Project Summaries for Summer 2010 Session
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Title: Isoproterenol-Induced Cardiac Injury and Mitochondria-Targeted Cardioprotective Strategies: Responses to Carvedilol
Medical Student Research Fellow: Charles Allderdice
Faculty Preceptors: Yao Sun, M.D., Ph.D.
Karl T. Weber, M.D.
Syamal K. Bhattacharya, Ph.D.
Robert A. Ahokas, Ph.D.
Acute stresses on the heart result in activation of the hypothalmic-pituitary-adrenal (HPA) axis. Such activation results in an excess release of catecholamines, driving excess calcium (Ca2+) into the cells and the mitochondria. The necrotic cell pathway that occurs in cardiomyocytes in response to these catecholamines has been shown to initiate in mitochondria due to such calcium overloading. This leads to a passive loss of membrane potential, ATP depletion, mitochondrial swelling, a rupturing of the outer membrane, and overproduction of reactive oxygen species (ROS)[1,2,3]. Such overproduction overwhelms the cells endogenous antioxidant defenses. Without these defenses, cardiomyocyte necrosis will set in.
Carvedilol is an alpha and beta-blocker that is commonly used for tachycardic treatment. In the current study, Carvedilol was used to examine its ability to inhibit Ca2+ entry via the mitochondrial Ca2+ uniporter. Eight week old Sprague-Dawley rats were pre-treated for ten days with Carvedilol. On day eleven, all rats were treated with Isoproterenol, a beta agonist. This synthetic catecholamine was used to simulate an acute tachycardic event. All rats were sacrificed two hours later. Left ventricular apexes were harvested to examine mitochondrial calcium overloading as well as ROS production. Results showed a reduction in mitochondrial calcium overloading and subsequent reactive oxygen species production.
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Title: Quantification and Evaluation of Patterns of Brain Injury Utilizing MRI
Segmentation Techniques in Children with Sickle Cell Disease and a
History of Prior Stroke
Medical Student Research Fellow: Patrick W. Aldred
Faculty Preceptor: Kathleen Helton, M.D.
The purpose of this study was to utilize objective quantitative magnetic resonance imaging (MRI) methods to differentiate white matter hyperintensities and regions of large or small strokes from normal brain and cerebrospinal fluid in children treated for Sickle Cell Disease. Whole brain volumetric segmentations were used to compare the efficacy of traditional transfusion with iron chelation therapy to alternative hydroxyurea therapy and phlebotomy for secondary stroke prevention of 149 SCA patients with prior history of stroke. A combined MRI set consisting of T1-weighted, T-2 weighted, and fluid-attenuated inversion recovery (FLAIR) images were mapped to a spacially normalized atlas and analyzed with a neural network segmentation based on a Kohonen self-organizing map (SOM). Segmented outputs were manually classified to identify the most hyperintense regions. A second SOM was trained to classify remaining normal brain regions as either normal brain white or grey matter, blood vessels or CSF. A pixel counting ImageJ script was used to classify segmented volumes by whole brain, quadrant, and vascular territory. Baseline studies were compared to off study images obtained up to three years following baseline. Preliminary data indicates no significant difference between the two segmentation outputs, implying no significant difference between the two treatment methods. Whole brain volumetric segmentations based on quadrant and vascular territory showed no significant difference in the patient population over the three year time course indicating hydroxyurea therapy may be equally effective therapy for SCA patients for secondary stroke prevention,while not causing the serious side effects of traditional transfusion therapy.
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Title:PCTV transport from ER to Golgi in Enterocytes
Medical Student Research Fellow: Matthew Barnes
Faculty Preceptor: Charles M. Mansbach, II, M.D.
The rate-limiting step in transporting fat across intestinal epithelial cells is the formation of pre-chylomicron transport vesicles (PCTV), which transport fat from the ER to the Golgi. It has been previously shown that recombinant liver fatty acid binding protein (L-FABP) is able to form PCTVs in the absence of ATP (1). However, initiation of PCTV budding in native cytosol is dependent upon ATP. Additionally, our lab has shown that L-FABP exists in cytosol as a ~75 kDa protein complex, but the protein that is primarily phosphorylated is small valosin interactive protein (SVIP) at ~9 kDa (2). This study addresses both the energy requirements for the formation of PCTVs, as well as an initial investigation into the association between L-FABP and SVIP. Using radioactively labeled ER to perform budding assays in the presence of an ATP generating system, the requirement of ATP for the formation of PCTV in native cytosol was confirmed. L-FABP was found to be associated with SVIP on SVIP immuno-precipitation from native cytosol suggesting that SVIP and L-FABP interact in the cytosol.
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Title: The effect of selective inhibition of NFκB on the anticancer activity of
IFN in glioblastoma multiforme
Medical Student Research Fellow: Jessica Bauman
Faculty Preceptor: Lawrence M. Pfeffer, Ph.D.
Brain tumors are among the leading causes of cancer-related death in the United States, with glioblastoma multiforme (GBM) being one of the most difficult subtypes to treat. Adjuvants to surgical resection, including chemotherapy and radiation, currently provide little improvement in the disease course and few patients are cured. Type I (α/β) interferons (IFNs) have long been recognized for their significant, pleiotropic anticancer activity in vitro against a variety of tumor types including gliomas, but the efficacy of IFNs in clinical trials has been disappointing. We believe that a significant contributing factor to the development of resistance to IFNs is through the activation of anti-apoptotic transcription factors, such as nuclear factor κB (NFκB). This finding suggests that the potency of IFNs may be limited by NFκB-mediated upregulation of anti-apoptotic and/or prosurvival genes. Based on these observations, we hypothesized that selective NFκB blockade will enhance the anticancer activity of IFNs in the treatment of glioblastoma multiforme. GBM cell lines U87 and MT330 were treated with the NFκB inhibitors Bortezomib and BMS-345541 singly or in the presence of Type 1 IFNs. We observed enhanced cell death in both lines with combination treatments compared to single agents, an effect that increased with increasing duration of treatment and concentrations of NFκB blockers. This study provides the first evidence of the effectiveness of combinations of NFκB inhibitors with IFN in long-term in vitro treatment of GBM cell lines, and provides rationale for the further examination of these drug regimens in preclinical models.
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Title: Molecular Mechanisms of Mitigation of Insulin Induction of SREBP-1c and Hepatic
Lipogenesis: The Effect of n-3 PUFA on LXRα-RXRα Heterodimer Formation and
Examination of Interactions Between LXRα and its Trans-acting Factors
Medical Student Research Fellow: Davis Berry
Faculty Preceptors: Marshall B. Elam, M.D., Ph.D., FAHA
Xiong Deng, Ph.D.
Regulation of SREBP-1c and its mechanisms are the primary focus of this laboratory. Specifically, the insulin responsive promoter of the rat SREBP-1c gene is in question. Tandem LXRα response elements (LXREs) are a primary mediating site. In previous studies, polyunsaturated fatty acids (PUFA), specifically n-3 PUFA, have been shown to reduce the transcription of SREBP-1c. This effectively inhibits the effects of insulin induced hepatic lipid production. The mechanism by which n-3 PUFA reduces the transcription of SREBP-1c, however, is not yet understood. In this case, we will determine if n-3 PUFA reduces the trans-activating capacity of LXRα by preventing the binding of oxysterol ligand activators by competitive displacement. We hypothesizes that this is one of the mechanisms by which n-3 PUFA may inhibit SREBP-1c transcription.
PCR was used to amplify cDNA for the LXRα ligand-binding domain (LXR LBD). The PCR product was ligated into pET-28a expression vectors. The pET-28/LXR LBD plasmid was transformed into Escherichia coli strain BL21. Cultures grown in a Luria-Bertani medium with 1% glucose added were induced 0.1 mM IPTG overnight at 20°C. Pellets were suspended in lysis buffer. The induced his tagged protein was recovered on a nickel column.
Summary of Results:
We obtained a purified protein at the conclusion of the summer.
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Title: Autoimmunity in Retinal Degenerative Diseases
Medical Student Research Fellow: Aleksandr Birg
Faculty Preceptor: Alessandro Iannaccone, MD, MS
Purpose: To report the clinical and functional characteristics of a retrospective case series of patients with Autoimmune Neuro-Retinopathy (AINR). Analyze imaging data to determine the type of retinal, nerve fiber layer, and RPE changes that may emerge as characteristic, if any, in AINR patients.
Methods: Twenty-five consecutive cases of AINR referred to rule out Retinitis Pigmentosa (RP) or other related disorders were ascertained. Complete optical coherence tomography (OCT) data were available for 22 of them [M=8; F=14; 21-89 years old (yo)] and the present analysis was limited to these cases. The diagnostic suspicion of AINR was based on the results of presentation, Goldmann visual fields, pattern visual evoked potentials (PVEPs) and flash electroretinograms (ERGs), and it was confirmed by diagnostic Western blots (WB) of serum samples for anti-retinal and anti-optic nerve auto-antibodies (Auto-Abs). Age of onset, modality of onset, and type of visual symptoms, history of cancer, and fundus features were analyzed. Custom segmentation analyses were performed on OCT retinal scans to ascertain specific layer differences.
Results: Clinical Findings - All patients presented with widely variable acute or sub-acute symptoms between age 21 and 89 yo (mean ± SD: 57±14.4). Photopsias at onset were seen in 43% of cases. Visual loss and/or fundus findings were asymmetric between eyes in 65% of cases. Seven of the 22 cases had history of, or subsequent diagnosis of cancer (paraneoplastic AINR). Anterior chamber cells, indicative of uveitis, were seen intermittently in 13% of cases. All but one case had changes (pallor, hyperemia/swelling) at or around the disc. Juxtapapillary pigmentation or atrophy were observed in 16 (70%) of cases, and punched-out peripheral retinal lesions in 10 (43%). Retinal vessels were attenuated only in 43% of cases and had sheathings in 9%. Macular changes were seen in 65% of cases. Peripheral changes included RPE hyperpigmentation or loss (67%), and bone spicule-like deposits, mainly paravascular and sparse, in only 22%. Functional Findings - Unlike RP, GVFs showed optic nerve (ON) and/or retinal ganglion cell (RGC)-related defects in 55% of cases and asymmetric loss in 55%, and ring scotomas and constriction with peripheral islands were absent in most (75-80%) cases. PVEPs were delayed in 85% of cases. Interocular timing and/or amplitude PVEP asymmetry was seen in 65% of cases each, and in all but one case (95%). Rod-driven and mixed ERGs ranged from non-recordable (30 and 10%, respectively) to normal but with asymmetric amplitudes (40-45%). Although photopic ERGs were reduced in 70% of patients and delayed in 60% (flicker) to 90% (transient) of cases, transient responses were always recordable. Asymmetric photopic amplitude loss was apparent in 35-40% of cases. Imaging Findings - ... Serological Findings - Numerous distinct reactive bands were identified by WB, α-enolase (46-kDa) being the most common (43%). More than half of the cases had ≥2 Auto-Ab bands, and as many as five. Patients testing positive for the autoantibody, α-enolase, were shown to have a statistically significant increase in retinal nerve fiber layer area when compared to control patients (p=0.0304). However, the average retinal thickness of AINR patients was comparable to control patients, meaning retinal thickness cannot be used to identify AINR.
Conclusions: Unlike patients with RP, patients with AINR present with the recognizable clinical and functional features of: late- and sudden-onset symptoms; inter-ocular asymmetry of visual loss; ophthalmoscopic findings most often characterized by (i) papillary and juxtapapillary changes, (ii) punched-out peripheral retinal lesions and (iii) macular punctate RPE dropout; GVF loss patterns attributable to ON/RGC damage, alone or combined with retina-driven changes; VEP timing delays; an unusually high proportion of normal rod/mixed but abnormal cone ERGs; and interocular asymmetry in visual function loss. Recognition of these highly suggestive features should prompt an Auto-Ab work-up and can aid in diagnosing these patients correctly and promptly. As more patients with AINR are recognized and serotyped, more accurate correlations could be drawn between autoantibodies and their effects on distinct retinal layers.
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Title: Does Thyrotoxicosis Predispose One to Cocaine Abuse?
Medical Student Research Fellow: Ryan Black
Faculty Preceptors: Vijaya Vasudevan, M.D.
Ben Bowman. M.D.
Solomon S. Solomon, M.D.
Purpose of Study: Thyrotoxicosis is characterized by increased levels of thyroid hormones (TH) and suppressed TSH. This produces increased sensitivity to catecholamines by TH mediated upregulation of beta adrenergic receptors. Thus, a person with thyrotoxicosis may both crave and be sensitive to adrenergic agonists. Cocaine is a sympathomimetic drug which mimics adrendergic agents. Hence, it is possible that subclinical thyrotoxicosis (i.e. suppressed TSH) predisposes individuals to cocaine abuse if they are in such a facilitated setting. In addition, thyrotoxicosis cause alterations in the hypothalamic-pituitary-adrenal axis which has also been associated with cocaine abuse in animal models.
Methods: Using the computerized patient record system (CPRS) at the VAMC in Memphis, TN we identified three groups of patients: a) cocaine abusers who are not thyrotoxic by clinical and/or laboratory measurements; b) subclinically or overtly thyrotoxic individuals; and c) thyrotoxic individuals with a history of cocaine abuse. Using 1191 patients taken from the same clinic population, we calculated the prevalence of cocaine abuse alone, thyrotoxicosis alone, and the prevalence of both diagnoses together to examine if a link exists between thyrotoxicosis and cocaine abuse.
Summary of Results: We found a prevalence of: a) 37.6/1000 patients with cocaine abuse; b) 18.8/1000 with thyrotoxicosis; and c) 65.4/1000 with both thyrotoxicosis and cocaine abuse. We found a highly statistically significant correlation between thyrotoxicosis and cocaine addiction in this balanced, heterogeneous VA population (p<0.0001 by Chi-square test). No other significant correlations were found. In addition, we found no significant differences in thyroid function tests or age-at-diagnosis of thyrotoxicosis between cocaine abusing and non-cocaine abusing thyrotoxics.
Conclusions: Because it has been previously demonstrated that cocaine abuse does not affect thyroid function, it appears that thyrotoxicosis is a highly significant risk factor for cocaine abuse in the right setting. While there are several theoretical mechanisms to explain this relationship, further prospective studies are needed before definitive conclusions can be drawn.
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Title: Inhibition of Rho-ROCK signaling is required for sAPPα-mediated neurogenesis
Medical Student Research Fellow: Alex Jordan Chinn
Faculty Preceptor: Francesca-Fang Liao, Ph.D.
Cholesterol-lowering statins have been shown in previous studies to increase the secretion of the soluble form of amyloid precursor protein (sAPP), which is known to have many functions, including synaptogenesis, neurite outgrowth, cell survival and cell adhesion. Statins also reduce the likelihood of developing Alzheimer's disease, but this clinical use is still debated. Statin drugs inhibit the production of cholesterol intermediate mevalonate, which is also used for isoprenylation of proteins, such as the Rho family of signaling proteins. This suggests that sAPP-mediated beneficial effects such as in adult neurogenesis may be caused by the disruption of Rho signaling after interruption of isoprenylation. The experiments performed in this study attempt to test this hypothesis.
This pathway was tested using Y-27632, an inhibitor of Rho-associated coiled-coil-containing kinase (ROCK), which is activated by binding Rho. The inhibitor was initially tested for its ability to increase expression of sAPP in cultured cells at doses of 10 μM and 20μM. The lower dose caused increased expression of sAPP within 24 hours, but the higher dose was effective within one hour. ROCK inhibitor Y-27632 was then tested in vivo by administering single doses at the above concentrations by intracerebroventricular (i.c.v.) injection into mouse CNS. No adult neurogenesis was found in either the control group or the treatment group at ages of 4 months (n=3). There was not sufficient time remaining in the training period to allow a second attempt at this experiment. However, since a basal level of neurogenesis is expected, and a link between Rho-ROCK inhibition and sAPP expression was established, it seems prudent to repeat this experiment in the future.
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Title: Isoproterenol-Induced Cardiac Injury and Mitochondria-Targeted Cardioprotective
Strategies: Responses to Quercetin
Medical Student Research Fellow: Brian H. Cohen
Faculty Preceptors: Robert A. Ahokas, Ph.D.
Karl T. Weber, M.D.
Syamal K. Bhattacharya, Ph.D.
Yao Sun, M.D., Ph.D.
An acute stressor event, i.e. head injury, car accident, or surgery, activates the hypothalamic pituitary adrenal (HPA) axis, which causes an increase in catecholamines and drives Ca+2 into cells. Isoproterenol, a synthetic catecholamine, simulates the neurohormonal activity and elevation of catecholamines observed during the activation of the HPA axis during an acute stressor state. This increase in catecholamines causes overloading of cardiomyocytes and mitochondria with Ca+2, which leads to reactive oxygen species (ROS), oxidative stress, mitochondrial permeability transition pore (mPTP) opening, and necrosis 5, 7. The ROS produced by Ca+2 overloading the cells potentiates the increase of intracellular Ca+2 in a feed forward, self-amplified loop causing more cellular damage 21. We examined the potential for mitochondria specific cardioprotective strategies in order to prevent cardiomyocytes' Ca+2 overloading and necrosis during a hyperadrenergic acute stressor state. Therefore, we propose that the pretreatment of rats with quercetin, a natural polyphenolic flavonoid with mitochondrial specific antioxidant properties, before introduction of a synthetic catecholamine, isoproterenol, will be cardioprotective and prevent cellular necrosis. Quercetin's proposed mechanism of action for cardioprotection is its antioxidant capacity to scavenge free radicals and ROS; therefore, attenuating cardiomyocytes and mitochondrial Ca+2 overloading that leads to necrosis. Our study observed that rats pretreated with quercetin compare to isoproterenol treatment showed a/an 1) decrease in mitochondrial free Ca2+, 3) decrease in mitochondrial H2O2, 3) attenuation of mPTP opening, 4) total antioxidant capacity (TAC) that was maintained near control levels. All data was analyzed and were significant with p values <0.05. We conclude that by using quercetin we canpharmacologically attenuate Ca+2 overloading in the heart during a hyperadrenergic acute stressor state. An application of this research might be the pretreatment with quercetin before the acute stressor stare of surgery in order to protect the heart from the damages of excess catecholamines.
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Title: β1 subunit of BK channel is essential for NO induced vasodilation of intact
Medical Student Research Fellow: Darren R. Cullinan
Faculty Preceptor: Jonathan H. Jaggar, Ph.D.
Trafficking of ion channel protein subunits to and from the plasma membrane is a novel regulatory mechanism. Blocking this transport with Brefeldin A yielded a reduction of myogenic tone of 6.5 μm. It is expected that both protein subunits that lead to vasoconstriction or vasodilation are transported in this manner. Since dilation was seen, the net effect of subunit trafficking leads to vasoconstriction.
When the β1 subunit is associated with the BK channel, it increases its Ca2+ sensitivity which increases the likelihood that the channel will be in the open position. By blocking transport of the β1 subunit to the membrane, the activity of the BK channel is reduced. It has been known for some time that NO activates BK channels, but the mechanism by which this happens has never been well understood. This experiment provides evidence that NO activates transport of vesicles containing the β1 subunit to the plasma membrane, increasing BK channel activity. Blocking transport of the β1 subunit with Brefeldin A produces a reduced vasodilation. When arteries are exposed to NO in the presence of Brefeldin A, vasodilation is reduced by 38% compared to NO alone.
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Title: Role of the Carboxy Terminus of Human Apolipoprotein A-IV in Modulating
Chylomicron Assembly and Secretion
Medical Student Research Fellow: Stanton Elseroad
Faculty Preceptor: Dennis D. Black, M.D.
A human apo A-IV mutant with a deleted EQQQ-rich carboxy terminus similar to native swine apo A-IV was previously shown to increase chylomicron triglyceride (TG) secretion 25-fold using IPEC-1 newborn swine intestinal epithelial cells and a Tet-On expression system, as compared to native human apo A-IV, suggesting an inhibitory function of this unique carboxy terminus. The observed increase was 2-fold for native human apo A-IV and 5-fold for native swine apo A-IV. This project demonstrated that a swine apo A-IV mutant containing the unique human EQQQ-rich carboxy terminus negatively modulated TG secretion in comparison to both native swine and native human apo A-IV. Additional mutants tested included a swine apo A-IV mutant with a randomized human carboxy terminus amino acid order and a human apo A-IV mutant with a human carboxy terminus containing all neutral amino acids. Both reduced chylomicron TG secretion, indicating that the unique amino acid order and charge of the EQQQ carboxy terminus are not necessary for the negative modulation of TG secretory enhancement observed for native human apo A-IV. These results significantly contribute to our understanding of the mechanism of regulation of intestinal lipid absorption by apo A-IV.
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Title: Targeting NF-κB and the Vitamin D Receptor in Melanoma Therapy
Medical Student Research Fellow: Emily Escue
Faculty Preceptor: Andrzej T. Slominski, M.D., Ph.D.
Melanoma, the most aggressive form of skin cancer, is highly resistant to current modalities of therapy, with melanogenesis playing an important role in resistance. NF-κB is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action. High NF-κB activity is present in many melanoma lines observed in this study, with some of them displaying higher nuclear NF-κB expression and others lower. Novel vitamin D derivatives 20-hydroxyvitamin D3 (20(OH)D3) and 20,23 dihydroxyvitamin D3 (20,23(OH)2D3) and classical 1α,25-dihydroxyvitamin D3, or calcitriol, (1,25(OH)2D3) inhibited melanoma cell proliferation, and this effect was seen only in the presence of interferon α (IFN). The NF-κB inhibition by Vitamin D3 (20,23(OH)2D3) and IFN was confirmed by an NF-κB DNA binding assay, while 1,25 (OH)2D3 and 20 (OH)D3 treatment of melanoma cells showed no effect. In summary, high basal NF-κB activity is seen in most of the melanoma cells, pigmented and nonpigmented. In addition, the induction of melanization is associated with the inhibition of proliferation of melanoma. Vitamin D derivatives, along with interferon, are a promising treatment for melanoma.
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Title: Oxidative Stress in Leukocytes from Non-Obese Diabetic Mice
Medical Student Research Fellow: Jamis Gouge
Faculty Preceptor: Ivan C. Gerling, Ph.D.
Insulin-Dependent Diabetes Mellitus (IDDM), or type 1 diabetes mellitus, is an autoimmune disorder in which leukocytes progressively destroy the pancreatic beta islet cells, eventually leading to the cessation of insulin production by the islets and subsequent insulin dependence. The process through which this occurs is not fully understood, but we believe it involves a defect in leukocyte mitochondria.
Our studies were carried out using Non-Obese Diabetic (NOD) mice, which spontaneously develop lesions in the Islets of Langerhans at about 5 weeks of age and diabetes from the age of 12 weeks. We measured 1) the levels of the pro-oxidant Ca2+ and the anti-oxidant Zn2+ in mouse spleen mononuclear leukocytes (PBLs) because they serve as indicators of mitochondrial damage in IDDM 2) evaluated the mitochondrial levels of radical oxygen species (ROS) in mouse PBLs because they serve as indicators of oxidative stress, and 3) evaluated the mitochondrial membrane potentials (MMP) within mouse PBLs because they serve as indicators of mitochondrial damage.
We found no differences in the levels of Ca2+ or Zn2+ in NOD compared to control mice. However, the levels of ROS were higher in NOD mice than in C57BL/6 controls. The MMP was also higher in NOD mice than in C57BL/6 mice. Those differences were particularly high in the B-lymphocyte subset of PBL's.
This data show that PBLs from NOD mice suffer an increased level of oxidative stress although this is not due to any abnormalities in the balance between the pro- and anti-oxidant divalent cations Ca2+ and Zn2+. In spite of increased ROS the mitochondria do not appear to sustain damage to the extent that it causes loss of membrane integrity (and MMP).
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Title: Comparison of High Protein and High Carbohydrate Diets on Levels of
Inflammation in Obese Pre-Menopausal Women
Medical Student Research Fellow: Mustafa Haddad
Faculty Preceptors: Frankie B. Stentz, Ph.D.
Abbas E. Kitabchi, M.D., Ph.D.
Background: Reduction of weight and inflammation is important in obese individuals in order to prevent complications such as diabetes, cardiovascular disease, and other metabolic complications. Literature shows high protein diets are more effective at reducing weight and inflammation while simultaneously providing greater satiety and compliance than the currently ADA accepted high carbohydrate diet. Some literature however shows that HP diets induce ketoacidosis. Little literature shows the long term effect of using high protein diets in comparison to high carbohydrate diets.
Hypothesis: High protein diets are more effective at reducing inflammation than high carbohydrate diets and both produce the similar levels of ketoacidosis and weight loss.
Specific Aims: To compare HP and HC diet levels of cardiovascular risk, ketoacidosis, oxidative stress, and insulin sensitivity measuring levels of E-selectin, β-hydroxybutyrate, malondialdehyde, and adiponectin, respectively.
Methods: 15 Pre-menopausal obese women were placed on HP(9) or HC(6) diets over 6 months. OGTT and MTT were performed before and 1,3 and 6 months after the onset of the diet. Blood was collected 15 minutes before and 120 minutes after each test. Serum concentrations of the above indicators were measured using enzyme assays in which data was interpolated from a standard curve.
Results: Over a six month period, both diets resulted in similar weight loss and levels of ketoacidosis ( HP=0.217 mM + 0.027, HC= 0.211mM + 0.043, p= 0.56) . The HP diet caused significantly higher levels of adiponectin ( HP=5840 ng/ml + 87, HC= 5327 ng/ml + 68, p≤0.05)and significantly lower levels of oxidative stress ( HP=0.7mM ± 0.05, HC=0.9 ± 0.07, p≤0.05)and E-selectin ( HP= 37.4 ng/ml + 1.2, HC=39.7 ng/ml + 1.6, p≤0.05).
Conclusions: HP and HC diets produce similar weight loss and ketosis, but the HP diet is more effective at increasing levels of adiponectin and lowering levels of oxidative stress and E-selectin. This study's long term results on ketosis differ from past studies. Although both diets were nutritious and palatable diets for long term use, higher levels of satiety and compliance were reported with the HP diet. These results suggest a HP diet could be more beneficial than a HC diet for weight loss, reduction of inflammation, and prevention of metabolic complications.
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Title: Effects of Ibuprofen & KF3589 on Pulmonary Contusions in Rats
Medical Student Research Fellow: Nickalus Khan
Faculty Preceptor: Timothy C. Fabian, MD, FACS
Pulmonary contusions remain associated with a high mortality (8). The current treatment for pulmonary contusions remains only supportive. There are currently no widely accepted pharmacologic treatments for pulmonary contusions. The effects of ibuprofen and KF3538 (P-selectin inhibitor) are investigated in this experiment as treatments for pulmonary contusions. Ibuprofen and KF3538 were administered separately to groups of laboratory rats given pulmonary contusions. Rats were treated post injury then harvested one, three, and seven days post contusion. The results from the treated and non-treated groups were analyzed using gross pathology, serum inflammatory markers IL-6 and TNF-alpha, arterial blood gas values, and bronchialveolar lavage fluid inflammatory markers using ELISA methods. The results of this experiment show that there is a significant difference in the inflammatory marker TNF-alpha from rat serum (p=0.000506<p=0.05). The results show a difference (70%) in serum between treated rats and non- treated for IL-6. However, the IL-6 results are not statistically significant likely due to the small sample size of the preliminary group tested. The remaining results are still in the data collection phase of the experiment and will be analyzed together to determine if the data can show that there is a difference in the outcome of treated and non treated rats. The results of this experiment could change the method in which pulmonary contusions are treated and be used in future clinical trials.
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Title: fMRI Analysis of Functional and Developmental Changes in the Primary Visual Cortex of Retinoblastoma Patients
Medical Student Research Fellow: Benjamin A. King
Faculty Preceptor: Robert J. Ogg, Ph.D.
Retinoblastoma is a rare malignant eye tumor which typically presents in children during the first year of life. Because these tumors arise during a period of rapid neural development, the abnormal sensory experience that results when these tumors eclipse a significant portion of the retina could have adverse consequences for the proper development and function of the visual system. Primary visual cortex (V1) is of particular concern because developmental irregularities in this region can lead to ablyopia-like symptoms which further exacerbate the visual deficits caused by the tumor itself.
We conducted a retrospective study of 90 Retinoblastoma patients to demonstrate the relationship between a given tumor's location on the retinal surface and the resulting functional deficits in V1. T2-weighted magnetic resonance imaging (MRI) of the eye and orbit were used to reconstruct the retinal surface topography on a standard polar coordinate system where the margins of the tumor could be accurately quantified. Functional MRI (fMRI) was used to analyze neural activity throughout V1 in response to photic stimulation presented during the scan. fMRI analysis showed consistently weaker activation signals in areas of V1 which receive inputs from areas of retina which are eclipsed by the tumor mass. These findings will shed further light on the abnormal sensory experience associated with this disease affects brain function and development in this particular patient population.
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Title: Effect of Diet Composition on Weight Change and Metabolic Parameters
Medical Student Research Fellow: Travis Littleton
Faculty Preceptors: Abbas E. Kitabchi, Ph.D., M.D.
Frankie B. Stentz, Ph.D.
Obesity is one of the most prevalent health hazards in the US today, and it causes an increased burden on the body, which increases the risk for a number of diseases. The primary way to lose weight is by diet and exercise. However, the optimal diet has still not been found that provides weight loss without negatively affecting the other metabolic parameters of the people on the diet. The current diet recommended by the American Diabetes Association (ADA) is a diet with reduced caloric intake composed of 55% carbohydrates, 30% fat, and 15% protein. The goal of this research study is to show that a diet with 30% protein, 30% fat and only 40% carbohydrates will show similar weight loss with greater benefit in other areas of health. We believe that 40% carbohydrate is adequate to avoid ketosis, but the increased protein component will provide greater satiety and improve other metabolic parameters. This was carried out by randomizing fourteen women ages 20 to 50 years with a BMI >30 and <55 to either a high protein (HP) diet or a high carbohydrate (HC) diet for 6 months. Weight, blood pressure, waist circumference, chemistry profile, CBC, lipids and oxidative stress markers [(dichlorofluorescein (DCF)] and lipid peroxidation [malondialdehyde (MDA)] were measured on the subjects before the start of the diet and at 1, 3, and 6 months. Glucose and lipids Area Under the Curve for OGTT (AUC) and meal tolerance were calculated for 0, 30, 60, 90, and 120 minutes.
Our study demonstrated significant weight loss of more than twenty pounds per patient and loss of total mass, fat and lean mass in 6 months in both diets. The HP diet appears to provide a greater improvement in oxidative stress and lipid peroxidation as well as reduction in glycemic and triglyceride excursions than the HC diet. These results are in addition to the already known effects of HP diets on greater satiety and decreased insulin release than HC diets.
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Title: Effects of Azithromycin on Macrophage Inflammatory Responses to Streptococcus
Medical Student Research Fellow: Matthew Marker
Faculty Preceptor: B. Keith English, M.D.
Streptococcus pneumoniae is a major cause of pneumonia, sepsis, and meninigits, with a high rate of morbidity and mortality in young children and the elderly. Pneumococcal pneumonia in particular can be devastating, especially when it follows an influenza infection. While lytic antibiotics such as beta-lactams have long been the accepted form of treatment for pneumonia and other serious pneumococcal infections, recent clinical studies have suggested that addition of a macrolide antibiotic to a beta-lactam such as penicillin results in improved outcomes for hospitalized adults with pneumococcal pneumonia. Some evidence from clinical studies and animal models of pneumococcal pneumonia indicates that the addition of azithromycin results in a less severe and less prolonged host inflammatory response. This may be because macrolides such as azithromycin, which inhibit protein synthesis in bacteria, kill the organisms but do not lead to bacterial lysis and thus trigger less release of pro-inflammatory bacterial components. Other studies (e.g., with cystic fibrosis patients worldwide and panbronchiolitis in Japan) have also suggested that macrolides may possess nonspecific anti-inflammatory effects independent of their antimicrobial activity. The goal of this study was thus to determine whether exposure of live pneumococci to the macrolide azithromycin, alone or in combination with the beta-lactam ampicillin, results in reduced secretion of tumor necrosis factor (TNF) by macrophages. Further, to determine if the anti-inflammatory effects of azithromycin are dependent on its antibiotic activity, I used the azithromycin-susceptible reference pneumococcal strain T3L and an azithromycin-resistant subclone of this strain. I found that while adding azithromycin to ampicillin resulted in lower amounts of macrophage TNF secretion in response to both strains, the difference was only statistically significant in response to the azithromycin-susceptible parental strain. I conclude that the anti-inflammatory effect of azithromycin in the in vitro model is at least partially dependent on the antimicrobial activity of the drug.
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Title: The relationship between FGF-23 and left ventricular mass and early atherosclerosis in children with chronic kidney disease.
Medical Student Research Fellow: Joseph Merriman
Faculty Preceptor: Deborah P. Jones, M.D., M.S.
FGF-23 has been shown to be an important prognostic factor for cardiovascular morbidity in adults with chronic kidney disease (CKD). Patients with CKD are at a higher risk for cardiovascular disease than the general population. Studies have shown that adult patients have elevated circulating levels of FGF-23 while studies relating FGF-23 to cardiovascular disease in children, however, are lacking. Thus, we conducted a cross-sectional study to test the hypothesis that serum FGF-23 levels are associated with left ventricular mass and arterial stiffness. In order to gain insight into the role of FGF-23 we will enroll a total of 60 children ages 8-21 years of age with CKD: ten patients in each stage of CKD 1-4, ten CKD stage 5 patients, and ten post-renal transplant patients. We expect to find that FGF-23 is independently associated with left ventricular hypertrophy and arterial stiffness.
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Title: Gluucoregulation in offspring of diabetic and non diabetetic parents
Medical Student Research Fellow: Cherechi C. Ogwo
Faculty Preceptor: Samuel Dagogo-Jack, MD, MBBS, FRCP
This study was performed in order to compare the glucose and insulin excursions during OGTT in 33 normoglycemic offspring of diabetic or non-diabetic parents that were matched in a case controlled fashion for age, race, gender and BMI. Normoglycemia was defined as fasting plasma glucose ≤99mg/dl and 2hour post glucose load plasma glucose level of ≤139mg/dl. The levels of insulin resistance and β-cell function were also compared between the two groups. We measured the plasma glucose and insulin levels at 0min, 30min and 120 min during 75g OGTT. Estimation of β-cell function and insulin resistance was made using the HOMA- B and HOMA- IR formulae respectively. Results showed no statistical difference in glucose and insulin excursions between the 2 cohorts (P= 0.38 and 0.68 respectively). The individuals with parental history of type 2 diabetes had a higher mean insulin resistance than the group without parental history but this difference didn't reach statistical significance (P= 0.06). HOMA-IR and HOMA-B correlated positively with plasma insulin levels in both groups (R= >0.99, P = <0.0001). HOMA-B correlated with BMI in subject without parental history (R=>0.48, P=<0.005), a relationship that was not evident in subject with parental history of diabetes (R= 0.21, P= 0.240). Furthermore while β-cell function in offspring of non-diabetic parents showed no correlation with age (R=0.06, P=0.729), offspring of diabetic parents demonstrated a negative correlation with advancing age (R=-0.370, P= 0.034). The findings of this study suggest that defects in insulin secretion and action may be discernible in the normoglycemic phase in offspring of diabetic parents.
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Title: Determination of Therapeutic Transgene Dose
Medical Student Research Fellow: Anand Patel
Faculty Preceptors: Tonia S. Rex, Ph.D.
Eldon E. Geisert, Ph.D.
The protein Erythropoetin (Epo) has been shown to have neuroprotective effects on many structures of the eye, including retinal ganglion cell axons and photoreceptor cells. Therapeutic use of Epo could be beneficial in the treatment of several conditions including Retinitis Pigmentosa, a genetic disease that affects Rhodopsin expression and causes photoreceptor cell death. The therapeutic effects of Epo are dose-dependent. Using transgene expression and a tetracycline-inducible promoter, Epo can be delivered directly to the eye in controlled amounts and avoid many harmful systemic effects.
VMD2.rtTA.RDS mice were subretinally injected with a viral vector, rAAV 2/1, which delivered the genes for Epo, eGFP, and the bi-directional Tet-response element promoter. The viral vector transduced these genes into photoreceptor cells as well as the retinal pigment epithelium. Five days after the subretinal injection of the viral vector, the mice were subject to intraperitoneal injections of doxycyline, a tetracycline analog. Doxycycline combines with the rtTA transcription factor to form an activated complex that acts as an inducer at the tet-response element. Activity at the bi-directional promoter drives expression of both Epo and the eGFP fluorescent marker.
Fluorescence quantification of eGFP was analyzed to determine the amount of transgene expression taking place. The amount of eGFP fluorescence was indicative of the amount of Epo expressed. However, auto-fluorescence from the mouse fur was also detected in the imaging, making it difficult to distinguish fluorescence originating from the eye or from other part of the body. For this reason, data analysis was impeded and definitive conclusions about transgene expression could not be made. Several modifications to the experiment were made and improvements are currently ongoing.
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Title: Caspase-3 Gene Silencing for Improved Islet Transplantation
Medical Student Research Fellow: Hashani Perkins
Faculty Preceptor: Ram I. Mahato, Ph.D.
Islet transplantation has been one avenue that offers type I diabetics the hope of sustained euglycemia. Nevertheless Up to 70% of islet cells undergo apoptosis within the first week of transplantation (Cheng et al., 2010). It would therefore be of great benefit to inhibit programmed cell death during this period. Apoptosis is a cascade of events due to the up-regulation of either caspase-8 extrinsically and caspase-9 intrinsically leading to increased caspase-3 production. Hypoxia and oxidative stress are involved in intrinsic/mitochondrial pathway. Bcl-2 family of proteins plays an intricate role in apoptosis.
Some (Bcl-2, Bcl-X and Blc-W and others) have anti-apoptotic properties while others (Bax, Bak, Bad, and Bok and others) have proapoptotic properties. The ratio of Bcl-2 to Bax and or Bak determines the cells fate (Mahato, 2009). Similarly elevated glucose and/ or cytokine levels up regulate Fas, which is known to activate caspase-8 through the extrinsic apoptotic pathway (Mahato, 2009). Silencing of caspase-3 alone has not provided a sufficient solution to difficulties in islet transplantation. Many other factors such as failure of islet matrix reformation may play a part in graft failure (Wang and Rosenberg, 1999). Ram Mahato noted that increases beta-cell proliferation and promotes revascularization of islets. With the work of these researchers in mind we contemplated many possible gene cocktails that would propose to improve viability. In this paper we discuss our work with adenoviruses containing X linked Inhibitor of Apoptosis Protein (XIAP) and hepatocyte growth factor (HGF).
Methods: Rat insulin cells (INS) were transfected with two different viral vectors, adv-XIAP and adv-XIAP-HGF-RGD at three different titers, 1x106, 1x107, 1x108. Media was then collected from the different titers for analysis of HGF. Cells were lysed and centrifuged for collection of intracellular proteins. A western blot was performed to visualize the presence and verify expression of XIAP, Caspase-3, and β-Actin. An Enzyme-linked immunosorbent assay (ELISA) was also performed to determine the concentrations of XIAP, and HGF produced by each viral titer.
Summary of Results: In both cases, a dose dependent increase in the production of XIAP was observed. Figure 1A shows a increased production as a result of transduction with Adv-XIAP. Figure 1B demonstrates the dose dependent increase in XIAP expression as a result of transduction with Adv-XIAP-HGF-RGD. Figure 2 demonstrates the dose dependent increase in HGF in cells transduced with adv-XIAP-HGF-RGD. Figure 3 shows a western blot performed on cells transduced with Adv-XIAP and Adv-XIAP-HGF-RGD demonstrating no change in β-actin and expression of XIAP corresponding to MOI.
Conclusion: By down regulating the apoptotic protein Caspase-3 through the anti apoptotic XIAP and improving angiogenesis with HGF we hope that islet transplants might be improved. Nevertheless, further experiments must be done to prove the viability of this option for improved islet transplants. The data produced is however encouraging for further trials.
Acknowledgements: Dr. Ram Mahato and Mr. Hao Wu for guidance and the NIH for financial support (1T35-DK-07405)
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Title: Defining loci that modulate glaucoma severity
Medical Student Research Fellow: KaiCee Ponds
Faculty Preceptor: Monica Jablonski, Ph.D.
Significance: Glaucoma is a heterogeneous set of diseases that cause permanent optic nerve damage and is the second leading cause of blindness in the United States. Genetic risk factors for pigmentary glaucoma (PG) are largely unknown.
Background: Elevated intraocular pressure (IOP), although not a prerequisite, is a significant risk factor for PG. Previous research in our lab indicates that there are genes modulating IOP and ganglion cell response independent of pigment dispersion. Crossing the D2 parental mouse strain (the mouse model for PG) with the B6 parental strain (containing genes that confer resistance to glaucomatous phenotypes), and comparing each clinical phenotype with a roughly matched cohort, will allow us to develop testable models for the complex polygenic causes of glaucoma.
Hypothesis: Mutant alleles of the genes Tryp1 and Gpnmb in the D2 strain are insufficient to cause glaucomatous damage to retinal ganglion cells and the optic nerve.
Methodology: The student's specific tasks involved morphometric analysis of proximal optic nerves of various strains of BXD mice. Categorical grading of degree of optic nerve damage and quantitative measurements of damage were completed.
Results: Preliminary analysis of optic nerve grade indicated that some strains appear to be resistant to optic nerve damage while others appear more prone.
Discussion: Following further analysis of tens of thousands of optic nerves from different BXD strains, using quantitative trait analysis we will be able to determine possible genetic loci, and candidate genes within these loci, that modulate glaucoma severity.
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Title: Theranostic Nanosomes for Osteoarthritis
Medical Student Research Fellow: James Tidwell
Faculty Preceptor: Karen Hasty, Ph.D.
Osteoarthritis (OA) is a common condition for which there are few preventive therapies. One obstacle to developing therapies is a reliable method of detecting and measuring progression in the early stages of disease when intervention may prove more beneficial. This application will address this question using fluorescent, targeted nanosomes (200 nm liposomes) that we have developed. The nanosomes incorporate a monoclonal antibody (Mab) to native type II collagen (CII) and a near infrared emitting fluorescent dye (NIF) that can be quantitatively visualized in vivo. CII in normal cartilage is not available for binding and is only unmasked when the surface of the cartilage is damaged. We observed targeted nanosomes will selectively bind this exposed CII and can then be quantitated using an external imaging system.
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Title: Phenotypic effects of Tetraspanin CD9 expression in HT1080 Fibrosarcoma Cells
Medical Student Research Fellow: Zhao Yunxiang
Faculty Preceptor: Lisa K. Jennings, Ph.D., Professor/Director
CD9 is an essential member of the tetraspanins, a group of integral membrane proteins with four hydrophobic transmembrane domains. CD9 regulates many cellular processes such as morphology, migration, proliferation, fusion, and tumor metastasis. Using HT-1080 fibrosarcoma cell line as a model, we studied the effects of ectopic expression of CD9 in the regulation of intracellular contractile proteins and in the regulation of matrix metalloproteinases (MMPs). A stable HT1080 cell line expressing CD9 was generated using pRC/CMV-CD9 expression construct and CD9 expression was confirmed by flow cytometry using monoclonal antibody specific for CD9 (mAb7). Expression of CD9 in HT1080 cells lead to dramatic increase in the expression of intracellular contractile proteins SM22α and calponin as confirmed by western blotting analysis of cell lysates. Furthermore, gelatin zymography results showed that increased CD9 expression leads to increased expression and secretion of MMP-9 into culture media supernatants. To further study the role of expression of SM22 α and calponin in HT1080 cells, we generated stable HT1080 cell lines that ectopically express these proteins. For these experiments, full-length human calponin and SM22α constructs were cloned into pEGFP plasmid and transfected into fibrosarcoma cells to express the proteins as GFP fusion proteins. Future studies are required to delineate specific role of aCD9 in the induction of SM22α, Calponin and MMP9 and overall significance of these changes in tumor cell metastasis.
Syamal K. Bhattacharya, Ph.D.
Professor of Surgery, Medicine and Neurology
Executive Director: MSRF Program
956 Court Ave, Suite B318
The University of Tennessee Health Science Center
Memphis, TN 38163