLNs were statistically indistinguishable.  After 12 weeks, diversity in the islets dropped precipitously while diversity in the PLNs was relatively unchanged.  The decrease in islet diversity was determined by both a declining overall species richness and an increasing frequency of the most abundant clonotypes.   Conclusions  After 10 weeks, IGRP206-214-specific CD8+ T cells concentrate in the islets and the pancreatic lymph nodes and this concentration increases disproportionately in the islets over time.

 
 

An Improved Technique of Use of the Temporary Keratoprosthesis in Eye Trauma Surgery

Watson J, Landers MB.
 

Use of a temporary keratoprosthesis (TKP) in eye surgery to establish both a water-tight seal and a clear view of the posterior segment of the eye can be helpful in cases of ocular trauma where assessment of the retina and vitreous is prevented by corneal edema, inflammation, or intraocular bleeding. Using a TKP during ocular trauma surgery may be perceived to be difficult because of the conventional wisdom that a water-tight seal must be maintained between the opposing corneal stroma and TKP trunk. Traditionally this is accomplished by the use of a TKP with a trunk diameter that is very slightly greater than the trephined opening in the injured cornea. However, we used the Landers TKP #2 to successfully establish a water-tight seal on the horizontal interface between the circumferential TKP flange and the anterior surface of the cornea rather than between the corneal stroma and TKP trunk, allowing us to use a TKP whose trunk was 0.8mm smaller than the diameter of the corneal opening. Twenty postmortem porcine eyes had a PMMA TKP #2 with a 7.2mm wide-field trunk secured into an 8.0mm trephination hole with six 5-0 Vicryl sutures after removal of the lens and capsule. Each eye was pressurized to 100mmHg and the seal between the TKP and corneal surface tested for leaking fluid. All twenty eyes were found to be water-tight. A standard pars plana vitrectomy was performed on sixteen of twenty eyes, and fifteen of these remained water-tight to 100mmHg. The Landers TKP #2 can be used to maintain a clear view of the posterior segment and a water-tight eye in cases where the corneal defect is up to 0.8mm greater in diameter than the TKP trunk, and/or where the original traumatic corneal injury precludes a tight seal between the corneal stroma and TKP trunk.

 
 

Risk of Nephrogenic Systemic Fibrosis: Evaluation of Gadolinium Chelate Contrast Agents By Four American Universities

 

Rebecca Wertman; Ersan Altun, MD; Richard Semelka, MD; et. al

 

Purpose: To determine the benchmark incidence of NSF in tertiary care centers of four different universities in the United States (University of North Carolina at Chapel Hill, Emory University, Thomas Jefferson University, and Wake Forest University) related to confirmed usage of different gadolinium chelate contrast agents.    Materials and Methods: Institutional review board approvals with waiver of informed consents were obtained for this HIPAA compliant retrospective multi-institutional study. Patient records from the databases of Dermatology, Pathology, Internal Medicine, Nephrology, Transplant Surgery and Radiology were cross-referenced to identify all patients with NSF at the four tertiary care centers ranging from January 2000 to December 2006. All centers had renal transplant and dialysis services. The type of agent, number of studies, and volume of gadolinium chelate contrast agent (GCCA) administered to the patients with NSF were determined in each center using a created checklist. The checklist included the search of technologists’ log book, clinical information system, MRI study headers, MR images and reports to confirm the GCCA used. Laboratory data including serum creatinine and estimated glomerular filtration rate (eGFR) were determined. Additionally, the benchmark incidence of NSF was determined for the same time period as the number of patients with NSF relative to the number of patients having undergone GCCA enhanced MRI in each tertiary care center.         Results: Two centers utilized gadopentate dimeglumine and two centers utilized gadodiamide as the solitary gadolinium agent during the study period. The incidence of NSF was 1 in 44,225 patients with gadopentate dimeglumine and 1 in 2,913 patients with gadodiamide.    Conclusion: The checklist verified the different GCCAs administered to the patients with NSF. We observed a benchmark incidence of NSF of 1 in 44,225 for gadopentate dimeglumine and of 1 in 2,913 for gadodiamide. 

 
 

Predictive Value of EBV Loads for Post-Transplantation Lymphoproliferative Disease in Lung Transplant Recipients

 
S. Wheless, M. Gulley, P. McNellie, I. Neuringer, and R.M. Aris
 

BACKGROUND: Post-transplantation lymphoproliferative disorder (PTLD) is a well-documented and often fatal complication of transplantation. Lung transplant recipients are at the greatest risk for PTLD. We aim to determine if elevated EBV loads, in combination with other predictive factors, may serve as a reliable diagnostic marker of PTLD risk in lung transplant recipients. METHODS: A retrospective, single-center study was performed using real-time TaqMan PCR EBV DNA load data in lung transplant recipients. The study population included all patients transplanted from 1990 to the present. Results were correlated with pre-transplant EBV status and development of PTLD. RESULTS: EBV load testing from 2000 forward has yielded approximately 1000 viral loads in 119 lung transplant recipients. Of these, 13 developed PTLD (12 confirmed histologically). Among pre-transplantation seropositive patients, 1 developed PTLD; the EBV load was >250copies/mL (true positive) (sensitivity and specificity 100%). Among the pre-transplantation seronegative patients, 12 developed PTLD. Of these, only 4 had EBV load data and all had detectable EBV DNA (sensitivity 100%, specificity 21.4%), but only 2 of 4 had EBV loads >250copies/mL (sensitivity 50%, specificity 57.1%). The lower rates of specificity were mostly due to false signal in non-PTLD cases and were load-level dependent [i.e. depending on whether one considered the positive as >250 (the assay positive) or as >0 (any detectable EBV)]. Thus, pre-transplant EBV status affects the performance of the assay. CONCLUSION: EBV loads are a useful but imperfect indicator of PTLD risk in lung transplant patients. Our EBV data are similar to published CMV PCR data; both tests have excellent negative predictive value. The main shortcoming of EBV viral load testing is that many EBV seronegatives who fail to develop PTLD have false positive results – likely due to primary EBV infection. EBV load testing requires further investigation and refinement before it can be used to accurately determine PTLD risk.

 
 

Effectiveness of the Protocol for the Assessment of Neurodevelopmental Function in Early Infancy (PANDI)

 
Stephanie Wolfe
 

Background: Although a significant portion of children have developmental delays, many are not diagnosed until school age, and when they are, it is difficult to translate this diagnosis into qualification for services. In addition, newborn screenings for genetic and metabolic disease are increasingly available, and early intervention services become more available, yet current assessments to determine the implications and recommended treatment for these diagnoses are lacking. Lastly, parents are not getting the information they need to help their children with these problems. The Protocol for the Assessment of Neurodevelopmental Function in Early Infancy (PANDI) was created to address these issues.      Aims: This study is to verify that the Protocol for the Assessment of Neurodevelopmental Function in Early Infancy (PANDI) can detect infants with developmental weaknesses, predict future developmental delay, and provide understanding of the infant’s status to clinicians, parents, and community providers.    Methods: Premature and full term babies will be assessed at 38-48 weeks gestational age using the PANDI. The Mullen Scale of Early Learning and the Cognitive Adaptive Test/Clinical Linguistic and Auditory Milestone Scale (CAT/CLAMS) will also be scored from this exam. Children will be reassessed using the Mullen and CAT/CLAMS at 12-18 months. Babies that have previously received the PANDI as part of their clinical care will also be contacted for follow-up using the Mullen and CAT/CLAMS. Surveys will be given to clinicians, parents, and community providers to determine if they thought use of the PANDI was helpful. Surveys and results from the PANDI will be compared to these other tests to determine aims.          Results: Preliminary data suggests that the PANDI can diagnose newborns with neurodevelopmental delays better and quicker than other assessments and that clinicians, parents, and community providers feel that the PANDI meets their needs more than current standards of care.