The function of voltage-gated sodium channels has been well characterized, however its potential role in producing a more metastatic phenotype of prostate cancer (PCa) is not well known. beta-subunits of sodium channels have immunoglobulin (Ig) loops of the V-set family that are known to interact homotypically and heterotypically as cellular adhesion molecules. We hypothesize that the beta subunits contribute to metastasis to the spine through perineural invasion by interacting with other V-set Ig-molecules. Western Blot for the SCN2beta subunit was performed on cells of the isogenic human PCa progression model: LNCaP, C4-2, and C4-2B4. Imunofluorescence also was performed on the C4-2B4 cell line. Digitonin-derived whole cell extracts from C4-2 cells were subjected to a wheat germ agglutinin (lectin) affinity column to enrich SCN2beta in the samples for western blot analysis. Western Blot analysis of the cells of the LNCaP PCa progression series showed an increasing expression of SCN2beta that corresponded to increasing tumorigenicity, invasiveness and metastatic potential. The protein was found predominantly in triton soluble fractions. Immunofluorescence showed little specific interaction. The C4-2 lysate subjected to lectin chromatography showed little SCN2beta. Although the results of the immunoblot suggest that the expression of SCN2beta increases as PCa progresses to a more metastatic phenotype, the functional significance of its location in triton soluble fractions remains to be determined. Additional troubleshooting or affinity purification of the antibody may be for specificity. Preliminary results with lectin chromatography were disappointing but this method may still prove efficient for enriching beta-subunits. Funded by the INBRE Program- P20RR016472.

Chronic Kidney Disease (CKD) patients are at a higher risk of cardiovascular (CV) mortality than progressing to end stage renal disease, and their risk of CV disorders is greater than would be predicted by traditional risk factors. The increase in CV disease in these patients may be the result of arterial stiffness secondary to endothelial dysfunction as a result of oxidative stress. To determine if oxidative stress is the mechanism underlying endothelial dysfunction and arterial stiffness, CKD patients and healthy control subjects are being administered acute intravenous infusions of saline or a supraphysiological dose of ascorbic acid, a potent antioxidant. Infusions are being performed in random order and the ascorbic acid dose is based on each subject’s fat free mass (FFM). Baseline and post infusion measurements of aortic augmentation index (AI), an index of wave reflection, and carotid-femoral pulse wave velocity (PWV) are being recorded using non-invasive applanation tonometry. Additionally, brachial artery flow mediated dilation (FMD), a measurement of endothelial dysfunction, is being assessed using high resolution ultrasound. To date baseline data has been collected on 9 CKD patients (age 62 ± 12 yr; 4 men, 5 women). We have found an AI of 25.4 ± 14.4 %, PWV of 7.9 ± 1.4 m/s, and FMD of 4.3 ± 2.2 % in this group of CKD patients. Infusion studies are ongoing and we hypothesize that ascorbic acid will increase FMD resulting in lower PWV and AI in CKD patients. Source of Funding: National Institutes of Health DK077306

In the recent decades obesity has become an epidemic. Due to the overwhelming effects of obesity on mortality and morbidity more people are turning to bariatric surgery. Recent studies have shown that patients who consistently attend follow-up visits with their surgeon and health care team were more successful with long-term weight loss. It has also been shown that telephone consultations can be used to increase compliance and decrease post-operative complications. Our goal was to determine if regular telephone consultations would increase compliance with follow-up appointments and therefore lead to improved bariatric surgery outcomes. Patients undergoing pre-operative counseling were consented and then randomly assigned to the experimental or control group. The experimental group would receive weekly telephone consultations encouraging them to attend follow-up appointments and addressing any questions. The control group would receive standard care post bariatric surgery. A power analysis determined that the appropriate sample size is 64 patients for both the control and experimental group. To date only 39 out of the required 128 patients are 6 months post-operation. Therefore, the preliminary results are inconclusive and research is continuing to be conducted. This project described was supported by Grant Number 2 P20 RR016472-08 under the INBRE Program of the National Center for Research Resources (NCRR), National Institutes of Health (NIH).

Regaining the ability to walk is a crucial component of stroke survivors’ rehabilitation. A recently funded NIH project, FastFES, combines fast treadmill training with functional electrical stimulus (FES) to improve the gait pattern of individuals who have suffered a stroke. Stroke subjects often experience an asymmetrical gait pattern due to the paretic leg’s decreased knee flexion and ankle dorsiflexion during the swing phase, and decreased hip extension during terminal stance. Stroke subjects also tend to walk at a slow speed and spend a decreased amount of time on their paretic leg. By training subjects at a faster pace, subjects will walk more symmetrically than at their self-selected pace and achieve greater hip extension. Stimulating the plantar of the paretic leg will increase the force generated at toe-off of the paretic leg to increase the knee flexion during swing, creating a more normal and efficient swing phase. Stimulating the dorsiflexors of the paretic leg will increase ankle dorsiflexion during swing phase to create a more efficient swing phase for that leg. The stance and swing phases of gait must be accurately identified to objectively evaluate each subject’s gait deviations and responses to treatment,. My role in this project has been to identify heel strike and toe off using Visual 3D Software to evaluate kinetic and kinematic data. Funding for this research has been provided by NIH Grant R01 NR010786 and the University of Delaware’s Science and Engineering Scholarship.

The bacterium Yersinia pestis causes pneumonic plague, a fatal infection for which no vaccine is currently available. Given that the current vaccine generates robust humoral immunity yet does not protect against pneumonic infection suggests humoral immunity alone is insufficient protection. We have hypothesized that cell-mediated immunity is required in addition to humoral immunity for complete protection. In order to study T cell mediated immunity, we have developed a vaccine model using an attenuated strain Y. pestis KIM D27 and convalescent serum that results in the production of T cell-meditated protection against infection. Convalescent serum is produced by low-dose Yersinia infected mice and most importantly, this protective response is only generated in the presence of convalescent serum. Our goal is to understand the development of this protection as well as investigate serum interaction with antigen-presenting cells and Yersinia pestis. Here, we developed an ELISA assay to determine Yersinia specific total IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM antibody titers. In addition, we are studying the portion of the antibody that is required to provide protection against infection. We have determined using in vivo experiment that the Fc receptor is not required for protection. Lastly, we are developing assays to digest antibodies from convalescent serum into F(Ab)’2 and column purify these fragments in order to further study the portion of the antibody that generates this protective response. Understanding this cell-mediated protective response will aid in the development of a vaccine that generates both humoral and cell-mediated immunity.

Understanding serotherapy-mediated cellular protection against Yersinia infection
Torsten A. Joerger, Sohil N. Golwala, and Michelle A. Parent
Department of Medical Technology

Yersinia pestis, a facultative intracellular pathogen, is the causative agent of plague and has been implicated in millions of deaths throughout human history. There are currently on going efforts investigating Yersinia infections with the hope of one day developing a vaccine. In many bacterial infections cytokines play an important role in host defense. It is a well known fact that TH1 cytokines such as IL-12 and IFN-gamma are required to protect against intracellular bacterial pathogens. Past experiments suggest that the cytokine IL-12 in the presence of convalescent serum is required to develop T cell mediated immunity against Yersinia infection. In order to conduct these experiments, we will use an attenuated strain of Yersinia pestis, KIM D27, which has spontaneous 102 kb chromosomal deletion and renders it unable to acquire iron. The Yersinia pestis KIM D27 previously was used as a vaccine strain and provided protection against pneumonic plague. However the vaccine caused a large percent of the healthy study group to become sick and thus cannot be used on the general population. Previous experiments have shown T cells are able to protect against KIM D27 pneumonic infection and that IL-12 is required for this protection. We hypothesize that the IL-12 P40 subunit is required to survive infection and that it has a role in IFN production. Here, we will study the role of these TH1 type cytokines in Yersinia pestis infection and confirm microarray data, which shows changes in gene expression of IL-12p40, p35.

Melanoma is a form of cancer that originates as a malignant tumor from pigment cells called melanocytes and aggressively metastasizes to other parts of the body. Disintegrins are composed of short amino acid sequences that bind to integrins and subsequently block interactions of ligands with receptors. Previous studies have shown that the disintegrin eristostatin increases lysis of SBcl2 melanoma cells by natural killer-like TALL-104 cells. In this study, cytotoxicity assays based on the release of adenylate kinase were performed on six melanoma cell lines to determine whether eristostatin has the ability to increase natural killer cell lytic ability. We expect that eristostatin will increase the lytic ability of natural killer cells on melanoma. Cell surface marker studies were also performed on all six melanoma lines to help characterize the mechanism of enhanced lysis. It is possible that eristostatin is capable of changing the cell surface repertoire. Understanding eristostatin’s impact on the innate immune system is an integral part of this project.

The ability of Natural Killer (NK) cells to lyse cancerous cells has been well documented. Interestingly, it has previously been shown that 4μM eristostatin, a snake (Eristocophis macmahoni) venom disintegrin, elicits a 2-fold increase in the lytic ability of TALL-104 cells, NK-like cells, towards SBcl2, a radial growth phase melanoma cell line. In order to translate this finding to NK cells, it is important to investigate the secretion of the cytokines Interferon-Gamma (IFN-γ) and Tumor Necrosis Factor-alpha (TNF-α) in the presence of melanoma cells. In this study, the effect of Eristostatin on Natural Killer cell secretion of IFN-γ and TNF-α in the presence of four different melanoma cell lines was examined; 1205Lu, M24Met, MV3 (metastatic growth phase) SbCl2 (radial growth phase) . When Natural Killer cells were incubated with 1205Lu there was a significant increase (14 fold with the 1:5 target cell to effector cell ratio) in the secretion of IFN-γ in the presence of 3μM Eristostatin. No significant TNF-α increase was observed with 1205Lu under the same conditions. Other cell lines tested (M24Met, MV3, SbCl2) did not show such an increase in TNF-α or IFN-γ.

Background: The National High School Federation (NHSF) recently reported a gain in soccer participation amongst high school females to over 330,000 players nationwide; pointing to the growing popularity. Soccer is a contact sport and therefore the participants risk injury, including head injuries. Interestingly, the activity most frequently associated with concussions is the act of heading the ball. Alarmingly, females respond differently to concussions when compared to their male counterparts. Objective: The purpose of this study is to determine if there are any changes in concussion symptoms, neuropsychological test performance, and balance after an acute bout of purposeful soccer heading in players with and without prior history of concussion. Methods: A total of 40 female soccer players will be recruited and divided into four groups based on concussion history. Subjects will complete a baseline Concussion Symptom Checklist, a computerized neuropsychological test, and a series of balance tests prior to performing the first soccer heading session (rotational or linear heading). During this session they will perform 15 purposeful “headers” over a 15 minute time frame. Afterwards, the subject will repeat the baseline tests described above. Following 7 days, subjects will return for another heading session (rotational or linear heading) utilizing similar test procedures. Data Analysis: For each subject we will measure the number of previous concussions, differences in concussion symptoms pre and post heading, the composite scores from the ImPACT testing, and the Balance Error Scoring System (BESS) scores. Analysis of variance (ANOVA) techniques will be used to test within subjects (type of heading [rotational vs. linear] & time [pre vs. post]) and between subjects (concussion grouping) data. This project is funded by a grant from the University of Delaware Women’s Studies Program.

Disintegrins are low molecular weight molecules derived from snake venom that bind to integins on a cell’s surface. Eristostatin is a disintegrin that prevents lung colonization of melanoma cancer in a murine model. It also inhibits human platelet aggregation. The actual mechanism that is involved in the inhibition caused by eristostatin is unknown; however there is confocal evidence that eristostatin is binding to the surface of melanoma cells. In this study, Crosslinking and Atomic Force Microscopy are used to try and determine with which integins on the surface of the melanoma cells eristostatin is interacting and at what degree of unbinding force. The crosslinker used was Sulfo-SBED, which is a photo-reactive crosslinker with a biotin tag. Strepavidin was used to isolate crosslinked material. With cross linking, we evaluated melanoma cell lines: WM164, MV3, and platelets from human blood to check the activity of the crosslinker. Crosslinking with the cell lines did not display any crosslinked proteins; suggesting that eristostatin was not binding or the crosslinker was not active. Using the Atomic Force Microscope (AFM), eristostatin was attached to the tip and WM164 melanoma cells coated the surface of a 35mm culture plate. As the tip was engaged with the surface and retracted, and unbinding force curve was calculated. We determined that the concentration of eristostatin plays a role in how strong the unbinding forces are. This could be due to the fact that when the protein is in higher concentration, we are visualizing more than one binding event.

The application of heat to skin tissue has many advantages in the wound healing process. Raising the tissue temperature promotes a variety of inflammatory processes, including leukocytosis, drainage, vasodilation, suppuration, and overall healing. Other studies have demonstrated that preoperative warming did decrease the incidence of wound infections following clean surgery. This knowledge laid the foundation for this research study in which the application of a heating pad prior to a Cesarean section (C-section) delivery was predicted to decrease the wound complication rate post procedure. Mothers scheduled for non-emergent C-sections were invited to participate. Once informed consent was obtained, subjects were randomized to one of two groups: routine care which involved no heating pad, or the application of a heating pad for 30 minutes within two hours of surgery. Subjects were called two weeks following their surgery to determine any incisional problems. To date, 161 subjects have been enrolled. Interim data analysis has shown no significant difference in wound complication between those subjects who received the heating pad and those who received standard care. However, the target sample size (N=250) has not been reached leaving the study underpowered and at risk for a type two error at this point in time. Supported by INBRE.

This study was conducted in an attempt to determine whether a higher quality of life is prevalent among those patients who have had pulmonary lobectomies using a video-assisted thoracic surgery (VATS) approach rather than a thoracotomy (THOR) approach. The research was done by evaluating information available in the Helen F. Graham Cancer Center thoracic surgery database and by surveying both THOR and VATS patients on aspects of quality of life. Once the two groups were finalized, the length of stay was analyzed using the information provided by the database. The VATS patients had a lower average stay than the THOR patients, a difference that showed to be statistically significant (p=0.007). Other collected data that also indicated a higher quality of life among the VATS patients included the length of time on pain medications, pain level one week after surgery, and length of time until able to return to work. The conclusions drawn from this study indicate that a higher quality of life during the initial post-operative period exists for patients who have pulmonary lobectomies via VATS rather than by thoracotomy. Supported by INBRE.

The National Center on Elder Abuse estimated 1-2 million Americans age 65+ were victims of abuse by a caregiver (2005). Elders are projected to comprise 20% of the US population in 2030. Frail elders with health care needs are at greatest risk for mistreatment by their caregivers. This study aims to elicit perceptions of elders about physical abuse as a kind of mistreatment. The first phase of the project, presented here, is the scale development. Kane’s (1991) family social support theory grounds construction of the scale and proposes that the more stable a family the more substantial the reciprocity, advice and feedback, and emotional involvement. This proposition will guide data interpretation with an assumption that the less stable the family the more likely they are to have destructive behaviors. A vignette design with Likert scaling method was chosen for the construction of the scale because it allows examination of context (Martin, 2004) while also presenting a sensitive topic in a non-threatening manner. A 30-item, 4-point Likert scale with three scenarios, each depicting a living situation is presented. In each scenario three different abusive themes and three different barriers to reporting are presented.  Respondents rate the abusiveness of a situation and their likelihood to reporting it. Perceived definitions and likelihood of reporting are important because whether or not elder victims would report “abuse” is meaningless without knowing how elders perceive abuse. Since it is unlikely elders would self-report (Fulmer et al, 2004), these data are essential to health care professionals in screening for abuse. Funding: NIH grant 2 P20 RR016472-08 from NCRR & Buxbaum Scholarship.