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Appalachian Student Research Forum

Office of Research and Sponsored Programs

Division V

Abstracts Submitted:Division V - Residents & Fellows

Measurement of Mast Cell Cytokine released by A Bio-Plex Assay

Jalilvand M, Bruel KF, De Ponti K, Saoudian M, Milhorn DM, Chi DS and Krishnaswamy G.

Rationale: The identification and quantification of mast cell cytokine production by enzyme linked immunosorbent assay (ELISA) has limitations. The development of flow cytometric assays using micro sphere technology allows the simultaneous detection of up to 100 analytes in a single micro titer well. Methods: Human cord blood-derived mast cells (CBDMC) were isolated and generated by established methods. Cells were activated with Phorbol 12-Myristate 13-Acetate + Ionomycin (PMA/Iono) or (IGE/anti-IGE [IgE]), supernatants were collected and cytokine levels assayed by Bio-Plex Assay (BA). In this technique, cytokine-specific antibodies were covalently coupled to coded beads and levels measured by cytokine binding. Levels were reported as median fluorescent intensity utilizing Bio-Plex software. Statistical analysis was preformed and p values reported. Results: Activation of CBDMC with PMA/I significantly increase the concentration of interlukin-1 beta [IL-1zn, interferon gamma [IFN-zntumor necrosis factor alpha [NF-], granulocyte macrophage colony stimulating factor [GM-CSF], G-CSF, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-13, and IL-17 as compared to those cultured with media alone. Interestingly, treatment of CBDMC with IGE/aIGE increased the concentration specifically of IL-8, monocyte chemo attractive protein (MCP)-1, and macrophage inflammatory protein 1 Beta [MIP-1nas compared with media alone.

Conclusion: Development of flow cytometeric micro sphere assays allows simultaneous detection of up to 100 analyte from CBDMC cultured. It allows us to identify and quantify mast cell cytokine levels and to evaluate the effect of medication or different stimuli on cytokine production. Our data suggests that binding the high affinity IgE on mast cells receptor results in selective signaling.

No bad effect of C-reactive protein on the atherosclerotic lesions in apolipoprotein E knockout mice

Sanjay K. Singh, Madathilparambil V. Suresh, *Zhihua Han and Alok Agrawal, Department of Pharmacology, and *Biochemistry and Molecular Biology, East Tennessee State University, College of Medicine, Johnson City, TN 37614

C-reactive protein (CRP) binds to modified low-density lipoproteins (LDL). However, the function of CRP in the uptake of modified LDL by macrophages and in the progression of atherosclerosis is not known. In the current study, we evaluated the impact of passively administered purified human CRP on the size of atherosclerotic lesions in apolipoprotein E knockout mice. Mice were fed on high-fat diet for 16 weeks and received 12 weekly injections of 100 g CRP each. Another group of mice was fed on chow diet for 20 weeks and received 20 weekly injections of 100 g CRP each. Control mice for both diets were without CRP. One week after the last CRP injection, the lesion size in the aortic root was measured on the basis of lipid staining. Under these conditions, on either diet, there was no difference in the lesion size between the CRP-treated and control mice. Immunochemistry showed positive staining for CRP in the lesions from CRP-treated mice indicating the effectiveness of CRP. The finding that CRP did not deteriorate the extent of atherosclerosis suggested a possible beneficial, but not deleterious, role of CRP. Consequences of CRP-LDL interaction and of the deposition of CRP in the lesions are not clear nevertheless these data provide support for CRP to maintain its status as a host-defense protein.

A novel action of transcription factor c-Rel on the C-reactive protein promoter

Prem Prakash Singh, Bhavya Voleti and Alok Agrawal, Department of Pharmacology, East Tennessee State University, TN 37614

Regulation of C-reactive protein (CRP) gene expression in hepatocytes by IL-6 and IL-1 occurs at the transcriptional level. In Hep3B cells, transcription factors C/EBPb(IL-6-activated) and NF-kB (IL-1-activated) play major role in this process. In mammalian cells, there are 5 NF-kB proteins: p50, p52, p65, Rel B and c-Rel; they form homodimers or heterodimers with each other, and bind tokB-sites on the promoter regions to induce transcription. We have shown previously that a C/EBP-site overlapping a nonconsensuskB-site on the CRP promoter is critical for CRP expression. On this critical site, a novel action of endogenous c-Rel was observed in IL-6-treated cells. c-Rel bound directly to C/EBPb-complexes formed on the C/EBP-site, without the requirement of thekB-site. In this investigation, we evaluated binding of endogenous C/EBPbto the CRP promoter in the Hep3B cells overexpressing c-Rel and p50 dimers. We employed electrophoretic mobility shift assay using nuclear extracts containing p50/p50, c-Rel/c-Rel, or p50/p50 + c-Rel/c-Rel + p50/c-Rel, and the C/EBP-site probe derived from CRP promoter.Binding of C/EBPbto its site was enhanced in the nuclei containing c-Rel/c-Rel but not in the nuclei with p50/p50. Presence of p50/c-Rel inhibited the c-Rel/c-Rel-mediated increase in C/EBPb-binding.This function of c-Rel/c-Rel was not seen when a consensus C/EBP-site-containing probe was used in the assay.We conclude that c-Rel homodimerincreases, and perhaps stabilizes, the binding of C/EBPbto its site on the CRP promoter. Transcription assays are in progress to determine the effect of c-Rel on CRP expression. Since the C/EBPb/c-Rel complex is only 20 bp away from the TATA box on the CRP promoter, we hypothesize that c-Rel may be acting as a bridge between C/EBPband TATA-box-binding proteins.

Myocardial Injury during Standard Treatment of Adults Status Asthmaticus

Said B. Iskandar, MD, Mathew Mathai, MD, Ryland P. Byrd, Jr., MD, FCCP, Thomas M. Roy MD, FCCP, Division of Pulmonary and Critical Care Medicine, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37604

Asthma is a chronic inflammatory disease of the airways that affects 5% of the total population in the United States and approximately 5% to 10% of adults. Although most asthmatic attacks can be easily treated in the outpatient setting, an intense asthma exacerbation can be life threatening and is the primary cause of death in 1% to 7% of asthmatics. Despite advancements in the understanding of the pathophysiology of asthma and more effective pharmacotherapy, the mortality rate from asthma is about 5,600 deaths per year in the United States (U.S.), which is approximately 15 patients dying from asthma each day in the U.S. The cause of death has been the subject of a number of observational studies that have attempted to identify the predisposing factors for fatal or near-fatal asthma. Adult asthmatics, especially those older than 65 years, are a subgroup primarily responsible for the increase in asthma mortality in the U.S. between 1979 and 1996. In adult asthmatics, the severity of reactive airways disease as determined by clinical criteria is the strongest independent risk factor for near fatal exacerbations and death. Additional factors such as low socioeconomic status, learning difficulties, and psychosocial issues are barriers to proper and timely care of the underlying inflammation that contribute to the high risk profile. In adults, these factors are often confounded by substance abuse and lack of compliance with medical therapy even when health care is available. Although adult asthma is often progressive and complicated by coexisting diseases, myocardial ischemia and infarction have not been identified as common occurrences. Making the diagnosis of myocardial injury in the context of status asthmaticus may be difficult without an understanding of the expected EKG changes and enzyme changes associated with standard treatment. The goal of our report is to alert the clinician treating adult asthmatics to the possibility of myocardial injury during standard therapy for status asthmaticus. The mechanisms for focal myocardial necrosis are multifactorial and include the potential of continuous inhaled, potent beta-2 agonists as a cause of myocardial ischemia. Regardless of whether beta-agonists are directly responsible for these adverse effects or are simply a marker for more severe asthma, heavy use of these agents should alert clinicians that it is necessary to reevaluate the patient's condition.


Turner J, Block W, Kalbfleisch J, Breuel K., Department of Obstetrics and Gynecology, East Tennessee State University, Johnson City, Tennessee. Department of Academic Affairs, East Tennessee State University, Johnson City Tennessee.

Pregnancy women are offered screening for Downs Syndrome and neural tube defects between 14 and 20 weeks gestation with risk calculations based on maternal blood values of alpha-fetoprotein, inhibin-A, beta subunit of human chorionic gonadotropin and unconjucated estriol. The risk assessment then guides if future intervention and further antenatal testing is warranted. No guidelines exists on how to treat these samples from time of being obtained until the actual laboratory assessment is performed possibly altering the various components of the sample and skewing the risk results. We enrolled six patients between 16 and 19 weeks gestation to have their routine quad screen performed and additional aliquots to be stored as whole blood at room temperature and serum stored at room temperature, refrigerated at four degrees Celsius, and frozen. These additional samples were tested at 24, 48 and 72 hours and the hormone levels and risk assessments were compared to the original quad screen values. The separate variables of time, temperature and storage medium were then adjusted against the original result. In our study, the adjusted Downs syndrome risk at different time or temperature did not vary from the original result in a statically significant manner (p = 0.676); the adjusted neural tube defect risk result also did not vary in a statically significant manner (p = 0.139). Our study shows that with routine handling of samples obtained within 72 hours of testing does not alter the risk assessments of Downs Syndrome or neural tube defects.


Madathilparambil V. Suresh, Sanjay K. Singh, Donald A. Ferguson Jr. and Alok Agrawal, Department of Pharmacology, East Tennessee State University, College of Medicine, Johnson City, TN 37614

C-reactive protein (CRP) is an acute phase protein in humans but not in mice. CRP binds to pneumococcal C-polysaccharide in vitro and subsequently activates classical pathway of complement in human serum. It has been shown previously that CRP protects mice from lethality following infection with Streptococcus pneumoniae, and complement activation is required for full protection. However, it is not known whether the complement-activating property of CRP per se is essential for this protection. In the current study, we investigated participation of CRP-mediated complement activation in the protection of mice from lethal infection with virulent S. pneumoniae type 3. We compared the protective capacity of wild-type CRP with that of a mutant CRP, Y175A. Like wild-type CRP, the Y175A mutant CRP binds pneumococcal C-polysaccharide, but unlike wild-type CRP, the mutant CRP does not activate complement in human serum. Passively administered mutant CRP protected mice from S. pneumoniae infection as effectively as the wild-type CRP did. Infected-mice injected with either wild-type CRP or with mutant CRP lived longer and had lower mortality compared to mice that did not receive any CRP. Increased median survival time correlated with reduced bacteremia in both cases. Thus, the protection of mice conferred by CRP was independent of its complement-activating function. Based on the assumption that CRP does not differentiate between human and mouse complement, we conclude that the complement activation by CRP is not necessary for the protection of mice from S. pneumoniae infection.

Humanin is a novel neuroprotective agent against stroke.

Xingshun Xu, Chu Chang Chua, Ronald Hamdy and Balvin Chua. Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614.

Humanin (HN) is a recently identified 24-amino acid peptide that is best known for its ability to protect neurons from damage caused by Alzheimer's disease-related proteins. Given the neuroprotective effects of HN, it is plausible that HN could protect against stroke; however, this possibility has not been fully explored to date. In this study, we examined the neuroprotective effects of HN on focal cerebral ischemia/reperfusion injury in mice. The animals underwent a sham operation or middle cerebral artery occlusion for 75 min followed by 24 h reperfusion. Mice were either pretreated with 0.1 ug HN (i.c.v.) 30 min before ischemia; post-treated at 0, 2, 4 h after ischemia; or pretreated with 1 ug HN (i.p.) 1 h before ischemia. The cerebral infarct volume was measured by triphenyl tetrazolium chloride staining after 24 h of reperfusion. In our experiments, HN pretreatment with 0.1 ug HN (i.c.v.) 30 min before ischemia reduced cerebral infarct volume from 56 +/- 7% to 25 +/- 6% (p<0.01). HN post-treatment reduced cerebral infarct volume from 56 +/- 7% to 34 +/- 6 % immediately after reperfusion (p<0.01), 42 +/- 6% at 2 h of reperfusion (p<0.01), and 46 +/- 7% at 4 h of reperfusion (p<0.05). HN pre-treatment with 1 ug HN (i.p.) 1 h before ischemia reduced cerebral infarct volume to 35 +/- 3% (p<0.01). HN also significantly improved neurological function. Extracellular signal-regulated kinase (ERK), a pathway involved in ischemic neuronal death, was rapidly activated after cerebral ischemia/reperfusion. A significant decrease of phospho-ERK was observed in mice treated with HN. Our results demonstrate that HN offers neuroprotection at least in part by inhibition of ERK activation. These findings have important implications for the therapeutic potential of HN in the treatment of stroke.


Semaan G. Kosseifi, MD., Thomas M. Roy, MD., Ryland P.Byrd, Jr., MD.,James H. Quillen VAMedical Center, Mountain Home, TN . Division Of Pulmonary Disease and Critical Care Medicine, James H.Quillen College Of Medicine, East Tennessee State University, Johnson City, TN.

Background: A One year review of the utility of unattended home sleep study in the diagnosis of obstructive sleep apnea in the VA population .

Objective: The main outcome is to understand the effectiveness of unattended home sleep study in the Veterans.

Design: This is a retrospective study. The data will be collected from the electronic charts of the VAMC population that underwent an unattended home sleep study during the previous year 2003 2004 and this data will be analyzed.

Setting: All electronic records of patients who were referred from different clinics and services at the Quillen VAMC for a sleep study will be reviewed. No patient identifiers will be recorded thus patient confidentiality will be assured.

Patients: Since unattended home sleep study is the utility currently used for the diagnosis of obstructive sleep apnea, we selected all patients who were referred from different clinics and services at the VAMC (N=480 patients) and we divided them into those who have a valid unattended home sleep study with the subsequent data to be analyzed and those whom a valid unattended home sleep study was not achieved for several reasons that will excluded from the data analysis (Table 1).

Measurements: The portable sleep monitoring measured the number of respiratory events per hour in bed (respiratory disturbance index [RDI]). Also, we reviewed associated comorbid conditions.

Results: Based on the Apnea Hypopnea Index (AHI) 38 patients (8.50%) didnt have a definable OSAS, 30 patients (6.71%) had an UARS, 161 patients (36.01%) mild OSAS, 88 patients (19.68%) had a moderate OSAS, and 130 patients (29.08%) had severe OSAS. The case recognition rate of sleep apnea among primary care physicians was 82.35%.

Conclusions: Portable monitoring was used as an initial utility in the diagnosis of OSA in our population, and despite technical sources of error, it has a reasonable accuracy for defining respiratory events since the access to diagnosis and treatment of sleep disorders is limited by overburdened sleep laboratories in the VHS and private sector. Another prominent finding is the awarness of the primary care physician of this disease and the early referral for unattended home sleep study for early diagnosis and treatment.


April Whitfield, Department of Obstetrics and Gynecology, East Tennessee State University, Johnson City, TN 37614

Purpose of study: To determine why professional women fail to receive annual gynecological examinations.

Material and Methods: A survey was created that included information about demographics (i.e.: age, education, income, insurance coverage, etc.) and key questions that included the following information: primary provider of annual exams, frequency of visits, last annual exam, reasons for lack of exams, and motivations to have annual exams. An IRB approval for expedited review was received 6/24/2004. The survey was distributed via coordinators of various departments to female health professionals (i.e.: residents, RNs, attending physicians, shift leaders, etc.).

Results: Approximately 184 surveys were distributed, 93 were returned with a 51% response rate. 56% of respondents were MDs (Family practice, Pediatrics, Internal medicine, etc.), 32% were RNs, and 21% other (clerical, LPN, ancillary services, etc.). Majority of respondents (27%) were in 26-30 y/o group, followed by 17% in 46-50 y/o group. 64% had greater than 12 years of education. 68% of the respondents were married, and 88% were Caucasian. 26% worked 36-40 hrs/wk, and 25% 50-80 hrs/wk. 67% of exams were performed by Obstetricians/Gynecologists, followed by Family Practice (18%), and 14% by other providers (nurse practitioners, internists, etc.). 42% had an annual in the last 6-12 months, and 40% had exams within the last 12-24 months, 7% in the last 24 months, 6% >24months, and 2% had no response. 93% reported having a pap and mammogram with their annual exam. 61% had no response for not having an exam, 13% claimed a busy schedule, 11% no particular reason, 6% problems with physician scheduling, 2 % cited a bad experience, and 5% other (being weighed, fear of exam, insurance issues, and illness). 31% of respondents had no response for motivation to see a physician, 20% cited preventative medicine, and13.9% having time in work schedule. Chi square analysis was insignificant when comparing RNs, MDs, and laymen in regards to obtaining an annual exam.

Conclusions: The majority of female health professionals have annual exams which include a pap smear and mammogram in the last 6-12 months. The individual who is most likely to perform these exams is an Ob/Gyn For those who have not an exam in the last year, common reasons include problems with scheduling at the office, bad experiences, busy work schedules, fear of the exam, and insurance issues Preventative medicine and time allotted in work schedule would be motivational factors to having an annual exam Weakness of this study included a low response rate. This could have been improved with phone surveys or one on one interview with the respondents. Further studies could be done to compare health habits of professional women versus the lay population. Hypotheses could include the following: Does having higher education mean that women are more likely to have an annual exam? Are there differences in the fears/concerns of professional women for exams versus the general population? Any information elicited could help health care providers and health educators to provide motivational impetus to their patients regardless of educational level.

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