Central Society for Clinical and Translational Research and Midwestern Section of American Federation for Medical Research

Id: 2 Microrna Expression Pattern in Patients with Ventricular Arrhythmias and End Stage Heart Failure

T. Calway, T. Bak, K. Furlough, G. Kim. Medicine, University of Chicago, Chicago, IL.

Introduction: Patients with heart failure (Hf) are among those at greatest risk for sudden cardiac death. Despite the advancement in medical and device therapy for Hf, there remains a high incidence of cardiac arrhythmias with a highly unpredictable nature. Ventricular tachyarrhythmias (VA) occur frequently in Hf, irrespective of the etiology, identifying an inseparable relationship between the arrhythmogenic and failing heart substrate. MicroRNAs (miRs) are critical regulators of diverse biological processes; however, studies of miRs and arrhythmia remain sparse, particularly in humans. Given their inherent pleiotropic actions to repress multiple gene targets simultaneously, microRNAs (miRNAs) are well poised to play a comprehensive and integrative role coordinating the interplay among electrical, contractile, and structural components that drive Hf-induced ventricular tacharrhythmias. We recently reported on the role of miR-130a in regulation of the gap junction protein, connexin43 (Cx43) and arrhythmogenesis in HF. Induction of miR-130a expression in murine heart in a cardiomyocyte-specific manner is sufficient to induce both atrial and ventricular tachyarrhythmias. We sought to determine whether elevated levels of miR-130a in explanted heart tissue from human patients was associated with the presence of VAs.

Methods: A prospective study of miR levels in myocardium was performed to determine the association between miR-130a levels and VAs. Subjects were enrolled between January 2013 and June 2014. Patients with high VA burden were defined as: 1) history of VA storm within the preceding 3 months, or 2) greater than five defibrillator shocks at the time of admission. Subjects with ischemic heart disease were excluded. Controls in this study were subjects listed for transplantation with a diagnosis of non-ischemic dilated cardiomyopathy with: 1) no prior history of VAs, or 2) not receiving antiarrhythmic medication at the time of admission. Quantitative PCR was performed for microRNAs with known pro-arrhythmic effects based on animal models.

Results: Seven patients with a history of VA were identified and compared to six patients with NIDCM. In both groups, the ejection fraction (Ef) was equally reduced (21.5 ± 1.4% (nonVT) vs. 21.9 ± 2.3% (VT), p=0.9). Using quantitative PCR, we found that miR-130a levels were increased by 2.9 fold in patients with VT storm compared non-VT controls (p=0.0012). Other known arrhythmogenic microRNAs, such as -1, 133, and 328 did not vary between groups.

Conclusion: Myocardial miR-130a levels are increased by nearly 3-fold in patients with VT and end-stage HF. These results provide important mechanistic insight into the possible role of miRs and VAs in human hearts.

Id: 3 Low Myocardial Infarction and Stroke Incidence in Hispanic Patients with Psoriasis

J. Nieves, C. Sulia, L. Figueroa, M. Crespo, N. Escobales. Medicine, Physiology and Dermatology, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico. P.I. Altieri, H.L. Banchs. Cardiovascular Center of Puerto Rico and the Caribbean, San Juan, Puerto Rico.

Purpose of Study: Psoriasis is an immune disorder characterized by a chronic inflammation which may produce myocardial infarction (M.I.) and strokes (S.). It is known that in Puerto Rico, a Hispanic Island has 20% less coronary disease. The leading cause of death in the Island is S. The purpose of this study is to find the incidence of M.I. and S. in patients (P.) with moderate –to-severe psoriasis.

Methods Used: We conducted a retrospective cohort study in which we analyzed the coronary angiography reports of 46 Hispanic P. with moderate-to-severe psoriasis. Findings were recorded and statistically analyzed using the student T test.

Summary of Results: The male to female ratio found was 7:3 and the median age was 56 years. Of the 46 P., 27 were found to have coronary artery disease (C.A.D.) (59%) by angiographic criteria, but without infarction. Twelve P. had obstructive C.A.D. requiring bypass graft surgery. Six P. underwent stent placement. None showed electrocardiographic changes suggesting a (M.I.). No clinical findings or history of S. were found.

Conclusion: Whether psoriasis can be considered a risk factor for developing C.A.D. or S. is still an unknown debate. At least 50% of our P. showed angiographic coronary artery disease, but no clinical M.I. or S., when the most frequent cause of death in the Island is S. followed by M.I. The HDLC and LDL levels were normal. Fifty percent had diabetes Type 2; when in P.R. is 16%. More research is needed to fully understand the atherogenetic mechanisms underlying this dermatologic condition, especially in the Hispanic population, probably inflammation is a factor more important than diabetes Type 2 or lipoproteins. In this study, we have successfully described the angiographic findings of P. with moderate-to-severe psoriasis having coronary disease, but no M.I. or S. This incidence is less than other series reported. This is due to the reported resistance of Puertorrican genes and possibly of the Hispanic population to the atherosclerotic risk factors.

Id: 4 Trpv1 Residues Vital to Protection against 4-Hne Modification, Prservation of Channel Activity and Microvascular Function

D.J. DelloStritto, P. Connel, I. Bratz. Integrative Medical Scineces, Northeast Ohio. Medical University, Rootstown, Ohio.

W. Geldenhuys. Pharmaceutical Sciences, Northeast Ohio. Medical University, Rootstown, Ohio.

We previously demonstrated enhanced 4-hydroxynonenal (4-HNE) post-translational modification (PTM) of TRPV1 decreases TRPV1 functional expression and contributes to microvascular dysfunction in diabetes. Accordingly, we hypothesized that manipulation of residues associated with 4-HNE PTM would preserve TRPV1 function and restore vascular integrity. 4-HNE decreased capsaicin mediated increases in myocardial blood flow and capsaicin-mediated relaxation in isolated coronary microvessels. TRPV1 functional analysis using electrophysiology revealed blunted capsaicin-mediated currents in the presence of 4-HNE which were reversed by the reactive carbonyl species scavenger aminoguanidine (AGD). Using computer modeling we identified three probable residues, previously identified to be important for oxidative modification, as potential sites for 4-HNE modification. The corresponding residues, C616, C621 and C634, were mutated to alanine (individually and in combination) and subsequently we examined the effects of 4-HNE on TRPV1 currents induced by capsaicin via electrophysiology. The mutation of the three pore cysteine (individually or in combination) abrogated the effects of 4-HNE on capsaicin-mediated currents. These data suggest that TRPV1 is targeted by redox-active substances that directly modulate channel activity at numerous sites in diabetes to decrease TRPV1 functional expression and contribute to microvascular dysfunction. The results obtained demonstrate an optimal redox state is critical for a properly functioning TRPV1 channel.

Id: 5 Amelioration of Nadph-Mediated Stress Reduces Cell Death following Blast-Induced Traumatic Brain Injury

B.P. Lucke-Wold, R. Turner, C. Rosen. West Virginia University School of Medicine, Morgantown, West Virginia.

Z. Naser. Drexel University, Philadelphia, Pennsylvania, UNITED STATES. A. Logsdon, K. Smith, J. Huber. West Virginia University School of Pharmacy, Morgantown, West Virginia. M. Robson. Vanderbilt University, Nashville, Tennessee. J. Bailes, J. Lee. Northshore University, Evanston, Illinois.

1.7 million traumatic brain injuries (TBIs) occur each year in the United States. Recent evidence suggests that repetitive TBIs can lead to chronic neurodegenerative changes over time. Currently, available pharmacologic options for the treatment of acute neurotrauma are limited. Oxidative stress is an important secondary mechanism of injury that can lead to cellular apoptosis and behavioral changes such as impulsivity. Utilizing a clinically relevant and validated rodent blast model, we investigated how NADPH oxidase expression and associated oxidative stress contributes to cellular apoptosis following single and repeat blast injuries. Nox4 forms a complex with p22phox following injury, both of which are important subunits of the NADPH oxidase system found within the brain. Using immunohistochemical-staining methods, we found a visible increase in Nox4 following single blast injury in Sprague Dawley rats. Interestingly, Nox4 was also increased in post-mortem human samples obtained from athletes diagnosed with chronic traumatic encephalopathy (CTE). Nox4 activity correlated with an increase in superoxide formation. Alpha lipoic acid, an oxidative stress inhibitor, prevented the development of superoxide acutely, and increased anti-apoptotic markers Bcl-2 (t = 3.079, p<0.05) and heme oxygenase 1 (t = 8.169, p<0.001) after single blast exposure. Subacutely, alpha lipoic acid treatment reduced pro-apoptotic markers Bax (t=4.483, p<0.05), caspase 12 (t=6.157, p<0.001), and caspase 3 (t=4.573, p<0.01) following repetitive blast, and reduced tau hyperphosphorylation indicated by decreased CP-13 and PHf staining. Alpha lipoic acid ameliorated impulsive-like behavior 7 days after repetitive blast injury (t=3.573, p<0.05) compared to blast exposed animals without treatment. TBI can cause debilitating symptoms, disability, and psychiatric disorders. Secondary mechanisms of injury, such as oxidative stress, are ideal targets for neuropharmacologic intervention. Alpha lipoic acid warrants further investigation as a therapeutic for the treatment of acute neurotrauma and prevention of chronic neurodegeneration.

Id: 6 Transgenic Or Pharmacological Inhibition of Sphk1 and S1Pr2 Prevents Hypoxiamediated Pulmonary Hypertension

J. Chen, J.R. Sysol, H. Tang, L. Moreno-Vinasco, K.M. Shioura, J.X. Yuan, J.G. Garcia, V. Natarajan, R.F. Machado. University of Illinois at Chicago, Chicago, Illinois. H. Tang, J.X. Yuan, J.G. Garcia. University of Arizona, Tucson, Arizona.

Previously we showed that Sphingosine kinase 1 (SphK1) and S1PR2 are up-regulated in patients with pulmonary hypertension (PH) and experimental PH models. We hypothesized that transgenic or pharmacological Inhibition of SphK1 and S1PR2 may prevent hypoxia-mediated PH (HPH). SphK1-deficient mice (SphK1KO) and C57Bl6 wild-type littermates (WT) were exposed to normoxia or 10% fiO2 for four weeks (n = 8 per group). In a separate experiment, C57Bl6 mice were injected with a S1PR2 inhibitor (JTE013, 8 mg/kg body weight every other day for 4 weeks). Additionally, male salt-sensitive rats (250–300 g, n= 6 per group) were exposed to normoxia or 10% fiO2 for 3.5 weeks and received SphK inhibitor (SKI2, 10 mg/kg body weight) every other day for 3.5 weeks. RVSP, right ventricle: left ventricle + septum (RV/LV+S) ratio and pulmonary vessel thickness were measured. After fourweeks of hypoxic exposure, SphK1KO mice developed significantly less severe PH (RVSP 30.09±0.68 vs. 36.77±1.07 mmHg, p<0.001) and right ventricular hypertrophy (RVH) (RV/LV+S 0.31±0.01 vs. 0.37±0.01, p<0.001), when compared to WT mice. After 3.5 weeks of hypoxic exposure, rats receiving SphKI2 developed significantly less severe PH (RVSP 45.30±1.20 vs. 30.10± 1.10 mmHg, p<0.01, figure B) and less RVH (RV/LV+S 0.34±0.04 vs. 0.26±0.01, p <0.05), when compared to vehicle treated rats. The similar data were observed in JTE013-treated mice after 4-week hypoxia exposure. All the vascular remodeling data are consistent with RVSP and RVH data. We concluded that transgenic or pharmacological Inhibition of SphK1 or S1PR2 blocker protects rodents against the development of HPH.

ID: 7 Exercise-Induced B-Natriuretic Peptide Elevation in Patients with and without Left Ventricular Hypertrophy

A. Abdul Jabbar, R. Markert. Internal Medicine / Cardiology Division, Wright State University Boonshoft School of Medicine, Dayton, Ohio. A. Abdul Jabbar, K. Makam, R. Mulamalla, C. Ahsan. Internal Medicine / Cardiology Division, University of Nevada School of Medicine, Las Vegas, Nevada.

Background: Serum BNP has cardio-protective effects in exercise that are consistent with its physiological actions. Neuro-hormonal mechanisms and gene expression control the level of natriuretic peptides and response to physical stress.

Methods: We investigated a prospective cohort referred for treadmill exercise test. Serum BNP was withdrawn before and after the test. Those with congestive heart failure, liver cirrhosis, renal failure, atrial fibrillation, uncontrolled hypertension, and valvular heart disease were excluded. Interpreting physicians were blinded to the results of BNP. The Mann-Whitney Test was used to examine the relationship between BNP and exercise time, and the Kruskal-Wallis Test for LVH status.

Results: Twenty-two patient (mean age 48.6, 22 with LVH), were prospectively enrolled in the study. All patients had normal LVEf and achieved >85% of their age-predicted maximum heart rate. Patients who exercised for >7 minutes showed a greater increase in exercise-induced BNP compared to those who exercised < 7 minutes (5.6 vs 2.5 pg/ml, p = 0.047). There was no relation between the degree of LVH (13 mild, 3 moderate, 6 normal) and the change in BNP following exercise (p = 0.64).

Conclusions: Exercise-induced BNP plays a role in cardiopulmonary adaptation to exercise. Serum BNP can be used as marker for response to therapy that alters the release and degradation of natriuretic peptides.

ID: 8 Right Ventricular Outflow to Aortic Valve Velocity Ratio: A Relative Dimensionless Index for Severe Aortic Stenosis

A. Abdul Jabbar, O. Ali, R. Markert, G. Broderick, B. White. Internal Medicine / Cardiology Division, Wright State University Boonshoft School of Medicine, Dayton, Ohio.

Background: The distinction between critical and noncritical aortic stenosis often requires an accurate calculation of the aortic valve area. Defined by the ratio of left ventricular outflow tract (LVOT) velocity to the aortic valve velocity, the dimensionless index (DVI) has an important role in evaluating the severity of aortic valve stenosis especially when there is a discrepancy among other Doppler measures of severity. In our study, we measured the right ventricular outflow (RVOT) to aortic valve velocity ratio in patients with aortic valve disease in order to derive a relative index (R-DVI) that can be used as a surrogate for severe aortic stenosis (AS).

Methods: We investigated a retrospective-cohort of patients who had surgical aortic valve replacement for severe AS between January 2010 and June of 2014 (n = 47). Those with contaminant surgery of other valves or significant valvular regurgitation were excluded. The RVOT TVI was measured from the base-short axis view with pulse-wave Doppler positioned just around 0.5 cm below the level of the pulmonic valve.

Results: Thirty-seven of 47 (79%) of patients had severe AS and 10 (21%) had mild to moderate AS. Using a cutoff of 0.25 for DVI to indicate severe AS (as defined by the ASE guidelines), the sensitivity was 76% (28 of 37) and the specificity was 100% (10 of 10). The positive and negative predictor values were 100% (28 of 28) and 53% (10 of 19), respectively. The likelihood ratio positive (sensitivity ¼[1 - specificity]) was ≈76 and the likelihood ratio negative ([1-sensitivity] ¼ specificity) was 0.243. Using the same cutoff of 0.25 for R-DVI, the sensitivity was 100% (37 of 37) and the specificity was 60% (6 of 10). The positive and negative predictor values were 90% (37 of 41) and 100% (6 of 6), respectively. The likelihood ratio positive was 2.5 and the likelihood ratio negative was 0.017.

Conclusions: Using the RVOT velocity to derive a relative dimensionless index (RDVI) to assess the severity of aortic valve disease is a novel concept. R-DVI ratio may be used to assess the severity of aortic stenosis when the measurement of other traditional indices is technically difficult.

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Id: 9 Hyperglycemia Induces Stress and Inflammatory Signaling that Leads to Hypertension in Pregnancy- a Translational Study

M.N. Uddin, C. Cawyer, N. Drever, S.R. Allen, T.J. Kuehl. Obstetrics and Gynecology, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. M.N. Uddin, M.A. Co, M.M. Beeram, T.J. Kuehl. Pediatrics, Baylor Scott & White Healthcare/Texas A&M Health Science Center College of Medicine, Temple, Texas A.F. Pantho, S. Munir. University of Texas at Austin, Austin, Texas.

Objective: Preeclampsia (preE) is characterized by hypertension and proteinuria during pregnancy. The frequency of preE in diabetes mellitus (DM) women who become pregnant is higher than in non-diabetics. However, the mechanism(s) contributing to this effect are unknown. Cytotrophoblast (CTB) cell dysfunction during placentation plays a role in the pathogenesis of preE. Hyperglycemia associated with diabetes is a risk factor for preE. Recently, we have described detrimental effects of excess glucose on CTB cells. In a recent study, we have shown that hyperglycemia impairs CTB function via stress signaling. In the present study, we analyzed the retrospective data to evaluate the percentage of DM pregnant women develop the PreE and also evaluated the CTBs migration and apoptotic signaling in hyperglycemia condition in vitro.

Study Design: In this study, we did the retrospective data analysis of 2684 consenting pregnant women in an IRB approved retrospective study from Scott & White hospital, Temple, Texas. In in vitro study, Human extravillous CTBs (Sw. 71) were treated with 0, 100, 200, 300 or 400 mg/dL glucose for 48h. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor gamma (PPARγ) ligand (rosiglitazone). While some other cells were treated with D-Mannitol to evaluate the osmotic effect. Cell migration study was performed by Matrigel migration kit. Cell lysates were utilized to measure PPARγ expression, p38 MAPK phosphorylation, and apoptotic and stress signaling proteins; Bcl-2-associated X protein (Bax), anti-apoptotic Bcl-2 and caspase-9 and pro-inflammatory cyclooxygenase-2 (Cox-2) expression by western blot. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.

Results: In retrospective data analysis, we found 32 out of 144 pregnant patients with DM (types I or II) developed PreE which is 22%; 8 out of 56 (14%) with gestational DM pregnant women developed PreE; and 152 out of 2684 (5.5%) without either gestational or DM developed PreE. In vitro study, we observed that CTBs migration was inhibited by ≥ 150 mg/dL of glucose compared to basal (100 mg/dL) (p<0.05). The p38 MAPK phosphorylation and PPARγ were upregulated (p<0.05) in CTBs treated with >150 mg/dL glucose compared to basal (100 mg/dL). The expression of Bax/Bcl-2, Cox-2, caspase-9 were up-regulated (p<0.05) in CTBs treated with ≥100 mg/dL glucose compared to basal. The SB203580 or rosiglitazone pretreatment showed an attenuation of hyperglycemia-induced apoptotic and stress signaling proteins. D-Mannitol had no effect on CTBs.

Conclusions: we have observed in our retrospective study that the frequency of preE in DM pregnant women is four fold higher than in non-diabetics. Hyperglycemia inhibited the migration of CTBs and induced the apoptotic signaling in CTBs by upregulating the Bax/Bcl2, caspase- 9 and Cox-2 expression. The attenuation of hyperglycemia-induced apoptotic signaling proteins by SB203580 or rosiglitazone pretreatment suggests the involvement of apoptotic signaling mechanisms in CTBs dysfunction. A prospective study is now underway with serial determinations of angiogenic and apoptotic signaling proteins and cytokines throughout the pregnancy to assess the relevance of these markers in early diagnosis to development of signs and symptoms of preE and to glucose levels.

ID: 10 Simvastatin Regulates Human Lung Endothelial Sphingosine1-Phosphate Receptor 1 Via Kruppel-Like Factor 2

X. Sun, B. Mathew, S. Sammani, J.R. Jacobson. Medicine, University of Illinois at Chicago, Chicago, IL. J.G. Garcia. The University of Arizona, Tucson, AZ.

Rationale: We have demonstrated that simvastatin and sphingosine 1−phosphate (S1P) each enhance endothelial cell (EC) barrier function, resulting in an attenuation of increased vascular permeability in acute lung injury or ischemia reperfusion injury. As statins are recognized to affect differential gene expression via modulation of specific transcription factors, we hypothesized that simvastatin enhances EC barrier via increased promoter activity of S1PR1 gene (S1PR1) through transcription factors.

Methods: S1PR1 promoter regulation by transcription factors was tested with in silico analysis. Mouse lungs were harvested for total RNA extraction and microarray analysis after 2.5 mg/kg LPS or 10 mg/kg simvastatin. Endogenous S1PR1 and lung Kruppel-like factor 2 (KLf2) were detected from human pulmonary artery EC lysates by immunoblotting. The S1PR1 promoter region from the human genome was cloned into pGL3 vector, and tested for promoter activity in response to simvastatin preincubation (5 μM, 12 hrs). Effects of simvastatin on KLf2 recruitment to the S1PR1 promoter were tested by chromatin immunoprecipitation assay (ChIP).

Results: In silico analyses identified S1PR1 promoter regulation by the transcription factor KLf2. Microarray assay demonstrated LPS significantly decreased KLf2 gene expression in mouse lungs. In contrast, simvastatin significantly increased human pulmonary artery EC KLf2 expression and enhanced S1PR1 protein expression while these effects were abrogated by transfection with siRNA specific for KLf2. Compared to basal levels, S1PR1 promoter activity was dramatically increased by simvastatin (120% increase, p < 0.05), which was also significantly attenuated by silencing of KLf2 expression with siRNA. Finally, ChIP assay indicated that simvastatin increased KLf2 recruitment to S1PR1 promoter.

Conclusion: While further echanistic studies are required, these results suggest that simvastatin upregulates S1PR1 expression via enhanced activity of the S1PR1 promoter through recruitment of the transcription factor KLf2. These data suggest a potential synergistic effect of simvastatin and S1P in endothelial signaling and barrier function which may lead to novel therapeutic strategies for lung inflammatory syndromes.

Id: 11 Cardiac Inflammation Increases with Contractile Dysfunction in Cardiomyopathies Caused by Sarcomere Protein Mutation

T.L. Lynch, S. Sadayappan. Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois.

Introduction: Cardiomyopathies are a leading cause of heart failure and are still associated with substantial morbidity and mortality despite optimal treatment. However, it is unclear whether a proinflammatory response resulting from contractile dysfunction and myocyte damage in sarcomere protein-mutated forms of cardiomyopathy perpetuates disease severity.

Hypothesis: Progressive cardiac dysfunction and damage resulting from sarcomere protein mutation promotes a robust proinflammatory response that exacerbates cardiomyopathy progression.

Methods and Results: A previously characterized mouse model of dilated cardiomyopathy expressing a homozygous cardiac myosin binding protein-C (MYBPC3) gene mutation (t/t) was used to determine whether cardiac inflammation elevated with contractile dysfunction at 2, 4, 8, and 12 weeks of age, compared to wild-type (WT) mice. M-mode echocardiographic analysis along the short-axis of t/t and WT hearts confirmed progressively increasing dilation and decreased contractile function exhibited by reduced ejection fraction and fractional shortening in t/t hearts. Compared to WT animals, H&E and Masson's Trichrome staining of t/t hearts revealed profuse myocardial disarray and fibrosis while second harmonic generation imaging depicted disorganized sarcomere structure and elevated collagen content that became more severe over time. Intriguingly, flow cytometry analysis indicated that T-cells, Bcells, monocytes, macrophages and neutrophils were elevated at various time-points in t/t hearts compared to WT hearts. Finally, immunofluorescence staining demonstrated progressively increased CD45+ leukocytes in t/t hearts compared to WT hearts confirming cardiac inflammation.

Conclusion: These results demonstrate that cardiac inflammation increases with the severity of contractile dysfunction in a mouse model of cardiomyopathy caused by sarcomere protein mutation indicating inflammation as a novel therapeutic target.

Id: 12 Urea and Thiourea-Based Tipodal Ligands Induce anti Angiogenic Profile on Human First Trimester Cytotrophoblast Cells

M.N. Uddin. Obstetrics and Gynecology, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. B.M. Madhava. Pediatrics, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. M.N. Uddin. Pediatrics, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, TX. A.F. Pantho. University of Texas at Austin, Austin, TX. J. Castor II, A. Ashraf, D.C. Sprague. Biotechnology, Temple College, Temple, TX. M. Hossain. Chemistry and Biochemistry, Jackson State University, Jackson, Mississippi.

Purpose: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy with abnormal placentation. We have demonstrated that marinobufagenin (MBG), a cardiotonic steroid (CTS), impairs cytotrophoblast (CTB) function, which is critical for placental development. However, the cell surface receptor for the CTS has not been identified yet. During this study, we have used highly functional tripodal ligands with three thiourea or urea groups that are either nitro-derivative or fluoro-derivative to explore the receptor/ligand affinity using CTB cells: Para Nitro Tripodal Urea (PNTU), Para Nitro Tripodal Thio-Urea (PNTTU), Penta fluoro Tripodal Urea (PfTU), and TRSPHNO2.

Design Method: The human extravillous CTB cells (Sw.71) utilized in these studies was derived from first trimester chorionic villus tissue. Cells were treated with 0.1, 1, 10, and 100 nM PNTU, PNTTU, PfTU, and TRSPHNO2 for 48 hours. CTB cells function was studied in the PNTU, PNTTU, PfTU, and TRSPHNO2-treated cells. The levels of angiogenic and anti-angiogenic factors were measured in the cell culture media, while the cell lysates were utilized to measure the VEGf-1, AT1 and AT2 receptors expression after PNTU, PNTTU, PfTU, and TRSPHNO2 treatment.

Results: The new compound impaired the CTB function. sflt-1 and sEnd secretion were significantly increased while VEGf and PIGf were decreased in the culture media of CTB cells treated with ≥ 1 nM PNTU, PNTTU, PfTU, and TRSPHNO2. The AT2 receptor expression was significantly upregulated in ≥ 1 nM PNTU, PNTTU, PfTU, and TRSPHNO2-treated CTB cells in compared to basal; however, the AT1 and VEGR-1 receptors expression was downregulated.

Conclusion: The anti-proliferative and anti-angiogenic effect of this compound on CTB cells are similar with those effect of CTS. The receptor/ligand affinity of urea and thiourea-based tipodal ligands on CTB cells provides us the clue to design a potent inhibitor to prevent the CTS-induced impairment of CTB cells.

Id: 13 L-Name Attenuates the Cardiotonic Steroids-Induced Monolayer Permeability in Lymphatic Endothelial Cells

T. Kuehl, M.N. Uddin. Obstetrics and Gynecology, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. J. Castor II, D.C. Sprague. Biotechnology, Temple College, Temple, Texas. W.E. Cromer, S.H. Afroze, D.C. Zawieja. Medical Physiology, Texas A&M Health Science Center College of Medicine, Temple, Texas. M.R. Beeram, T. Kuehl, M.N. Uddin. Pediatrics, Baylor Scott & White Health and Texas A&M Health Science Center College of Medicine, Temple, Texas.

Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CTS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na+/K+ ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome in vivo and increases endothelial cell monolayer permeability. Edema is a common syndrome of preE. To assess whether CTS are involved in the leakage of lymphatic endothelial cells (LECs) lining during preE, we evaluated the effect of these CTS on monolayer permeability of LECs in culture.

Methods: LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (vehicle), MBG, CINO, or OUB (1, 10 or 100 nM). Some LECs were pretreated with L-NAME (N-Nitro-L-Arginine Methyl Ester) at a concentration of 1μM before treatment with 100 nM MBG or CINO. Monolayer permeability of CTS-induced LECs was measured by using a fluorescent dye that was quantified on a fluorescence plate reader. The expression of b-catenin and VE-cadherin in the CTS-treated LECs was measured by immunofluorescence. Western blot was performed to measure b-catenin, VE-cadherin, and LYVE-1 protein levels. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests.

Results: MBG (≥ 1 nM, p<0.05) and CINO (≥ 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to DMSO while OUB had no effect. Pretreatment of LECs with 1μM L-NAME attenuated the monolayer permeability of LECs treated with either 100 nM of MBG (p<0.05) or 100 nM of CINO (p<0.05). The b- catenin protein expression in LECs was downregulated by both MBG (p<0.05) and CINO (p<0.05) treatment. However, CTS did not cause any disruption of the LECs tight junctions. CINO (p<0.05) downregulated the VEcadherin and LYVE-1 protein expression, but MBG did not.

Conclusions: We have demonstrated that bufadienolides, MBG and CINO, caused an increase in the monolayer permeability of LECs which was attenuated by L-NAME pretreatment. Moreover, the b-catenin protein expression was downregulated by MBG and CINO treatment with no significant effect on tight junctions. These data suggest that CTS may be involved in the vascular leak syndrome in the LEC lining in preE.

Id: 14 Feasibility of Transcutaneous Aortic Valve Replacement in a Non-Academic Community Hospital

D. Pappas, K. Hungate, R. Graf, T. Alexander, S. Damaraju. Cardiology, Spohn Shoreline, Corpus Christi, Texas. A.M. Damaraju. W B Ray High School, Corpus Christi, Texas.

Introduction: Transcutaneous Aortic Valve Replacement (TAVR) has been approved by the fDA since 2012 for patients who are high risk or inoperable for open-heart surgery. The procedure has been shown to improve patients’ quality of life and improve survival. It was introduced as an option at the Christus Spohn Shoreline Hospital in Corpus Christi Texas from february 2014. This is a 330 bed community hospital and primary cardiac center for a city of 280 thousand. The procedure is performed by a core group of interventional cardiologists, cardiothoracic surgeons and cardiac anesthesiologists. All potential candidates are screened by a multidisciplinary team of specialists and TAVR is performed twice a month on selected patients. We present the first 17 patients who underwent the procedure in this hospital.

Baseline Characteristics: Out of the 17 patients, 7 were male and 10 were female, the average age of the patients was 87, the average BMI was 25.2, the Society of Thoracic Surgery Risk Score (STS) was 12, The average New York Heart Association (NYHA) Class was >3, 5 previously had Coronary Artery Bypass Graft Surgery and 3 had End Stage Renal Disease.

Results: No operative mortality was recorded. 100 percent operative success was obtained with insertion of 3 sizes of Sapien Valves (8 were 23 millimeter, 6 were 26 millimeter, 3 were 29 millimeter). 3 were performed by transapical (TA) and 14 by transfemoral (TA) approach. 7 peri-procedural complications were encountered, none of which were fatal. The average length of stay was 9 days (range: 2–51 days).

Conclusion: TAVR is a viable treatment option for high risk and inoperable patients with severe aortic valve stenosis. This can be performed safely in a community hospital with a multidisciplinary approach.

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TABLE

TAVR Patients

Id: 15 the Role of Endothelial Microrna-130A in Angiogenesis

K. Furlough, T. Calway, S. McSharry, C. Harmon, D. Kim. University of Chicago-Molecular Cardiology, Chicago, IL.

Angiogenesis, the growth of new capillaries from preexisting blood vessels, is a complex process involving endothelial cell (EC) activation, disruption of vascular basement membranes, and migration and proliferation of ECs. Angiogenesis, which serves an important role in embryonic and postnatal development, is vital for the growth of new blood vessels from pre-existing vasculature and is subject to complex regulation in health and disease. MicroRNAs, short noncoding RNAs that downregulate gene expression at the post- transcriptional level, are important for proper organ function. Given their inherent pleiotropic actions to repress multiple gene targets simultaneously, microRNAs (miRNAs) are well poised to play a comprehensive and integrative role coordinating the interplay among the diverse pathways critical for angiogenesis. There has been emerging evidence that miR-130a plays a role in angiogenesis, though this role remains largely undefined. To study the effects of miR130a on angiogenesis, we created an inducible mouse model to express miR-130a, under the control of the VE-cadherein promoter, in ECs (VEcad- miR130a). Overexpression of miR-130a was initiated in adult ECs at weaning, approximately 4 weeks post-birth. We conclude that the overexpression of miR-130a in endothelial cells plays a role in the regulation of angiogenesis with likely anti-angiogenic effects. Loss of capillary density may be a mechanism for the induction of heart failure in this model.

Results: Through the use of fluorescence in situ hybridization, we identified the presence of miR-130a in endothelial cells of the adult mouse. Endothelial cells in the VE-cad-miR130a mouse were found to have a 3.6 fold elevation of miR-130a compared to controls. Survival analysis demonstrated a 50% mortality by 9 weeks of miR-130a overexpression. Using echocardiography, VE-cad-miR130a mice demonstrated a reduction of left ventricular function by 8 weeks of overexpression (% fractional shortening (%fS) 35.2 vs 23.1, p<0.001, n=8). We used immunofluorescence (If) to evaluate capillary density in the myocardium. By 6 weeks of overexpression, VE-cadmiR130a hearts demonstrated a significant loss of CD-31+ cells, supportive of a reduction in capillary density.

Id: 16 Deficiency of Raptor in Smooth Muscle Cells Attenuates Hypoxia-Induced Pulmoanry Hypertension

H. Tang, Y. Gu, J.J. Yuan. Department of Medicine and Physiology, University of Arizona, Tucson, Arizona. D.R. Fraidenburg. Department of Medicine, University of Illinois, Chicago, IL. A. Makino. Department of Physiology, University of Arizona, Tucson, AZ.

Rational: Pulmonary arterial hypertension (PAH) is a rare but progressive and fatal disease characterized by a persistent increase in pulmonary arterial pressure associated with vascular remodeling due to excessive pulmonary vascular rcell proliferation and migration. Our previous data showed the importance of PTEN/Akt/mTOR signaling pathway in which increased PTEN, or knockout of AKT1 can attenuate the development of hypoxia induced pulmonary hypertension (HPH). However, the molecular mechanism and the implication of its downstream components in PH remain elusive.

Objective: The aim of this study is to examine whether mTOR complexes in pulmonary arterial smooth muscle cells (PASMCs) are involved in the pathogenesis of PAH.

Methods and Results: The mice with smooth muscle cells conditional and inducible knockout of Raptor were used in hypoxiamediated Pulmonary Hypertension (PH) model. Right ventricle systolic pressures (RVSP) and right ventricular hypertrophy, by RV/ (LV+S) ratios, were measured. Baseline pulmonary hemodynamic measurements, fulton's index, and pulmonary artery wall thickness were unchanged between Cre+/Raptorf/f mice treated with vehicle control (oil) or tamoxifen. Exposure of mice to normobarci hypoxia (10% O2) for 4 weeks resulted in significantly increased RVSP, fulton Index and pulmonary arterial wall thickness. However, Raptor conditional KO in smooth muscle cells displayed significantly lower RVSP, less right ventricular hypertrophy and less pulmonary vascular remodeling compared with WT mice.

Conclusion: Raptor play significant roles in pulmonary vascular remodeling associated with PH. The mTOR complexes will continue to be a focus of research aimed at determining factors which affect pulmonary vascular remodeling and focusing on both complexes of mTOR may lead to novel therapeutic targets for PAH.

Id: 17 Cha2Ds2Vasc Score in Predicting Future Development of Atrial Fibrillation after Successful Atrial Flutter Ablation

S.M. Hussain. Internal Medicine, UIC/AdvocateChrist Medical Center, Oak Lawn, Illinois. A. Bhan, M. Duggal, P. Dunskis. Cardiology, UIC/AdvocateChrist Medical Center, Oak Lawn, Illinois. A. Bhan, M. Duggal, P. Dunskis, H. Jibawi. Cardiology, Advocate South suburban hospital, Hazel crest, Illinois.

Introduction: After a successful catheter ablation for typical atrial flutter, long term development of atrial fibrillation is common. No definite recommendation exists about long-term anticoagulation therapy after successful catheter ablation for Atrial flutter. The aim of this study was to evaluate whether CHA2DS2VASc score can predict future occurrence of Atrial fibrillation after successful catheter ablation of Atrial flutter and help guide anticoagulation therapy.

Methods: Consecutive patients with isolated Atrial flutter who underwent successful catheter ablation and completed at least 18 months of follow-up subsequently were included. Chart review was performed to assess CHA2DS2VASc score at time of catheter ablation. Long term follow-up comprised of multiple transtelephonic electrocardiograph monitors and / or office EKGs, cardiac implantable electronic device interrogations and clinic visits. Additional data was collected on anticoagulation use and occurrence of Atrial fibrillation. We compared and contrasted our data using descriptive statistics as well as the two-tailed independent t-test and binary logistic regression.

Results: One hundred and five patients meeting inclusion and exclusion criteria were included. 75 (71%) were male and 30 (29%) female with a mean age 66 + SD 11.7. Mean follow-up duration was 25 months. Mean CHA2DS2VASc risk score for the cohort was 2.9 (range: 0 - 7). Atrial fibrillation occurred in 35 patients (34%) at 2 years. The average CHADS2VASc in patients experiencing Atrial fibrillation was 3.23. This was not found to be significant in predicting Atrial fibrillation occurrence at 1 year but when followed for 2 years (90 patients) it showed a trend towards significance. Higher CHA2DS2VASc scores were more likely to be associated with future development of Atrial fibrillation although this did not reach statistical significance (3.23 + 1.44 vs 2.64 + 1.42; p=.058).

Conclusions: The incidence of Atrial fibrillation in patients following catheter ablation of Atrial flutter is common. CHA2DS2VASc score of >3 may help predict the occurrence of Atrial fibrillation at 2 years and help guide long term anticoagulation therapy. This needs further validation in a larger prospective study.

Id: 18 Interactions of Acidic Glycosaminoglycans with Basic Proteins and Peptides

S. Woodburn, A. Ellington. Chemistry, Bowling Green State University, Bowling Green, Ohio.

Heparin, the common sulfated glycosaminoglycan, has been in long-time clinical use as an anticoagulant prior to surgical procedures. Subsequent to surgery, it is neutralized by administration of the basic, low m.w. polypeptide, protamine, which is derived from salmon sperm. In past years, it has been reported that protamine itself exhibits a small anticoagulant effect when tested with a prothrombin time (PT) assay. In the current study, this laboratory reports that pharmacological quantities of protamine display considerable anticoagulant effects in PTassays and that the anticoagulant effect is readily neutralized by Dermatan Sulfate (DS). Whereas 2, 4, 8, 20, and 40 ug protamine generate PTs of 15.1, 17.3, 19.8, 24.0, and 31.1 seconds relative to a control of 10.3 seconds, addition of 90 ug DS to plasma/protamine mixtures reduces PT to 13.2, 13.0, 13.6, 24.0, and 14.5 seconds, respectively. The secondary control for 90 ug DS was only 13.6 seconds. Furthermore, the basic protein, calf thymus histone, displays a very slight anticoagulant effect in PT assays. However, it exhibits a significant inhibition of the anticoagulant effect of heparin when added sequentially to plasma. Relative to PTs of 12.3, 24.8, 47.3, and 53.3 seconds for addition of 2, 4, 8, and 10 ug heparin to plasma, the corresponding values are 10.4, 15.4, 42.8, and 48.3 seconds with the further sequential addition of 2 ug of calf thymus histone. Histone alone prolongs plasma by 2.5 seconds, from 10.3 to 12.8 seconds. More profound neutralizing results are obtained with 5 ug histone. Hence, the characteristic reaction of the basic proteins and peptides in their interaction with sulfated glycosaminoglycans such as heparin and DS may be a far more common phenomenon in nature than heretofore perceived. (The authors acknowledge, with profound appreciation, the contribution of the Medical Technology Program at BGSU for its donation of coagulation proteins in this study).

Id: 19 Dual - Energy Ct Myocardial Perfusion Imaging of Vasodialator Induced Myocardial Ischemia

M. Sidhu. Cardiovascular Medicine, University of Missouri, Columbia, Missouri. S. Uthamalingam, L. Engel, A.M. Lee, B. Ghoshhajra. Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

Aims: To evaluate the feasibility of vasodilator induced myocardial ischemic perfusion defect detection using Dual energy CT and to evaluate accuracy of detecting these myocardial perfusion defects utilizing different energy spectra (100 kVp vs. 120 kVp vs. iodine overlay maps) of Adenosine stress Dual-Energy CT Myocardial Perfusion Imaging (DE-CTP).

Methods: 12 prospectively enrolled patients (Men: 83%, Age 63 ± 13 years) had both DE-CTP and SPECT-MPI, of which 10 also underwent invasive coronary angiography. DE-CTP data was processed using 1) only 100 kVp energy spectra, 2) 120 kVp energy spectra and 3) DECT-based iodine maps. In a blinded fashion, two independent readers analyzed all 3 data sets (i.e. for each reconstruction method) to identify stress induced perfusion defects. Each segment of DE-CTP rest and stress data sets were scored a severity and transmurality score (range: 0-3). Myocardial ischemia was derived from difference of stress (S) and rest score (R). SR >3 was considered moderate to severe ischemia.

Results: Based on per patient analysis (n=10), the highest sensitivity (100%), specificity (75%), positive (50%) and negative (100%) predictive value for the detection of myocardial ischemic perfusion defect was observed with100 kVp energy spectra DE-CTP images for patients with ≥ 70% stenosis in invasive coronary angiography (figure). Similarly per vessel analysis showed the 100 kVp energy spectra DE-CTP images had the highest overall sensitivity (80%), negative predictive value (95%) and diagnostic accuracy (83%) for patients with ≥ 70% stenosis in invasive coronary angiography. Per patient analysis (n=12) comparing SPECT-MPI with Summed Difference score > 3 versus DE-CTP S-R score >3; Iodine overlay maps showed the highest sensitivity (100%), specificity (89%), positive (75%) and negative (100%) predictive value.

Conclusion: Adenosine stress DE-CTP can detect presence of vasodilator induced myocardial ischemic perfusion defects. 100 kVp energy spectra and Iodine map provide superior results for the detection of these perfusion defects compared to 120 kVp energy spectra.

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Comparative analysis of Stress and Rest images of Cardiac CT using different energy spectra (Panel AYD) with Stress (Panel E) and Rest (Panel F) Sestamibi Nuclear scan and a left coronary angiogram (Panel G). A thin perfusion defect (arrows) is more obvious on a 100 kVp scan (Panel A) when compared to 120 kvp energy scan (Panel C). Perfusion scan is absent on the rest scan (arrows Panel B and D). Panel E. Nuclear stress scan shows thin area of reversible perfusion defect when compared to the rest image (Panel F) that matches the area on stress CT scan Panel A. Panel G. Left coronary angiogram showing a significant hemodynamic lesion in the high diagonal branch supplying that area of reversible perfusion defect.

Id: 20 Isolated Aneurysms of the Ventricular Membranous Septum

A. Abdul Jabbar, O. Mufti, B.K. S. Internal Medicine / Cardiology Division, Wright State University Boonshoft School of Medicine, Dayton, Ohio. W. M. Cardiology, The Christ Hospital, Cincinnati, Ohio. B.M. Q, V. Tivakaran. Cardiology, Dayton VA Medical Center, Dayton, Ohio.

Background: Ventricular septal defect is the most common congenital cardiac abnormality encountered in infants and children who may survive through adulthood before diagnosis. Two third of the cases involve the membranous part of the septum. In the absence of inter-ventricular shunt or concomitant cardiac surgery, guidelines for surgical intervention or resection of isolated aneurysms of the membranous septum (AMS) are not well established. Cases Description: The first patient is a healthy 65 year-old male evaluated for exertional dyspnea. Physical exam was unremarkable. Echocardiography with agitated saline injections showed a 1.4 X 1.2 X 2.2 cm perimembranous ventricular septal aneurysm. The second patient is a 65-year male with history of colon cancer and rheumatoid arthritis was evaluated for recurrent unexplained ventricular tachycardia. His nuclear stress images revealed a fixed defect in the inferoseptal wall but no reversible ischemia. Echocardiographic examination showed a giant aneurysm of the membranous septum protruding to the right atrium and ventricle. The third patient is a middle age male with intermediate-risk probability for coronary artery disease who had an incidental finding of AMS during a coronary CT Angiogram testing for chest pain evaluation. The classical “windsock” appearance of the aneurysm is demonstrated again on the reconstructed images. The appearance is similar to the TEE image for our first case. There were no shunts detected in any of the cases with Doppler echocardiography.

Discussion: VSDs are classified hemodynamically based on the ratio of right and left ventricular systolic pressures, cardiac output, and the vascular resistance between the systemic circulation and the pulmonary circulation. Defect Diameter and Indexed crosssectional area of the AMS were found to be predictive factors for prognosis, spontaneous closure and successful surgical closure. The current guidelines recommend a 3 to 5 year interval follow-up with echocardiography to screen for possible complications in the absence of underlying intra-cardiac shunt. Similarly restrictive VSDs, with or without AMS, are managed conservatively in the absence of symptoms. Some authors recommend early operation during the childhood to prevent enlargement and future complication of asymptomatic AMS. While some think that intervention should be reserved to treat complications or concomitant with other surgical procedures. The argument becomes challenging when other valvular structures are involved. Even with spontaneous closure of aneurysm, there is still potential aortic regurgitation. Owing to the rapid progression from mild to severe regurgitation, an early surgical intervention is a consideration in the presence of aortic valve involvement. Because of its anatomical location, resection of the aneurysm may cause significant tricuspid regurgitation and can create AV block if the aneurysmal tissue is resected in its entirety. Membranous VSD with associated aneurysmal tissue represent an anatomically favorable defect for percutaneous transcatheter closure and carries acceptable efficacy and safety.

Conclusion: The management of asymptomatic cases is controversial. In the absence of major complications or concomitant cardiac surgery, surgical correction carries risk of injury to the valvular apparatus and conduction system. Closer follow up is advised due to potential complications, and early repair is a consideration with evolving device-based interventions.

Id: 21 Severe Mrsa Endocarditis Treated with Veno-Venous Extracorporeal Membrance Oxygenation

J. Vargha, S. Mihalov, T. Yousuf. Department of Internal Medicine, Advocate Christ Medical Center, Oak Lawn, Illinois.

Introduction: Chronic intravenous drug users are at a high risk of developing tricuspid infective endocarditis. In many cases, this course of endocarditis can become severe leading to septic shock, multiorgan failure, and respiratory failure. Veno-venous Extracorporeal Membrane Oxygenation (VV-ECMO) is a treatment in cases of cardiac and respiratory failure where a damaged cardio-pulmonary system is bypassed and allowed to repair. In this rare case, VV-ECMO was used to treat a IV heroin abuser with severe cardio-pulmonary failure secondary to MRSA endocarditis.

Case Presentation: Our case is of a 25 year old female with a past medical history of type 1 diabetes mellitus and chronic daily heroin abuse with visible needle marks including along her internal jugular vein who presented with worsening chest pain and shortness of breath. She was found to be in diabetic ketoacidosis, and was admitted to the ICU for DKA treatment. She reported a progressively worsening productive cough along with multiple cutaneous abscesses that were located on both of her wrists, left forearm, right elbow and left lower extremity. She also reported insulin noncompliance for the previous two weeks prior to admission. She denied fevers or chills prior to admission. After initiation of treatment for DKA, she began to have worsening dyspnea and was subsequently intubated for respiratory failure. Blood cultures and culture of her multiple abscesses grew MRSA so Vancomycin was initiated. A transesophageal echocardiogram showed vegetations on the tricuspid valve and a CT chest demonstrated both pulmonary and splenic septic emboli. The patient continued to have refractory hypoxia on the mechanical ventilator despite maximal medical treatment and was medically paralyzed to reduce hypoxia and overcome respiratory asynchrony. She developed bilateral pneumothraces requiring two right chest tubes and a left chest tube. She was then transferred to the adult surgical heart unit for emergency peripheral veno-venous ECMO placement. Ventilation was an ongoing issue which required multiple bronchoscopies due to severe mucous plugging with frank pus. The patient eventually underwent a tracheostomy due to the development of critical illness polyneuromyopathy. The patient was eventually weaned off the paralytics and the VV-ECMO. Her case was further complicated by a diaphragmatic perforation with fistula formation between the left lower lung and left upper quadrant of the abdomen requiring drainage. The patient's clinical status eventually improved and she was discharged to a rehabilitation center on IV Vancomycin therapy.

Discussion: This case shows a great example of tricuspid MRSA endocarditis with septic emboli in an IV drug abuser with diabetic ketoacidosis. It also exemplifies the use of ventilator paralysis and VV-ECMO in decompensations of the cardio-pulmonary system. The use of ECMO is becoming more common in respiratory failure patients that continue to be hypoxic and hypercapnic despite maximal medical and ventilator treatment. Cases have shown similar treatment for MRSA infections in pediatric cases, but not adults, especially MRSA endocarditis. VV-ECMO is useful for severe cases of MRSA endocarditis.

Id: 22 a Case of Polythemia Vera with Pulmonary Hypertension, Pulmonary Embolism, Hypertension and Renal Artery Stenosis

H. Tang. University of Arizona, Tucson, Arizona.

Q. gu, D. Yang, C. Xiong, Z. Zhao, Q. Luo, J. He, Z. Liu. fuwai Hospital National Center for Cardiovascular Diseases, Beijing, CHINA.

Introduction: Polycythemia vera (PV) is a chronic myeloproliferative disorder characterized by excess erythrocytosis. We present an unusual case of PV patient with hypertension, renal artery stenosis, pulmonary hypertension and pulmonary embolism. Analytic study was performed on the clinical features, imaging, laboratory test, treatment and follow up of this patient. We investigated the mechanism according to the literature.

Case report: A 47- year-old male was found hypertension 12 years ago. Renal artery angiography revealed severe stenosis at bilateral renal arteries. He took three kinds of antihypertensive agents (included nifedipine) but his blood pressure did not been satisfactorily controlled. Erythrocytosis was been neglected. The patient was diagnosed as PV after 2 years. His hemoglobin and red blood cell count were normal after treatment with interferon and hydroxycarbamide. Meanwhile, his blood pressure decreased after PV treatment. However, he felt a progressive worsening of physical capacity occurred these two years. His echocardiography showed dilated right ventricular chamber and estimated PASP 100mmHg. Computed tomography pulmonary angiogram showed no evidence of thromboembolic disease but pulmonary ventilation-perfusion scintigraphy showed pulmonary embolism. Pulmonary hypertension was confirmed at RHC with mPAP of 57 mmHg, cardiac index of 2.33L/min/m2, pulmonary artery wedge pressure of 1 mmHg, and pulmonary vasoreactivity testing was positive. His serum immunological markers were normal. He could not been treated with interventional therapy because of his distal renal artery lesion. He was started on warfarin and continued on calcium antagonist treatment. Three months followed, his physical capacity and heart function were improved. His 6-minute walk distance increased from 479m to 510m and N-terminal prohormone of brain natriuretic peptide decreased from 3357.5 pmol/L to 1316.9 pmol/L. His PASP on echocardiogram decreased from 100mmHg to 73mmHg. Repeat RHC 1 year later showed mPAP of 43 mmHg, cardiac index of 4.07L/min/m2, and pulmonary vasoreactivity testing was still positive. Pulmonary ventilation-perfusion scintigraphy showed no change.

Discussion: The patient showed pulmonary hypertension, pulmonary embolism, hypertension and renal artery stenosis, which were related to PV. PV can cause hypertension because of hypervolemia. Furthermore, the mechanism may be due to prothrombotic state leading to repeated endothelial injury, thrombosis and resultant intimal hyperplasia. In addition, interferon also can induce pulmonary arterial hypertension. PV with pulmonary hypertension was caused by multiple factors. In this case, the patient developed pulmonary arterial hypertension, but not CTEPH, because that pulmonary vasoreactivity testing was positive. PV may account for the incidence of pulmonary hypertension, pulmonary embolism, hypertension and renal artery stenosis. A comprehensive analysis should be performed to get an early diagnosis and treatment. Key words: Polycythemia vera; Pulmonary hypertension; Pulmonary embolism.

ID: 23 High Heart Rate Variability; a Marker of Healthy Longevity. Do Women have an Advantage over Men?

U. Zulfiqar. Internal Medicine, Peninsula Regional Medical Center, Salisbury, Maryland.

D.H. Singer. Dept of Medicine, Section of Cardiology, University of Illinois at Chicago, Chicago, IL.

Background: Women (W) tend to outlive men (M), world wide. Findings that aging is associated with a decline in heart rate variability (HRV), a marker of autonomic function & mortality risk, suggest that gender differences in longevity relate to differences in autonomic preservation.

Methods: We compared HRV using 4 time domain measures [rMSSD, pNN50, SDNN, SDANN] from 24 hour Holter records of 344 [159 M, 185 W], 10-99 year old healthy subjects. HRV of M and W was compared using two sample t-test. While SDNN and SDANN measure sympathetic function (Sf), rMSSD and pNN50 are indices of parasympathetic function (Pf).

Results: M have higher Pf than W at baseline. Pf then declines with aging, being more rapid in M than W, with disappearance of gender difference by age 30. The decline continues, until it reaches its nadir by late 70's, the average life span of U.S. population. Surprisingly, by early 80's the pattern reverses itself and Pf begins to increase progressively in M and W (Table 1), reaching high levels, more typical of younger individuals. Baseline Sf is higher in M than W. It then begins to decline in both with aging, but at slower rate than PF. Contrary to late reversal and increase in Pf, Sf continues to decline throughout life in M and W.

Conclusions: High Pf is cardio-protective. Slower rate of decline in Pf of W, on top of late reversal and increase in Pf, contribute to protection against arrhythmias, sudden cardiac death and hence increased longevity over M. Late life reversal & increase in Pf underscores its importance as a marker of healthy longevity.

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Table 1

Gender Effects on 24-hr HRV, Across Nine Decades

Endocrinology/Metabolism ID: 24 RELATIVE ETHNIC AND RACIAL fREQUENCY OF GCK-HYPERGLYCEMIA (GCK-MODY)

S.W. Greeley, R.N. Naylor. Pediatrics/Medicine, University of Chicago, Chicago, Illinois, UNITED STATES. D. Carmody. Medicine, University of Chicago, Chicago, Illinois. L.H. Philipson. Medicine/Pediatrics, University of Chicago, Chicago, Illinois.

Introduction: GCK-MODY leads to stable, mildly elevated blood glucose typically not requiring therapy. As GCK-MODY is frequently misdiagnosed as type 1, type 2 or gestational diabetes, we established the U.S. Monogenic Diabetes Registry to facilitate identification of cases. Within the Registry racial/ethnic minorities are underrepresented, and it is unclear if this is due to ascertainment bias or racial differences in GCK-MODY prevalence. We sought to estimate the relative frequency of GCK-MODY across racial/ethnic groups using the Exome Aggregation Consortium (EAC) in order to compare it to our Registry demography and to determine if a large dataset suggests that race specific differences exist in the prevalence of heterozygous mutations likely to cause GCK-MODY.

Methods: The EAC includes exome data from large-scale sequencing projects as part of disease-specific and population genetic studies. We derived the relative allele frequency of all heterozygous missense or loss of function variants in exons 2- 10 (or adjacent splice sites) of GCK within the EAC. Tissue specific promoter regions were excluded from analysis due to the possibility of benign polymorphisms in these regions. The allele frequency in each ethnic group was calculated using the average number of alleles assessed across each of the variants found. Novel nonsense or frameshift variants were classified as highly likely to be pathogenic if they affected a conserved amino acid.

Results: African American, Latino and Asian subjects made up only 6.0, 6.8, and 7.7% respectively of Registry probands with a GCK-MODY phenotype, despite these groups having a higher prevalence of diabetes within the US population (12.8,13.2, 9.0% respectively). The proportion of probands found to have a GCK mutation through research based testing was similar across each ethnic/racial group. Of 61,486 unrelated individuals in the EAC, there were 178 identified with mutations known or predicted to be associated with GCK-MODY, and 55 of 91 variants in the EAC dataset were novel. The average allele frequency across the entire populations was 0.001548 giving a predicted incidence of GCK-MODY of 3.096 per 1000 individuals. Deleterious mutations were found in each ethnic/racial group. Relative to the European population studied, GCK mutations were found at a higher allele frequency in Latinos and Africans (Table 1).

Conclusions: Using the largest publically accessible exome database, we have identified GCK variants in a diverse population. It is difficult to estimate the true frequency of GCK-MODY from this dataset as it included a number of diabetes-specific studies. However, the relative frequency of GCK-MODY causing mutations in this cohort would suggest that GCK-MODY is at least as prevalent in racial and ethnic minorities as in Caucasians. This suggests ascertainment bias as a cause of low rates of racial/ethnic minorities followed within the Registry with GCK-MODY and requires additional examination to ensure there is equitable utilization of diabetes genetic testing resources.

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TABLE

Racial and ethnic distribution within the Exome Aggregation Consortium

ID: 25 Testosterone Therapy Interacts with Previously Undiagnosed Familial Thrombophilia, Facilitating Development of Osteonecrosis

R. Riaz, M. Prince. Internal Medicine Residency Training Program, Jewish Hospital of Cincinnati, Cincinnati, Ohio. C.J. Glueck, P. Wang. Cholesterol, Metabolism, and Thrombosis Center, Jewish Hospital of Cincinnati, Cincinnati, Ohio. R.A. Freiberg. Department of Orthopedics, VA Hospital, Cincinnati, Ohio.

Background: Thrombophilia is etiologic for development of osteonecrosis (ON). In 12 men and 2 women who developed idiopathic osteonecrosis (ON) 6 months (median) after starting testosterone treatment (TT), we examined the interaction between TT and previously undiagnosed thrombophilia facilitating development of ON.

Methods: Measures of thrombophilia-hypofibrinolysis included PCR measures of the factor V Leiden (fV), Prothrombin, and PAI-1 gene mutations, factor VIII, factor XI, and homocysteine. We compared the 14 cases with 95 healthy normal controls without ON, and to 20 subjects on TT, without venous thromboembolism (VTE) (TT-noVTE controls).

Results: ON was “idiopathic” in the 14 cases, who not taken high dose-long term corticosteroids, were not alcoholic, and had no hip dislocation or pinning. In 10 of the 14 cases, ON developed < 6 months after starting TT. Four of the 14 cases (29%) were fV heterozygotes vs 0/95 healthy controls (p=.0002), vs 0/20 TT-noVTE controls (p=.02). Four of the 14 cases (29%) had high (>150%) factor VIII vs 7/95 (7%) healthy controls (p=.034), 3/14 (21%) had high (>150%) factor XI vs 3/95 (3%) healthy controls (p=.027), vs 0/20 TT-noVTE controls (p=.06). Of the 14 ON patients, 11 (79%) had ≥ 1 abnormal procoagulant (of the 6 measured) vs 34/95 (36%) of healthy controls (p=.003). Of the 5 cases where serum estradiol (E2) was measured while on TT, E2 was high (≥ 42.6 pg/ml) in 4. By logistic regression, of the 14 cases and 95 normal controls, with the dependent variabile ≥1 procoagulant abnormality vs no abnormality, and explanatory variables case/normal control, age, gender and BMI, the only significant explanatory variable was case/normal control, OR=7.86, 95% CI 1.58-39.15, p=.01.

Conclusions: When TT is given to patients with familial thrombophilia, especially the factor V Leiden mutation, risk of developing ON is increased. E2 is often high in men on TT, via aromatization, and, speculatively, is the mechanism of the interaction of TT with thrombophilia producing ON. Screening for common familial thrombophilias before starting TT should optimize estimation of the benefit/risk ratio of TT.

Id: 26 Pancreatic Islet Glp-1 Secretion is Increased in Non-Diabetic Obesity and Glp-1 Directly Regulates Beta-Cell Cholecystokinin Production

A.K. Linnemann, T.J. Battiola, M.E. Kimple, D.B. Davis. Medicine/Endocrinology, University of Wisconsin-Madison, Madison, Wisconsin. J.C. Neuman. Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin. D.B. Davis. William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin.

finding ways to protect pancreatic beta-cells from apoptosis is of particular interest in the prevention and treatment of diabetes. The use of glucagon-like peptide-1 (GLP-1) mimetics for the treatment of type 2 diabetes is increasing rapidly. However, despite in vitro and ex vivo evidence that GLP-1 protects beta-cells from apoptosis, the exact mechanism is not fully understood. Although GLP-1 is classically produced by intestinal L-cells after a meal, it was recently demonstrated that islet alpha-cells can also produce GLP-1 in response to stimuli such as cytokines and nutrients. Furthermore, islets isolated from obese mice express increased levels of prohormone convertase 1/3, the enzyme responsible for processing of GLP-1. This raises the question of whether islet production of GLP-1 is increased as a function of obesity in vivo. We now show that active GLP-1 is also secreted from human pancreatic islets as a function of BMI (R2=0.406, p=0.01). Similar to human, islets from obese C57BL/6J mice with a leptin mutation (ob/ob mice) secrete significantly more GLP-1 than islets from lean controls (p=0.0016). Considering the short half-life of GLP-1, it is likely that this islet-produced GLP-1 acts in a paracrine fashion to regulate a signaling network that is activated in obesity and may function to preserve beta-cell mass. Similarly, it was recently discovered that the classic gut hormone cholecystokinin (CCK) is also expressed in pancreatic beta-cells and is the most highly upregulated islet gene in response to obesity. Loss of CCK in obese mice results in decreased beta-cell mass and increased beta-cell death, leading to elevated fasting blood glucose. Additionally, we have recently found that beta-cell specific overexpression of CCK in lean mice confers protection from streptozotocin (STZ) induced apoptosis. This suggests that CCK intra-islet signaling is also important for apoptosis protection. As both GLP-1 and CCK are made in the obese islet, we hypothesized that GLP-1 regulates CCK in non-diabetic obesity. Indeed, GLP-1 treatment of betacells stimulates both CCK production (p=0.047) and secretion (p=0.004) by ∼2-fold. GLP-1 signals through cyclic AMP (cAMP) and treatment of beta-cells with a membrane permeable cAMP analogue stimulates Cck transcription by up to ∼9-fold (p=0.0005) and secretion by ∼8-fold (p=0.004). We used chromatin immunoprecipitation (ChIP) to show that both cAMP and GLP-1 treatment promote recruitment of RNA polymerase 2 and the cAMP-modulated transcription factor, CREB, to the promoter of the Cck gene in beta-cells (p<0.05 for all experiments). Inhibition of cAMP with sulprostone reduces Cck transcription by ∼50% indicating that cAMP-mediated signaling is required. However, reduction of glucose does not abolish the ability of cAMP to stimulate CCK production or secretion. Importantly, we find that CREB recruitment to the Cck promoter is present in the islets of ob/ob mice, but not in lean mice.

In summary, we provide a novel description of how islet production of incretins is regulated. We show that both human and mouse islets secrete active GLP-1 as a function of BMI/obesity. In turn, GLP-1 can promote beta-cell CCK production and secretion through direct transcriptional regulation of the gene in a cAMP-dependent manner. This mechanism of regulation likely explains the increase in islet CCK production seen in obesity. As both CCK and GLP-1 offer protection of beta-cells from apoptosis, we propose that these hormones may be co-regulated within the islet and exert a local effect of beta-cell protection.

Id: 27 Differential Effects of Leptin on Adiponectin Expression with Weight Gain versus Obesity

P. Singh, P. Sharma, K.R. Sahakyan, D.E. Davison, F.H. Sert-Kuniyoshi, A. Romero-Corral, F. Lopez-Jimenez, T. Kara, V.K. Somers. Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota. J.M. Swain. Department of Surgery, Mayo Clinic, Rocehster, Minnesota. M.D. Jensen. Endocrinology Research Unit, Mayo Clinic, Rochester, Minnesota.

Background/Objective: Adiponectin exerts beneficial effects by reducing inflammation, and improving lipid metabolism and insulin-sensitivity. Although adiponectin is lower in obese individuals, whether weight gain reduces adiponectin expression in humans is controversial. We sought to investigate the role of weight gain, and consequent changes in leptin, on altering adiponectin expression in humans.

Methods/Results: forty four normalweight healthy subjects were recruited (mean age 29 years; 14 women) and randomized to either gain 5% of body weight by 8-weeks of overfeeding (n=34) or maintain weight (n=10). Modest weight gain of 3.8 ± 1.2 kg resulted in increased adiponectin (p=0.03) while weight maintenance resulted in no changes in adiponectin. Further, changes in adiponectin correlated positively with changes in leptin (p=0.0085). In-vitro experiments using differentiated human white preadipocytes showed that leptin increased adiponectin mRNA and protein expression, while a leptin-antagonist had opposite effects. To understand the role of leptin in established obesity, we compared adipose tissue samples obtained from normal weight versus obese subjects. We noted, first, that leptin activated cellular signaling pathways and increased adiponectin mRNA in adipose tissue from normal-weight participants, but did not do so in adipose tissue from obese participants; and second, that obese subjects had increased caveolin-1 expression, which attenuates leptin-dependent increases in adiponectin.

Conclusions: Modest weight gain in healthy individuals is associated with increases in adiponectin, which correlate positively with changes in leptin. Leptin induces adiponectin expression in-vitro. Additionally, adipose tissue from obese subjects show increased caveolin-1 expression, which contributes to impaired leptin signaling. This leptin signal impairment may prevent concordant increases in adiponectin in obese subjects despite their high levels of leptin. Therefore, impaired leptin signaling may contribute to low adiponectin expression in obesity. Improved leptin signaling in obese subjects may be a target for increasing adiponectin expression, and hence improving insulin sensitivity and cardio-metabolic profile, in obesity.

Id: 28 Targeted Disruption of Atp Sensitive Potassium Channel Expression in Skeletal Muscle Promotes Energy Consumption during Physical Activity

S. Koganti, D. Zhu, D. Subbotina, D. Gao, D. Sierra, M. Proenza, L. Yang, D. Zingman. Internal Medicine, The University of Iowa, Iowa City, Iowa. D. Zingman, A. Alekseev. Mayo Clinic, Rochester, Minnesota.

Despite the medical, social and economic impact of obesity, only a few therapeutic options, focused largely on reducing caloric intake, are currently available and these have limited success rates. A major impediment is that any challenge by caloric restriction is counterbalanced by activation of systems that conserve energy to prevent body weight loss. Therefore, targeting energy-conserving mechanisms to promote energy expenditure is an attractive strategy for obesity treatment. Skeletal muscles account for about 40-50% of body mass and their function relies on the energy demanding processes of ion homeostasis and actin-myosin cycling. Even under sedentary conditions, spontaneous physical activities required to maintain muscle tone, body posture and “fidgeting” account for about 7- 10% of daily body energy usage. During exercise, energy consumption by skeletal muscles increases 20-100 times over the basal level. Considering this level of muscle metabolic activity, even small changes in energy efficiency could have a substantial effect on bodily energy consumption. Here, in order to suppress muscle energy efficiency, we target sarcolemmal ATP-sensitive potassium (KATP) channels which have previously been shown to be important in maintaining muscle energy economy. Specifically, we employ intramuscular injections of cell-penetrating oligonucleotides to prevent translation of the channel pore-forming subunit. This intervention results in significant reduction of KATP channel expression and function in treated areas, without affecting the channel expression in nontargeted tissues. Furthermore, suppression of KATP channel function in a group of muscles causes a substantial increase in activity-related energy consumption, with little effect on exercise tolerance. These findings establish a proof-of-principle that selective skeletal muscle targeting of sarcolemmal KATP channel function is possible and that this intervention can alter overall bodily energetics to treat or prevent obesity.

Id: 29 Ethanol Activates Midkine and Alk Signaling in Neuroblastoma Cells and in the Brain

D. He, H. Chen, H. Muramatsu, A. Lasek. Psychiatry, University of Illinois at Chicago, Chicago, Illinois.

Background: Midkine (MDK) is a secreted growth factor whose expression is higher in the prefrontal cortex of chronic alcoholics and in mice genetically predisposed to consume large amounts of alcohol (ethanol). One of the receptors for MDK is anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase that regulates behavioral responses to ethanol in mice. Although MDK and ALK activity appear to modulate behaviors related to alcohol abuse, it is not known whether ethanol activates signal transduction through these proteins. We investigated whether ethanol exposure alters MDK and ALK gene expression and signaling pathways in neuroblastoma cells in culture. To extend our studies to the brain, we determined if MDK and ALK are important for the ability of ethanol to increase phosphorylation of extracellular signal-regulated kinase (ERK), a downstream transducer of MDK and ALK signaling.

Methods: The human neuroblastoma cell lines SH-SY5Y and IMR-32 were treated with various concentrations of ethanol and tested for gene expression changes using quantitative real-time PCR (qPCR) and protein expression using western blotting. Activation of ALK signaling was examined in IMR-32 cells treated with 100 mM ethanol in the presence and absence of the ALK inhibitor TAE684 or a small-interfering RNA (siRNA) targeting MDK. Phosphorylation of ALK, ERK, STAT3, and AKT were measured after ethanol exposure by western blotting. IMR-32 cells were also treated with recombinant MDK protein and examined for protein phosphorylation. To test if ALK is important for ERK phosphorylation in response to ethanol in the brain, mice were treated with 3 g/kg ethanol intraperitoneally after pretreatment with TAE684. Thirty minutes after ethanol treatment, brains were removed and processed for immunohistochemistry using antibodies to phosphorylated ERK. ERK phosphorylation in response to ethanol treatment was also examined in the brains of MDK knockout mice.

RESULTS: MDK and ALK gene and protein expression increased in response to ethanol treatment in SH-SY5Y and IMR-32 cells. Ethanol also caused a rapid increase in ALK, ERK, and STAT3 phosphorylation in IMR-32 cells. Treatment of IMR-32 cells with recombinant MDK had a similar effect on protein phosphorylation compared to ethanol. These effects were blocked by pretreatment with TAE684 or MDK siRNA. ERK phosphorylation in the amygdala was increased after ethanol treatment, an effect that was attenuated by pretreatment with TAE684 and in MDK knockout mice.

CONCLUSIONS: These results suggest that ethanol activates signaling in the brain through the MDK and ALK pathways and that the MDK and ALK genes are ethanol-responsive. Since these genes are important for modulating behavioral responses to ethanol, targeting ALK and MDK signaling pathways may represent a novel therapeutic strategy for the treatment of alcohol use disorders.

Id: 30 Prevalence of Preeclampsia in Pre-Gestational Diabetic Pregnancy in Bangladeshi Patients

T.J. Kuehl, M.N. Uddin. Obstetrics and Gynecology/Pediatrics, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. G.U. Ahsan, M. Jahan, M. Hasanuzzaman, S. Mahbuba, H. Jahan, S. Chowdhury, K. Leena, M.N. Uddin. Public Health, North South University, Dhaka, Dhaka, BANGLADESH.

Objective: Preeclampsia (preE) is a pregnancy disorder characterized by the de novo development of hypertension and proteinuria with multiple path physiologic triggers and mechanisms. Approximately 20% of the diabetic pregnant women develop preE. The mechanisms contributing to this effect is not well characterized. In a recent study, we have shown that hyperglycemia impairs cytotrophoblast (CTB) function via stress signaling. Several researchers demonstrate a direct link between preE and diabetes. The objective of the study was to evaluate potential linkage between the risk of developing preE and the presence of diabetes in pregnant patients in Bangladesh.

Methods: This is a cross-sectional study of 406 pregnant women performed to evaluate the prevalence of PreE with respect to different risk factors such as previous pregnancy, presence of Antiphospholipid antibodies, pre-existing diabetes (before this pregnancy), multiple gestation / singleton, family history of preE in first degree relative (mother, sister and daughters; most commonly mother), maternal age of 40 or greater. The study was conducted in selected hospitals of Dhaka city, Bangladesh during October 2012 to December 2013. We gave special emphasis on the occurrence of PreE in pre-gestational diabetic patients. This study was approved by the Ethical review committee of Bangladesh Medical Association.

Results: The key study findings revealed that the rate of development of PreE in Bangladeshi pre-gestational diabetic patients is 23%.

Conclusions: There is an association has been found between the risk of developing preE and the presence of diabetes in pregnant patients in Bangladesh.

Id: 31 the Extracellular Matrix Regulates Adipose Function and Expansion

M.K. Vaicik, E.M. Brey. Biomedical Engineering, Illinois Institute of Technology, Chicago, Illinois. M.K. Vaicik, E.M. Brey. Research Service, VA Hines, Hines, Illinois. M. Morse, A. Blagajcevic, R. Cohen. Section of Endocrinology, Department of Medicine, University of Chicago, Chicago, Illinois.

Obesity is a global epidemic that contributes to the increasing medical burdens related to type 2 diabetes, cardiovascular disease and cancer. The extracellular matrix (ECM) has been shown to regulate the development and function of numerous tissues and organs. An understanding of the role the ECM plays in adipose tissue function and expansion could lead to new therapeutics that eliminate or reduce obesity-associated morbidity and mortality. Laminin a4 is upregulated during adipogenesis and is present in the ECM surrounding fully differentiated adipocytes. However, there is little understanding of its function in adipose tissue. We have found that mice with a null mutation of the laminin α4 gene (Lama4−/−) exhibit reduced weight gain and fat mass accumulation in response to both aging and high-fat diet when compared to wild-type (Lama4+/+) animals. However, the underlying mechanisms by which Lama4 regulates fat mass have not yet been defined. We have now found that physical activity and food intake does not differ between Lama4+/+ and Lama4−/− mice. However, Lama4−/− mice have a significantly increased metabolic rate at 25C (room temperature) and 16C (cold) compared to Lama4+/+ mice. Interestingly, Lama4−/− mice exhibit significantly increased UCP-1 expression in subcutaneous adipose tissue [18.79 ± 4.97% UCP-1 positive compared to 2.62 ± 1.63% (n=5, p£0.01)]. In contrast, in thermoneutral conditions at 30C both Lama4+/+ and Lama4−/− mice exhibit equivalent metabolic rates. These results suggest that beiging of subcutaneous adipose tissue in Lama4−/− mice may lead to decreased adipose tissue accumulation and potentially improved metabolic function. Thus, alterations in laminin composition suggest that the ECM plays a role in modulating cellular behavior in adipose tissue expansion in a temperature- and depot-specific manner.

Id: 32 Significant Differences in Fecal Microbiota Are Associated with Various Stages of Glucose Tolerance in African-American Male Veterans

I. Ciubotaru, S. Kukreja, E. Barengolts. Endocrinology, University of Illinois at Chicago, Chicago, Illinois. I. Ciubotaru, A. Akbar, S. Kukreja, E. Barengolts. Endocrinology, Jesse Brown VA, Chicago, Illinois. S. Green. CRC, DNA Services, University of Illinois at Chicago, Chicago, Illinois.

There is emerging evidence that intestinal microbiota is a contributor to the metabolic/glycemic phenotype. While changes in microbiota have been described in obesity and diabetes, little is known about microbiota composition in various dysglycemic states. This study aimed to investigate the relationship between microbiota and changes in the glycemic control of prediabetic subjects. Stool was collected from African American men participating in a randomized controlled trial of vitamin D Intervention at VA. Four groups (Gr) were characterized based on changes in OGTT between baseline (T0) and the study completion at 12 months (T12): Gr 1- stable normal glucose tolerance (GT); Gr 2- stable impaired fasting glucose or stable impaired GT; Gr 3 - worsened GT; and Gr 4 - improved GT. Microbiota DNA was extracted from stool collected at T12, analyzed using high-throughput next-generation sequencing of microbial rRNA genes and data processed using established bioinformatics pipelines. Microbiota (composition, alpha diversity, abundance) was analyzed in 116 subjects: Gr 1= 35, Gr 2 = 27, Gr 3 = 24, and Gr 4 = 29. At Phylum level significant differences in bacterial composition were observed between Gr 1 and Gr 2 (p= 0.03) and a trend to significance for Gr1 vs Gr3 (p= 0.06), and Gr 1 vs Gr4 (p= 0.06). Bacteroidetes was higher, firmicutes lower, and hence the Bacteroidetes/firmicutes ratio (B/f) was lower with worsening glycemic control (B/f: Gr 1 vs Gr 2 = 1.9 vs 0.9, p= 0.01; and Gr 1 vs Gr 3 = 1.9 vs 1.1, p= 0.04). Proteobacteria decreased in Gr 2 and Gr 4 compared to Gr 1 (p=0.01 for both). Similarly, there were significant differences in microbiota at the family and Genus levels. In Gr 2 vs Gr1 there was less Prevotella (hence higher Bacteroides/Prevotella ratio, 5.6 vs 2.7, p= 0.05), less Enterobacteriaceae (p=0.03), and more Ruminococcaceae (p= 0.01) and Veillonellaceae (p= 0.02). Notably, Akkermansia was more abundant in Gr 4 vs Gr 1 (p=0.04). We speculate that lower abundance of Prevotella may be associated with worsening glycemia, and conversely higher abundance of Akkermansia might be associated with improving glycemia, thus corroborating suggestions from previous studies. Ruminococcaceae might be associated with higher insulin level (seen in previous research), and therefore conducive to maintenance of stable glycemia. Observed association of Veillonellaceae and glycemia was novel. In Conclusion, significant differences in microbiota (composition, alpha diversity, abundance) were observed in various GT states. Interesting most of the differences were seen between normoglycemic subjects and those who were remained prediabetics at the end of the study. These findings suggest that there may be a certain makeup of the gut microbiota associated with steady glycemic states. Further studies are needed to evaluate whether causative relationship exist between microbiota and changes in GT.

Id: 33 Roles of Growth Hormone/Insulin-Like Growth Factor-I Axis in Prostate Stem/Progenitor Cells Amplification

J.D. Rinaldi, D. Hu, W. Hu, G. Shi, L. Birch, G.S. Prins. Urology, UIC, Chicago, Illinois. J.D. Rinaldi, L. Justulin. Morphology, UNESP, Botucatu, Sao Paulo, BRAZIL.

Background: Growth hormone (GH) promotes insulin-like growth factor-I (IGf-1) production and GH/IGf-1 axis has been implicated in cancer including prostate cancer. Further, rodent models lacking GH or its receptor are resistant to the induction of a wide range of cancers. IGf-I is a mitogen for prostate epithelial cells and its serum level has been associated with prostate cancer risk in human. In rodent, both IGf-1 and IGf-1 receptor (IGf- 1R) were found to increase in SV40-T prostate cancer. Cancer may originate from the transformation of stem cells and previous studies shown that stem cells may be the direct target of hormones.

Aim: The aim of the current study was to investigate the roles of GH/IGf-1 axis in prostate stem/progenitor cell amplification.

Methods: By using a 3-D prostasphere culture system, adult prostate stem/early progenitor cells were isolated from ventral (VP), dorsal (DP) and dorsolateral (DLP) prostates obtained from Sprague–Dawley (WT) and Spontaneous Dwarf rat (DW, which lacks GH due to a mutation (dr) in its GH gene). Gene expressions of GHR and IGf-1R were evaluated by real-time qPCR and GH effects on prostate stem/progenitor cell proliferation were measured by prostasphere number and size.

Results: Both GH and IGf-1R were expressed in prostate stem/progenitor cells. Although VP from DW rats expressed lower levels of GHR, IGf-1R was increased in both VP and DP from DW rats as compared to WT rats. Further, VP from DW rats generated an increased number of prostaspheres than VP from WT rats. Finally, treatment of GH (10 and 100 nM) dose-dependently increased the prostasphere number in DLP from DW but not from WT Prostate stem/early progenitor cells were isolated from ventral (VP) and dorsal (DP) prostate using 3-D Matrigel culture for 7 days. GHR and IGf-1R gene expressions were quantified by real-time qPCR (N=5). Of note, both genes were expressed in prostate stem/progenitor cells from WT and DW rats. Although rat prostate expressed lower levels of GHR, IGf-1R was increased in both VP and DP from DW rats when compared to WT rats.

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Figure 1

Prostate stem/early progenitor cells were isolated from ventral (VP) and dorsal (DP) prostate using 3-D Matrigel culture for 7 days. GHR and IGF-1R gene expressions were quantified by real-time qPCR (N=5). Of note, both genes were expressed in prostate stem/progenitor cells from WT and DW rats. Although rat prostate expressed lower levels of GHR, IGF-1R was increased in both VP and DP from DW rats when compared to WT rats.

ID: 34 the Impact of Prostaglandin E2 Levels on Glycemic Control and Therapeutic Response in Human Subjects with Type 2 Diabetes Mellitus

A.C. Weeks, D.B. Davis, M. Kimple. Endocrinology, Diabetes & Metabolism, University of Wisconsin, Madison, Wisconsin. M. Dart. Department of Surgery, University of Wisconsin, Madison, Wisconsin. X. Li. Department of Biostatistics and Medical Informatics, University of Wisconsin, Madison, Wisconsin.

The varied response of patients with type 2 diabetes mellitus (T2DM) to therapeutic agents is well recognized, but the underlying etiology remains unclear. Given the substantial morbidity and mortality associated with T2DM, as well as the cost of pharmacotherapy, elucidating causal factors is of great interest. As incretin mimetics function in part by directly stimulating the insulin-producing b-cells, factors down-regulating b-cell function are of particular concern. Using islets isolated from diabetic mice and humans, we showed prostaglandin E2 (PGE2) acts through a specific b-cell receptor to reduce insulin secretion, directly competing with incretin signaling[i]. We hypothesized (1) individuals with high PGE2 production have poorer glycemic control and a weaker response to incretin-based therapeutics than those with low PGE2 production and (2) plasma PGE2 level can be a biomarker of T2DM status and therapeutic response. In a preliminary study using 75 anonymized patient samples, individuals with T2DM (n=65) had significantly elevated plasma PGE2 levels (18.0 ± 22.4 pg/ml) as compared to non-diabetics (ND) (11.0 ± 7.3 pg/ml, n=10). Subjects with diabetic nephropathy, indicating poorer glycemic control, demonstrated the greatest elevation (19.9 ± 11.6 pg/ml, n=16, p=0.04 vs. ND). This data gave support to a new cohort study of T2DM and ND subjects recruited from the UW Endocrinology and Diabetes Clinic. Exclusion criteria included age <18 or>70, chronic or recent use of most COX inhibitors or oral steroids, active infection, anemia of any type confounding hemoglobin A1c measurement, and most autoimmune diseases. A power analysis with α=0.05 and b=0.08 indicated 35 ND and 137 T2DM subjects (with 20-30% on incretin therapy) needed. PGE2 levels are correlated with (1) donor demographics and measures of (2) diabetes/obesity status, (3) glycemic control, and (4) inflammation. Chart review is performed at 6 and 12 months to record diabetes status, glycemic control and medication failure. We performed an interim analysis of 62 subjects (14 ND and 47 T2DM, with 19 T2DM subjects on incretin–based therapy). Multivariable analysis of all subjects adjusted for covariates demonstrated no significant effects on PGE2 levels with respect to age, BMI, triglycerides, HgA1c, ESR, or aspirin use; however, age (p=0.0795) and TG (p=0.0588) demonstrated a strong trend. Within the T2DM group alone, triglycerides again showed a strong trend (p=0.0678), as did ESR (p=0.1363) and age (p=0.18). The correlation of ESR with PGE2 levels in the T2DM group appeared to be more relevant with incretin therapy (p=0.2398) than without (p=0.9732), whereas the opposite was true for triglyceride levels (p=0.3262 with incretin therapy vs. 0.0489 without). Although results did not show a significant correlation between PGEM levels and T2DM status, this may reflect additional unidentified covariates rather than a true rejection of our hypothesis, providing guidance for the full analysis.

Id: 35 Hypoglycemia in Sulfonylurea-Treated Kcnj11-Related Diabetes is Infrequent and Usually Mild

D. Carmody, M. Lanning, L. Szczerbinski, L.H. Philipson, S.W. Greeley. Section of Pediatric and Adult Endocrinology, Diabetes and Metabolism, University of Chicago, Chicago, Illinois.

Background: Persistent hyperglycemia requiring treatment diagnosed within the first six months of life is referred to as neonatal diabetes mellitus (NDM) and has an incidence of 1 in 90,000-160,000 live births. Mutations in KCNJ11, one of the genes encoding the ATP-sensitive potassium channel, are the most common monogenic cause of permanent NDM. The great majority of patients with KCNJ11-related diabetes are able to transition from insulin therapy to high dose oral sulfonylureas and experience excellent glycemic control; however, little data has been available on the risk of any hypoglycemia.

Objective: To determine the frequency, severity, and clinical significance of hypoglycemia in subjects with KCNJ11-related diabetes in order to better inform clinical decision making regarding risks and benefits of increasing sulfonylurea dosage.

Design/Methods: Subjects consented for participation through the University of Chicago Monogenic Diabetes Registry. Parents completed a 36 item online questionnaire addressing detailed aspects of hypoglycemia. Continuous glucose monitoring (CGM) data was analyzed for any subject who had previously utilized CGM.

Results: 29 subjects with sulfonylurea-treated KCNJ11-related NDM (16 females, 13 males; mean age = 10.5 years) were assessed. The average dose of sulfonylurea (glyburide) was 0.58 ± 0.54 mg/kg/day. The average HbA1c was 5.72 ± 0.75%. Respondents expressed wide variation on what they considered to be low blood glucose (range 50-80 mg/dL; average 62 mg/dL) and the frequency of glucose monitoring (>3 times daily to less than once a week). Clinical hypoglycemia (<70 mg/dL) was reported as once monthly or less frequently in 81.5% of individuals, while 6 individuals (20.7%) reported at least one episode ever of severe hypoglycemia (defined as partially or fully unconscious, having a seizure, or otherwise unable to assist with their own care). 7 individuals wore a CGM for a total of 912 hours with blood sugars falling below 70 mg/dL for 6% of the time recorded.

Conclusions: families report infrequent and usually mild hypoglycemia in those treated with sulfonylureas for KCNJ11-related diabetes. Severe hypoglycemia was rarely reported despite the high doses of sulfonylurea used and near-normal level of glycemic control seen in this unique population. CGM may be a useful tool when adjusting doses in those with KCNJ11-related diabetes.

ID: 36 Expression Of Receptors For Growth Hormone-Releasing Hormone (Ghrh-R) In Human Papillary Thyroid Cancer Cells: Effects Of Ghrh Antagonists On Matrix Metalloproteinase-2

P. Catanuto, J. Tashiro, M.K. Glassberg, N.L. Block. Medicine, University of Miami Miller School of Medicine, Miami, florida. P. Catanuto, J. Tashiro, J.I. Lew, S.J. Elliot. Surgery, University of Miami Miller School of Medicine, Miami, florida. F.G. Rick. Urology, florida International University, Miami, florida. A.V. Schally. Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center and South florida Veterans Affairs foundation for Research and Education, Miami, florida.

Papillary thyroid cancer (PTC) is the most prevalent of all endocrine cancers. In recent studies, the presence of receptors for pituitary-type growth hormone-releasing hormone (pGHRH-R) has been demonstrated in various human cancers, including human prostate, brain, and other cancer lines. Thyroid malignancies however, have not yet been investigated in this regard. In this study, we found that pGHRH-R and its functional splice variant, SV1, are present in normal thyroid and PTC cells. We also treated seven normal and PTC tumor thyroid cells in vitro with a GHRH antagonist, MIA-602, to compare its anti-proliferation and anti-invasion potential against untreated cells. We found that treatment with GHRH antagonist increases the expression of SV1 and pGHRH-R in tumor cells compared to tumor cells exposed to vehicle only, a response which may alter the sensitivity of signaling kinases within the cells. GHRH antagonist treatment of tumor cells also reduced activity of the tumor invasion marker, matrix metalloproteinase (MMP)- 2, compared to tumor cells exposed to vehicle only. The expression of pGHRH-R and SV1, as well as MMP-2 activity, in normal thyroid cells remained unaffected by GHRH antagonist treatment. Similarly, cell proliferation rates for tumor or normal thyroid cells were not affected by GHRH antagonist treatment. Our findings have important implications for the therapeutic use of GHRH antagonist in cases of aggressive PTC refractory to conventional treatment modalities, and in which protein expression and MMP-2 activity in normal thyroid tissue is left unaltered.

Id: 37 Pituitary Tumor Apoplexy and Influenza: Case Report

T.M. Yousuf, N. Libo, A. Lara, A. Bacal, T. Yasmeen. Internal Medicine, Advocate Christ Medical Center, Oak Brook, Illinois.

INTRODUCTION: Pituitary adenomas have a population prevalence of less than 0.1%. They can rarely present with serious complications such as pituitary tumor apoplexy which occurs in 1.6-2.8% of patients with a macroadenoma. Apoplexy occurs more frequently in men than women, with a ratio of 2:1. The most common symptoms of pituitary tumor apoplexy are sudden severe headache, vision impairment, nausea and vomiting. These are due to sudden expansion of the pituitary mass which most commonly arises secondary to an acute hemorrhage or infarction. We report a case of pituitary tumor apoplexy in a female patient incidentally found to have a macroadenoma.

CASE DESCRIPTION: Our case is of a 44 year old female with past history of hypertension and diabetes mellitus who presented to the emergency room with a 2 day history of left anopsia and right hemianopsia along with progressively worsening headache and dizziness for the past 1 month. She also reported amenorrhea for the past four years and she gained 30 pounds over the past 6 months. She denies the use of any anticoagulants or OCP. On physical exam, she was found to be in no acute distress, febrile with a temperature of 39.0 and hypertensive at 166/80. Pertinent laboratory studies included: influenza A positive, fSH 0.7 munit/ml, LH <0.1 munit/ml, prolactin 0.6 ng/ml (low), TSH 0.597 mcunit/ml, fT4 0.6 ng/dl (low), hemoglobin A1c 7.2%, cortisol 187 mcg/dl (measured after 100 mg of intravenous hydrocortisone was started) and insulin like growth factor 90 ng/ml. CT and MRI revealed a 2.9 cm x 3.1 cm suprasellar and sellar mass with high density components and mass effect on the optic chiasm. MRI of the brain revealed heterogeneous appearance of a large suprasellar mass compatible with macroadenoma with internal hemorrhage. In addition, there was also restricted diffusion present in the left caudate head and adjacent anterior putamen concerning for acute ischemia from vascular compression. She underwent emergent transsphenoidal excision of the pituitary tumor, with pathology confirming the presence of an adenoma with apoplexy. Her steroid regimen was quickly tapered to oral hydrocortisone 30 mg/day on post op day 4. Urine output, sodium level and frequent neurological checks were normal postoperatively. On postoperative day 5, the patient's right sided vision began to improve but her left sided vision was still completely absent. On postoperative day 6, she was able to count fingers with nasal field on right side with very poor vision on right temporal field. She received a five day course of oseltamivir and was discharged on levothyroxine 75 microg/day.

DISCUSSION: Pituitary tumors can present with either symptoms due to hormonal imbalances or mass effect on adjacent structures. Our case of pituitary apoplexy had the unique presentation of severe mass effect and stroke. Prompt recognition of the mass effect is needed in order to prevent permanent neurological damage. The association of influenza A with pituitary tumor apoplexy has not been described before. While a causative link cannot be firmly established, our patient was not on anticoagulants and influenza infections have been described to increase the risk of bleeding in general.

ID: 38 Design Of Pilot Study To Improve Quality-Of-Life In Chronic Diseases Using Custom Web-Based Educational And Monitoring Platform

O. Oni. Clinical Research, Kansas City VA Medical Center, Kansas, Missouri. A. Goel, V. Savin. Nephrology, Kansas City VA Medical Center, 4801 E Linwood Blvd, Kansas City, Missouri. K. Gandy. Play-it Health, LLC, 1800 Baltimore Avenue, Kansas City, Missouri. E. Witten. Witten and Associates, LLC, 8318 Connell Street, Overland Park, Kansas.

Background: The Veterans Health Administration system serves a cohort of Veterans who are aging and affected by chronic diseases. A large proportion of Veterans live far from medical centers, and in remote places. A web-based application will potentially bridge the gaps in the care of individuals with chronic conditions who would otherwise make frequent hospital visits, require closer monitoring, and are always in need of information regarding their conditions. Chronic Kidney Disease (CKD) is of particular interest to us because it is a risk factor for progression of vascular disease and to debility and dependence on costly renal replacement therapy (RRT). The disease burden represented by CKD is great and people with CKD experience a diminished Health-related quality of life (HRQOL).

Objectives: The question to be tested is whether a web-based platform can provide tools that engage Veterans and increase their HRQOL. Specific Aim 1. Customize and implement internet-based informational modules and patientprovider interaction devices for use by individuals with CKD. Modules will include facts about kidney disease and other chronic conditions. Medication reminders and adherence monitoring will be included. We will explore the use of several mobile devices and determine effective ways to introduce Veterans with limited computer experience to these devices. We will test several game formats and reward systems. Specific Aim 2. Assess baseline HRQOL and activation using the VR-36 Health Survey and the Patient Activation Measure (PAM-13) which have been validated in chronic illnesses. HRQOL is a predictor of medical well-being and the PAM-13 is also an indicator of patient outcomes. We will perform testing at the beginning and end of the study. The data will define baseline values in our CKD patients, reveal trends for change and provide data to assist in power analyses in the design of a larger study. Increase in VR-36 and PAM-13 will be the primary end-points. Specific Aim 3. Assess changes in physical activity and medical resource utilization. We will use direct measurement by accelerometer and will review medical charts. We will assess physical activity at the beginning and end of the study and will compare medical utilization in the 6 months prior to the period and during the study. Any changes in activity or medical outcomes will be secondary outcomes. Data from the pilot study will establish feasibility for a future study.

Methods: Twenty subjects with CKD will be randomized in a prospective 2-arm cohort study. Experimental subjects will receive an activity monitor and an electronic device with access to study educational modules. We will partner with Play-it health©, to adapt existing software. Control subjects will receive activity monitors and computer tablets and publicly available on-line and printed resources. Exclusion criteria are only unwillingness to participate and inability to learn to use the tablet format. Subjects will be recruited from our Renal clinics. HRQOL, patient activation surveys, and clinical parameters will be recorded at baseline and end-of-study. Differences in study endpoints between the two arms will be determined. We will generate descriptive statistics for each measure and analyze changes in serial measurements of endpoints. Mixed modelling analysis will be performed to compare endpoints.

Anticipated impact: Demonstrating that a mobile webbased platform can be used to engage Veterans, enhance knowledge and improve HRQOL, will be a significant contribution to care of Veterans with multiple chronic medical conditions.

Id: 39 An Online Discussion Group for Rare Monogenic Diabetes Patients: Supporting Families and Fueling New Research

M.E. Perrone, D. Carmody, L.H. Philipson, S.W. Greeley. Section of Pediatric and Adult Endocrinology, Diabetes and Metabolism, University of Chicago, Chicago, Illinois.

Background: Online discussion forums are a popular resource for patients with rare diseases. There is a paucity of data on the degree to which such groups provide useful information or valuable support to their participants and whether any additional utility is gained by physician/researcher participation.

Objective: To assess the utility of the Kovler Monogenic Diabetes Registry online discussion group for families affected by a rare form of diabetes – ATP-sensitive potassium channel (KATP)-related diabetes – in providing psychosocial support and information, and in identifying concerns unique to these patients.

Design/Methods: We qualitatively analyzed all 1,410 messages generated during 2010-2013 by the group, which consisted of 64 people with a family member affected by KATP-related diabetes and 11 researchers. We used the Social Behavior Support Code to characterize the type of support each message contained (information, networking, esteem, emotional, or tangible support). Deductive thematic analysis was used to identify discussion topics addressed by each message.

Results: The most prevalent type of support was found in the 44% of messages that were informational (e.g. “Hearing so much about the other kids is invaluable in helping us to nail down [my child's] best way to take his meds.”), while 17.7% of the messages involved networking (e.g. “When you're here for a visit let me know. I would love to meet you.”); 17.5% of messages contained esteem support (e.g. “These are amazing progress reports all! Makes me so excited and hopeful!”); 13.9% contained emotional support (e.g. “I know it must have been a difficult day for you. I hope you know that you were not alone that day, or now.”); and 9.2% of messages contained tangible support (e.g. a parent offered to help write IEPs (individualized education programs) for others’ children). The most common topics of discussion were diabetes management (503 messages) and neurodevelopmental concerns (472 messages). Researcher participation in the discussion led to modifications of survey instruments and new studies focusing on specific novel topics of concern, such as abnormal sleep patterns mentioned by participants.

Conclusions: We demonstrate that an online discussion group for a rare disease is a valuable tool for informational and psychosocial support. Notably, participation by researchers in this group proved to be invaluable in that it informed future research directions.

Id: 40 Effect of Vitamin D Supplementation during Pregnancy on Maternal and Neonatal Outcomes: A Systematic Review and Meta-Analysis of Randomized Clinical Trials

P. Thota. Infectious Diseases, Case Western Reserve University, Cleveland, Ohio. FR. Perez-Lopez. University of Zaragoza, Zaragoza, SPAIN. V. Pasupuleti. Case Western Reserve University, Cleveland, Ohio. E. Mezones-Holguin, V. Benites-Zapata. Instituto Nacional de Salud, Lima, PERU. A. Deshpande. Medicine, Cleveland Clinic, Cleveland, Ohio. A.V. Hernandez. Cleveland Clinic, Cleveland, Ohio.

Objective: To assess the effects of vitamin D supplementation during pregnancy on obstetric outcomes and birth variables.

Design: Systematic review and meta-analysis of randomized controlled trials (RCTs). Patient(s): Pregnant women and neonates Intervention(s): PubMed and 5 other research databases were searched through March 2014 for RCTs evaluating vitamin D supplementation ± calcium/vitamins/ferrous sulfate vs. a control (placebo or active) during pregnancy.

Main Outcome Measure(s): Circulating 25-hydroxyvitamin D [25(OH)D] levels, preeclampsia, gestational diabetes (GDM), small for gestational age (SGA), low birth weight (LBW), preterm birth (PB), birth weight, birth length, cesarean section. Mantel-Haenszel fixed-effects models were used due to expected scarcity of outcomes. Effects were reported as relative risks (RR) and their 95% confidence intervals (CIs).

Result(s): Thirteen RCTs (n=2,299) were selected. Circulating 25(OH)D levels were higher at term in comparison to the control group (mean difference [MD] 66.5 nmol/L, 95%CI 66.2-66.7, p<0.0001). Birth weight and birth length were significantly greater for neonates in the vitamin D group: MD 108 g (95%CI 60-155) and 0.3 cm (95%CI 0.10-0.41), respectively. Incidence of pre-eclampsia, GDM, SGA, LBW, PB, and cesarean section were not influenced by vitamin D supplementation. Across RCTs, there was heterogeneity of doses and types of vitamin D supplements, gestational age at first administration, and outcomes.

Conclusions: Vitamin D supplementation during pregnancy was associated with increased circulating 25(OH)D levels, birth weight and birth length, and was not significantly associated with other maternal and neonatal outcomes. Larger, better designed RCTs evaluating clinically relevant outcomes are necessary to reach a definitive Conclusion.

Id: 41 the Utility of Commonly Used Laboratory Tests to Screen for Excessive Alcohol Use in Clinical Practice

G. Gough, S. Liangpunsakul. Medicine, Indiana University, Indianapolis, Indiana.

Background: Screening for ongoing and excessive alcohol use is of importance in general medicine practice and in those with underlying chronic liver diseases. Early detection can lead to early behavioral interventions which will reduce the risk for subsequent adverse outcomes. This current study was undertaken to carefully assess the accuracy of routinely used laboratory tests in detecting excessive/recent alcohol use. We also determined the kinetics of these markers in subjects who underwent intensive alcohol rehabilitation program.

Methods: To determine the performance of commonly used lab tests and levels of alcohol consumption, 272 subjects with history of alcohol use disorder who were admitted for alcohol rehabilitation and 210 non-excessive drinkers were recruited. To determine the kinetics of these markers after alcohol abstinence, we prospectively followed 45 subjects with history of excessive alcohol use for 12-week during the intensive alcohol treatment program.

Results: During the study period, 482 subjects were recruited (210 non-excessive drinkers/272 excessive drinkers). Excessive drinkers were older (39 vs. 33 yrs), had higher percentage of divorce/separation (28%vs.18%), and had lower BMI (27.9 vs. 29.9), when compared to those of non-excessive drinkers. Excessive drinkers had higher AUDIT scores (26.9 vs. 4.7, p<0.001), and greater total standard drinks in the past 30 days (257 vs. 13 drinks, p<0.001). Excessive drinkers had significantly higher levels of serum AST (39 vs. 26 U/L), ALT (50 vs. 36 U/L), GGT (85 vs. 36 U/L), MCV (93 vs. 89 fL) and % CDT (2.6 vs. 1.9). % CDT provided the highest diagnostic performance with an AUC of 0.77, followed by ALT (AUC=0.74), GGT (AUC= 0.68), MCV (AUC=0.66), and AST (AUC=0.61) (fig 1A). In the multivariate analysis, we found that only the levels of GGT and % CDT were associated with the level of alcohol consumed during the past 30 days. To study the utility of these markers as for follow up of abstinence in clinical practice, we found that the levels of GGT, MCV, and % CDT were significantly lower compared to those at baseline before alcohol rehabilitation. When compared the decrease in these values to the average levels of these markers at the time of enrollment (MCV 93 fL, GGT 74 U/L, and % CDT 2.8%), the reduction was ∼ 2.7% (for MCV), 19% (for GGT), and 43% (for % CDT) at the end of 12-week follow up compared to the baseline. During the course of the follow up, the average levels of AST and ALT were unchanged (fig 1B).

Conclusions: Our data showed that the non-hepatic enzyme, % CDT, is the most useful marker to screen for excessive alcohol use and follow up for abstinence. However, the performance of the % CDT to screen for heavy alcohol use is still not ideal. Further research to identify the non-invasive marker(s) (i.e. using proteomic or metabolomics approach) should be considered.

Funding: K08 AA016570 (NIH),1I01CX000361 (VA Research and Administration), and W81XWH-12-1-0497 (DoD).

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Id: 42 Comparative Effects of Protamine and Poly-L-Lysine upon Prothrombin Time

A. Brecher, A. Riaz. Chemistry, Bowling Green State University, Bowling Green, Ohio.

Alcoholism is responsible for the observance of prolonged blood coagulation times in a significant fraction of the alcoholic population. This laboratory has reported earlier that acetaldehyde, the primary intermediate in ethanol metabolism, readily prolongs coagulation times upon covalent interaction with thrombin, prothrombin, factors Xa, IXa, XIa, XIII, and fibrinogen, among the proteins tested. In this communication, it is reported that protamine, the basic polypeptide isolated from salmon sperm and utilized to neutralize the anticoagulant effect of heparin, has anticoagulant properties which are enhanced upon exposure to acetaldehyde. It is further demonstrated that the basic polypeptide, poly-L-lysine, similarly prolongs prothrombin time (PT), and that mixtures of plasma, poly-L-lysine, and acetaldehyde have additional PT times at 4.47mM acetaldehyde, and synergistic anticoagulant effects at 44.7 mM acetaldehyde. The pattern for protamine and poly-L-lysine are quite analogous. Whereas 9mM protamine prolongs PT at 30 sec relative to 11.5 sec for the control, 4.4 mM acetaldehyde prolongs PT at 13.2 sec, and 44.7 mM acetaldehyde extends PT at 33.7 sec. Pre-mixtures of 9 mM protamine/4. 4 mM acetaldehyde produce an additive 33.1 sec PT, while a 9 mM protamine/44.7 mM acetaldehyde generates a synergistic 91.2 sec PT. Comparable numbers with poly-L-lysine generate values of 11.3, 15.1, 34.8, 12.4, 17.1, and 45.9 sec, respectively. Poly-L-lysine is a bacterial fermentation product. The commercial source was tested at concentrations from 0.035 to 0.64 mM/L, and mixtures ranging from 0-0.5, 1-5, and 4-15 kDa/mol. The higher molecular weights produced prolonged PTs for the same concentrations. These data may reflect the decreased solubility of the poly-L-lysine-fibrinogen complexes. (The authors appreciate the generous donation of reagents by the BGSU Medical Technology program to this study).

Id: 43 Correlation of Foxm1 and Oxidative Stress Induced Dna Damage in Human Hepatocellular Carcinoma

B.L. Sun, N. Ronquillo, P. Raychaudhuri, G. Guzman. University of Illinois Hospital & Health Sciences System, Chicago, Illinois.

Background: forkhead box protein M1(fOXM1) transcription factor is essential for the development of hepatocellular carcinoma (HCC). Previous in vitro studies showed that foxM1is critical for malignant transformation and tumor cell survival by inhibiting premature senescence and apoptosis in response to DNA damage under oxidative stress. This study has investigated fOXM1 and its correlation with DNA damage induced by oxidative stress in humanHCC.

Design: One hundred one subjects with diagnosed HCC were collected in the University of Illinois Hospital & Health Sciences System. HCC was morphologically graded as 1 to 3 by using Edmondson-Steiner grading system. Eight subjects without significant liver diseases were collected as normal controls. Tissue microarrays were generated by selecting representative tissues with hepatocellular carcinoma, dysplasia, or cirrhosis from HCC subjects and normal tissue from controls. FOMX1 and 8-Hydroxy-2′-deoxyguanosine (8-OHdG, an oxidative stress induced DNA damage marker) were detected on tissue microarrays by immunohistochemical assay (IHC). The intensities of fOMX1 and 8-OHdG staining were digitalized by using Aperio Image Analysis and Vectra Automated Multispectral Imaging System. SAS and Pearson product-moment correlation were used for statistic analysis.

Results: In HCC group, fOMX1 was significantly upregulated and co-existed with DNA damage in the nuclei of malignant cells. Staining for 8-OHdG was negative or faint in normal cells, but variant from weak to strong in HCC cells. Ninety percent of grade 3 HCCs presented with intensive staining for 8-OHdG; consistently, all grade 3 HCCs were moderately to strongly positive for fOMX1. Significant staining for 8-OHdG was observed in 38% of grade 2 HCCs and 44% grade 1 HCCs. The cells with intensive DNA damage appeared to have higher level of fOMX1 in the nuclei. Pearson productmoment correlation analysis showed significant correlation between fOMX1 expression and 8-OHdG intensity in the nuclei of HCC cells.

Conclusions: The upregulation of fOXM1 in the nuclei and oxidative stress induced DNA damage are highly correlated in human HCCs, especially high grade HCCs. These results are consistent with our previous in vitro study and suggest that fOXM1 plays a key role in oncogenesis by maintaining tumor cell survival against DNA damage. Targeting fOXM1 may provide therapeutic strategy for prevention and treatment of HCC.

Id: 44 Cholestatic Drug-Induced Liver Injury Treated with Corticosteroid in a Patient with Miliary Tuberculosis and Associated Adrenal Insufficiency

S. Lee, P. Khankhanian, A. Schneier, O.N. Machado. Medicine, Icahn School of Medicine at Mount Sinai, Elmhurst, New York. S. Liu. Pathology, Icahn School of Medicine at Mount Sinai, Elmhurst, New York.

Drug-induced liver injury (DILI) in a patient with multiple comorbidities is challenging to diagnose because liver injury can be attributed to multiple disease processes. Many medications have been reported to cause DILI, but the pathophysiology is multifactorial and difficult to pinpoint. However, delayed treatment of DILI could have fatal consequences, and therefore, understanding the features and risks of DILI is crucial. A 82 year-old woman a remote history of cholecystectomy presented with a pre-syncopal episode. Two days prior, she was discharged from another hospital after prescribed ciprofloxacin and metronidazole for colitis diagnosed by abdominal computational tomography (CT). She took ciprofloxacin and metronidazole for 3 days in total. At admission, she was found to have severe sepsis with elevated AST (111 IU/L) and ALT (450 IU/L) with normal alkaline phosphatase (AP) (111 IU/L) and hyponatremia to 125 mEq/L. She was treated with vancomycin and imipenem/cilastatin in addition to fluid resuscitation. She became hemodynamically stable after three days. At this time, vancomycin and imipenem/cilastatin were discontinued, and cefepime was started, given negative cultures. However, AP began to increase (figure A). After five doses of cefepime, it was discontinued as a possible cause of liver injury. She was restarted on imipenem/cilastatin for two more days for a total of 10 days of antibiotic treatment, but her AP continued to rise. A search for common non-drug causes of acute liver injury was all negative. Liver biopsy revealed cholestasis and noncaseating granulomatous disease, suggesting DILI or infectious etiology (figure B). An ACTH stimulation test for persistent hyponatremia and fatigue confirmed the diagnosis of adrenal insufficiency for which corticosteroids were started. The AP peaked at 1,004 IU/L and started to trend down two days after steroid was started. Chest CT revealed multiple subcentimeter central and peripherally located densities. A diagnosis of miliary tuberculosis with associated adrenal insufficiency was made, and the patient improved with anti-TB medications and corticosteroids. A few weeks after discharge, induced sputum cultures grew mycobacterium tuberculosis confirming the diagnosis. This patient received five different antibiotics including ciprofloxacin, metronidazole, vancomycin, imipenem/cilastatin, and cefepime. Danan et al. provide a consensus method for assessing the causal role of a drug in liver injury. Only ciprofloxacin and metronidazole are “suggestive” agents for cholestatic liver injury in terms of both the onset and cessation of medications. Metronidazole is known to cause hepatocellular liver injury, and pharmacology of each drug implies that ciprofloxacin was the most likely antibiotic causing DILI. This case is unique because miliary TB was complicated by adrenal insufficiency and drug-induced cholestatic liver injury, but acute liver injury was fully reversed after corticosteroid treatment. This implies an immune-mediated etiology of DILI, especially ciprofloxacin-induced cholestatic liver injury.

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Figure 1

(A) Daily trend of alkaline phosphatase (AP). Day 1 is the first day of ciprofloxacin and metronidazole. Liver injury is cholestatic when AP is greater than two times the upper limit, represented as blue horizontal dotted line. The date on which corticosteroid was given is represented as the red vertical line. Liver injury started on day 8. “cm” stands for ciprofloxacin and metronidazole (for 3 days); “vic,” vancomycin and imipenem / cilastatin (for 3 days); “cef,” cefepime (for 5 days); “ic,” imipenem / cilastatin (for 2 days). (B) Liver biopsy of this patient, demonstrating a non-caseating epithelioid granuloma involving both portal tracts and lobules with evidence of cholestasis. Scale bar = 150 μm.

Id: 45 Spirochetes in the Gut! a Case Report of An Hiv Related Chronic Diarrhea

L. dakhoul, F. Abou Obeid, H. Raddawi. Internal Medicine, Advocate Christ Medical Center, Oak Lawn, Illinois.

Introduction: Diarrheal disease in human immunodeficiency virus (HIV)-infected individuals is frequently caused by infectious agents. Infectious organisms may vary with the degree of immunocompromise in the host. They can include routine enteric pathogens or opportunistic pathogens. Prospective series have implicated a wide variety of protozoal, viral, and bacterial organisms as diarrheal pathogens. Some of them, like Mycobacterium avium-complex (MAC), are unique to HIV disease. Others, like Cryptosporidium, cause self-limited diarrheal illness in healthy hosts but chronic diarrhea in immunosuppressed patients. Rarely, intestinal spirochetosis has been described in those patients. We present a case of a young patient presenting with chronic diarrhea, newly diagnosed with HIV and was found to have mixed organisms including cryptosporidiosis and spirochetes.

Case presentation: A 28 year old male presented to the emergency department (ER) for lightheadedness and generalized weakness. On further questioning, the patient reported loose stools and 30 pounds weight loss over the last 6 months. His symptoms were mainly exacerbated in the last month. The patient was hemodynamically stable and his physical exam was unremarkable. Laboratory data revealed hypokalemia at 2.6, mildly elevated creatinin at 1.26 and leukopenia at 2.9. Viewing his clinical presentation and his history of homosexual lifestyle, HIV screen obtained was positive and it was confirmed later with western blot. Specific Stool studies were sent. Meanwhile, a Colonoscopy and an ileoscopy were performed which revealed mild non specific proctitis and nodular terminal ileum. Biopsies were taken from both sites. Stool culture grew Campylobacter. Giardia and Cryptosporidium specific antigens were also positive. The pathology reported terminal ileum biopsy positive for cryptosporidiosis; edematous colonic mucosa with chronic inflammation and spirochetosis. After the initiation of treatment with azithromycin and metronidazole, the patient was feeling better and his diarrhea was improving. The patient was discharged to an HIV specialized center on azithromycin and metronidazole for a full course of 7 and 10 days respectively.

Discussion: Chronic diarrhea in a young patient with history of homosexual activity should raise the concern for an underlying immunodeficiency state with unusual infectious etiologies. However, HIV enteropathy is not uncommon and must be also included in the differential diagnosis. In this case, the diarrheal illness was the result of mixture of pathogens including spirochetes. Primary intestinal spirochetosis is a rare entity. It generally involves the rectum. Literature review reported HIV patients with Syphilitic proctitis as the sole culprit of their GI symptoms. Manifestations are chronic and symptoms can mimic those of inflammatory bowel diseases (IBDs) or HIV enteropathy. Therefore, it is important in this situation to broaden the differential diagnosis as the diagnostic tests and management strategies are quite different. In Conclusion, specific stool studies and endoscopic biopsies should be mandatory in patients with suspected HIV related infectious etiology. Stool culture is not enough and unusual organisms can be identified on microscopic examination using special stains. Pathology reports can be surprising!

Id: 46 Your Appendix Can Tell Your Story! An Unusual Cause of Appendicitis

L. dakhoul, H. Raddawi. Internal Medicine, Advocate Christ Medical Center, Oak Lawn, Illinois.

Introduction: Appendicitis is an acute inflammation of the appendix whose pathogenesis starts with luminal obstruction. Several materials, including calculi, and benign or malignant tumors can constitute the etiology of obstruction. Malignant etiologies include villous adenomas, small cell cancer, and prostatic adenocarcinoma. These tumors, either primary or metastatic, grow insidiously on the appendix, obstruct its lumen and cause gangrene and perforation, presenting clinically as appendicitis. We report a case of acute appendicitis provoked by a combination of tumors in a 61-year-old man.

Case presentation: A 61 year old male, presented to the Emergency Department for sharp right lower quadrant (RLQ) pain of one day duration. He developed a low grade fever of 38.1°C in the hospital. Abdominal exam was remarkable for mild RLQ tenderness and guarding. Laboratory data revealed a white cell count (WBC) of 14000. Computed Tomography (CT) of the abdomen and pelvis reported diffuse thickening of the retrocecal appendix with mild periappendiceal fat stranding. A laparoscopic appendectomy was performed revealing acute perforated appendicitis with gangrene. Postoperatively, the patient had uncomplicated hospitalization course. Due to suspicion for cecal malignancy, the patient underwent outpatient colonoscopy showing only sigmoid diverticulosis with negative biopsies. Surgical pathology was subsequently reported as tubular adenoma with areas of high grade dysplasia and small foci of invasive high grade neuroendocrine carcinoma. Based on the surprising pathology results, a CT chest-abdomino-pelvis was done that showed an enlarged lymph node (LN) 2.5cm x 1.5 cm located anterior to the kidney with no other masses. Another CT was repeated after few months showed an increase in the size of the LN and a right hemicolectomy with LN resection was performed. LN pathology reported small cell carcinoma. A repeat CT chest-abdomino-pelvis showed scattered multiple lymph nodes and liver lesions. The patient is currently still receiving systemic chemotherapy.

Discussion: Tubular adenomas and neuroendocrine tumors are two different entities that are well defined in the literature. However, their concomitant presence on the appendix is extremely rare. To our knowledge, this is the first reported case of these tumors combined together. In a case of appendicitis, it is important to determine whether the underlying cause is benign or malignant, as the management and the prognosis are quite different. A more aggressive attitude should be taken in the pre and postoperative management of any elderly patient who presents with appendicitis, viewing the increased risk of tumoral infiltration of the appendix. A careful histopathological examination of all appendectomy specimens should be mandatory. Despite the fact that a combination of tumors of the appendix is rare, our case illustrates that these must be included in the differential diagnosis of acute appendicitis, as the latter can be the initial manifestation.

Id: 47 Primary Amyloidosis Diagnosed by Endoscopic-Ultrasound Guided Celiac Lymph Node Biopsy

N. AKBAR, V. Puri, A. Kubbara, U. Ahmad, A. Nawras. Internal Medicine, University of Toledo medical Center, Toledo, Ohio.

Introduction: Amyloidosis is a rare array of diseases resulting from the deposition of extracellular tissue protein fibrils composing variety of normal serum proteins. However, for these proteins to be deposited in a lymph node is extremely rare. Confirming the diagnosis can be very challenging and mandates tissue biopsy and histopathological examination. In our case we diagnosed a rare involvement of primary amyloidosis in a peripancreatic lymph node confirmed by Endoscopic Ultrasound guided fine needle biopsy (EUS-fNA).

Case Stem: A 64-year-old Caucasian gentleman with history of MGUS presented to our facility with severe recurrent abdominal pain. The patient had previously presented to an outlying facility with severe right upper quadrant abdominal pain, nausea and vomiting of one week duration. Abdominal CT then showed 59 by 39 mm peri-pancreatic mass/lymph nodes. CT guided percutaneous biopsy of the mass was performed to rule out malignancy which was negative. One week later, he presented to our emergency room with recurrent abdominal pain. CT of abdomen & pelvis showed diminished attenuation in a mass between the pancreatic head, body and liver consistent with interval hemorrhage within the mass due to the previous biopsy. Upon presentation elevated white blood cell count, alkaline phosphatase level of 288 IU/L (normal 44 to 147 IU/L), Aspartate transaminase 58 IU/L (normal 14-20 IU/L) were noticed. Patient underwent EUS that revealed multiple celiac lymph nodes. The largest celiac lymph node measured 20.0 x 12.0 mm, and peripancreatic lymph node measured 55.0 x 54.0 mm. EUS-fNA of the celiac lymph node revealed amorphous material with bland spindle cells on smear slides. Similar amorphous material was present on cell block. Congo red and Sulfate Alcian blue stains were examined which confirmed the diagnosis of amyloidosis.

Summary: Amyloidosis is an uncommon disease caused by abnormal protein deposition within the tissues. Primary amyloidosis presenting only as lymphadenopathy is an extremely rare entity and confirming the diagnosis through lymph node biopsy has only been reported very few times in the literature. We present a case of primary amyloidosis successfully diagnosed by EUSfNA involving the celiac lymph node

Id: 48 Herpes Esophagitis in An Immunocompetent Teenager

T.M. Yousuf, S. Wang, A. Khan, B. Blumenstein. Internal Medicine, Advocate Christ Medical Center, Oak Brook, Illinois.

Herpes esophagitis is a common infection in the immunocompromised host. However, it is a rare condition in the immunocompetent population, and usually presents with the constellation of symptoms of odynophagia, fever and retrosternal chest pain. In immunocompetent patients, symptoms of odynophagia and chest pains are usually attributed more to pill induced esophagitis, toxic ingestion or reflux esophagitis. Rarely, if ever, is infectious esophagitis from HSV, CMV or candida considered. HSV-1 is more commonly the cause of esphoagitis, but typically as a reactivation. In very rare cases does it present as a primary infection in the esophagus. We describe a case of HSV esophagitis in an eighteen year old immunocompetent host with no significant past medical history. He initially presented to his primary care physician with complaints of odynophagia and was prescribed a course of amoxicillin and prednisone syrup. He presented four days later to the emergency room with worsening odynophagia, retrosternal chest pain and anorexia. He was evaluated by the gastroenterology team and was taken for esphsophagogastroduodenoscopy (EGD), which revealed diffuse bleeding superficial ulcerations along the entirety of his esophagus. Biopsies were taken and subsequently found to be HSV positive. The patient was treated with intravenous acyclovir, proton pump inhibitor, and sucralfate. HIV status on the patient was found to be negative, and no other causes of immunosuppression was found. We believe the patients’ initial presentation was in fact, HSV esophagitis, which was exacerbated by his prednisone use. The purpose of this report is to highlight the rare occurrence of infectious esophagitis in otherwise healthy, young individuals and to be able to promptly diagnose and begin treatment on such patients. EGD with biopsy is the gold standard diagnostic modality for HSV esophagitis and should always be considered in young patients who present with odynophagia with or without other alarm features. EGD findings often can point to the cause as HSV has a very typical appearance on EGD. Often we see superficial, well-demarcated ulcers, typically along the mid to distal esophagus. The treatment of choice for HSV esophagitis is intravenous acyclovir 5mg per kg every eight hours for seven to fourteen days. Treatment should be initiated promptly following the EGD to expedite resolution. Symptoms for most immunocompetent patients resolve spontaneously in about one to two weeks. Although rare, HSV esophagitis should be entertained especially given the correct constellation of history and symptoms. In confirmed cases of HSV esophagitis, it is important to reassess the patient for any underlying immunodeficiencies, especially HIV as a cause for the infection.

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ID: 49 Elevated Liver Transaminases; One More Etiology To Look For

N. AKBAR, A. KUBBARA, R. QADIR. Internal Medicine, University of Toledo medical center, Toledo, Ohio.

Introduction: Different eating disorders have been found to cause elevated liver transaminases. The exact mechanism is not well understood. We are reporting a case of a 67-years old female who presented with loss of appetite and found to have elevated liver transaminases.

Case Stem: 67-years old white female with past medical history of hypertension presented to our outpatient clinic with anorexia, poor oral intake, and change in food taste secondary to decrease appetite. She was found to have 20 pounds weight loss over the previous 6 weeks and laboratory investigation showed increased liver transaminases. Her BMI at that time was 22 kg/ṁ. Her physical examination did not reveal any abnormality. Her laboratory workup showed: ALT 524 IU/L (normal 7-52), and AST 228 IU/L (normal 13-39). However her Total Bilirubin, alkaline phosphatase, & albumin were all within normal limits. Further workup revealed the following; Hemoglobin 11.8 g/dL (normal 12-15), HCT 33.4 (normal 36-48), MCV 90.2 fL (normal 80-100), ferritin: 444 ng/ml (normal 11-307), Iron 45 mcg/dl (normal 50-212), and Lipase 154 Units/L (upper limit of normal being 35) In searching for an etiology for her abnormal transaminases, we ruled out viral Hepatitis A, B, and C. In addition, TSH, ANA, as well as Urine toxicology where normal. She denied any history of acetaminophen ingestion and other hepatotoxic drugs. Additionally, she does not drink alcohol. Not to mention that her pattern of transaminases elevation was inconsistent with alcoholic liver disease. Abdominal CT, and hepatic ultrasound were both unremarkable. Colonoscopy performed “as an age appropriate cancer screening” revealed only a sigmoid stricture for which we did air barium enema. There was no abnormality detected. Upper endoscopy and biopsy ruled out celiac disease as the cause of the anemia. Electrolytes where corrected and nutrition was strongly encouraged resulting in the correction of liver transaminase over a few days as in table 1.

Conclusion: Elevated liver transaminases have a wide differential diagnosis. And it is not uncommon to reach a dead end. However, correlating a dietary history with this laboratory change can establish the diagnosis of the specific eating disorder responsible for such abnormalities. The exact mechanism is not well understood however hepatic autophagocytosis is thought to be the culprit in the setting of severe nutrient deficiencies.

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TABLE

AST and ALT progression in our patient

Id: 50 a Rare Cause of Heartburn and Abdominal Pain in a Teenager Female

N. AKBAR, A. KUBBARA. Internal Medicine, University of Toledo medical center, Toledo, Ohio. A. NAWRAS. Gastroenterology, University of Toledo medical center, Toledo, Ohio.

Introduction: Abdominal pain is a common medical problem seen in all ages including teenagers. Trichobezoar means a ball of hair in the gastrointestinal tract, it is associated with trichotillomania (obsessive hair pulling), and trichophagia (hair swallowing). Bezoar originates from “bazahr” used by Arab medical writers to describe antidotes to poisons extracted from animals’ abdomens. Trich means hair in Greek. Human hair is not digestible and it resists peristalsis. Accumulation occurs in the stomach and can extend to the intestines.

Case stem: We present an 18 years-old white female who was referred to us from an outlying facility for evaluation of heartburn and chronic abdominal pain. She stated that she started experiencing the pain for several months which is cramping, intermittent, occurring 3 times/week, lasting for a few hours with no relieving or aggravating factors and no change in bowel habits. Patient had no previous surgical or psychiatric problems. However on further questioning she gave a history of trichophagia. Abdominal exam showed mild diffuse tenderness, normal bowel sounds, and no palpable masses. On further evaluation, microscopic anemia and mild leuckocytosis where detected. Upper gastrointestinal x-ray with air contrast was performed at an outlying facility showing distended stomach appearing to be filled with debris, with a normally filled duodenum. Esophagogastroduodenoscopy was performed at our institution which showed trichobezoar occupying 75% of the gastric lumen and extending through the pylorus and into the duodenal bulb. A few attempts to remove the hair with scissors and forceps were not successful. Exploratory laparotomy & gastrostomy with removal of the bezoar were performed successfully without complications. The mass measurements were 17x6x7 cm and weighed 2.33Ibs. The patient recovered well and was discharged home with psychiatric follow up.

Conclusion: Trichobezoar could be the cause of abdominal pain in a young woman particularly those with psychiatric disorders or with a history of trichotillomania and trichophagia.

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Id: 51 Irony of Transfusional Support for Refractory Anemia in Hemophagocytic Lymphohistiocytosis

S. Lee. Medicine, Icahn School of Medicine at Mount Sinai, Elmhurst, New York. M. Chary. Icahn School of Medicine at Mount Sinai, New York, New York.

Hemophagocytic lymphohistiocytosis (HLH) is an aggressive disorder from immune system derangement with uncontrolled macrophages engulfing patients’ own blood cells. Patients with myelodysplastic syndrome, leukemias, sickle cell disease, or beta-thalassemia major depend on chronic transfusion, requiring 10 - 20 units of RBC transfusion a year. However, these patients do not develop iron deposition in the organ parenchyma until the late stage of diseases. Here we report a unique case of a patient with HLH and associated severe refractory anemia, who developed rapid iron deposition in the liver and pancreas parenchyma after 16 units of RBC transfusion. This case shows the irony of managing refractory anemia with transfusional support because patients with HLH require continuous transfusional support and are simultaneously more susceptible to developing iron overload. A 44 year-old woman with no significant past medical history presented with fatigue, fevers, and jaundice. She was found to have hepatosplenomegaly and severe jaundice at admission. Her blood tests showed hemoglobin of 8.4 g/dL, mean corpuscular volume 85.0 fL/cell, iron 138 μg/dL, ferritin 4,170 ng/mL, transferrin saturation 93%, aspartate transaminase/alanine transaminase 123/191 U/L, gamma-glutamyl transpeptidase 57 U/L, lactate dehydrogenase 1,310 U/L, and total/conjugated bilirubin 20.7/18.7 U/L. Computerized tomography (CT) of the abdomen and pelvis showed hepatosplenomegaly and a 4.7 cm hemangioma (figure A). Liver biopsy showed hemophagocytosis with negative iron staining. Bone marrow biopsy showed hypercellular marrow with a large number of CD4- and CD68- positive histiocytes and hemophagocytosis: she was diagnosed with HLH, for which steroid and chemotherapy were started. Sixteen units of RBC transfusion were provided for refractory anemia, and MRI of the abdomen was performed: severe iron deposit in the liver and pancreas (MRI T2* relaxation time of 8 msec and R2* (1/T2*) = 125 Hz) was revealed, which was not shown at admission on CT scan or liver biopsy (figure B). Per Wood et al., the liver iron concentration (LIC) can be estimated, based on the shortening of T1, T2, and T2* relaxation times on MRI: her LIC was estimated to be 13 mg/g dry weight. Drasar et al. demonstrated the followings: 1) patients with sickle cell disease who had serum ferritin > 1,000 ng/mL and received less than 20 units (range 12 - 19) of RBC transfusions showed a mean LIC of 2.1 - 2.3 mg/g dry weight; 2) the total unit of transfusion correlates more strongly with LIC than the rate of transfusion. This result demonstrates that patients with HLH could reach the iron overload state more rapidly than patients with other diseases requiring transfusional support. A large amount of iron from breakdown of phagocytosed RBCs by reticuloendothelial macrophages is stored as ferritin, resulting in a very high transferrin saturation in HLH. Therefore, the reticuloendothelial system in HLH has a low iron binding capacity to store extra iron generated by transfusional products, triggering more rapid iron deposition in the organ parenchyma.

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Abdominal CT and MRI images. (A) is an abdominal CT image at the early stage of hospital admission, showing splenomegaly and hepatomegaly with a 4.7 cm irregular mass at the posterior right hepatic lobe. (B) is an abdominal MRI imaging after the patient received 16 units of RBC transfusional supprt with diffuse signal dropout due to iron desposition in the liver and pancreas (T2* = 8 msec).

Id: 88 a Gene Regulatory Network Shared between Neurulation and Orofacial Development

Y.A. Kousa, R.R. Roushangar. Biochemistry and Molecular Biology Department, Michigan State University, East Lansing, Michigan. H. Zhu, Y. Lei, R.H. Finnell. Dell Pediatric Research Institute, Department of Nutritional Sciences, University of Texas at Austin, Austin, Texas, UNITED STATES. W.D. Fakhouri. Department of Diagnostic and Biomedical Sciences, University of Texas at Houston, Houston, Texas. T.D. Busch, J.C. Murray, A. Bassuk. Department of Pediatrics, University of Iowa, Iowa City, Iowa. G.M. Shaw. Department of Pediatrics, Stanford University School of Medicine, Stanford, California. A. Ashley-Koch, S. Gregory. Duke Molecular Physiology Institute, Department of Medicine and Molecular Genetics and Microbiology, Duke University, Durham, California. N. Patel, B.C. Schutte. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan. E.J. Leslie. Center for Craniofacial and Dental Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania. T.J. Williams. Department of Craniofacial Biology, University of Colorado Denver at Anschutz Medical Campus, Aurora, Colorado. Y. Chai. Center for Craniofacial Molecular Biology, University of Southern California Ostrow School of Dentistry, Los Angeles, California.

IRf6, TfAP2A and GRHL3 encode transcription factors that are required for orofacial development in humans and mice. We showed that rs642961 is highly associated with orofacial clefting and perturbs a TfAP2A binding site in an enhancer element (MCS9.7) that recapitulates Irf6 expression. In keratinocyte culture, TfAP2A regulates IRf6 expression via MCS9.7. We also showed that Irf6 regulates Grhl3 expression in zebrafish and that mutations in IRf6 and GRHL3 cause nearly identical human phenotypes. These observations suggest that TfAP2A, IRf6 and GRHL3 form a network in craniofacial morphogenesis. However, mice that lack either Tfap2a or Grhl3 also have neural tube defects. These neurulation defects are located rostrally (exencephaly) and caudally (curly tail) in both mutant mice. In addition, Tfap2a knockout embryos have an exceptional ventral wall defect involving both the thoracic and abdominal cavities. Therapeutically, neurulation defects in Grhl3 knockout embryos are rescued by inositol supplementation, but not folate. In this study, we discover a critical role for Irf6 in neural tube and ventral wall morphogenesis by characterizing an allelic series, including both loss- and gain-of-function alleles. We find that reducing Irf6 expression leads to a curly tail and reductions in both Tfap2a and Grhl3 expression. Remarkably, reducing Irf6 expression in Tfap2a haploinsufficient embryos rescues exencephaly. Irf6 over-expression leads to a ventral wall defect that is highly analogous to Tfap2a knockout embryos. Molecularly, we find that Irf6 is expressed in neural ectoderm and early migrating neural crest cells and that MCS9.7 is active in these cells. Strikingly, we show that Tfap2a regulates MCS9.7 in multiple tissues, including tail and skin. Consistently, we find that Tfap2a and Grhl3 interact genetically in rostral and caudal neurulation and ventral wall development in a dose-dependent manner. Therefore, Irf6 homeostasis is required for at least three ectoderm derived tissues via positive and negative regulation of Tfap2a and Grhl3. Lastly, we sequence IRf6 in 96 individuals with spina bifida and find a rare variant previously shown to be disease causing in orofacial clefting. We further genotype common SNPs in 2,500 individuals with spina bifida to cover 28% of the genetic variation at the IRf6 locus but do not detect a common association. With roles in neural tube, ventral wall and craniofacial morphogenesis, Tfap2a-Irf6-Grhl3 appear to be a key gene regulatory network in ectoderm development. Considering that Grhl3 is a distal node in this pathway, inositol supplementation and inhibition may provide an environmental lever to alter multiple developmental programs of the ectoderm based on genetic risk.

Id: 89 An Epigenetic Map of Age-Associated Autosomal Loci in Northern European Families at High Risk for the Metabolic Syndrome

D. Cerjak, R. James, Y. Zhang. Medicine, Medical College of Wisconsin, Brookfield, Wisconsin. O. Ali Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin. J.W. Kent, J. Blangero, M.A. Carless. Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin. Genetics, Texas Biomedical Research Institute, San Antonio, Texas.

Background: The prevalence of chronic diseases such as cancer, type 2 diabetes, metabolic syndrome (MetS) and cardiovascular disease increases with age in all populations. Epigenetic features are hypothesized to play important roles in the pathophysiology of age-associated diseases but a map of these markers is lacking. We searched for genome-wide age-associated methylation signatures in peripheral blood of individuals at high risks for MetS by profiling 485,000 CpG sites in 192 individuals of Northern European ancestry using the Illumina HM450 array. Subjects (ages 6-85 years) were part of 7 extended families and 73% of adults and 32% of children were overweight or obese. Results We found 22,122 genome-wide significant age-associated CpG sites (pα=0.05=3.65 x 10-7 after correction for multiple testing) of which 14,155 are positively associated with age while 7,967 are negatively associated. By applying a positional density-based clustering algorithm, we generated a map of epigenetic “hot-spots” of age-associated genomic segments, which include 290 age-associated differentially methylated CpG clusters (aDMCs), of which 207 are positively associated with age. Gene/pathway enrichment analyses were performed on these clusters using fatiGO. Genes localized to both the positively (n=241) and negatively (n=16) age-associated clusters are significantly enriched in specific KEGG pathways and GO terms. The most significantly enriched pathways are the hedgehog signaling pathway (adjusted p=3.96x 10-3) and maturity onset diabetes of the young (MODY) (adjusted p=6.26 x 10-3) in the positive aDMCs and type I diabetes mellitus (adjusted p=3.69 x 10-7) in the negative aDMCs. We also identified several epigenetic loci whose age-associated change rates differ between subjects diagnosed with MetS and those without.

Conclusion: We conclude that in a family cohort at high risk for MetS, age-associated epigenetic features enrich in biological pathways important for determining the fate of fat cells and for insulin production. We also observe that several genes known to be related to MetS show differential epigenetic response to age in individuals with and without MetS.

ID: 90 Liver Eqtls for Warfarin dose Response Genes Reveal Susceptibility to Venous Thromboembolism among African Americans

W. Hernandez, E.R. Gamazon, A. Konkashbaev, M.A. Perera. Deparment of Medicine, Section of Human Genetics, The University of Chicago, Chicago, Illinois. L.H. Cavallari. University of florida, Gainesville, florida. R.A. Kittles. University of Arizona Cancer Center, Tucson, Arizona.

Venous thromboembolism (VTE) is a chronic disease encompassing deep vein thrombosis (DVT), pulmonary embolism (PE), or both. In the US, African Americans (AAs) have the highest incidence and mortality rates of DVT/PE. Warfarin is used to treat and prevent DVT/PE and the dose requirement has been shown to be higher among AAs as well as for DVT/PE patients regardless of ethnicity. Because genetic variation within cis-regulatory elements or trans-acting regulators can affect gene expression in a cell type specific manner, we aimed to investigate the role between VKORC1, CYP2C9, and CALU eQTLs and DVT/PE susceptibility using liver eQTL data. We identified and genotyped 72 cis and trans eQTLs in a study population of 462 AAs on stable warfarin dose; of which 256 individuals were treated with warfarin due to DVT/PE (cases) and the remaining due to a variety of other conditions (controls). We found a significant decrease in risk of DVT/PE for carriers of the minor allele of two VKORC1 eQTLs and one CALU eQTL (rs9925964, OR=0.53, P=0.01; rs12597511, OR=0.48. P=0.02; and rs11054879, OR=0.61, P=0.03 respectively). We also found our DVT/PE patients had a higher percentage of West African ancestry and were younger compared to controls (t=-1.991, p=0.04 and t=2.720, p=0.007 respectively). The frequency of these protective alleles were much lower (10%) in our study population and among the HapMap Yourbans (YRI) but significantly higher in the HapMap European Americans (CEU) at approximately 40%. By investigating eQTLs for genes known to contribute to warfarin dose requirement we have uncovered novel disease loci involved in the risk of DVT/PE. These findings may help explain the increased risk of DVT/PE seen in the African American population.

Id: 91 Interstitial Triplication of 15Q11-Q13: An Assessment of Methylation Status and Gene Expression

A.M. Goetjen, N. Germain, S. Chamberlain. University of Connecticut Health Center, Farmington, Connecticut.

The majority of genes in the human genome are biallelically-expressed, but this is not true for a number of genes that are located at the 15q11-q13 locus, which are regulated by a process called genomic imprinting. Genomic imprinting is a phenomenon in which genes are expressed in a parent-of-origin specific manner. Imprinted genes are functionally haploid. The master regulator of gene expression from this imprinted region is a differentially methylated region known as the Prader-Willi imprinting center (PWS-IC). A number of genes are selectively expressed from the paternal allele, whereas one gene (UBE3A) is maternally-expressed in neuronal cells. There are three clinical syndromes that result from duplication or deletion of these mono-allelically expressed genes: Prader-Willi syndrome, Angelman syndrome, and 15q duplication syndrome. Here I focus on 15q duplication syndrome. Children with 15q duplication syndrome present with cognitive dysfunction, speech/language disorders, and autism. Duplication of the 15q11-q13 locus may occur as an interstitial duplication or as an isodicentric chromosome 15 (idic(15)) that contain two or three copies of the maternal locus, respectively. Those with paternal interstitial duplication are clinically unaffected. An increased number of maternal copies of the locus correlates with a greater severity of the clinical phenotype, however little is known about the effect that chromosome structure has on the phenotypic presentation and gene expression. Thus, the focus of this project was to characterize gene expression and methylation status of the 15q11-q13 locus in a patient with a maternal interstitial triplication. Induced pluripotent stem cells (iPSCs) and iPSC-derived neurons were used as the model system. Methylation at the PWS-IC, was shown to be 75%, consistent with the individual's cells having three maternal copies and one paternal copy of the 15q11-q13 locus. Based on observations from iPSCs generated from patients with maternal interstitial duplication or idic(15), it was hypothesized that there would be a positive correlation between the level of gene expression and copy number in iPSCs generated from the patient with the maternal interstitial triplication. Indeed, 15q gene expression from the maternal interstitial triplication clones is positively correlated with the number of maternal copies of the locus. We observe minor differences in gene expression between interstitial triplication and idic(15) iPSCs or neurons, suggesting that different structural presentations of the three maternal copies of 15q11-q13 may be regulated similarly.

Id: 92 Effects of Rpd3 Mutation on Mitochondrial Function and Metabolism in Aging Drosophila

J. Woods, B. Rogina. Genetics and Genome Sciences, University of Connecticut Health Center, West Hartford, Connecticut.

Previously our lab showed that mutations in rpd3 (Drosophila HDAC1 homologue) extend the lifespan of Drosophila through a mechanism that overlaps with caloric restriction (CR). CR is known to increase lifespan by altering many physiological processes, including mitochondrial function and nutrient metabolism. The objective of this project is to determine the mechanism of lifespan extension in heterozygous rpd3-mutant flies as it relates to mitochondrial function, stress response, and metabolic homeostasis. The mitochondrial-to-nuclear gene ratio, level of spargel (Drosophila PGC-1 homologue) mRNA, and quantification of electron micrographs were examined as indicators of mitochondrial biogenesis and quantity. Mitochondrial respiration was examined to analyze differences in mitochondrial function. qPCR and mRNA sequencing were used to examine changes in genes responsible for metabolism, as well as other cellular pathways that affect longevity. We have found significant differences in mitochondrial respiration in fruit flies with heterozygous mutations of rpd3. However, our results indicate no difference in mitochondrial biogenesis. Many genes of the insulin-signaling pathway are differentially expressed in rpd3-mutant flies. Based on these results, we conclude differences in mitochondrial biogenesis are not the reason for lifespan extension in rpd3-mutant flies as initially hypothesized. The insulin-signaling pathway remains a candidate pathway for the lifespan extending effects. Future work will be needed to determine how targeting this pathway could promote healthy aging and lifespan extension.

Id: 93 Heterozygous Mutations in Aggrecan Cause Short Stature, Accelerated Bone Maturation, and Early Growth Cessation

M. Guo, C. Jacobsen, J. Hirschhorn. Division of Endocrinology, Boston Children's Hospital, Boston, Massachusetts. O. Nilsson, J. Lui, J. Baron. Program in Developmental Endocrinology and Genetics, National Institutes of Health, Bethesda, Maryland. J. Quintos. Warren Alpert Medical School of Brown University, Providence, Rhode Island. J. Popovic, D. Flynn. University of Pittsburgh Medical Center, PIttsburgh, Pennsylvania. A. Dauber. Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. N. Dunbar. Connecticut Children's Medical Center, Hartford, Connecticut.

Short stature is a common presentation to pediatric endocrinology clinics and is frequently associated with delayed skeletal maturation. In contrast, short stature with advanced skeletal maturation is a rare presentation. We recruited four families with autosomal dominantly inherited short stature and advanced skeletal maturation. Affected family members presented with childhood short stature (height SDS -1.9 to -3.5), advanced bone age, early growth cessation, and adult short stature (height SDS -2.6 to -4.7). Additional features that were variably present include osteochondritis dissecans, early onset osteoarthritis, macrocephaly, midface hypoplasia, and exaggerated lumbar lordosis. To identify the genetic cause of this phenotype, we performed whole exome sequencing in selected individuals from each family. In all four families, we identified novel heterozygous variants in the aggrecan gene (ACAN), which encodes a proteoglycan that serves as a major structural component of the extracellular matrix in growth plate and other cartilaginous tissues. The sequence variants were present in all affected but in none of the unaffected family members. Each of the mutations identified was predicted to result in loss of protein function. Our study indicates that heterozygous mutations in ACAN cause a mild skeletal dysplasia that presents as short stature with advanced bone age and early growth cessation. This contrasts with previous reports of ACAN mutations identified in individuals with severe skeletal dysplasias. Our findings thus expand the spectrum of ACAN defects and provide a new molecular genetic etiology for the unusual child who presents with short stature and accelerated skeletal maturation.

Id: 94 Central Retinal Artery Occlusion Associated with Familial Thrombophilia in Young Healthy Females

P. Shah, C.J. Glueck, J. Patel, Schockman, D. Smith. Internal Medicine/Endocrinology, Jewish-Mercy Cholesterol and Metabolism Center, Cincinnati, Ohio.

Background: Central retinal artery occlusion (CRAO) presents with acute painless monocular loss of vision. CRAO incidence is approximately 1 in 10,000 with mean age of 60-65, men are commonly more affected than women. CRAO is rare before age 40. In the absence of carotid artery atherosclerosis with cholesterol emboli to the retinal artery, familial thrombophilia can cause CRAO.

Specific Aim: We assessed thrombophilia-hypofibrinolysis as pathoetiologies of CRAO in two otherwise healthy women who developed CRAO at ages 18 and 31.

Methods: Complete measures of thrombophilia-hypofibrinolysis.

Results: Case 1: A thin (BMI 24.5) previously healthy 18 year old Caucasian female reported seeing “a little spot” in her right eye after having a picture taken and was found to have CRAO of the right eye. There was no previous history of blood clots, pregnancy, or estrogen-progestin oral contraceptive use. Her mother had two pregnancies with 2 live births, but required 3 months bed rest for preeclampsia in one pregnancy, suggestive of familial thrombophilia. The patient denied tobacco, alcohol, and recreational drug use. Carotid-vertebral sonography and echocardiography were normal. Evaluation for thrombophiliahypofibrinolysis revealed homozygosity for the C677T mutation in the MTHfR gene with concurrent elevated homocysteine (12.8 umol/L, laboratory upper normal limit 10.4umol/L), known to be pathoetiological for CRAO. Treatment was started with L-methylfolate 5 mg, vitamin B6 100 mg, and vitamin B12 2000 mcg/day, to normalize homocysteine levels, as an approach to prevent further CRAO. Case 2: A previously healthy, non-smoking 31yr. Caucasian female developed CRAO at 13 weeks gestation in her first pregnancy. Carotid-vertebral sonography and echocardiography were normal. Family history was positive for deep venous thrombosis and lethal pulmonary embolus in her maternal grandmother. Coagulation studies revealed high factor VIII (165%, upper normal limit 150%) and low protein S (44%, lower normal limit 50%). She was prescribed Enoxaparin 40mg subcutaneously daily by her gynecologist. At her follow-up visits, at 20 and 23 weeks gestation, vision had improved significantly, and, as per her retinal specialist, the CRAO was significantly diminished.

Discussion: When CRAO occurs in young cases, in the absence of carotid atherosclerosis-cholesterol emboli or paradoxical embolus via an atrial or ventricular septal defect, familial and/or acquired thrombophilia is a major pathoetiology. When CRAO is caused by hyperhomocystenemia, future CRAO can, to a large degree, be prevented by normalization of homocysteine by L methylfolate, B6-B12. When CRAO is caused by thrombophilic low protein S-high factor VIII, both of which increases risk for placental insufficiency and miscarriage, Enoxaparin therapy can both protect the pregnancy and the retinal artery.

Conclusions: When CRAO occurs in young, non-smokers, with normal carotid arteries and normal hearts, it is commonly caused by treatable familial thrombophilia.

Id: 95 Falls and Fractures in Patients with Breast Cancer on Aromatase Inhibitors Compared with Their Age-Matched Controls

M. Williams, P. Choksi, K. Kidwell, C. Van Poznak. Internal Medicine, University of Michigan, Ann Arbor, Michigan. J. Stella School of Medicine, University of Michigan, Ann Arbor, Michigan. D. Hanauer. Pediatrics, University of Michigan, Ann Arbor, Michigan.

Background: Aromatase Inhibitors (AIs) are the treatment of choice for estrogen receptor positive breast cancer (BCA) in post-menopausal women, as AIs reduce recurrence compared with tamoxifen alone. AI toxicity includes myalgias, arthralgias, bone loss resulting in osteopenia and osteoporosis, and fractures. Data comparing the incidence of fractures in patients (pts) with BCA on AIs compared with pts without BCA are lacking. To define the risk of fall and fracture in pts on AIs, we investigated the incidence in pts on AIs compared to aged-matched controls.

Methods: Institutional Review Board approval was obtained for this retrospective chart review. Women with hormone receptor positive early stage BCA on AI were identified and matched to controls based on age, race and gender using the University of Michigan Cancer Registry. Then, fall and fracture data were abstracted from the medical record from the time of initiating AI to 3 months after stopping AI and for controls using the Electronic Medical Records Search Engine (EMERSE). Conditional logistic regression was used to determine differences in cases versus controls.

Results: 332 pairs of women were matched on age and race. In each pair, one pt took an AI (case) while the other pt did not and did not have BCA (control). 59 pairs (17.8%) consisted of patients in which the control fell but the case did not and 61 pairs (18.4%) consisted of women where the case fell but the control did not. There was no statistically significant difference in the rate of falls in age-matched cases and controls (p=0.86). For pairs in which control fell but case did not, median age at fall was 67, compared with median age of 64 for pairs in which case fell but control did not. For pairs where both patients fell (n=22), there was no difference in the age of pts at time of fall, p=0.19 using sign test. Similarly, we found no statistically significant difference in the rate of fractures between cases and controls (p=1.0). There were 35 pairs (10.5%) in which the case had a fracture and the control did not and another 35 pairs (10.5%) where the control had a fractured but the case did not. There was low power to detect differences in age at time of fracture as there were only 6 pairs in which both patients had fractures; thus we found no significant difference in the age at fracture between cases and controls. (p=0.22) using the sign test. For pairs in which the control fractured but the case did not, median age at fracture was 71 as compared with median age of 63 for pairs in which case fractured but control did not.

Conclusions: This retrospective analysis did not identify a difference in falls or fractures in women on AIs compared to their age matched controls. Falls and fractures remain a public health concern for post-menopausal women. Although this retrospective study does not show a significant difference in incidence of fall and fracture in AI patients compared with age matched controls, there are significant limitations in our study design. To better define the risk of fall and fracture in AI pts, a prospective study is warranted.

Acknowledgments: EMERSE support from UM Biomedical Informatics Core and National Institutes of Health CA46592.

Reference: Burstein et al. Journal of Clinical Oncology 28.23 (2010): 3784-3796.

Id: 96 Improved Outcomes of Autologous Hematopoietic Cell Transplantation (Ahct) for Light Chain Amyloidosis: A Center for International Blood and Marrow Transplant Registry (Cibmtr) Study

A. D'Souza, P. Hari. Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, UNITED STATES. A. D'Souza, J. Huang, M. Zhang, P. Hari. Center for International Blood and Marrow Transplant Registry (CIBMTR), Milwaukee, Wisconsin.

Background: Light chain (AL) amyloidosis is a rare plasma cell neoplasm associated with systemic amyloid deposition leading to organ dysfunction and death without treatment. The use of AHCT in AL amyloidosis remains controversial as a prospective randomized control trial suggested inferior outcomes when compared with standard chemotherapy, driven primarily by high peri-transplant mortality (TRM) up to 24%. Improved patient selection criteria, supportive care and risk-adapted therapy have reduced TRM in recent single center studies. We analyzed trends and prognostic factors associated with AHCT outcomes in AL amyloidosis patients.

Methods: We identified 1532 AL amyloidosis patients who underwent AHCT following high dose melphalan (MEL) within 24 months of diagnosis between 1995 and 2012 from the CIBMTR database. A subset of patients with more complete level of research data reported between 2001 and 2012 was analyzed for multivariate analysis (n=354). The primary endpoints were day30 and day100 mortality, hematologic progression free survival (PfS), hematologic relapse/progression and overall survival (OS). Data regarding cardiac, renal, hepatic and neurologic amyloid involvement was collected. Hematologic and organ response and progression were defined based on the 2004 uniform consensus criteria.

Results: The median age at transplant was 57 years, with 61% males. AHCT was performed within 6 months of diagnosis in 66% patients. Karnofsky performance score (KPS) was <80 in 14%, HCTCI was ≥ 3 in 20% and 69% had a lambda isotype. Organ involvement included renal, cardiac, hepatic and autonomic nervous system involvement in 74%, 38%, 16% and 11% respectively. Coexistent myeloma (>10% bone marrow plasma cells) was seen in 14%. Progressively higher numbers of patients received AHCT from 1995-2000 (n=140) to 2001-2006 (n=595) and 2007-2012 (n=800). The majority were untreated pre-transplant (77%) while 8% received melphalan, 6% thalidomide and 4% each received lenalidomide and bortezomib based pre-AHCT therapy. The median CD34 cell dose infused was 4.4 X 106/kg cells (IQR 3.3-6.2). MEL dose reduction was common (60% received < MEL 180 mg/m2 and 38% < MEL 140 mg/m2). The median length of hospital stay was 17 days (IQR 13- 23). The median follow-up of patients from the time of transplant was 61 months (range 3-145). Best response was hematologic CR (33%), PR (28%), SD (19%) and renal response in 31%. Table 1 shows day 30 and day100 mortality and OS at 1, 3 and 5 years showing steady declines in mortality rates and improvements in survival in successive time cohorts. The figure (supplemental data) shows 5 year OS in each of the time cohorts. On multivariate analysis, a number of prognostic factors were identified to be associated with mortality, relapse/progression and progression-free and overall survival (will be shown in final presentation).

Conclusions: There has been a significant improvement in survival of AL patients after AHCT driven primarily by a reduction in early peri-transplant mortality. Better KPS, renal function, pre-transplant chemotherapy and absence of cardiac amyloid involvement were associated with good outcomes confirming that a risk-adapted therapy is necessary for superior outcomes.

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TABLE

Outcomes of AHCT in AL amyloidosis. Values are expressed as probabilities with 95% confidence intervals

Id: 97 Diagnostic Ramifications of Ocular Vascular Occlusion as a First Thrombotic Event Associated with Factor V Leiden and Prothrombin Gene Heterozygosity

S. Schockman, M. Prince, R. Riaz. Internal Medicine Residency Program at Jewish Hospital- Mercy Health, Cincinnati, Ohio. C.J. Glueck, P. Wang. Cholesterol, Metabolism, and Thrombosis Center of the Jewish Hospital- Mercy Health, Cincinnati, Ohio. C.J. Glueck. Mercy Medical Physicians, Cincinnati, Ohio. R. Hutchins. Cincinnati Eye Institute, Cincinnati, Ohio. R. Hutchins. Ophthalmology, University of Cincinnati College of Medicine, Cincinnati, Ohio.

Purpose: Assess the diagnostic ramifications of ocular vascular occlusion (OVO) as a first thrombotic event associated with factor V Leiden (fVL) and/or Prothrombin Gene (PTG) heterozygosity.

Methods: Patients with OVO, free of cardio-embolic etiologies (n=264), were sequentially referred from vitreoretinal specialists for measurement of thrombophilia-hypofibrinolysis, and compared to 111 healthy normal controls.

Results: Of the 264 patients, 27 (15 women, 12 men), 10% of all referred OVO cases, were found to have familial thrombophilias (fVL and/or PTG), including 15 with fVL, 11 PTG, and 1 with both fVL and PTG. Compared to controls, the 264 cases were marginally more likely to have ≥1 abnormality of fVL and/or PTG mutation (10% vs 5%, p=.071). Patients with OVO, compared to controls, were more likely to have low free protein S (9% vs 2%, p=.048), high homocysteine (21% v 5%, p<.0001), high factor VIII (19% vs 7%, p=.007), and high anticardiolipin IgM antibody (ACLA IgM) (8% vs 2%, p=.027). Of the 264 cases, 48% had ≥1 thrombophilic abnormality (fVL, PTG, free protein S, homocysteine, factors VIII and XI, and/or ACLA IgM), vs 19% of normal controls, p<.0001. Of the 27 cases with fVL and/or PTG, 15 had retinal vein occlusion (RVO), 5 non-arteritic anterior ischemic optic neuropathy (NAION), 4 retinal artery occlusion (RAO), 2 amaurosis fugax (Af) and 1 had both RVO and RAO. Of the 15 fVL cases, 14 (93%) had OVO as a first thrombotic event without prior DVT or PE, 5 (33%) had other thrombotic events including recurrent miscarriages, osteonecrosis, ischemic stroke and/or ischemic colitis, and 5 (33%) had immediate family members with previous venous thromboembolism (VTE). Of the 11 PTG cases, 8 (73%) had OVO as a first thrombotic event, 5 (45%) experienced VTE other than DVT or PE, and 5 (45%) had immediate family members with VTE. In 1 patient with both fVL and PTG, DVT occurred before RVO. Of the 15 women with fVL and/or PTG mutations, 7 (47%) experienced ≥1 miscarriage, 4 (27%) were on estrogen, and 1 (7%) was on Clomiphene.

Conclusion: Of the 264 patients with OVO, 27 (10%) had fVL and/or PTG, and 81% of these cases presented with OVO as their first thrombotic event. By diagnosing thrombophilia as an etiology for OVO, the ophthalmologist opens a window to family screening and preventive therapy.

Id: 98 Central Retinal Vein Thrombosis 3, 3, and 4 Months after Starting Testosterone Therapy in Men with Previously Unrecognized Familial Thrombophilia

S. Schockman, R. Riaz, M. Prince. Internal Medicine Residency Program at The Jewish Hospital – Mercy Health, Cincinnati, Ohio. C.J. Glueck, P. Wang. Cholesterol, Metabolism, and Thrombosis Center of the Jewish Hospital- Mercy Health, Cincinnati, Ohio. C.J. Glueck. Mercy Medical Physicians, Cincinnati, Ohio.

Background: Testosterone therapy (TT) can cause venous thromboembolism (VTE) in men subsequently found to have previously unrecognized familial thrombophilia, but central retinal vein occlusion (CRVO) has not previously been recognized as a complication of TT.

Materials and Methods: Thrombophilia and hypofibrinolysis was studied in 3 non smoking, Caucasian men, free of cardio-embolic disorders, who developed CRVO 3, 3, and 4 months after starting TT.

Results: Case #1, age 72, BMI 53, developed CRVO 3 months after starting TT 200 mg/wk intramuscularly (IM). He was found to be heterozygous for the prothrombin gene mutation, had high factor VIII (186%, UNL 150%), and high homocysteine (14.1 nmol/L, UNL 13.5). On TT his serum T was supranormal (1500 ng/dl, UNL 800) as was estradiol (E2, 144 pg/ml, UNL 42.6). Later, he also developed deep venous thrombosis and pulmonary embolus. Case #2, age 50, BMI 25.1, developed CRVO 3 months after starting T gel 50 mg/day, and was found to have 4G4G homozygosity for the PAI-1 gene and high lipoprotein (a) (150 mg/dl, UNL 30). Case #3, age 32, BMI 22.7, developed CRVO 4 months after starting HCG injections to increase serum T, and was found the have factor V Leiden heterozygosity.

Discussion: TT is esterified to E2, particularly in the obese, and high E2 on TT appears to interact with familial thrombophilia to produce CRVO.

Conclusion: When CRVO is diagnosed by vitreo-retinal ophthalmologists in cases receiving TT, studies of thrombophilia and hypofibrinolysis should be carried out, with diagnostic benefit to the affected proband and first degree relatives who have a 50% chance of also being thrombophilic and are at high risk for VTE and CRVO.

Id: 99 Identification of Signaling Lipid Lysophosphatidic Acid as a Critical Link between Diet-Induced Obesity, Angiogenesis and Breast Cancer Progression

L. Dong, Y. Chen, R. Yuan, R. Silverstein, B. Ren. Blood Research Institute, Blood Center of Wisconsin, Milwaukee, Wisconsin. Y. Yuan, S. Wu. Edison Biotechnology Institute and Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio. I. Aguilera-Barrantes. Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin. A. Sturich, R. Silverstein, B. Ren. Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin.

Obesity increases cancer risk including breast cancer (BC) but the direct link and mechanisms by which obesity promotes BC progression remain largely unknown. Nutritional obesity is accompanied by autotaxin (ATX)-mediated synthesis of the bioactive signaling phospholipid, lysophosphatidic acid (LPA). We have shown that LPA stimulates angiogenesis by turning off CD36 antiangiogenic switch in microvascular endothelial cells (MVECs) via protein kinase (PKD-1) signaling pathway that may be associated with mitochondrial bioenergetic metabolism. We hypothesize that LPA-PKD-1 signaling is a key BC promoter by modifying mitochondrial bioenergetics in both tumor and endothelial compartments. Using the Seahorse Bioscience Extracellular flux Analyzer, we showed that LPA enhanced mitochondrial respiration in human breast adenocarcinoma MDA-MB231 cells transduced with wild type PKD-1 (PKD-WT) in the presence of high glucose concentration, resulting in elevations in both oxygen consumption rate (OCR) and ATP-linked OCR, but not in the extracellular acidification rate (ECAR). The conditional medium from human MVECs transduced with PKD-WT reduced levels of basal OCR, ATP-linked OCR, and ECAR in breast cancer cells when compared with the control medium. However, in tumor-associated ECs, exposure to LPA decreased not only CD36 expression but also basal OCR, ATP-linked mitochondrial respiration and basal ECAR. Furthermore, overexpressing PKD-WT or constitutively active PKD-1 decreased basal OCR and ATP-linked mitochondrial respiration. To determine in vivo mechanisms of obesity-derived LPA, we established a syngeneic breast adenocarcinoma model in diet induced obese female mice. BC growth was significantly larger in the diet-induced obese mice than in the lean controls after tumors were subcutaneously implanted for 21 days (3884.6±804.4 mm3 vs 843.4±392.1 mm3). The tumor endothelium demonstrated increased LPA receptor 1, along with reduced CD36 expression and increased periphery vessels when compared to the lean control group (81.5±12.75vs 34.5±7.25 mm²). Proteomic angiogenic profiling showed significant elevations in several angiogenic proteins, particularly leptin level in the serum of the obese mice. Our study indicates that LPA changes cellular bioenergetics in both endothelial and BC cells. This may be associated with the metabolic switch between mitochondrial oxidative phosphorylation and aerobic glycolysis. The switch could be regulated by PKD-1 signaling and associated with proangiogenic responses and BC initiation and progression. Targeting LPA/PKD-leptin-metabolic signaling axis in the tumor microenvironment could provide a novel therapeutic strategy against breast cancer.

Id: 100 Deregulated Cell Cycle Confers Resistance to Aromatase Inhibitors in Postmenopausal Breast Cancer Patients

I. Doostan, C. Karakas, K. Keyomarsi. Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. I. Doostan, K. Keyomarsi. Graduate School of Biomedical Sciences, The University of Texas at Houston, Houston, Texas. S. Moulder. Department of Breast Medical Oncology, MD Anderson, Houston, Texas. K. Hunt. Department of Breast Surgical Oncology, Houston, Texas.

Doostan I1, Karakas C2, Moulder S3, Hunt KK4, Keyomarsi K5 1,5 University of Texas, Graduate School of Biomedical Sciences & 1,2,5 Department of Experimental Radiation Oncology, 3Department of Breast Medical Oncology and 4 Department of Breast Surgical Oncology, MD Anderson Cancer Center, Houston, Texas, 77030 Contact author: idoostan@mdanderson.org

Key Words: low molecular weight cyclin E, aromatase inhibitors, resistance Almost seventy percent of all breast cancer patients have estrogen receptor (ER) positive tumors requiring hormonal therapy. Aromatase inhibitors (AIs) are considered as the first line hormonal therapy for ER+ postmenopausal patients. However, resistance to these drugs remains a major challenge in clinic and the biology of such resistance is not clear. Previous studies have shown that cyclin E pathway, a key regulator in the G1 to S transition of cell cycle, is deregulated in breast cancer. Full-length cyclin E is abnormally cleaved into low molecular weight isoforms (LMW-E) that renders patients to poor survival. Here we hypothesize that cyclin E deregulation can confer resistance to AIs. To address this, we engineered aromatase overexpressing MCf7 cells to overexpress LMW-E under doxycycline inducible promoter. Full-length cyclin E, GfP and empty vector transfected cells were also generated and used as controls. Our results indicated that AIs inhibited proliferation by arresting the cells at G1 phase of the cell cycle. However, LMW-E expression significantly enhanced proliferation of the cells when treated with AIs. In addition, LMW-E bypassed G1 arrest following AI treatment. At the molecular level, AIs decreased CDK2, pCDK2, and Rb levels and attenuated Rb phosphorylation. However, these effects were completely rescued only when LMW-E was expressed. Moreover, using an in vitro kinase assay we indicated that AIs decreased CDK2 kinase activity while LMWE expression reversed this effect by increasing CDK2 enzymatic activity. To confirm our findings, we downregulated CDK2 and Rb using independent shRNAs and showed that LMW-E mediated resistance is dependent on CDK2/Rb pathway. Taken together, these results suggest that LMW-E inactivates Rb protein as a tumor suppressor and renders the cells to bypass G1 checkpoint following AI treatment. In addition, this study provides early evidence that CDK2 inhibitors could be beneficial in combination with AIs for LMW-E expressing tumors. Currently we are investigating whether LMW-E can bypass the activity of AIs using inducible breast cancer cell line xenograft. We are also examining the correlation between cyclin E status and response to treatment in a cohort of patients who received AIs in the neo-adjuvant setting

Id: 101 Histone Deacteylase Inhibition Regulates Inflammation and Enhances Tregs after Allogeneic Hematopoietic Cell Transplantation in Humans

E. Gatza, G. Hou, Y. Song, S. Choi. Pediatrics-Hematology/Oncology, University of Michigan, Ann Arbor, Michigan. Y. Sun, K. Oravecz-Wilson, P. Reddy. Internal Medicine- Hematology/Oncology, University of Michigan, Ann Arbor, Michigan. J. Whitfield. Comprehensive Cancer Center Immunology Core, University of Michigan, Ann Arbor, Michigan. C. Dinarello. Internal Medicine-Infectious Diseases, University of Colorado, Aurora, Michigan.

Background: We recently reported that patients treated with histone deacetylase (HDAC) inhibition in a first-in-human allogeneic hemato-poietic cell transplantation (HCT) study had reduced acute GVHD (Choi et al The Lancet Oncology 2014), similar to its effects in preclinical models. The impact of HDAC inhibition in directly regulating immune responses in human is not known.

Objective: We therefore set out to examine the immunologic effects of HDAC inhibition on human immune responses after clinical allogeneic HCT.

Design/Methods: Innate and adaptive immune responses were analyzed in peripheral blood samples acquired from 50 patients who underwent allogeneic HCT in a prospective, single-arm Phase 1/2 trial (#NCT00726375). HDAC inhibition was administered daily (D-10 thru D100 post-HCT). Controls consisted of healthy donors and post-HCT patients who were similarly transplanted, but not treated with HDAC inhibition. The Mann-Whitney non-parametric test was used. A two-sided p-value of <0.05 indicated statistical significance.

Results: HDAC inhibition was associated with prolonged increases in acetylation of histones H3 and H4, and increased expression of indoleamine-2,3-dioxygenase (IDO) in peripheral blood mononuclear cells (PBMC). HDAC inhibition caused significant reductions in plasma pro-inflammatory cytokines, IL-1b (p=0.006), TNf-α (p=0.0005), IL-6 (p=0.02) and IL-8 (p=0.007) 14 days after HCT. HDAC inhibition also decreased secretion of IL-1b (p=0.06) and TNf-α (p=0.01) production by CD11c+ PBMC 30 days after HCT. The number of peripheral blood Tregs (CD4+CD25+CD127-) was increased 1.7-fold 30 days post-HCT (p=0.01) and 2.7-fold on day 100 (p=0.01) in study patients compared with controls. Tregs remained >2-fold higher in study patients 180 days after HCT (p=0.06). Tregs showed that patients treated with HDAC inhibition had greater demethylation of the foxp3 T regulatory-specific demethylation region (TSDR; p=0.03), demonstrating naturally occurring Tregs with stable foxP3 expression. Furthermore, HDAC inhibition increased co-expression of CD45RA and CD31 (p=0.03) compared with control patients, suggesting increased de novo generation of Tregs. Finally, Tregs isolated from participants treated with HDAC inhibition were more suppressive on a per-cell basis than Tregs from control patients.

Conclusions: These data demonstrate for the first time, to our knowledge, that HDAC inhibition reduces inflammatory responses of PBMC but enhances Tregs after allogeneic HCT in humans. These effects might have been crucial for the ability to reduce clinical GVHD in our first-in-human clinical trial.

Id: 102 Ceramide Mediated Lethal Mitophagy is a Novel Cell Death Mechanism in Flt3 Targeted Therapy of Acute Myeloid Leukemia

M. Dany. Medical Scientist Training Program, Medical University of South Carolina, Charleston, South Carolina. B. Ogretmen. Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina. M. Dany, B. Ogretmen. Medical University of South Carolina, Hollings Cancer Center, Charleston, South Carolina.

Mutations in fLT3 receptor tyrosine kinase are common in Acute Myeloid Leukemia (AML) and confer a worse prognosis. FLT3 inhibitors are promising therapeutic agents; however, clinical trials show limited success due to development of resistance. A better understanding of the cell death mechanism in response to fLT3 inhibitors helps in identifying alternative therapeutic strategies. Ceramide, a bioactive sphingolipid, is synthesized de novo by Ceramide Synthases (CerS) and mediates cancer cell death in response to chemotherapeutic agents. This study investigates the biological role of ceramide in the response of AML to fLT3 targeted therapy. We show that AML patient samples and cell lines expressing fLT3 have suppressed CerS1 expression and lower levels of its product C18-ceramide. Silencing fLT3 expression or its pharmacological inhibition increased CerS1 and C18-ceramide levels while fLT3 overexpression suppressed them. Mechanistically, fLT3 signaling increases the activity of Histone Deacetylase 1 (HDAC1) that prevents the recruitment of Sp1 transcription factor to CerS1 promoter. However, upon fLT3 inhibition, CerS1 promoter becomes associated with acetylated histones leading to the recruitment of Sp1 and resulting in increased levels of CerS1 and C18-ceramide. The increase in C18-ceramide mediates cell death as silencing CerS1 expression or inhibiting its enzymatic activity protected from fLT3 inhibitors-induced cell death. Mass spectroscopy, confocal, and electron microscopy reveal that the increase in C18-ceramide occurs in mitochondria that form contact sites with autophagosomes. Indeed, fLT3 inhibitors resulted in the formation of LC3B-II containing autophagosomes that co-localize with ceramide and was accompanied by mitochondrial depolarization and decreased ATP generation. LC3B-II has a hydrophobic domain at the amino terminal that binds to ceramide. This ceramide binding domain is required for execution of cell death since disrupting it by overexpressing LC3B-I35A and LC3Bf52A mutants failed to sensitize the cells to cell death mediated by fLT3 inhibition. Interestingly, treatment with C18- pyridinium-ceramide, which accumulates selectively in mitochondria due to the conjugated pyridinium ring, is able to induce cell death in cells sensitive or resistant to fLT3 targeted therapy, through the same mechanism of LC3B-II dependent lethal mitophagy. In summary, our novel data highlights the importance of ceramide metabolism in AML by showing that fLT3 suppresses CerS1 expression in HDAC1 dependent manner, while its inhibition reactivates it leading to the generation of C18-ceramide in mitochondria. C18-ceramide then orchestrates the mechanism of lethal mitophagy by binding to LC3B-II to recruit autophagosomes to mitochondria. The ability of C18-pyridinium-ceramide to target the mitochondria and bypass fLT3 signaling highlights its potential of being established as an alternative therapeutic drug for AML patients regardless of whether patients are sensitive or resistant to fLT3 targeted therapy.

Id: 103 Fatty Acid Synthase, Cyclooxygenase-2 and Osteoprtegerin Expression in Invasive Breast Cancer: Implications in Carcinogenesis

N. Sharma-Walia, S. Goswami. RFUMS, North Chicago, Illinois.

Breast cancer, the leading cause of death among women with an onset frequency of one in eight, is the most common type of cancer among women. However, despite the advancement in therapy, the mortality rate in breast cancer patients still remains high. The role of various different genes, directly or indirectly, regulating these processes have tremendously added to our knowledge of the complex process of proliferation, which has remained the most important prognostic factor so far. In our previous study, we established Osteoprotegerin (OPG)'s role in aneuploidy, cell proliferation and angiogenesis in breast cancer. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to human normal mammary epithelial cells (HMEC). Here, we demonstrated by mass spectrometry that OPG pulls down fatty acid synthase (fASN), a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. We observed large number of lipid droplets in SUM149PT and SUM1315MO2 cells in comparison to HMEC by electron microscopy. Human breast cancer tissue samples showed high expression of fASN. Treatment with fASN inhibitor C75 decreased the number of lipid droplets/cell, altered expression of genes involved in cell proliferation, cell signaling (Akt/PI3K, ERK, GSK3b) apoptosis (caspase-3 and caspapse-9) and cell cycle. Treatment with C75 lowered the inflammatory pathway enzyme cyclooxygenase-2 (COX-2) expression and reduced secretion of its inflammatory metabolite prosataglandin E2 (PGE2). We reasoned that there might be crosstalk between OPG, fASN, and COX-2, that sustains the inflammatory pathways, and drives the progression of breast cancer. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 by recombinant human OPG. Collectively, our study indicates the crosstalk between fASN, COX-2 and OPG. Combinatorial drug treatment of these main players of carcinogenesis can be used as a potential therapeutic target to treat highly invasive breast cancer.

Id: 104 Sphingosine Kinases Play Important Role in Breast Cancer Cell Survival

L. Yogendran, N. Sharma-Walia. RfUMS, North Chicago, Illinois.

Breast cancer is the most common cancer and the second leading cause of death from malignancy in women in the United States. Highly metastatic Inflammatory Breast Cancer (IBC) is an extremely rare and lethal form of breast cancer, affecting roughly 1-5% of all breast cancer patients. The spread of IBC is, in part, regulated by a series of signaling lipids. We discovered high gene expression and protein levels of sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2) in SUM149PT and SUM1315MO2 inflammatory and invasive breast cancer cell lines as compared to human mammary epithelial cells (HMEC). Sphingolipid pathway centers on three molecules: the cell growth inhibiting/arresting sphingosine and ceramide (N-acyl sphingosine), and the pro-cell survival bioactive sphingosine 1 phosphate (S1P). These sphingolipids with opposing functions are interconvertible inside cells, suggesting that a finely tuned balance between them can determine cell fate. Imbalances in sphingolipid levels, especially with an excessive production of S1P correspond to unhealthy cell proliferation and differentiation. We observed high secretion of lipid messenger SIP and its receptors S1PR2 and S1PR3 in SUM149PT and SUM1315MO2 cells when compared to HMEC cells. Targeting SPHK mediated signaling in SUM149PT and SUM1315MO2 cells using clinically relevant pharmacologic inhibitor ABC294640, induced dose dependent caspase-3 cleavage, G2-M cell cycle blockage and apoptosis in IBC cells. SUM149PT and SUM1315MO2 cells revealed activation of protective autophagy under nutrient starvation when compared to HMEC cells. Since S1P promotes cancer cell survival or protective autophagy under nutrient starvation via activation of antiapoptotic signal transduction, we evaluated the effect of SPHK inhibition on autophagic pathway. Treatment with SPHK inhibitor resulted in accumulation of proapoptotic ceramides and reduction of intracellular S1P. SPHK inhibition downregulated autophagic pathway suggesting that autophagic survival pathways during starvation are dependent upon the induction of SPHK mediated signal transduction events. Collectively, these results implicate interrelated mechanisms and SPHK inhibition in the induction of IBC cell death ABC294640 and rationalize evaluation of novel SPHK inhibitor in future studies clinical trials for IBC. Since SPHK1, SPHK2, and S1PRs play multiple roles in cancers, effective inhibition of these pathways via their targeted inhibitors could potentially be tested in breast cancer progression.

ID: 105 Red Blood Cells Store And Release Interleukin-33

J. Wei, J. Zhao, Y. Zhang, M. Gladwin, Y. Zhao. Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. V. Schrott, M. Gladwin, G. Bullock. Vascular Medicine, Pittsburgh, Pennsylvania. Y. Zhao, V. Schrott, G. Bullock. Department of Pathology, Pittsburgh, Pennsylvania.

Interleukine-33 (IL-33) is a member of the IL-1 cytokine superfamily that potently drives production of a variety of cytokines and contributes to the pathogenesis of inflammatory diseases. Serum IL-33 levels are increased in various diseases, such as atopic dermatitis, chronic hepatitis C infection, and asthma. IL-33 is a nuclear protein and is released from apoptotic or necrotic cells. Here, we show that red blood cells (RBCs) are one of the major sources of plasma IL-33. IL-33 levels are significantly increased in supernatants from lysed RBCs. Plasma IL-33 levels are increased in patients during hemolysis and plasma IL-33 levels show a positive correlation with degree of hemolysis. IL-33 protein and mRNA levels were detected in the late stages of differentiation in ex vivo primary human erythroid progenitor cell cultures, suggesting that IL-33 is expressed during maturation of RBCs. Furthermore, hemoglobin depleted red cell lysates induced IL-8 expression in human epithelial cells. These results suggest that erythroid progenitor cells produce IL-33 and circulating RBCs represent a major source of IL-33 that is released upon hemolysis.

Id: 106 the Role of Sirt2 in Regulating the Critical Basal-Like Breast Cancer Determinant Slug

W. Zhou, C. Kuperwasser. Developmental, Molecular, and Chemical Biology, Tufts University, Boston, Massachusetts.

Breast cancer is a strikingly heterogeneous disease comprised of a multitude of discrete molecular subtypes. Basal-like breast cancer (BLBC) represents the most deadly form of these subtypes, and affects 20% of breast cancer patients. Unlike other subtypes, few driver mutations have been identified and, thus, no targeted therapies have been established. Moreover, knowledge of the molecular processes underlying BLBC is limited. We previously reported that the transcriptional repressor Slug is frequently overexpressed in BLBC in patients harboring BRCA1 mutations, and may in fact represent an important determinant in BLBC genesis and progression. Notably, Slug is a short-lived protein that is strictly regulated by proteasomal degradation in normal breast tissue. The mechanism by which it becomes deregulated in cancer remains unknown. To identify potential homeostatic regulators of Slug, we performed a chemical inhibitor screen and found that sirtinol, a drug causing Sirt2 inhibition, leads to rapid turnover and depletion of Slug protein. Conversely, Sirt2 overexpression stabilizes Slug and increases its level and activity. Mechanistically, we showed that Sirt2 binds to and deacetylates Slug protein to protect it from ubiquitinationmediated proteasomal degradation, thereby stabilizing Slug protein and increasing its bioavailability. Analysis from The Cancer Genome Altas (TCGA) dataset revealed that basal-like breast tumors frequently exhibited Sirt2 copy number amplification compared to other breast cancer subtypes (23%, p<0.05). Furthermore, we found that knocking down of Sirt2 in BLBC cell lines abolishes Slug stability leading to decreased Slug abundance, as well as suppression of metastasis-associated markers of the epithelial-to-mesenchymal transition (EMT). Taken together, these results illuminate an important role of Sirt2-dependent deacetylation in controlling Slug abundance. Co-opting this molecular mechanism to aberrantly stabilize transcription repressors such as Slug could be a general feature of cancer cells for adopting malignant behaviors. As such, targeting Sirt2 may be a rational strategy to molecularly dampen the Slug hyperactivity and to halt BLBC progression.

Id: 107 Impact of Insurance Status on the Day of Admission and Clinical Outcome in Acute Myeloid Leukemia (Aml) Patients

B. Ahmad, N. Pierson, H. Maymani, M. Khawandanah, S. Srour, A. Asch, M. Cherry. Hematology/Oncology, University of Oklahoma health sciences center, Oklahoma city, Oklahoma, UNITED STATES. H. Saeed. Internal Medicine, University of oklahoma health science center, Oklahoma city, Oklahoma, UNITED STATES. M. Machiorlatti. Biostatistics and Epidemiology, Research Design and Analysis Center, University of Oklahoma health science center, Oklahoma city, Oklahoma.

Background: In patients with acute myeloid leukemia (AML), insurance status has not been demonstrated to adversely impact outcomes. However, insurance status appears to be an independent factor in healthcare utilization. University of Oklahoma Health Sciences Center (OUHSC) is the main tertiary hospital in the State of Oklahoma treating patients with acute leukemia. We hypothesized that treatment patterns might be different between the insured and uninsured patients. We hereby attempt to analyze the association between insurance status, week day of admission and outcomes.

Methods: We retrospectively analyzed patients from January 2000 to June 2012 diagnosed with AML over 18 years of age, who were treated at OUHSC with induction chemotherapy. We retrospectively analyzed patients from January 2000 to June 2012 diagnosed with AML over 18 years of age, who were treated at Oklahoma University Health Sciences Center (OUHSC) with induction chemotherapy. Patients were divided into two groups: Group 1 included patients who were admitted earlier in the week (Sun-Wed) and group 2 included patients admitted towards the weekend (Thurs-Sat). Patients were also sub-classified as having private insurance, public insurance (Medicaid and Medicare) or no insurance. Primary outcomes were overall survival at follow up (OS), complete remission (CR) and relapse. Chi-Square analysis was utilized to assess if day of admission and insurance status was related to OS, CR and relapse. Cox proportional hazards model was used to measure association of insurance status, day of admission and their interaction and Kaplan Meir Survival curves were used to estimate survival rates for day of admission by insurance status.

Results: We analyzed total of 161 patients, 157 met inclusion criteria with 69 (44%) having private insurance, 58 (37%) with public insurance and 30 (19%) were uninsured. Group 1, with 94 (60%) patients, was admitted earlier in the week (Sun–Wed), and group 2, with 63 (40%) patients, was admitted later in the week (Thurs-Sat). The median age at diagnosis was 49 years, 63.7% male 36.3% female. 77.0% white, 10.6% African American, 6.2% Native American and 3.7% Hispanic. 63% of uninsured patients were admitted later in the week (Thurs-Sat) compared to only 35% of insured patients (p=0.0385). There was an interaction between insurance and day of admission with OS (p=0.0378). Patients with insurance who were admitted later in the week Thurs-Sat (Group 2) had a hazard ratio (HR) of death 3.09 (95%CI 1.76-5.42) relative to those admitted earlier in the week Sun-Wed (Group 1) (p<0.0001). Median overall survival (OS) for uninsured patients in Groups 1 and 2 was 0.77 and 1.47 years as compare to median OS for insured patients in Groups 1 and 2 of 1.47 and 0.42 years with a p-value=0.0006 respectively. The proportion of patients achieving CR did not differ by day of admission (p=0.6389) and insurance type (p=0.3611). Relapse was not associated with day of admission (p=0.7316) or by insurance type (p=0.1806).

Conclusions: for the patients with the diagnosis of AML who presented to our institution, uninsured patients were admitted later in the week in comparison to insured patients. The overall survival was lower for the patients who were admitted towards the weekend as compare to patients who were admitted earlier in the week. This trend is both noteworthy and significant and due to its possible impact on standard of care warrants further investigation.

Id: 108 Time from Hospital Admission to Induction Chemotherapy Adversely Affects Outcomes in Patients with Acute Myeloid Leukemia

H. Maymani. Internal Medicine, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma. B. Ahmad, S. Srour, A. Asch, G. Selby, M. Cherry. Hematology Oncology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma. M. Machiorlatti, S. Vesely. Biostatistics, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma.

Background: The duration of time from the diagnosis of acute myeloid leukemia (AML) to the initiation of chemotherapy is dependent on multiple factors. Previous studies suggest that delays in the time from diagnosis to treatment do not impact overall survival (OS) and complete remission (CR). As a result, awaiting laboratory analysis of molecular targets for therapy, specifically fLT3, has become more commonly adopted by clinicians. However, this strategy can lead to significant delays in initiation of chemotherapy. The aim of this study is to analyze the impact of delaying chemotherapy on OS and CR in AML patients.

Methods: We performed a retrospective analysis on adult patients with AML who were treated with induction chemotherapy at The University of Oklahoma Health Sciences Center from January 2000 to June 2012. Time from admission to treatment (TAT) was calculated from the date of admission to the date of initiation of chemotherapy. In addition, we analyzed the admission day of the week and its association with TAT (days). Association between CR and TAT was assessed using ANOVA and Chi-Square tests. Kaplan-Meier estimates of median OS were calculated for groups defined by categorical variables. A Cox Proportional Hazards model on OS was implemented using TAT, age, risk status (favorable, intermediate and unfavorable risks), day of admission, distance to hospital, and white blood cell (WBC) count. Interaction was assessed and a backward selection procedure was used to find the covariates associated with OS. Statistical analysis was performed using SAS 9.3 software.

Results: A total of 160 patients with AML received induction chemotherapy at our institution during the defined time, with 137 meeting inclusion criteria. The median age at diagnosis was 51 years with 63.7% being male and 36.3% being female. Of these patients, 77.0% were white, 10.6% African American, 6.2% Native American and 3.7% Hispanic. The median TAT for all patients was 3.0 days. There were 116 (84.7%) patients treated within 0-4 days (Group 1) and 21 (15.3%) patients treated beyond 4 days (Group 2). Patients in Group 1 had a median survival of 252.5 days compared to those in Group 2 of 188.5 days (p = 0.0958) when analyzed univariately. Multivariable analysis demonstrated TAT of 0-4 days was independently related to OS with a hazard ratio of .604 (95% CI 0.369- 0.990, p = 0.0451). The CR rate for Group 1 was 69.8% compared to Group 2 of 54.6% (p = 0.0692). In addition, patients admitted on a weekday (Monday-friday) were more likely to initiate chemotherapy within 0-4 days as compared to patients admitted on the weekend (Saturday-Sunday) with a p = 0.0102.

Conclusion: AML patients treated more than 4 days following admission have decreased OS and a trend toward decreased rate of CR as compared to patients treated within 0-4 days. This finding is independent of age, risk status, WBC count, and distance to hospital. Also, patients admitted on the weekend were more likely to experience delays in initiating chemotherapy compared to those admitted on the weekday. Although a larger sample size and testing in other clinic settings needs to be done to confirm this relationship, this study suggests treating AML patients within 4 days of hospital admission may be associated with improved outcomes.

Id: 109 Malignant Salivary Gland Tumors: A Single Institution Epidemiological Report

B. Ahmad, S. Nabeel, M. Khawandanah, W. Razaq, M. Razaq. Hematology/Oncology, University of Oklahoma health science center, Oklahoma city, Oklahoma. H. Saeed, M. Muqeet Adnan. Internal Medicine, University of oklahoma health science center, Oklahoma city, Oklahoma. M. Machiorlatti, S. Vesely. Department of Biostatistics and Epidemiology, University of oklahoma health science center, Oklahoma City, Oklahoma.

Background: Salivary gland tumors comprise a rare heterogeneous group of tumors, making up less than 1% of all cancers, occurring at a rate of about 1 case per 100,000 per year in the United States. These tumors represent 6-8 % of all head and neck cancers. We present a single institution retrospective analysis of epidemiology of salivary gland tumors from 1976 to 2013.

Methods: We included all salivary gland tumors between June 1976 - Oct 2013 at Oklahoma university health science center. All benign tumors were excluded. We stratified patients in different groups according to age, gender, ethnicity, location of primary tumor, histology and grade of tumor. Simple descriptive statistics on counts and percentages for categorical covariates were created and medians were calculated for the continuous variables. Fisher's Exact tests were performed to examine the relationships between sex, age, race, histology and grade of tumors due to low expected cell counts.

Results: A total of 276 cases of malignant salivary gland tumors were identified. 117 cases of age <60 years and 159 cases of age ≥60 years were identified. Median age at the time of diagnosis was 60 years with 170 (62%) males and 106 (39%) females. With regards to ethnicity, 242 (88%) (95% CI 84.2-92.0) were Whites, 26 (9%) were African Americans (AA), 6 (2%) were Native Americans and 2 (<1%) were Asians. Based on primary site of tumor location, 226 (82%) were located in parotid gland, 35 (12.4%) in submandibular gland, 2 (<1%) in sublingual gland and 13 (5%) in salivary glands not otherwise specified (NOS). Based on histological subtype, 68 (25%) were muco-epidermoid carcinoma (CA), 57 (21%) were squamous cell CA, 30 (11%) were adenoid cystic CA, 26 (9%) were acinar cell CA, 25 (9%) were adenocarcinoma, 20 (7%) were CA NOS and 50 (18%) were other less common CA which included 14 (5%) lymphoma, 4 (1%) large cell CA, 2 (<1%) amelanotic melanoma, 2 (<1%) small cell CA, 2 (<1%) sarcoma, 2 (<1%) neuroendocrine CA, 1 (<1%) malignant fibrous histiocytoma, 2 (<1%) papillary cystadeno-CA, 2 (<1%) merkel cell CA, 2 (<1%), myoepithelial CA, 2 (<1%) malignant mixed tumor, 1 (<1%) adenocarcinoma and 15 (5%) unknown CA. Tumor grade was also studied: Refer to table 1. Males and females differed statistically by grade and histology of tumors. (P=0.0179 and 0.0017 respectively). Histology was statistically different in two age groups (<60 y and ≥ 60 y) as well (p=<0.0001). Ethnic groups differed statistically by site (P= 0.0386) and grade of tumors (p=0.0556).

Conclusions: Salivary gland tumors are a rare group of cancer. Based on our epidemiological study we concluded that the median age was 60 years at the time of diagnosis. The majority of cases were diagnosed ≥ 60 years of age. Males were at 1.6 times higher risk as compared to females. The most common histology among males was squamous cell CA while muco-epidermoid CA among females. The most common histology in ≥ 60 years age group was squamous cell CA while muco-epidermoid CA in <60 years age group. Parotid gland was the most common site of primary tumor with muco-epidermoid carcinoma being the most common histology among these tumors. Grade III cancer with poorly differentiated histology was the most common of all tumors. This large retrospective data provides the epidemiology and bio-pathological characteristics of salivary gland tumors.

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Id: 110 Predicting Response to Histone Deacetylase Inhibitors Using High-Throughput Genomics

P. Geeleher, D. Lenkala, F. Wang, B. LaCroix, J. Wang, S. Karovic, M.L. Maitland, R. Huang. Univ of Chicago, Chicago, Illinois.

A. Loboda, M. Nebozhyn, M. Chisamore, J. Hardwick. Merck Research Laboratory, North Wales, Pennsylvania.

BACKGROUND: Histone deacetylase inhibitors (HDIs) have diverse effects on the tumor and host tissues. Although approved for some indications in oncology, HDIs have had highly variable effects in clinic. The mechanisms-of-resistance and mechanisms-of-action of this class of drugs remain unclear. A plethora of previous studies have implicated many disparate biological processes as putative molecular markers of HDI response. However, none have been successfully applied clinically.

METHODS and RESULTS: We showed that rather than this being entirely an issue of reproducibility, response to vorinostat, the first fDA approved HDI, is determined by the additive effect of multiple molecular factors, many of which have previously been demonstrated. Using machine learning, the small effects that aggregate, resulting in sensitivity or resistance, can be recovered from high-throughput gene expression data from a large panel of cancer cell lines. In this pre-clinical model, we showed that this approach can predict vorinostat response extremely accurately in an out-of-batch set of samples, whereas single gene or pathway markers cannot. Our analyses recapitulated and contextualized many previous findings and suggest an important role for processes such as chromatin remodeling, autophagy and apoptosis. As a proof-of-concept, we also discovered a novel causative role for CHD4, a helicase involved in the histone deacetylase complex that is associated with poor clinical outcome. As a clinical validation, we demonstrated that a common dose limiting toxicity of vorinostat, thrombocytopenia, can be predicted several days before it is detected clinically.

CONCLUSIONS: Our work suggests a paradigm shift from the traditional single gene/pathway evaluation to simultaneously evaluating multiple independent high-throughput gene expression datasets. Taken together, these results will bring this drug closer to being used in a much broader clinical context. The methodologies developed here can be easily extended to other investigational compounds, where similar issues are hampering clinical adoption.

Id: 111 Healthcare Disparities According to Insurance Status among Patients with Salivary Gland Tumors

B. Ahmad, S. Nabeel, M. Khawandanah, W. Razaq. Hematology/Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. M. Razaq, B. Ahmad, H. Saeed, M. Muqeet Adnan. Internal Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. M. Machiorlatti, S. Vesely. Department of Biostatistics and Epidemiology, University of Oklahoma Health Sciences center, Oklahoma City, Oklahoma.

Background: Insurance status appears to be an independent factor in healthcare utilization in common cancers according to some studies. However, there is no data available in this regard for rare tumors such as salivary gland tumors. University of Oklahoma Health Sciences Center (OUHSC) is the main tertiary hospital in the State of Oklahoma, treating patients with rare tumors like salivary gland tumors which comprise a group of heterogeneous cancers making less than 1% of all cancers. As healthcare reforms, and affordable care act (ACA) proceeds in the United States aiming to cover a large number of uninsured population, it is important to know the trends and outcomes in the setting of cancer specially in the rare tumors. We describe a retrospective analysis of insurance status and distance to travel for treatment of patients with malignant salivary gland tumors at our institution evaluating impact on the clinical outcomes.

Methods: from June 1976 to October 2013, we identified a total n=276 patients who met the inclusion criteria for the study. There were n=117 cases of age <60 years and n=159 cases of age ≥ 60 years were identified. Median age at the time of diagnosis was 60 years with n=170 (62%) males and n=106 (39%) females. Patients were divided into two groups on the basis of insurance status: Group 1 included patients with Medicare, Medicaid, private and military insurance total n=184 (67%), Group 2 included uninsured patients total n=92 (33%). Distance to travel for treatment was assessed in n=276 patients using a continuous variable dividing in four groups: 0- 40 miles n=82 (30%), 41-75 miles n=107 (39%) and >75 miles n= 84 (31%), three patients were excluded as their residential address was not available. A Chi-Square test of independence was performed on insurance status and distance groups. A Kaplan-Meier method was used to estimate survival rates for categorical variables. A Cox Proportional Hazards model on overall survival (OS) was implemented to assess continuous covariates as well as create a final multivariable model. Interaction for age, distance, insurance, and gender were assessed with all covariates. All statistical analysis was performed using SAS 9.2 software (SAS Institute Inc.).

Results: There was no association between insurance status and distance groupings (Log Rank p=0.8740). We found no statistical difference in survival rates (Log-Rank p-value =0.5905) in overall survival (years) between insured and uninsured groups. There was no statistical difference among groups with varying distance to travel for treatment (Log-Rank p-value=0.4497). In the presence of other variables and subgroup analysis, using the Cox Proportional Hazards model, the variables that were influential and statistically significant included age ≥60 years (HR=1.86 95% CI 1.25 – 2.80) and gender female (HR=0.58 95% CI 0.40 – 0.86).

Conclusions: Based on our retrospective analysis insurance status did not impact survival outcomes. Similarly, distance travelled to receive treatment did not alter survival outcomes. However, the survival analysis showed a statistically significant OS benefit in patients with age less than 60 years and female gender. Further studies are warranted for a better understanding of healthcare disparities and subsequently proper implementation of affordable care act in rare tumors like salivary gland tumors.

Id: 112 Cinobufotalin Inhibits Ovarian Cancer Cells Proliferation, Migration and Invasion

A.B. McDowell Jr, T.C. McCormick, T.J. Kuehl, M.N. Uddin Obstetrics and Gynecology, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. M. Co, M.R. Beeram, M.N. Uddin. Pediatrics, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. S.H. Afroze, D.C. Zawieja. Medical Physiology, Texas A&M Health Science Center College of Medicine, Temple, Texas.

Objective: Cinobufotalin (CINO), a cardiotonic steroids or bufadienolides, is extracted from the skin secretions of the traditional Chinese medicine giant toads (Chan Su). Cinobufotalin has been used as a cardiotonic, diuretic and a hemostatic agent. We have shown that CINO inhibits the cytotrophoblast cells function. Recently, it has been shown that CINO inhibits the lung cancer cells A549 function. In the present study, we proposed to pursue this potential application of CINO using ovarian tumor cell lines SK-OV-3.

Study Design: We evaluated the effect of CINO on ovarian cancer, SK-OV-3 cell function in vitro. Cell proliferation was measured using a CellTiter Assay (Promega), which is a colorimetric method for determining the number of viable cells. Cells were treated with 0.1, 1, 5, and 10 μM CINO. After CellTiter 96 was added to the cells, absorbance at 490 nm was measured on a plate reader (SPECTRAmax 340PC384 Microplate Spectrophotometer, Molecular Devices). Cell migration was measured using a CytoSelect Assay (Cell Biolabs). Cells were treated with 0.1, 1, 5, and 10 μM CINO. After the CyQuant GR Dye solution was added to the cells, fluorescence was measured at 480 nm / 520 nm on a fluorescence plate reader (Cytofluor Series 4000 fluorescence Multi-Well Plate Reader, Applied Biosystems). Cell invasion was measured using a fluoroBlok Assay (BD). Cells were treated with 0.1, 1, 5, and 10 μM CINO. After Calcein-AM was added to the cells, fluorescence was measured at 490 nm / 700 nm on a fluorescence plate reader (Cytofluor Series 4000). Cell viability was measure using a CellTiter Assay (Promega). Cells were treated with 0.1, 1, 5, and 10 μM CINO. After the CellTiter-Blue reagent was added to the cells, absorbance at 520 nm was measured on a plate reader (SPECTRAmax 340PC384).The effect of CINO on p38 phosphorylation was evaluated by a cellular activation of signaling ELISA (CASE) kit (SuperArray). Cell cycle progression was evaluated by a Cell Cycle Phase Determination Kit (Cayman Chemical) and apoptosis was evaluated by an Apoptotic Blebs Assay Kit (Cayman Chemical). Cell cycle arrest and apoptotic signaling was determined by fluorescence-activated cell sorting (fACS) analysis.

Results: CINO at ≥ 0.5 μM inhibited CTB cell proliferation, migration, and invasion (p < 0.05) but had no effect on cell viability. There was a higher (p < 0.05) percentage of G0/G1 phase cells in groups treated with CINO at ≥ 0.5 μM. CINO caused an increase in stress signaling p38 MAPK and a positive annexin-V staining in SK-OV-3 cells, indicating the activation of apoptotic signaling.

Conclusion: This data demonstrates that CINO impairs SK-OV-3 cell function via cell cycle arrest and apoptotic signaling. The results of this study suggest that CINO might be further investigated as a novel anti-ovarian cancer agent.

Id: 113 Acute Myeloid Leukemia: Comparison of Epidemiology and Treatment Outcomes between Insured and Uninsured Patients

S.A. Srour, U. Bhutta. Medical Service, Department of Veterans Affairs, Oklahoma City, Oklahoma. M. Machiorlatti, D. Thompson. Biostatistics and Epidemiology, OUHSC, Oklahoma City, Oklahoma. U. Bhutta. Internal Medicine, OUHSC, Oklahoma City, Oklahoma. S.A. Srour, N. Pierson, M.A. Cherry, C. Kurkjian. Medicine, Hematology/Oncology Section, The University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, Oklahoma.

Background: The overall prognosis for most acute myeloid leukemia (AML) patients remains poor. The majority of adult patients (pts) with AML will ultimately need allogeneic stem cell transplant (allo-SCT). Limited data is available comparing epidemiology and treatment outcomes between uninsured and insured pts with AML. We describe a retrospective analysis of adult pts with AML treated at our institution.

Methods: from January 2000 to June 2011 we identified 239 pts with AML, of which 185 met inclusion criteria. Patients were classified as having private insurance, public insurance (Medicaid and Medicare), or no insurance. Primary outcomes were overall survival at follow up (OS), complete remission (CR), and percent of pts receiving allo-SCT. Kaplan-Meier analysis was used to estimate survival rates.

Results: Of the 185 pts studied, 146 (81%) were white, 16 (9%) African-American and 11 (6%) were Native American. The median age at diagnosis was 49 years. 75 pts had private insurance at the time of diagnosis, 70 had public insurance, and 33 were uninsured. Of those receiving allo-SCT (n=60; 38 male) 48% had private insurance, 30% public, and 22% were uninsured. Whether a patient underwent allo-SCT did not differ according to insurance source (p=0.2189) nor to status at follow-up (p= 0.6480). The proportion of patients achieving CR was higher among those undergoing allo-SCT (p=0.0002). Median OS was 920 days for those who underwent allo-SCT and 192 days for those who did not (p< 0.0001). Median OS did not differ by insurance type (p = 0.1454). The proportion of patients achieving CR did not differ by insurance type (p=0.2665) (66.7% of those with private, 62.7% of those with public, and 78.8% of those with no insurance).

Conclusions: for the population referred to our institution, a tertiary referral center for the state, there were no differences in treatment outcomes or in the percent of pts receiving allo-SCT when pts were stratified according to insurance source. Further analysis of this dataset will identify the impact of pretreatment variables stratified by type of insurance.

ID: 114 Arsenic Dysregulates Human Prostate Stem/Progenitor Cell Homeostasis, Perturbs Autophagy And Drives Transformation Through Accumulation Of SQSTM1/P62

L. Xie, D. Hu, W. Hu, J. Yang, G.S. Prins. Urology, Univ of Illinois at Chicago, Chicago, Illinois.

Inorganic arsenic (iAs) is an environmental toxin which increases cancer risk, including prostate cancer, for chronically exposed populations worldwide. Presently, the underlying biological mechanism for iAs-induced prostate carcinogenesis is poorly understood. Since recent studies indicate that iAs can target tissue stem cells, we sought to determine whether iAs can modify normal human prostate stem and progenitor cell homeostasis and drive their transformation. Primary prostate epithelial cells (PrEC) of young, disease-free donors were utilized and stemprogenitor cells isolated using fACS and 3D prostasphere (PS) culture. Treatment with 1 mM iAs significantly increased stem-like cell numbers in 2D PrEC cultures and PS formation in 7-day 3D cultures whereas 5 mM iAs markedly reduced these parameters indicating a biphasic effect. Furthermore, iAs at either dose hindered differentiation of PS and in vitro derived prostate organoids. Together these results show that short-term iAs exposure perturbs homeostasis and differentiation of normal human prostate stem-progenitor cells. Autophagy is a cell selfprotective mechanism that plays a critical role in self-survival of stem/progenitor cells and suppression of tumorigenesis. While iAs had no effect on autophagy in PrEC parental cells, it markedly increased LC3B-II and p62 protein levels (but not mRNA) in day 7 PS in a dose-dependent manner (0, 0.1, 1, 5 mM iAs), suggestive of autophagy flux blockade. Serial passage of PS through passage 4 showed continued elevation of SQSTM1/p62(hereafter referred to as p62), presumably due to p62 mRNA induction with 4-5 weeks exposure. p62 is known to be involved in tumorigenesis and p62 protein levels are positively associated with prostate cancer Gleason score; however, it is not known whether p62 can transform the normal prostate epithelial cells. To test this, we stably overexpressed p62 in the normal human prostate cell line RWPE-1 by lentiviral vector and grew the cells as subcutaneous xenografts in nude mice for 3 months. Overexpression of p62 transformed the RWPE1 cells, forming high Gleason grade tumors with faster growth and size vs empty vector cells. Immunohistology and qRT-PCR data revealed EMT (elevated vimentin, Twist, Snail, N-cadherin, reduced E-cadherin), nuclear localization of YAP, and induction of YAP and b-catenin downstream genes suggesting that these pathways are involved in p62-induced tumorigenesis. In summary, iAs disturbs the autophagy flux in prostate stem/progenitor cells leading to chronic accumulation of p62. Since p62 overexpression can transform the normal human prostate RWPE1 cell line, we propose that chronic iAs exposure may drive carcinogenesis of the human prostate through chronic p62 accumulation in the prostate stem-progenitor cells that maintain the prostate epithelium. This work was supported by NIH R01 ES022071.

Id: 115 Cell-Free Hemoglobin, Hmox1 and Apol1 in Sickle Cell Nephropathy

S.L. Saraf, X. Zhang, B. Shah, J. Arruda, R.F. Machado, V.R. Gordeuk. Medicine, University of Illinois at Chicago, Chicago, Illinois. K.P. Gudehithlu, A. Singh, John H Stroger Jr. Hospital of Cook County, Chicago, Illinois. R. Kittles. University of Arizona, Tucson, Arizona. M.T. Gladwin. University of Pittsburgh, Pittsburgh, Pennsylvania.

We recently reported that hemoglobinuria is associated with chronic kidney disease (CKD) stage and progression in sickle cell disease (SCD). To further investigate a potential role of cell-free hemoglobin in SCD nephropathy, we measured urine concentrations of kidney injury molecule-1 (KIM-1), a biomarker of tubular injury, and nephrin, a biomarker of glomerular injury, in 25 University of Illinois at Chicago (UIC) SCD patients. Urine KIM-1 concentration directly correlated with increasing urine cell-free hemoglobin concentration (P=0.002)(figure 1) but urine nephrin did not. We then added lyophilized hemoglobin to cultured human kidney-2 (HK2) tubular cells (n=5). Supernatant KIM-1 concentrations increased progressively with increasing cell-free hemoglobin exposure (P=0.01). To determine whether enzymes for metabolizing hemoglobin and protecting from reactive oxygen species are affected by exposure of HK2 cells to cell-free hemoglobin, we evaluated candidate gene expression using rt-PCR. The expression of HMOX1 (P=0.0001)(figure 2) progressively increased with increasing cell-free hemoglobin dose while expression of SOD1, SOD2, CAT, GSR, GSS, and NfE2L2 did not change. We then focused on variants of HMOX1 in a cohort of 247 UIC SCD patients. Genotyping was carried out using Affymetrix Axiom genome-wide Pan-African array in DNA isolated from peripheral blood mononuclear cells. Examination of 11 tag SNPs within +/-10 kb of HMOX1 identified a SNP in HMOX1 (rs743811, minor allele frequency 0.14) that had a significant association with CKD stage (OR=3.0, P=0.0001) and non-significant associations of the same direction with end stage renal disease (ESRD)(OR=2.5, P=0.2). Validation studies were conducted in 517 SCD patients from the Walk-Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk-PHaSST) cohort. Genotyping was performed using the Illumina Human 610-Quad SNP array and imputed using the HapMap II reference panels for HMOX1 tag SNPs. HMOX1 rs743811 had a significant associated with ESRD (OR=9.8, P=0.0003) and non-significant associations of a similar direction with CKD stage (OR=1.4, P=0.2). Homozygosity or compound heterozygosity for the G1/G2 variants of APOL1 have been implicated in CKD in African Americans with and without SCD and we therefore examined their association with hemoglobinuria-associated CKD in the UIC cohort. S342G and I384M substitutions are in almost complete linkage disequilibrium and are termed G1; deletion of two amino acids, N388 and Y389, is termed G2. The G2 variant was further imputed using reference panels of the 1000 Genomes Project. Homozygosity or compound heterozygosity of the G1/G2 variant was associated with hemoglobinuria (OR=7.3, P=0.0002), CKD stage (OR=2.6, P=0.02), and ESRD (OR=6.5, P=0.04). For validation studies in Walk-PHaSST, the G1 and G2 variants of APOL1 were imputed using the 1000 Genomes Project. Homozygosity or compound heterozygosity of G1/G2 in the Walk-PHaSST cohort was also significantly associated with hemoglobinuria (OR=2.6, P=0.003). Our findings are consistent with the possibility that cell-free hemoglobin contributes to sickle cell nephropathy through tubular injury. A tag-SNP in HMOX1 is associated with CKD stage in the UIC cohort and ESRD in the Walk-PHaSST cohort, raising the possibility that altered HMOX1 expression or function can have a role in SCD-associated CKD. Our results also point to a novel association of the G1/G2 variants of APOL1 with cell-free hemoglobin-mediated CKD in SCD subjects. Future studies to explore the roles of HMOX1 and APOL1 in cell-free hemoglobin-associated sickle cell nephropathy are warranted.

Id: 116 Corticosteroid-Induced Morphological Changes in Cells of the Myeloid Lineage

S. Lee, P. Khankhanian. Medicine, Icahn School of Medicine at Mount Sinai, Elmhurst, New York. J. Mascarenhas. Tisch Cancer Institute, Hematology and Oncology, Icahn School of Medicine at Mount Sinai, New York, New York.

A 60 year-old previously healthy woman presented with generalized body swelling, found to have IgA nephropathy. She received intravenous pulse corticosteroids, followed by prednisone 60 mg daily. Her symptoms improved, and she was discharged on a monotherapy of prednisone 60 mg daily for a six month course. The onemonth blood smear showed morphological changes in myeloid cells as follows, which were not present on the initial blood smears and smears after steroid therapy was discontinued. In response to chronic steroid exposure, peripheral smears displayed a high degree of heterogeneity in cells of the myeloid lineage. (A) shows ring-shaped nucleus of a neutrophils. (B) demonstrates a nuclear club in the upper lobe of the nucleus previously reported in patients with androgen-secreting tumors. The neutrophil in (C) shows pseudo-Pelger-Huët anomaly (PHA) previously reported in myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with MDS-related feature (AML-MDS). (D) is an example of hypersegmented neutrophils reported in B12 or folate deficiency. A neutrophil in (E) is mono-lobed with a highly clumped chromosome previously reported in MDS or AML-MDS. Nuclear lobes in (f) are interconnected via thin filaments, resembling myelokathexis in the WHIM syndrome. (G) shows dysmorphic a nucleus with clubs/hooks in eosinophils, previously not reported. The cytoplasm reveals cytoplasmic clearing and basophilic granules reportedly observed in myeloid leukemia or hypereosinophilic syndrome. The eosinophil in (H) demonstrates pseudo-PHA with an indented single-lobed nucleus as reported in MDS or AML-MDS. (I) shows a hyper-segmented eosinophil also reported in B12 or folate deficiency. This cell displays basophilic cytoplasmic granules. (J) is an example of a large number of eosinophils demonstrating a “satellite” nuclear lobe that is highly clumped, lightly stained, and smaller than other lobes. This finding was never reported in other pathologies. This cell also shows a small number of basophilic cytoplasmic granules. (K) is an eosinophil with a highly clumped chromosome and a large degree of cytoplasmic clearing as reported in MDS or AML-MDS. (L) is a bi-lobed eosinophil with cytoplasmic clearing, basophilic granules, and a nuclear hook in the lower lobe. A basophil in (M) reveals the cytoplasmic granules displaying a large degree of heterogeneity in size with empty granules near the plasma membrane, suggesting partial degranulation, reported in AML. A monocyte in (N) shows a highly dysmorphic nucleus, previously reported as monocytic pseudo-PHA observed in leukemoid reactions or severe inflammation. (O) displays a highly clumped nucleus with a nuclear club in a monocyte, which was never reported in other pathologies.

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Id: 117 Primary Cardiac Prosthetic Valve-Associated Lymphoma: Case Report and Literature Review

S. George, S.A. Srour. Hematology and Oncology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma.

Introduction: Primary cardiac tumors are extremely rare with an incidence of < 0.1%. Primary cardiac lymphomas, majority of which are non-Hodgkin's lymphomas, account for 1-2% of cardiac tumors. Primary lymphoma involving cardiac valves has been described in scattered case reports and case series. Most cases, especially prosthetic valve-associated lymphomas (PV-AL), present with thromboembolic manifestations diagnosed incidentally during valve replacement, and have been associated with EBV. We provide a literature review and report an unusual case of primary cardiac, prosthetic-associated, large B-cell lymphoma successfully treated by adjuvant chemotherapy with an excellent outcome. Case A 65 year-old male with a history of ascending aortic aneurysm and secondary aortic insufficiency status post composite valve replacement of aortic valve (AV) and ascending aorta with St Jude Toronto root in 2004. In June 2011, he presented with fatigue and headache, found to have a right parietal intraparenchymal hemorrhage(IPH). In March 2012, he presented with left frontal IPH followed by ischemic stroke in 10/2012. Recurrent strokes were attributed to thromboembolic events secondary to prosthetic AV. Echocardiography showed a mass consistent with intraluminal thrombus in ascending aortic graft. Surgical excision and replacement of AV conduit explant was performed in 11/2012. Final pathology was consistent with EBVassociated large B-Cell lymphoma. Complete staging including bone marrow biopsy was negative. He was treated with 4 cycles of RCHOP and he is 24 months post treatment without evidence of disease. Discussion Little is known about the pathophysiology of PV-AL. It has been thought, as described with other prosthetic-related neoplasms (metallic implants, breast implants), that chronic inflammation and EBV infection may play an essential role in pathogenesis of this entity. Only 6 cases of PV-AL were reported in 2010 as reviewed by Miller et al. Including our patient 11 cases were identified. As detailed in table 1, PV-AL patients has a median age of 60, predominates among males, and has a median duration of 8 years for development from date of prosthesis placement. Surgical resection and repair of primary involved prosthetic valve is essential for initial treatment. However, there is no consensus about best approach for subsequent therapy. We speculate that poor outcomes for the described cases are mostly related to primary cardiopulmonary deterioration in first 6 months, but are likely to be related to recurrent lymphoma and suboptimal treatment for patients who survive over 6 months. Of the five patients who survived over 6 months, 2 out of the three patients who didn't receive adjuvant chemotherapy died with recurrent lymphoma. This is in contrast to the 2 patients who received adjuvant chemotherapy and reported to be alive at 16 and 24 months. Increased awareness of clinicians about this entity, early diagnosis, and appropriate timely surgical intervention are essential for early survival. We believe that adjuvant chemotherapy should be an integral part for these patients for better long term survival outcomes.

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Table 1

Patient characteristics and outcome of reported cases

ID: 118 Saved From Hit

S.S. Tirunagari, D. Frieri. Medicine, Nassau University Medical Center, Westbury, New York.

Introduction: Heparin-induced thrombocytopenia Type II is one of the most serious life-threatening adverse reactions that may occur in certain patients exposed to heparin and is caused by the formation of anti-platelet factor 4 (Pf4)/heparin antibodies which are also known as chemokine CXCL4/heparin complexes within 5-14 days of exposure or in remote exposure (more than 100 days). 1-5% of patients receiving heparin will progress to develop HIT II and a third of them may develop serious complications. The authors report a case of HIT type II that was diagnosed promptly, successfully treated and averted serious complications. This case report contributes to a better understanding of immune mediated adverse drug reaction due to heparin.

Case discussion: A 57 year old Afr-Am female presented with past medical history of schizophrenia and hypertension who forgot to bring her medication Risperidol on a trip to USA. Physical examination: Tachycardia, BP 164/89, RR 18, temp 98. Patient was obese, confused, agitated, GCS 13-14 with pinpoint pupils, not responding appropriately to commands and yelped each time she was touched. Patient was admitted and catheterized. EEG showed no change in mental status and LP was not significant. Patient was instituted Acyclovir, Risperidol and Ativan. Within a few days, patient was communicative and responsive. Day 14, left ankle swelling with tenderness was noted, ultrasound Doppler study of the left lower extremity showed left proximal common femoral occluded, DVT, non-occlusive DVT close to R proximal common femoral. Patient was started on Lovenox subcutaneously. Day 16, patient became very critical with platelets trending down, CBC showed 48. Lovenox was discontinued and Argatroban started on day 17, after diagnosing HIT. The goal was to maintain PTT 60-80, and to start Coumadin 5mg. Argatroban with warfarin was to be bridgesd, follow up CBC, PT/PTT/INR.Day 20, patient tolerated sitting for 30 minutes. Patient was carefully monitored with labs, PTT stabilized to 88.6, with plan to titrate Argatroban and eventually discontinue. Day 26, patient was discontinued with Argatroban, INR was 2.7. Patient was discharged with Coumadin 3mg with target INR 2-3.

Discussion: Heparin induced thrombocytopenia occurs in two distinct forms: type I and type II. Type I (non-immune) manifests within 48-72 hours of administration of heparin, results from the platelet clumping or aggregationd due to heparin. Platelet count returns to normal with discontinuation of heparin within 4 days, with no complications. HIT type II should be suspected if the drop in platelet count is over 30-50% of baseline count. Studies have recommended that pre-test Warkentin 4Ts clinical score (Thrombocytopenia, Timing of platelet decrease, Thrombotic complications and other causes for thrombocytopenia) in combination with the Pf4/heparin immunoassay, in which case the sensitivity and specificity of HIT testing is greater (100% and 70% respectively) compared with the Pf4/heparin immunoassay alone (100% and 26%) may be used to diagnose HIT II. treatment may be initiated pending lab confirmation. This case illustrated that a high index of suspicion of HIT and prompt substitution of heparin with alternative direct thrombin inhibitor reduced the risk of serious thromboembolic events. Direct thrombin inhibitor (e.g., argatroban or lepirudin) or the heparinoid danaparoid may be used to reduce the risk of thromboembolic events such as severe thrombocytopenia, thrombosis related incidents such as stroke, pulmonary embolism, limb ischemia, myocardial infarction and death. Repeated lab measurements are necessary.

Id: 119 Retinal Artery Occlusion as a First Thrombotic Event Associated with High Factor Viii and Low Free Proteins in the First Trimester of Pregnancy

S. Schockman, M. Prince, R. Riaz. Internal Medicine Residency Program at The Jewish Hospital – Mercy Health, Cincinnati, Ohio. C.J. Glueck, P. Wang. Cholesterol, Metabolism, and Thrombosis Center of the Jewish Hospital- Mercy Health, Cincinnati, Ohio. C.J. Glueck. Mercy Medical Physicians, Cincinnati, Ohio.

Background: Central retinal artery occlusion (CRAO) during pregnancy is a marker for maternal familial thrombophilia and for placental insufficiency, secondary to thrombotic obstruction of the spiral arteries of the uterusplacenta mediated by the physiologic hyperestrogenemia of pregnancy interacting with underlying thrombophilia.

Case Description: A healthy 31 year-old Caucasian female was evaluated after an ophthalmologist diagnosed CRAO at 13 weeks gestation in her first pregnancy. Cardio-embolic etiologies were ruled out by cardiac echocardiogram and carotid ultrasound. The patient had no previous thrombotic events, however her maternal grandmother had multiple episodes of venous thromboembolism (VTE), including a lethal pulmonary embolus (PE). Thrombophilia and hypofibrinolysis studies were carried out and she was found to have elevated factor VIII (165%, UNL 150%) and low free Protein S (44%, LNL 50%). She maintained vision loss in the affected eye. Thromboprophylaxis with Lovenox (60 mg/day) and 81 mg aspirin was initiated.

Discussion: CRAO during pregnancy as a first thrombotic event heralds the presence of familial or acquired thrombophilia, here represented by elevated factor VIII and low free Protein S. During normal pregnancy, particularly comparing the last to the first trimester, factor VIII increases, and total and free protein S fall, making the diagnosis of familial high factor VIII and familial protein S deficiency difficult during pregnancy. However, the rare development of CRAO during the first trimester in the presence of a strong family history of VTE, points to the presence of familial thrombophilia. The physiologic hyperestrogenemia of pregnancy promotes thrombosis, including ocular events, in patients with major gene familial thrombophilias.

Conclusion: When CRAO occurs during pregnancy, a complete thrombophilia and hypofibrinolysis evaluation should be done. The goal is to protect against a second thrombotic event during pregnancy in the mother and to protect the fetus from thrombotic placental insufficiency, both achieved by low molecular weight heparin therapy throughout pregnancy. CRAO during pregnancy should also lead to screening of first-degree relatives, to minimize risk of VTE for the patient and kindred.

Id: 120 More than a Headache: A Case of Cetuximab-Induced Aseptic Meningitis

D. Prasanna, E. Shum, T. Elrafei. Jacobi Medical Center, Bronx, New York.

Introduction: Cetuximab is a recombinant human/mouse chimeric monoclonal antibody which binds specifically to the epidermal growth factor receptor (EGfR, HER1, c-ErbB-1) and competitively inhibits it. In this report we present a rare case of aseptic meningitis induced by intravenous Cetuximab administration.

Case report: 58 year old man with right tonsillar Squamous Cell Cancer was emergently admitted for complains of headache and fever which started one hour after his first dose of Cetuximab (800 mg), after Benadryl premedication. His remote history included HIV on HAART, hypertension and CKD stage V. On admission, the patient was febrile to 102f, with chills and a persistent headache. The headache was described as frontal and 10/10 in severity. There was no neckstiffness or photophobia, but given his immunocompromised state, there was a high suspicion for infectious meningitis. Computed tomography scan of the head was non-revealing and cerebro-spinal fluid (CSf) studies were promptly sent, showing a neutrophil predominant pleocytosis to 473 WBCs/ cu. mm (80% neutrophils) in CSf tube 1 and 500 WBCs/ cu. mm (62% neutrophils) in CSf tube 4. RBCs were 150 and 50 cells/ cu. mm respectively. CSf protein was elevated to 128 mg/dL and the glucose was normal at 66 mg/dL. Other labs including complete blood count, serum comprehensive metabolic panel and coagulation profile were within normal limits. The patient was empirically treated with Dexamethasone, Vancomycin, Ceftriaxone and Ampicillin, while awaiting CSf cultures. CSf bacterial antigens for Nisseria meningitidis, Streptococcus pneumonia, Haemophilus influenza and Cryptococcus were negative, along with a viral encephalitis panel. Symptoms resolved with supportive care. Antibiotics and dexamethasone were discontinued on Day 2 when cultures turned negative and the patient was discharged home with no additional medications on Day 4. On Day 7, patient received his second dose of Cetuximab at 250mg/m2 per planned his chemotherapy regimen, with no adverse reaction.

Discussion: Aseptic meningitis in the setting of Cetuximab therapy has been reported on 6 occasions previously. In contrast to bacterial meningitis, most cases of aseptic meningitis are self-limited and resolve with supportive care. While the wide belief is that monoclonal antibodies, such as trastuzumab and cetuximab would not be able to penetrate the BBB due to their larger size, there have been reports of trastuzumab levels measured in the CSf after intravenous administration. Monoclonal antibodies have a significant role in treatment of diseases outside Oncology and Multiple Sclerosis (MS) is one such area where extensive research has been performed on the ability of these antibodies to cross the BBB while tagged to T-cells. Further molecular research is needed to understand the mechanism of the leak of Cetuximab into the CSf in the absence of cerebral disease and the resultant inflammatory cascade.

Id: 121 Tretinoin-Induced Thrombocytosis, a Rare Finding with Potential Complications: Two Case Reports

S.A. Srour, M. Cherry. Medicine, Hematology/Oncology Section, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. S.A. Srour, U. Bhutta. Medical Service, Department of Veterans Affairs, Oklahoma City, Oklahoma. U. Bhutta. Internal Medicine, OUHSC, Oklahoma City, Oklahoma.

Introduction: Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia, is characterized by an arrest of leukocyte differentiation at the promyelocyte stage. APL is confirmed by presence of PML-RARA fusion gene or the associated chromosomal translocation t(15;17). All-trans retinoic acid (ATRA; tretinoin) induces differentiation through maturation of the primitive promyelocytes. ATRA, combined with cytotoxic chemotherapy, is the first-line therapy for patients with APL. ATRA has been rarely reported to cause thrombocytosis. We report 2 cases of APL where patients treated with ATRA developed thrombocytosis and deep venous thrombosis (DVT). Case 1 A 31 year-old male patient was referred to our institution for newly diagnosed APL. Initial presentation included generalized fatigue and petechiae/ecchymotic patches. CBC revealed pancytopenia with platelets of 11 K/cmm. Patient received ATRA combined with chemotherapy with excellent response; achieved remission from first cycle. However, platelets progressively increased peaking at 874 K/cmm in 5 weeks after initiation of ATRA. Platelets dropped during cycle#2 chemotherapy, but progressively escalated up to 924 K/cmm in 3 weeks after starting 2nd course of ATRA. Secondary/reactive causes of thrombocytosis were ruled out. Interleukin-6 was elevated at 83.6 pg/mL. Patient did otherwise well except for moderate headaches. Right upper-extremity (UE) DVT was diagnosed around 5 weeks from first dose ATRA. Case 2 A 22 year-old male patient was referred by his PCP for leukocytosis (74.4 K/cmm) and severe thrombocytopenia (12 K/cmm). Clinically, patient was complaining of multiple bruises, nose bleeds, and mild-moderate headaches. Patient was confirmed to have APL. Initial labs revealed DIC, a commonly associated manifestation of APL. Started on ATRA combined with chemotherapy; achieved remission from first round. However, as noted in case 1, he developed as well marked thrombocytosis with platelets peaked at 1200 K/cmm in 4 weeks. Platelets dropped with starting cycle#2 (patient is currently on chemotherapy). Of notice, similar to case 1, patient developed left UE DVT in 3 weeks from start of ATRA.

Discussion: Scattered reports are available in literature about drug-induced thrombocytosis. Only few drugs has been noted to potentially cause thrombocytosis compared to the commonly described thrombocytopenia associated with many different drugs. ATRA was first demonstrated to be effective for APL in China in the mid-1980s. Few reports described thrombocytosis associated with the use of ATRA. The mechanism of thrombocytosis is unknown, but has been associated with elevate interleukin-6. ATRA has been associated with increased incidence of arterial and venous thrombosis. Both of our patients achieved remission, but developed UE DVTs. Whether thrombocytosis is considered an additional risk for DVT in patients receiving ATRA and/or positive marker for enhanced response for ATRA in APL remains unknown. Multivariate analyses from large studies are needed to prove the assumed correlation between ATRA-induced thrombocytosis and DVTs and/or enhanced response.

Id: 122 Pseudo-Pelger-Huët Anomaly and Granulocytic Dysplasia Associated with Human Granulocytic Anaplasmosis

S. Lee, P. Khankhanian, C. Salama, M. Brown, J. Lieber. Medicine, Icahn School of Medicine at Mount Sinai, Elmhurst, New York.

Human granulocytic anaplasmosis (HGA) is a rare tick-borne disease caused by Anaplasma phagocytophilum, a Gram negative cocco-bacilli residing in the endoplasm of granulocytes. Hematological abnormalities associated with HGA including reactive leukopenia, leukocytosis, thrombocytopenia, thrombocytosis, or anemia occur during the early stage of infection and normalize by the end of second week. Pseudo-Pelger-Huët anomaly (PHA) designates mono- or bi-lobed granulocytes, reportedly shown in patients with severe infections and inflammation or hematological malignancies including myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Dysplastic granulocytic changes are also typical manifestations in MDS and acute or chronic granulocytic leukemias. Here, we report a unique case of a male patient who presented with fever, maculopapular rash sparing face, palms and soles, lower extremity edema, headache, arthralgias, and unilateral vision loss for 10 days. He was found to have HGA confirmed by the peripheral smear finding (morulae in neutrophils, figure H), who also showed striking hematological manifestations including a large number of pseudo-PHA (figure A-C), a severe degree of a left shift (figure D and E), and dysplastic granulocytes (figure A-C, f and G). These hematological presentations on the peripheral smear all resolved with doxycycline treatment, implying that the changes are most likely reactive manifestations secondary to HGA, rather than underlying hematological malignancies including MDS or AML. Dysplastic eosinophils included hypodense eosinophils with both basophilic and eosinophilic granules (proeosinophilic granules) reported in myeloid leukemia, as well as cytoplasmic clearing with reduced cytoplasmic areas occupied by cytoplasmic granules as previously reported in hypereosinophilic syndrome (figure f). Karyorrhexis of neutrophils, a nucleic fragmentation in apoptotic cells, is a dysplastic feature when it is revealed on the peripheral smear, as reported in AML or MDS (figure G). In the natural disease course of HGA, reactive leukocytosis, leukopenia, thrombocytosis, or thrombocytopenia resolve by two weeks after the onset of symptoms. Therefore, this patient's clinical presentation at hospital admission is a unique feature of HGA: no reactive leukocytosis, leukopenia, thrombocytosis, or thrombocytopenia with “reactive” pseudo-PHA and dysplastic granulocytes.

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Figure 1

Pseudo-Pelger-Huët anomaly and a toxic left shift of granulocytes with dysplastic features, associated with human granulocytic anaplasmosis. (A) and (B) demonstrate Pseudo-Pelger-Huët anomaly in a bi-lobed neutrophil (A) and a mono-lobed neutrophil with toxic granules and Döhle bodies (B). (C) is a reactive monocyte with an irregular nuclear membrane contour typically shown in Pelger-Huët anomaly. (D) and (E) demonstrate a left shift showing immature granulocytes. (F) shows a dysplastic hypodense eosinophil having cytoplasmic clearing and basophilic granules in the cytoplasm. (G) is an example of karyorrhexis of neutrophils, a dysplastic feature of granulocytes when displayed on peripheral smears. (H) shows a morula, Anaplasma phagocytophilum in the endosome of granulocytes. Distinct cocco-bacilli are shown in the cytoplasm of the neutrophil.

ID: 123 Paraneoplastic Orthostatic Hypotension Associated With Acute Myeloid Leukemia With Trisomy 8

S. Lee, M. Chary. Medicine, Icahn School of Medicine at Mount Sinai, Elmhurst, New York. B. Petersen, J. Mascarenhas. Tisch Cancer Institute, Hematology and Oncology, Icahn School of Medicine at Mount Sinai, New York, New York.

Paraneoplastic neuropathies (PNP) associated with leukemia are rare, and consensus guidelines for the treatment of PNP are lacking. Since those associated with leukemia are not mediated by autoimmune antibodies, therapeutic approach is focused on treatment of the underlying leukemia. Most PNP cause irreversible neurologic damage, and early diagnosis and treatment are crucial to lessen the morbidity associated with PNP. This is the first report to describe severe paraneoplastic orthostatic hypotension which resolved after treatment of acute myeloid leukemia (AML). A 76 year-old woman presented with progressive dizziness and fatigue. She had severe orthostatic hypotension (supine blood pressure 186/82 mmHg; standing 79/53 mmHg). Flow cytometry demonstrated an aberrant myeloid phenotype: CD33+, CD13 dim+, HLA-DR+, CD56+, CD4+, CD38+, CD11b dim+, CD15 dim+, CD7 partial dim+, CD34-, CD117-, CD14-, CD3-, CD5-, CD10-, and CD19-. Bone marrow showed blasts comprising approximately 90% of overall cellularity (see figure); immunoperoxidase stains performed on the biopsy revealed that neoplastic cells were also positive for CD43 and CD68, and negative for CD123; labeling for myeloperoxidase was equivocal. Trisomy 8 was also detected. She was diagnosed with AML, for which azacitidine was initiated. Orthostatic hypotension resolved after initiation of treatment. The diagnosis of PNP in leukemia is usually based on clinical signs and symptoms due to the lack of detectable autoimmune antibodies. The resolution of symptoms after azacitidine is sufficient to diagnose this autonomic instability as a PNP: this patient's neurological disorder is categorized as a “definite” paraneoplastic neurological syndrome, based on the criterion established by Graus et al.: “A non-classical syndrome that resolves or significantly improves after cancer treatment without concomitant immunotherapy, provided that the syndrome is not susceptible to spontaneous remission.” This case demonstrates a unique example of paraneoplastic sequelae remitting with treatment of the underlying hematologic neoplasm. Paraneoplastic autonomic dysfunction has been reported concurrently with other irreversible neurological disorders, which attributes to increased mortality complicating the disease course of a primary neoplasm. This patient presented with severe orthostatic hypotension, which resolved after treatment of the leukemia. This report demonstrates that neurological deficits associated with leukemia require immediate attention, and treatment of the hematologic malignancy can reduce the morbidity associated with PNP. Physicians should be aware of this unusual occurrence of autonomic neuropathy with AML as delay in treatment of the hematologic malignancy can lead to irreversible neurologic deficit and increased morbidity and mortality.

Reference: Graus f, Delattre JY, Antoine JC, et al. Recommended diagnostic criteria for paraneoplastic neurological syndromes. J Neurol Neurosurg Psychiatry 2004;75:1135-1140.

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Peripheral blood and bone marrow aspirate smears. (A) Peripheral blood smear showing a blast cell with a nucleolus and multiple cytoplasmic vacuoles (Wright Giemsa stain, ×1,000). (B) Bone marrow aspirate smear with sheets of blasts similar to those in the peripheral blood (Wright Giemsa stain, ×1,000).

Id: 124 Fulminant Hepatic Failure from Hemophagocytic Lymphohistiocytosis Secondary to Hepatosplenic T-Cell Lymphoma, Mimicking Chinese Herb-Induced Liver Failure

S. Lee, A. Schneier. Medicine, Icahn School of Medicine at Mount Sinai, Elmhurst, New York. B. Petersen. Pathology, Icahn School of Medicine at Mount Sinai, New York, New York. J.L. Gabrilove, J. Lin. Tisch Cancer Institute, Hematology and Oncology, Icahn School of Medicine at Mount Sinai, New York, New York.

A 40 year-old previously healthy women presented for jaundice and fatigue. Two weeks prior, she had rashes on lower extremities, spreading to the entire body, for which she received Chinese herbs. Her rashes resolved, but she developed progressive jaundice and fatigure. At admission, she was found to be febrile with hepatosplenomegaly and severe jaundice on physical exam. Initial laboratory results are as follows: white blood cell 3,800 /μL with neutrophil 78.0% and lymphocytes 12.0%, hemoglobin 8.4 g/dL, platelets 35,000 /mm3, MCV 85.0 fL/cell, transferring saturation 93%, TIBC 189 μg/dL, iron 138 μg/dL, ferritin 4,170 ng/mL, haptoglobin < 7 mg/dL, negative direct Coomb's test, folic acid 12.35 ng/mL, vitamin B12 592 pg/mL, INR 1.4, AST/ALT 123/191 U/L, GGT 57 U/L, total bilirubin/conjugated 20.7/18.7 mg/dL, LDH 1,310 U/L, triglyceride 384 mg/dL, and creatinine 0.4 mg/dL. Liver biopsy showed lymphocyte infiltration and mild hemophagocytosis. A search for common causes of liver failure including infectious etiologies was all negative. Bone marrow biopsy (figure) was performed for severe pancytopenia requiring continuous transfusional support and showed a hypercellular marrow although she was profoundly pancytopenic in peripheral blood count. It showed hemophagocytosis (figure A) with 73% histiocytes positive for CD4, CD68, and CD163 (figure B) and 27% T-lymphocytes positive for CD3 and CD8, localized interstitially and in sinusoids (figure C). Staining and flow cytometry came back positive for CD3, CD5 (dim to moderate), CD7, CD8, CD38, and CD52. The T-cell receptor gene rearrangement study sent when she was severely neutropenic (WBC 100 /μL) came back indeterminate. Based on bone marrow biopsy and flow cytometry result, a diagnosis of hemophagocytic lymphohistiocytosis (HLH) secondary to hepatosplenic T-cell lymphoma (HSTL) was made. Because of elevated total bilirubin (48.7 mg/dL), etoposide was not started; instead methylprednisolone, pentostatin, and alemtuzumab were started. She deteriorated with worsening pancytopenia up to as low as WBC of 0.1 /μL and platelets of 15 /mm3, ferritin 56,856 ng/mL, LDH 2,334 U/L, and total/direct bilirubin 71.3/66.6 mg/dL. On the fifth day after these medications were started, she developed cardiopulmonary arrest and died. This case demonstrates fulminant hepatic failure from HLH and HSTL, mimicking acute hepatotoxicity attributed to Chinese herbal ingestion. She was pancytopenic with very high bilirubin, all of which seemed to be consistent with acute hepatotoxicity from Chinese herbs per the patient's history. However, given constant fever, very high ferritin, and pancytopenia, bone marrow biopsy was performed and confirmed HLH. This case suggests that diagnostic tests, especially bone marrow or liver biopsy, should not be delayed if a patient has unexplained fever, pancytopenia, and evidence of transaminitis or very high MELD scores, even though the patient's history suggests other toxic or infectious etiologies.

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Bone marrow biopsy of this patient with HLH secondary to HSTL. (A) is a bone marrow smear displaying a histocyte with red blood cells in the cytoplasm. Wright-Giemsa stain, ×1,000. (B) show a large proportion of CD163-positive histiocytes. (C) shows CD3-positive T-cells localized in sinusoids of the bone marrow. (B) - (C) ×1,000.

ID: 125 Navigating Care in a Challenging Case of T(6;9) and fLT3 Positive Aml

Y. Song, D. Bixby, D. Roulston, J. Magenau, S.W. Choi. University of Michigan, Ann Arbor, Michigan.

Translocation t(6;9)(p23;q34) is a rare cytogenetic abnormality found in fewer than 5% of pediatric and adult cases of acute myelogenous leukemia (AML). This translocation results in the formation of chimeric fusion gene DEK-NUP214 on the der(6) chromosome, which is translated to nucleoporin fusion proteins and leads to altered nuclear transport and upregulated myeloid protein synthesis. The outcomes of t(6;9) AML are generally poor, with lower five-year overall survival (OS) (£40%) and higher relapse risk (RR) (>50%). Patients with t(6;9) AML demonstrate lower response rates to induction chemotherapy and higher relapse rates despite achieving remission. Additionally, t(6;9) is often seen in AML with the fMS-like tyrosine kinase 3 gene (fLT3) internal tandem duplication (ITD) mutation, one of the most common abnormalities in AML associated with increased risk of treatment failure and mortality, detected in 15 to 25% of patients. FLT3-ITD with a high ITD allelic ratio has been associated with five-year OS rates <35% and RR >60%. We report a challenging case of t(6;9) and fLT3-positive AML in a young adult male. The patient was a 23-year-old Caucasian male who initially presented to his primary care physician with fever, night sweats, and bilateral lower extremity rash. Laboratory testing revealed white blood cells 8.5 x103/μL, hemoglobin 10.4g/dL, and platelets 41.0 x103/μL, with a manual differential count showing 66% blasts, 10% neutrophils, 1% myelocytes, 1% bands, and 18% lymphocytes. Bone marrow biopsy showed 74% blasts with Auer rods. Flow cytometry detected an immunophenotype of CD13, CD33, CD38, CD117, and HLA-DR. These cells also expressed low levels of CD4 and CD45; partially expressed CD7, CD25, and CD64; and variably expressed CD34. Cytogenetic and molecular testing confirmed a diagnosis of AML with karyotype 46, XY, t(6;9)(p23;q34), DEK/NUP214 and the presence of the fLT3-ITD mutation, respectively. The patient responded poorly to multiple standard induction and intensification regimens, demonstrating persistent disease. Morphologic remission was eventually achieved with a fLT3 inhibitor (sorafenib) and a hypomethylating agent (azacytidine), a novel investigative combination therapy with a reported response rate of >40%. The patient proceeded with a 9 of 10 HLA matched (HLA-B mismatched) unrelated donor peripheral blood hematopoietic cell transplantation (HCT) (10 months from initial diagnosis). His post-HCT course was complicated by grade I acute graft-versus-host disease of the skin (palms and soles, non-biopsied) requiring high-dose steroids. Bone marrow testing on days 30 and 100 showed no morphologic, flow cytometric, cytogenetic, or molecular evidence of AML. However, despite re-initiation of sorafenib in the post-HCT setting, he experienced early relapse on day 139 with the original [fLT3-ITD and t(6;9)] and new (fLT3-D835 and +8) molecular and cytogenetic markers, respectively, perhaps reflecting selection of these sub-clones within the preexisting disease or clonal evolution. This case highlights the need for improved strategies in the post-HCT setting for high-risk AML. While the incorporation of non-cytotoxic targeted post-HCT maintenance agents is currently being explored, the type of drug, disease-specific characteristics, timing of initiation, dosing/schedule, and hold/re-start parameters for myelosuppression need to be better defined and studied in appropriate clinical trials to ensure uniform practices across institutions.

References: Tarlock et al. BJH 2014. 166:254-9. Slovak et al. Leukemia 2006. 20:1295-7. Kottaridis et al. Blood 2001. 98:1752-9. Ravandi et al. Blood 2013. 121:4655-62.

Id: 126 Endothelial Cell Cd36 Transcription is Downregulated by Lpa/Pkd-1-Hdac7-Foxo1 Signaling Axis and is Associated with Proangiogenic Reprogramming

B. Ren, R. Silverstein. Blood Research Institute/Medicine, BloodCenter of Wisconsin/Medical College of Wisconsin, Milwaukee, Wisconsin. B. Best, D. Ramakrishnan. Blood Research Institute, Blood Center of Wisconsin, Milwaukee, Wisconsin. B. Walcott. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts. P. Storz. Cancer Biology, Mayo Clinic, Jacksonville, florida.

CD36 is a scavenger receptor that plays an important role in athero-thrombotic diseases, diabetes and cancer. Lysophosphatidic acid (LPA), a bioactive phospholipid signaling mediator, abolishes endothelial cell responses to antiangiogenic proteins containing thrombospondin type 1 homology domains by down-regulating CD36 transcription. The mechanism as to how angiogenic signaling is integrated to regulate endothelial specific CD36 transcription remain unknown. We here describe that in microvascular endothelial cells (MVECs) LPA represses CD36 transcription by activating a PKD-1 signaling pathway that induces the formation of a HDAC7/NCoR1-foxO1 complex in the nucleus. Intriguingly, we show that turning off CD36 transcription is associated with reprogramming MVECs to express ephrin B2, a critical “molecular signature” involved in angiogenesis and arteriogenesis, and phosphorylation of Erk2, an important MAP kinase in arterial differentiation. We also demonstrate that activation of this signaling pathway in the vasculature of mice with transplanted Lewis lung carcinomas is associated with low CD36 expression or with CD36 deficiency, as shown by tumor vessel staining for phospho-PKD-1, arterial markers ephrinB2 and smooth muscle cell actin in the tumor vasculature of both wild type and CD36 deficient mice. Functionally, turning off CD36 transcription promotes angiogenesis in a PKD-1-dependent manner as an in vivo Matrigel assay showed that pharmacologic inhibition of PKD signaling significantly reduced the inhibitory effect of LPA on TSR/CD36-mediated anti-angiogenic effects in response to VEGf in vivo (p<0.01). These studies suggest that a LPA/PKD1-HDAC7-foxO1 signaling axis regulates endothelial CD36 transcription and mediates silencing of the antiangiogenic switch, resulting in proangiogenic reprogramming and arteriogenic responses, and a HDAC7-mediated epigenetic mechanism may be involved in angiogenesis regulated by LPA. Targeting this signaling cascade could be a novel approach for cancer, cardiovascular complications of diabetes and athero-thrombotic diseases as well as obesity.

Id: 127 Antibiogram of Vre Causing Urinary Tract Infections as a Restrospective Study from Mmc, Springfield Illinois

M. das, V. Sundareshan. Internal Medicine, Southern Illinois University, Springfield, Illinois.

The prevalence of Vancomycin-resistant Enterococci (VRE) has steadily been increasing in the hospital setting for nosocomial infections. Data from the Centers for Disease Control and Prevention reported that during 2006 and 2007, 30% of all enterococci infections within hospitals in the United States are VRE. Of the total percentage of VRE, approximately 80% are species E. Faecium. These organisms can be extremely difficult to treat due to their high resistance levels to various antimicrobial agents. E. Faecium in particular usually have higher levels of resistance to beta lactams and aminoglycosides compared to E. Faecalis. The objective of our study was to determine the prevalence of VRE at our institution and determine the best therapy for treatment based on the susceptibilities to a panel of antimicrobials. We performed a retrospective analysis of any enterococci isolate found in the urine from August 2012 to August 2014 using the VITEK system. The isolates were then broken down into those susceptible and those resistant to a select group of antimicrobials, thus generating an antibiogram for each isolate. The antimicrobials which were tested included: gentamycin, streptomycin, nitrofurantoin, daptomycin, linezolid, ciprofloxacin, levofloxacin, erythromycin, tigecycline, tetracycyline, and doxycycline. The results showed a total of 180 enterococci isolates collected from in urine cultures. Of these 180, nearly 60% were E. Faecium and 40% were E. Faecalis. Of the E. Faecium group, only 94% were resistant to ampicillin and 100% produced beta lactamase. From the E. Faecalis group 100% were susceptible to ampicillin and 100% were beta lactamase producers. As noted in from our results, E. Faecium was the predominant organism isolated which was also found to be more resistant to ampicillin compared to E. Faecalis. The higher resistance pattern is thought to be due in part to the low affinity of Penicillin Binding Protein and beta lactamase production. Ampicillin resistant strains, which produce beta lactamase, can be treated with ampicillin with sulbactam, and some case reports have noted treatment with high doses of ampicillin. Some of these resistant strains are treated with daptomycin. However, there are many downsides to the use of it such as, it is not fDA approved for E. Faecium infections, and its inability to concentrate in the genitourinary system. Similarly, the side effects and drug interactions limit the use of linezolid in treatment of VRE, especially with prolonged use. In summary, the frequent occurrence of VRE bacteruria, specifically with E. Faecium, and the increasing antimicrobial resistance, makes extremely challenging to treat. There are many newer antimicrobials available which could potentially treat these infections, however VRE associated UTI treatment guidelines would help in clinician awareness as well as aiding in treatment decisions.

Id: 128 Kaposi—s Sarcoma-Associated Herpesvirus (Kshv) Latently Infected Cells Derived Nanovesicles Promote Endothelial Mesenchymal Transitions (Endmt) and Vascular Permeability

N. Sharma-Walia, K. Patel. RfUMS, North Chicago, Illinois.

Nanovesicles (NVs) are nano-sized extracellular vesicles that are released from a wide range of mammalian cell types such as fibroblast, epithelial, and endothelial cells. NVs are abundant in proteins, mRNAs, DNA fragments and microRNAs, which help NVs act as intercellular communicasomes. NVs production, transport and their significance in tumor microenvironment has been demonstrated in several cancer models but their prevalence and role(s) in Kaposi's sarcoma herpes virus (KSHV) associated human malignancies have not yet been studied extensively. KSHV is associated with a neoplastic angioproliferative endothelial malignancy called Kaposi sarcoma (KS) and two other AIDS-related lymphoid cell malignancies called primary effusion lymphoma and multicentric Castleman's disease. KS progression and pathogenesis has been linked to a number of critical events such as endothelial mesenchymal transition (EndMT), cell migration and invasion. The role(s) of NVs derived from KSHVinfected cell conditioned medium was investigated in this study. NVs from KSHV latently infected primary effusion lymphoma (PEL) cells (KSHV+/EBV-; BCBL-1, BC-3), non-infected (KSHV-/EBV-; BJAB) human Burkitt's lymphoma (BL) cells, KSHV latently infected endothelial cells (TIVE-LTC), and their control counterpart TIVE cells were isolated and purified. Our study revealed that KSHV+/EBV- PEL and KSHV latently infected endothelial cells secrete NVs. Fluorescently labeled NVs derived from either KSHV+ or KSHV- cell lines were efficiently/rapidly internalized by primary human microvascular dermal endothelial cells (HMVEC-d). KSHV+ cell line derived NVs were enriched in inflammatory pathway proteins such as cyclooxygenase-2 (COX-2), 5 lipoxygenase (5LO), and leukotriene A4 hydrolase (LTA4H). We hypothesized that infected cell derived NVs are taken up by surrounding uninfected endothelial cells and induce oncogenesis. The effect of infected cell derived NVs on phenotypic changes and endothelial cell biology controlling factors such as angiogenesis, vasoconstriction, vasodilation, inflammatory response, apoptosis, cell adhesion, and EndMT was studied. Uptake of KSHV+ cells derived NVs by recipient uninfected endothelial cells reduced the expression of proteins such as VE-cadherin, Tie-1/2, VEGfR-1/2, PECAM/CD31, upregulated smooth muscle actin, type I and III collagens, N-cadherin, and vimentin, and also upregulated the expression of EndMT regulating transcriptions factors, including Snail, Slug, Twist, ZEB1 and ZEB2. Furthermore, this uptake of NVs by endothelial cells induced Rac1-GTPase, and enhanced the migration and invasive potential of uninfected endothelial cells. KSHV+ cell line derived NVs, when compared to non-infected BJAB derived NVs induced vascular permeability leading to the disassembly of cell junctions. Together, this study for the first time reports that NVs secreted from KSHV+/EBV- cells have a unique inflammatory signature and these NVs induce EndMT in primary endothelial cells. The crucial role of NVs in KSHV pathogenesis via manipulation of extracellular vesicles in the infected cell microenvironment is highlighted in this study. Thus, KSHV infection induced NVs represent a novel strategy employed by KSHV to promote inflammation, migration, invasion, and vascular permeability.

Id: 129 Dissection of Usher-Chaperone-Subunit Interactions during Pilus Biogenesis in Escherichia Coli

G.T. Werneburg, D.G. Thanassi. Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, New York. G.T. Werneburg. Medical Scientist Training Program, Stony Brook University, Stony Brook, New York.

The chaperone/usher (CU) pathway is a conserved secretion system employed by many species of Gram-negative bacteria to construct virulence-associated surface structures known as pili or fimbriae. Our model system, type 1 pili, facilitates colonization of the bladder by uropathogenic Escherichia coli, and subsequent urinary tract infection. The CU pilus biogenesis pathway requires both a periplasmic chaperone protein and an integral outer membrane protein, the usher. The chaperone facilitates proper folding of pilus subunits in the periplasm and prevents premature interactions. The usher catalyzes the exchange of chaperone-subunit for subunit-subunit interactions, and promotes ordered polymerization and secretion of the pilus fiber. The usher, a putative multimer, has 5 domains: a periplasmic N-terminal domain (N), a transmembrane beta-barrel domain that is gated by an internal plug domain, and two periplasmic C-terminal domains (C1 and C2). Chaperone-subunit complexes first bind to the N domain of the usher and then move to the C domains as the plug is expelled and the pilus subunits are inserted into the barrel channel. To understand the mechanism of the usher (fimD) in catalyzing the assembly and secretion of pili and to identify mechanistic targets for pharmacological inhibition, we monitored interactions of chaperone-subunit (fimCfimH) complexes with usher domains during pilus assembly using fluorescence-based approaches. We calculated an overall affinity for fimC-fimH binding to the full length fimD usher that is in agreement with previous surface plasmon resonance measurements. We then combined our approach together with domain deletion mutants of fimD to reveal the contributions of individual usher domains to chaperone-subunit binding and gain insight into the sequential order of chaperone-subunit-usher interactions. Our data suggest that the C domains are fimD's high-affinity binding site for fimC-fimH complex. However, in the absence of the usher's N domain, no binding was detected. These findings support a model wherein the high affinity C domains are only accessible once the chaperone-subunit complex binds the usher's N domain, and the N to C domain handoff is then driven by differential affinity. We show that in the context of the full-length usher, the plug domain does not contribute directly to fimD's affinity for chaperone-subunit complexes, but likely “masks” fimD's C domains prior to usher activation. Further, in vivo we show that fimD is functional in trans, suggesting that the non-translocating fimD protomers of the multimer do not remain idle during pilus biogenesis, but rather actively recruit and contribute to incorporation of chaperone-subunit complexes. Based on these findings, we are currently developing and implementing additional fluorescence and biochemical assays to further our understanding of the mechanism of the usher in the assembly and secretion of pili, and to identify targets and test inhibitors of our newly elucidated mechanistic steps of pilus assembly.

Id: 130 Lipoxins: Implications in Kaposi—s Sarcoma Associated Herpesvirus (Kshv) Biology

J.A. Chandrasekharan, N. Sharma-Walia. RfUMS, North Chicago, Illinois.

Kaposi's sarcoma-associated herpesvirus (KSHV), a γ-2 herpesvirus, is etiologically associated with angioproliferative endothelial Kaposi's sarcoma (KS), primary effusion body cavity B-cell lymphoma (PEL or BCBL) and B cell-lymphoproliferative multicentric Castleman's disease (MCD). Our exciting paradigm shifting previous studies established that KSHV utilizes host pro-inflammatory pathways such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and 5-lipoxygenase (5LO) to promote its latent gene expression and consequent pathogenesis. COX-2 and 5-LO inhibitors are associated with gastrointestinal side effects and adverse cardiovascular effects at high doses; additionally, these inhibitors do not completely eradicate the viral load. Therefore, there is a need for targeted, endogenous, and safe therapeutics with high antiviral potential. Lipoxins are host cell endogenous anti-inflammatory, pro-resolving and short-lived bioactive lipids. Here, we discovered that KSHV infection of human dermal microvascular endothelial cells (HMVEC-d) significantly reduces the secretion of anti-inflammatory lipoxin (LXA4). We demonstrate that KSHV infected HMVEC-d cells and human KS lesion cells express lipoxin receptor ALXR, and more importantly incubation of KSHV infected cells with stable small molecule analogs of lipoxins and aspirin triggered lipoxins significantly downregulated KSHV's latent promoter activity and latent gene expression. Thus, the anti-inflammatory lipoxin pathways offer possibilities for regulating KSHV biology. Further studies on therapeutic potential of lipoxin in KS would offer novel and effective treatment method.

Id: 131 Leukotriene A4 Hydrolase Inhibitors: Potential Chemotherapeutic Modality for Primary Effusion Lymphoma

P. Sowa, N. Sharma-Walia. RfUMS, North Chicago, Illinois.

Kaposi's sarcoma-associated herpesvirus (KSHV), a γ-2 herpesvirus, is etiologically associated with angioproliferative endothelial Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and B cell-lymphoproliferative multicentric Castleman's disease (MCD). The significance of leukotriene A4 hydrolase (LTA4H) in KSHV biology prompted us to examine the role of LTA4H inhibitors in PEL, an aggressive AIDS-linked KSHV-associated non- Hodgkin's lymphoma using Bestatin, well-known LTA4H inhibitor. We demonstrate that (1) Bestatin is efficacious in specifically inducing proliferation arrest in PEL cells (KSHV+ BCBL-1 and BC-3) when compared to non-infected (KSHV-; BJAB) Burkitt's lymphoma cells; (2) Bestatin treatment reduced the number of KSHV + PEL cells in S phase; (3) Bestatin treatment induced ER stress proteins such as calnexin, BiP, CHOP. Overall, our study provides a comprehensive molecular framework that links LTA4H with ER stress induced PEL cell death and identify the chemotherapeutic potential of Bestatin in treating PEL.

Id: 132 Accuracy of Loop-Mediated Isothermal Amplification for the Diagnosis of Clostridium Difficile Infection: A Systematic Review

P. Thota, C.J. Donskey. Infectious Diseases, Case Western Reserve University, Cleveland, Ohio. A. Lloyd, V. Pasupuleti. Medicine, Case Western Reserve University, Cleveland, Ohio. C. Pant. University of Kansas Medical Center, Kansas City, Kansas. D.D. Rolston. Geisinger Medical Center, Danville, Pennsylvania. A.V. Hernandez, T.G. Fraser, A. Deshpande. Cleveland Clinic, Cleveland, Ohio. V. Benites-Zapata. Universidad Peruana de Ciencias Aplicadas (UPC), Lima, PERU.

Objective: Nucleic acid amplification tests including real-time polymerase chain reaction and loopmediated isothermal DNA amplification (LAMP) are currently used as standalone diagnostic tests of C. difficile infection (CDI) in the United States. These assays are reported to have similar sensitivity and specificity to toxigenic culture. We assessed the diagnostic accuracy and clinical value of LAMP for the diagnosis of CDI.

Methods: We searched PubMed and 4 other databases to identify diagnostic accuracy studies that compared LAMP with culture cytotoxicity neutralization assay (CCNA) or anaerobic toxigenic culture (TC) of C. difficile from database inception to 2014. We used the random-effects model to calculate pooled sensitivities, specificities, likelihood ratios, diagnostic odds ratios and their 95% CIs. Hierarchical summary receiver operating characteristic curves were constructed.

Results: A search of the databases yielded 16 studies (6,979 samples) that met the inclusion criteria. When TC was used as the gold standard (6,572 samples), bivariate analysis yielded a mean sensitivity of 0.95 (95% CI, 0.93-0.97; I2 = 67.4) and a mean specificity of 0.99 (95% CI, 0.96-1.00; I2 = 97.0). With CCNA as a gold standard (407 samples), the mean sensitivity was 0.93 (95% CI, 0.85-0.97; I2 = 68.6) and mean specificity, 0.91 (95% CI, 0.87-0.94; I2 = 90.7). The studies had substantial heterogeneity. None of the subgroups investigated could account for the heterogeneity.

Conclusions: LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting CDI. The results should however be interpreted only in the presence of clinical suspicion and symptomatic diarrhoea.

Id: 133 Evolution of Peripherial Blood Smear and Lymph Node Histology in Miliary Tuberculosis

S. Lee, D. Chang. Medicine, Icahn School of Medicine at Mount Sinai, Elmhurst, New York. M. Chary. Icahn School of Medicine at Mount Sinai, New York, New York, UNITED STATES. F. Salem. Pathology, Icahn School of Medicine at Mount Sinai, New York, New York. J. Lin. Tisch Cancer Institute, Hematology and Oncology, Icahn School of Medicine at Mount Sinai, New York, New York.

A 40 year-old previously healthy man presented with an enlarging left neck mass. He denied difficulty breathing, fever, chills, night sweats, or weight loss. On admission, he was febrile and tachycardic. Physical exam was notable for a firm globular mass on his left neck and hepatomegaly with no oropharyngeal exudate or uvular deviation. Computerized tomography of the neck and chest demonstrated diffuse lymphadenopathy of the left neck and multiple lung nodules all less than 1 cm. Initial laboratory studies were remarkable for leukocytosis with white blood cell (WBC) 22,200 /μL, alkaline phosphatase 217 U/L, and gamma glutamyl transpeptidase 521 U/L. An initial peripheral blood smear (figure A) revealed many pseudo-Pelger-Huët neutrophils with toxic granules and vacuoles, previously reported in acute myeloid leukemia (AML) or severe infections. Hypersegmented neutrophils were noted, an extremely rare finding in a patient with no evidence of underlying B12 or folate deficiency. Nuclear projections in neutrophils were noted, previously reported in patients with androgen-producing tumors. Eosinophils had cytoplasmic clearing with reduced cytoplasmic areas occupied by cytoplasmic granules, as reported in hypereosinophilic syndrome. Both basophilic and eosinophilic granules were observed in eosinophils, previously reported in myeloid leukemia. Basophils revealed a reduced number of granules with vacuoles, signifying partial degranulation as reported in AML. Two days later, a fine needle aspiration and core biopsy of the left neck mass demonstrated well-formed granulomas with negative fungal and acid-fast bacilli (AfB) staining (figure C). Empirical itraconazole, vancomycin and piperacillintazobactam were initiated. Blood and urine cultures remained without growth. Ten days after admission, a peripheral smear (figure B) showed more pseudo-Pelger-Huët anomaly and thrombocytosis (1,200,000 /μL). Twenty five days after admission, he developed pancytopenia (WBC 3,200 /μL and platelets 80,000 /μL). Subsequently, he developed blisters and ulcers on the tongue, and its biopsy showed ulcerated squamous mucosa with acute inflammatory exudate and reactive histiocytosis (figure D). Suspicion for military tuberculosis (MTB) grew given his clinical manifestations, for which rifampin, isoniazid, moxifloxacin and ethambutol were started. He became afebrile. A peripheral smear at discharge demonstrated no Pelger-Huët anomaly or dysplastic granulocytes. The culture of neck lymph node biopsy came back with Mycobacterium tuberculosis (figure E). This case report shows a unique presentation of MTB and associated hematological manifestations including dysplastic granulocytes and alternating leukocytosis, leukopenia, thrombocytopenia, and extreme thrombocytosis. These hematological presentations seem to be reactive responses to MTB. This report suggests that severe infections can trigger striking hematological manifestations previously reported in myeloid leukemias.

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Figure 1

(A) Peripheral blood smear at admission; (B), day 10 after admission. Insets in (A) and (B) showfurther examples of neutrophils, eosinophil, basophil on the indicated day. (C) Fine-needle aspiration of a left neck lymph node (Diff-Quik stain) showing a well-formed granuloma consisting of epithelioid cells characterized with ovoid to elongated nuclei. (D) Tongue biopsy showing a granuloma (H&E stain). The inset in (D) shows CD68+ histiocytes in the granuloma. (E) Lymph node culture growing Mycobacterium tuberculosis six weeks after the lymph node biopsy was performed (Ziehl-Niessen stain). Scale bar in (A) and (B) = 100 μm; (C) = 20 μm.

Id: 134 Cyp2C19*17 Genetic Polymorphism and Therapeutic Challenges in An Allogeneic Stem Cell Transplant Recepient with Invasive Pulmonary Aspergillosis

M. Abidi, A. D‘Souza, P. Hari. of Medicine, Medical College of Wisconsin, Brookfield, Wisconsin. K. Kuppalli. Department of Medicine, Loyola University, Chicago, Illinois.

A 48-year-old Caucasian male was seen in consultation for pulmonary Invasive Aspergillosis. He had undergone allogeneic peripheral blood hematopoietic stem cell transplant for myelodysplastic syndrome 6 months. Post-transplant course was complicated by extensive graft versus host disease. He was treated with prednisone, tacrolimus and tocilizumab and photophoresis. Upon presentation, he was intubated secondary to multifocal pneumonia and respiratory failure. A chest computerized tomography (CT) scan showed bilateral upper lobe lung opacities. Empiric antimicrobial coverage included intravenous cefepime, azithromycin and voriconazole 300mg Q12h. Bronchoalveolar lavage (BAL) fluid cultures grew 10,000 cfu/ml Pseudomonas aeruginosa and 1,000 cfu/ml Aspergillus fumigatus. Nucleic acid amplification testing was positive for Mycoplasma pneumoniae. He was switched to doxycycline and he completed a 14-day course of doxycycline and cefepime. He improved clinically, was extubated and switched to oral voriconazole 300 mg q12h a week. However, his respiratory status worsened a week after this transition. Chest CT showed worsening opacities bilaterally in the upper and lower lobes. Voriconazole serum level was undetectable, as was a repeat voriconazole level a week later. We suspected that he might be an ultra metabolizer of voriconazole and a cytochrome assay was requested from an outside laboratory (LabCorp, Burlington NC). The methodology employed was DNA analysis of the cytochrome P450 2C19 gene including the alleles for poor metabolizers *1, *2, *3 *4, *5, *6, *7 and *8 as well as the ultra-metabolizer alleles *17. This was performed on the Tm Bioscience /Luminex Universal Array Platform using primer extension chemistry. Multiplex PCR amplifies DNA fragments containing mutations associated with the alleles mentioned above. Primer extension then generates a biotinlabeled product that hybridizes to complementary, bead-immbolized probes to permit flow-sorted detection of both normal and mutation sequences. We increased the dose of intravenous voriconazole to 400 mg every 12 hours and added intravenous micafungin. Repeat imaging one week after the addition of micafungin showed improvement in the bilateral ground glass opacities. CYP2C19 genotype testing was positive for the *17 translocation indicating he was an ultra metabolizer for voriconazole. Voriconazole was discontinued and he was discharged home on intravenous micafungin 150 mg daily. A month later, chest CT demonstrated slow improvement. He was continued on micafungin with plans to switch him to liposomal amphotericin B if there was any worsening. He was readmitted with recurrent pulmonary infections with evidence of aspergillus infection by BAL culture. He was treated with antibiotics and continuation of intravenous micafungin, and each time he improved clinically. He had multiple bronchoscopies with BAL including lung biopsies in order to differentiate between a new bacterial superinfection, pulmonary aspergillus, drug toxicity and pulmonary GVHD. We considered oral posaconazole as his gut GVHD improved but given cost he remained on intravenous micafungin. Nine months after his original presentation, he developed respiratory failure with new areas of lung consolidation and evidence of acute eosinophilic reaction. Aspergillus fumigatus was detected on BAL cultures and he was switched fto liposomal amphotericin B and posaconazole. sirolimus was discontinued given concern that lung pathology could be due to sirolimus toxicity. While work up was underway the patient suffered a cardiac arrest and died.

Id: 52 Discovery of Mesencephalic Astrocyte-Derived Neurotrophic Factor as a Urine Biomarker for Endoplasmic Reticulum Stress-Related Kidney Diseases

Y. Kim, H. Lee, J. Miner, Y.M. Chen. Renal Division, Department of Internal Medicine, Washington University in St. Louis, St. Louis, Missouri. F. Urano. Division of Endocrinology, Metabolism and Lipid Research, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri.

Accumulating evidence has highlighted the important role of endoplasmic reticulum (ER) stress and disrupted proteostasis in the pathogenesis of a variety of glomerular and tubular diseases. Thus, it is imperative to develop non-invasive biomarkers for detecting ER stress in podocytes or tubular cells in the incipient stage of disease, when clinically a kidney biopsy is not yet indicated. Moreover, restoration of ER homeostasis before irreversible kidney cell injury may hold significant promise as an attractive therapeutic strategy. However, there are no known biomarkers for detecting ER stress in associated kidney disease to date. Here, using mouse models of human nephrotic syndrome caused by mutant laminin b2 protein-induced podocyte ER stress and of acute kidney injury triggered by the ER stress inducer tunicamycin in renal tubular cells, we identified mesencephalic astrocyte-derived neurotrophic factor (MANf) as a potential urine biomarker for detecting ER stress in podocytes or renal tubular cells respectively. We found that MANf was upregulated by ER stress in the podocytes and tubular cells. Most importantly, urinary MANf excretion concurrent with podocyte or tubular cell ER stress preceded clinical or histological manifestations of the corresponding disease. Thus, MANf can potentially serve as a urine diagnostic or prognostic biomarker to help stratify disease risk, predict disease progression, and monitor treatment response in the ER stress-related kidney diseases.

ID: 135 Focal Segmental Glomerulosclerosis And Papillary Renal Cell Carcinoma: Causal Link?

M. Haroon Al Rasheed, S. Setty. Department of Pathology, University of Illinois Hospital and Health Sciences System, Chicago, Illinois. A. Dudek. Department of Medicine, University of Illinois Hospital and Health Sciences System, Chicago, Illinois. M. Abern. Department of Urology, University of Illinois Hospital and Health Sciences System, Chicago, Illinois.

Background: focal Segmental Glomerulosclerosis (fSGS) is associated with a significantly increased risk of Renal Cell Carcinoma. However, there are only a handful of reported cases in the literature that show this association. We describe perhaps the first known case of papillary renal cell carcinoma with fSGS and discuss the significance of this association.

Method: We report the case of a 26 year old African American male with diabetes mellitus, hypertension and chronic kidney disease who presented with weight loss and hematuria. Imaging revealed a renal mass for which he underwent a hemi-nephrectomy. Histopathology showed papillary renal cell carcinoma along with fSGS in the non-neoplastic renal tissue. Electron microscopy revealed mainly intact foot processes, expanded mesangial areas and variable tubular and glomerular basement membrane thickening.

Conclusion: There are multiple highpoints discussed within this report. Firstly, we highlight the need to identify the possible association between fSGS and papillary renal cell carcinoma, perhaps as a hereditary link between the two entities, papillary renal cell carcinoma as a cause of secondary fSGS, or fSGS as a paraneoplastic syndrome of papillary renal cell carcinoma. Secondly, this case calls for the need to detect the prevalence rate of fSGS in patients with renal malignancies. Thirdly, patients with known fSGS should be closely investigated for accompanying malignancies and vice versa with a lower threshold for the clinical suspicion of fSGS. And finally, routine assessment of the non-neoplastic renal tissue within a nephrectomy specimen could avoid a potentially risky separate renal biopsy of the remaining solitary kidney in a patient with chronic kidney disease. Original magnification 100x view of papillary renal cell carcinoma forming papillae lined by malignant epithelial cells with hemorrhage and necrosis.

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Figure 1

Original magnification 100x view of papillary renal cell carcinoma forming papillae lined by malignant epithelial cells with hemorrhage and necrosis.

ID: 136 Medullary Tophi in Chronic Kidney Disease: A Causal Link between URIC ACID and Progressive Renal Failure?

I. Ayoub, L. Hebert, B. Rovin. Medicine/Nephrology, The Ohio State University Wexner Medical Center, Columbus, Ohio. S. Brodsky, T. Nadasdy. Pathology, The Ohio State University Wexner Medical Center, Columbus, Ohio.

Background: It is well established, but not widely appreciated, that gouty tophi can form in the kidney, particularly in the renal medulla and papillae. More recently, hyperuricemia has been identified as a possible risk factor for progression of chronic kidney disease (CKD). Because a single collecting duct serves more than 2000 nephrons, we postulated that obstruction and damage to collecting ducts by medullary tophi may explain, in part, the emerging association between uric acid and progressive CKD. To address this question we examined our pathology database to understand the association of medullary tophi and CKD.

Method: We conducted a retrospective case series analysis. A 10-year cross-section of our nephropathology database was queried for native kidney biopsies that had medullary tophi. The presence or absence of CKD and uric acid levels were determined by chart review.

Results: Medullary tissue was captured in 2878 biopsies out of 7850 biopsies. Medullary tophi were seen in 36 of these biopsies, suggesting a minimum prevalence of tophi to be 1.25%. Uric acid levels were available in 16 patients within 30 days of the biopsy, and of these, uric acid was abnormally elevated in 11 patients (table1). Most of these patients had asymptomatic hyperuricemia. Of 36 patients with medullary tophi, 35 had CKD.

Conclusion: The present work shows medullary tophi occur not-infrequently in kidneys of CKD patients. They can occur regardless of the serum uric acid level or whether the patient has symptomatic hyperuricemia. Given their strategic location and bulk, it is possible that medullary tophi contribute to CKD progression by causing upstream nephron damage. These findings suggest the practice of limiting urate-lowering therapy in CKD only to those patients with symptomatic hyperuricemia may need to be re-examined.

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Table 1

Clinical characteristics of patients with medullary/papillae tophi

Id: 137 Acute Renal Infarct Case Series

T.M. Yousuf, H. Iqbal, A. Said, A. Chadaga, J. Oyama, E. Lerma. Internal Medicine, Advocate Christ Medical Center, Oak Brook, Illinois.

Acute Renal Infarction is a commonly under-diagnosed condition. Acute renal infarction usually presents with the constellation of signs and symptoms of abdominal or flank pain, elevation of LDH, elevation of white blood count, hematuria, oliguria or anuria and fever. Differential diagnosis includes acute appendicitis, nephrolithiasis, cholecystitis, urinary tract infection and biliary pathology. This report will hopefully highlight the rarity of acute renal infarction and the importance of considering the diagnosis of acute renal infarction in patients presenting with acute kidney injury. The myriad of findings of 1) acute abdominal pain or flank pain, 2) elevation of LDH and 3) a high risk of thromboembolism in a patient presenting with acute kidney injury should make the astute clinician consider the possibility of acute renal infarction in the differential diagnosis. Case 1 is a 55 year old male with a past history of coronary artery disease, AAA and atrial fibrillation who presented to the emergency room with a 2 day history of intractable abdominal discomfort localized to the left flank and right lower quadrant (RLQ) with radiation to the right femoral area. On physical exam, the patient was in moderate distress and had significant tenderness over the RLQ. In addition, he also had notable left sided costovertebral angle tenderness. Pertinent laboratory studies included: WBC of 8.0, creatinine of 1.41mgs/dL and INR of 1.2. He was known to be non-compliant with warfarin anticoagulation. CT of abdomen revealed areas of decreased perfusion involving the lower pole of the right kidney with perinephric stranding, new occlusion of the left internal iliac artery and aneurysms involving the right common iliac artery as well as both common femoral arteries. Duplex ultrasonography revealed the presence of aneurysms in both common femoral arteries and increased velocity in the right proximal third of the renal artery. Patient was afforded supportive measures and an adequate analgesic regimen and subsequently showed significant clinical improvement as well as resolution of laboratory abnormalities. Case 2 is that of a 53 year old male who has a past history of chronic kidney disease and atrial fibrillation who presented to the emergency room with an acute onset of abdominal discomfort localized to the left flank with radiation to the back. Physical exam revealed stable vitals and an irregular pulse. On examination, he had left CVA tenderness. Initial labs revealed creatinine of 3.5mgs/dL, INR of 1.7 and significantly elevated LDH (1323). CT scan of the abdomen demonstrated the absence of the right kidney. A MAG3 nuclear medicine scan revealed diminished flow to the left kidney. A CT angiogram revealed evidence of thrombus in the left renal artery. He was then started on anticoagulation with heparin and a wire guided thrombolysis with thrombectomy was performed. Although there was brisk urine output production post-procedure, there was no evidence of renal recovery in the ensuing days; therefore, he was subsequently initiated on hemodialysis. Acute renal infarction is a commonly missed phenomenon and it should be high on the list of differential diagnosis if a patient presents with abdominal pain, elevated LDH and predispositions to increased risk of thromboembolism. The mainstay of treatment remains to be appropriate anticoagulation. Our cases demonstrate acute renal infarction likely secondary to atrial fibrillation with and without therapeutic anticoagulation. Atrial fibrillation, as the most common arrhythmia identified in hospitalized patients, warrants increased awareness of its association with acute renal infarction so that it can be promptly recognized and managed appropriately.

Id: 53 Preeclampsia Has Detrimental Impact on Health of Offspring

S. Munir, N. Drever, S.R. Allen, T. Kuehl, M.N. Uddin. Obstetrics and Gynecology, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas, M.A. Co, M.R. Beeram, T. Kuehl, M.N. Uddin. Pediatrics, Baylor Scott & White Health/Texas A&M Health Science Center College of Medicine, Temple, Texas. S.H. Afroze. Medical Physiology, Texas A&M Health Science Center College of Medicine, Temple, Texas.

Objective: Preeclampsia (preE), a syndrome of hypertension and proteinuria in pregnancy, is thought to be initiated with alterations of placental function. Hypoxia and oxidative stress can lead to placental apoptosis. preE is the most frequent complication of pregnancy. It is associated with endothelial dysfunction in the mother, which is related to the release of circulating vasculotoxic factors and the induction of augmented oxidative stress by the diseased placenta. These circulating factors may pass the placental barrier and leave persistent defect in the circulation of the offspring that may predispose to a pathological response later in life. We assessed apoptotic signaling in umbilical cord from patients with or without preE. We also evaluated the status of these stress signaling markers in a rat model of preE.

Methods: In this study, we recruited 20 normal pregnant (NP) and 20 preE consenting patients in an IRB approved prospective study from Scott & White hospital, Temple, Texas. Inclusion criteria for determination of preE patients include blood pressure >140/90 and presence of proteinuria >300 mg of protein/24h urine. Samples of placenta and umbilical cord were collected from those subjects after deliveries. Two groups of rats were also used in this study to assess the stress signaling markers. These rats were: normal pregnant (n=10) and preE rats (n=10) which were given weekly injections of desoxycorticosterone acetate and 0.9% saline to drink. Apoptotic and stress signaling proteins; Bcl-2-associated X protein (Bax), pro-apoptotic Bcl-2 protein (Bad) and proinflammatory protein cyclooxygenase-2 (Cox-2) expression were assayed both by western blot and immunohistochemistry. The p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation was evaluated by western blot. Comparisons were performed using ANOVA with Duncan's post-hoc test.

Results: Expression of Bax (Placenta: 1.2 fold, Cord: 1.5 fold), Bad (Placenta: 1.7 fold, Cord: 1.7 fold), Cox-2 (Placenta: 0.8 fold, Cord: 2.5 fold) and p38 MAPK phosphorylation (Placenta: 1.5 fold, Cord: 1.7 fold) were up-regulated (p<0.05) in placental tissues and umbilical cords of preE compared to NP patients. Apoptotic signaling was upregulated in preE rat placentas (Bax: 1.4 fold, Bad: 2.3 fold, Cox-2: 2.5 fold, p38: 3.0 fold) compared to NP. We did follow up examination of the babies for both groups of patients to assess the pregnancy outcome. Average hospital stay for PreE babies were significantly longer than those of NP babies (3.4 vs 6.1). There were no complications has been reported for the NP babies. However, Out of 20 babies: 2 had hypoglycemia; 8 had hyperbilirubinemia; 6 had respiratory distress syndrome; 2 had bilateral polydactyly with bilateral syndactyly; 2 had late gestational age; 2 had fetal ascites; 2 had intrauterine growth restriction.

Conclusions: Apoptotic signaling is augmented in preE which may lead to reduced nutrient transport capacity triggering placenta release of vascular factors that produce maternal vascular responses characteristic of this syndrome. PreE alters the intrauterine environment by modulating the pattern of hormonal signals and activating the detrimental cellular signaling that has been transported to the fetus. The fetus has to adapt to this impending intrauterine environment and detrimental signaling and this adaptation increases the risk of disease to the offspring.

Id: 54 Cigarette Smoking Inhibits Rig-I and Interferon Responses to Influenza in Human Small Airway Epithelial Cells

W. Wu, W. Zhang, J. Booth, J. Metcalf. Internal Medicine, University of Oklahoma Health Sciences Center, Edmond, Oklahoma.

RATIONALE: Cigarette smoking (CS) is the main risk factor for the development of COPD and 64% of COPD exacerbations are caused by respiratory infections including influenza. Influenza infections are more severe in smokers. The mechanism of increased susceptibility to infections in smokers is likely multifactorial, but clearly includes alteration of immunologic host defenses. Retinoic acid-inducible protein I (RIG-I) is believed to play an important role in the recognition of, and response to, influenza A virus (IAV) in most types of cells. Our group has shown that RIG-I induction in the lung is inhibited by CS in our human lung organ culture model and two animal models.

METHODS: We investigated RIG-I and interferon (IFN) induction by IAV in human small airway epithelial cells (SAEC) from smokers and non-smokers. Freshly isolated human SAEC cells were infected with A/Puerto Rico/8/1934 (PR8) H1N1influenza virus at an MOI of 1. Virus diluent (mock) was used as a negative control. After 24 h of infection, cells and supernatants were collected for qRT-PCR and ELISA to determine RIG-I and IFN expression levels.

RESULTS: As expected, we found that IAV PR8 exposure induced a vigorous antiviral response in SAEC from non-smokers as RIG-I was induced 10 fold over mock by virus. Expression of the antiviral cytokines IFN-b, IFN-λ 1 and IFN-λ 2/3 mRNA were also increased 6 fold, 300 fold and 150 fold, respectively, over baseline. However, in cells from smokers, viral RIG-I induction was only 40% of the non-smoker response, and smoking inhibited viral induction of the IFN-b, IFN-λ1 and IFN-λ 2/3 mRNA response by 79%, 88% and 97%, respectively, from that seen in SAEC from nonsmokers.

CONCLUSIONS: Our results suggest that active smoking reduces expression of antiviral cytokines in primary SAEC cells. This effect likely occurs via down-regulation of RIG-I. Reduction in lung epithelial cell RIG-I responses may be one major mechanism contributing to the increased incidence of viral respiratory infections in smokers and to viral induction of acute exacerbations of COPD.

Id: 55 Refractile Foreign Material Deposits and Alveolar Hemorrhage in Crack Cocaine Smoker

R. Sogomonian, V. Gandhi, T. McGarry, M. Moezzi. Internal Medicine, Icahn School of Medicine at Mount Sinai Elmhurst Hospital Center, Elmhurst, New York.

Introduction: Recreational use of alkaloid free-base cocaine, also known as crack cocaine, has reached epidemic proportions in the United States. Inhalation of crack cocaine is known to cause a variety of pulmonary complications. Herein we present a case of diffuse alveolar hemorrhage (DAH) and particulate foreign matter deposition in the setting of crack cocaine inhalation.

Case Report: A 50 year-old African-American female prisoner with history of Human Immunodeficiency Virus infection, diabetes mellitus and asthma presented with acute onset of subjective fever, shortness of breath and cough productive of blood-streaked sputum. She reported tobacco smoking as well as cocaine and marijuana use, including binge crack inhalation 2 days prior. She was febrile with normal pulmonary exam. Laboratory results were significant for a CD4+ count of 592 cells/μL, serum lactate dehydrogenase level was 252 U/L. Chest radiography showed diffuse interstitial and alveolar infiltrate involving the entire right lung with right upper lobe predominance. Empiric antibiotics were started for community-acquired pneumonia. Chest computed tomography showed an extensive right upper lobe quasi-nodular ground-glass infiltrate and a similar but less severe process in the right lower lobe (figure 1). Microbiologic evaluation, including routine cultures, legionella and histoplasma urine antigens, as well as sputum mycobacterial smears, were ultimately negative. Bronchoscopy showed diffuse edema of the airways without active hemorrhage. Brochoalveolar lavage (BAL) fluid samples were non-hemorrhagic. Transbronchial biopsy demonstrated alveolar hemorrhage with refractile foreign material deposits. There was no evidence of pneumocystis.

Discussion: Inhalation of crack cocaine exposes the lungs to its alkaloid vapor as well as to impurities used to “cut” the original substance. Heavy and repeated use has been associated with a wide spectrum of pulmonary complications such as asthma exacerbations, pneumothorax, pulmonary edema, diffuse alveolar hemorrhage (DAH), hypersensitivity pneumonitis, and so-called “crack lung.” DAH has been associated with “crack lung syndrome,” which is characterized by dyspnea, cough, fever, pulmonary infiltrates and hypoxemia. Of note, BAL samples can often be non-hemorrhagic in DAH caused by crack owing to resolution of bleeding by the time of bronchoscopy in the absence of repeated exposure. Transbronchial biopsy in this case demonstrated alveolar hemorrhage with refractile foreign material compatible with substances used to cut cocaine. It is important for the pulmonologist to be familiar with complications of crack cocaine use and to obtain a thorough substance abuse history.

Conclusion: Our patient received treatment with albuterol, acetaminophen, and codeine. Antibiotics were stopped after biopsy results were obtained. Lack of further exposure to cocaine while in hospital facilitated the resolution of her DAH. She was successfully discharged to the jail's infirmary.

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Id: 56 Stability of Protein Inhibitor of Activated Sata1 (Pias1) is Regualted in the Ubiquitinproteasome System

J. Zhao, B. Chen. Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.

The ubiquitin-proteasome system regulates intracellular protein stability, thus modulating cellular responses including cytokine release. PIAS1 (protein inhibitor of activated STAT1) exhibits an anti-inflammatory property via negatively regulating pro-inflammatory pathways such as JAK-STAT and Nf-kB. However, the stability of PIAS1 has not been revealed. Here we demonstrate that a novel ubiquitin E3 ligase, HECTD2, targets PIAS1 for its ubiquitination and degradation, thus increasing inflammatory responses. Glycogen synthase kinase (GSK3b) binds and phosphorylates PIAS1 serving as a phosphodegron motif for HECTD2 interacting with PIAS1. We identified a naturally occurring HECTD2 polymorphism (HECTD2A19P) in 8.5% of the population that functions to blunt inflammatory responses by exclusively causing the mislocalization of HECTD2A19P in the cytosol preventing nuclear interaction with PIAS1. We also developed a small molecule inhibitor, BC-1382, to target HECTD2 and attenuates proinflammatory stimuli-induced Nf-κB activation. These studies provide a new innate immunity pathway suggesting that mutation or antagonism of E3 ligase HECTD2 results in reduced severity of inflammation by selectively modulating the abundance of anti-inflammatory protein PIAS1.

Id: 57 Glycogen Synthase Kinase 3Beta Regulates Il-33 Receptor Internalization and Il-33 Signaling

J. Zhao, J. Wei, R.K. Bowser, R.S. Traister, M. Fan, Y. Zhao. Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.

IL-33, a member of the IL-1 cytokine family, contributes to the pathogenesis of lung inflammatory diseases including asthma, fibrosis, and lung injury. ST2L, the membrane bound receptor for IL-33, is expressed on immune effector cells and lung epithelia, and plays a critical role in triggering inflammation. We have previously shown that ST2L is an ubiquitinated protein and its protein level is regulated by the ubiquitin-proteasome system. Here, we investigate the molecular mechanisms of ST2L internalization and demonstrate that glycogen synthase kinase 3b (GSK3b) regulates ST2L internalization and IL-33 signaling. Immunofluorescence staining, flow cytometry analysis, and cell surface protein isolation and detection kit were used to determine cell surface ST2L expression. Plasmids containing GSK3b wild type and site-directed mutants were generated and transfected to mouse lung epithelial cells. IL-33 treatment reduces ST2L cell surface expression, an effect was attenuated by inhibition or down-regulation of GSK3b. GSK3b was phosphorylated and interacted to ST2L in response to IL-33 treatment. Serine residue 446 in ST2L was identified as docking stie for GSK3b. GSK3b binding site mutant (ST2LS446A) and phosphorylation site mutant (ST2LS442A) are resistant to IL-33-induced ST2L internalization. IL-33 also activated focal adhesion kinase (FAK), a protein tyrosine kinase. Inhibition of FAK impaired IL-33-induced GSK3b activation and ST2L internalization. Further, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that FAK/GSK3b modulates ST2L internalization and IL-33-induced inflammatory responses. Activation of FAK/GSK3b might serve as a unique strategy to lessen pulmonary inflammation.

Id: 58 Lysocardiolipin Acyltransferase (Lycat) Regulates Tgf-B-Induced Differentiation of Human Lung Fibroblasts

L. Huang, S. Reddy, V. Natarajan. UIC, Chicago, Illinois. C. Feghali-Bostwick. Medical University of South Carolina, Charleston, South Carolina. J. Garcia. University of Arizona, Tuscon, Arizona.

Rationale: Lysocardiolipin acyltransferase (LYCAT), a cardiolipin remodeling enzyme, plays a key role in normal mitochondrial function and vascular development. We previously reported that LYCAT mRNA levels in PBMCs directly and significantly correlated with pulmonary function outcomes and overall survival in IPf patients. In murine models of pulmonary fibrosis, overexpression of LYCAT reduced several indices of lung fibrosis, whereas knockdown native LYCAT expression accentuated fibrogenesis. In this study, we investigated the role of LYCAT in TGf-b mediated differentiation of human lung fibroblasts to myofibroblasts.

Objectives: To define roles of LYCAT in TGf-b-induced lung fibroblast differentiation.

Methods: The mRNA and protein expression of LYCAT was determined deter in lung tissues and fibroblasts from control and IPf patients. Human lung fibroblasts were infected with vector control and adenoviral hLYCAT vector (10 MOI) for 48 h and the effect of LYCAT over-expression on the regulation of TGf-b-induced fibroblast differentiation was assessed by Western blotting for fibronectin and α-smooth muscle actin.

Results: LYCAT expression was up-regulated in lung fibrotic foci and primary lung fibroblasts isolated from IPf patients and bleomycin-challenged mice, compared to controls. In vitro, TGf-b induced LYCAT expression in human lung fibroblasts through activation, translocation and binding of SMAD2/3 to its binding motif in LYCAT promoter. Over-expression of LYCAT attenuated TGf-b-induced differentiation of human lung fibroblasts through regulation of the Akt and ERK signaling pathways and inhibition of TGf-b-induced ROS generation.

Conclusion: These studies suggest that LYCAT regulates TGf-b-induced lung fibroblast differentiation, and may serve as a potential therapeutic target for human lung fibrosis. This work was supported by P01 HL098050 (VN) and R03HL096933 (SPR).

Id: 59 Genetic and Epigenetic Regulation of Group V Phospholipase A2 in Pulmonary Inflammation

X. Sun, D. Adyshev, W. Zhang, M. Wade, S.M. Dudek. Medicine, University of Illinois at Chicago, Chicago, Illinois. A.R. Leff. The University of Chicago, Chicago, Illinois.

Rationale: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is characterized by inflammatory disruption of the alveolar-vascular barrier, resulting in severe respiratory compromise. We previously have demonstrated that Group V phospholipase A2 (PLA2G5) expression is increased in cultured lung endothelium and mouse lungs by LPS and augments pulmonary vascular permeability in vitro and in vivo.

Objective: To explore the regulation of PLA2G5 gene expression in lung inflammation by disease-associated genetic variants, microRNA and transcription factors.

Methods: A combination of genomic wide association study (GWAS), in silico analysis, transient transfection of PLA2G5 3’ UTR luciferase reporter assay, protein-DNA mobility shift, and chromatin immunoprecipitation assay of the PLA2G5 promoter was utilized. We investigated samples from 206 sickle cell disease patients with and without acute chest syndrome in Chicago, and analyzed PLA2G5 with Genomatix and Haploview for gene regulation and allele linkage disequilibrium, respectively. The interactions of PLA2G5 promoter with lung inflammatory transcription factors, and PLA2G5 3’ UTR with microRNAs, were studied for PLA2G5 epigenetic regulation.

Results: In silico analyses identified binding elements for lung inflammatory early response transcription factors NfkB and NfAT in the promoter of PLA2G5, while miR-688-3p, miR-501-5p and miR-200c binding sites were identified in the 3'UTR region. GWAS identified several intronic genetic variants of PLA2G5 that were significantly associated with acute chest syndrome, and these were in high linkage disequilibrium with promoter variants rs11573185 and rs11573188. Protein-DNA mobility shift assays indicated that lung inflammatory early response transcription factors NfkB and NfAT bind to the PLA2G5 promoter with high affinity. Exposure to LPS significantly increased NfkB recruitment to the PLA2G5 promoter. SNP rs11573188 significantly influenced lung transcription factor HDGf recruitment to PLA2G5 promoter. ALI/ARDS-relevant stimuli LPS and 18% cyclic stretch significantly increase PLA2G5 promoter activity. PLA2G5 3'UTR reporter activity is increased by LPS, and significantly attenuated by miR-688-3p, miR-501-5p and miR-200c.

Conclusions: In vitro and in vivo data identify PLA2G5 as a promising candidate gene in inflammatory lung disease. The current study characterizes the regulation of PLA2G5 gene expression by inflammatory disease-associated polymorphisms, microRNA, and acute inflammatory stimuli and advances our understanding of how PLA2G5 levels are regulated during lung injury pathogenesis.

Id: 60 the Lysophosphatidic Acid Receptor 1 Antagonist Ki16425 Blunts Abdominal and Systemic Inflammaton in a Mouse Model of Peritoneal Sepsis

A.M. Jacko, J. Zhao. Medicine, University Of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. J. Wei, N. Weathington, Y. Zhao, H. Huang, A. Tsung. Surgery, University Of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator of inflammation via the LPA receptors 1-7. We and others have previously described pro-inflammatory and pro-fibrotic activity of LPA signaling in pulmonary systems in bleomycin- or lipopolysaccharide (LPS)-induced lung injury models. In this study, we investigated if LPA signaling is active in the pathogenesis of systemic sepsis from an abdominal source. We report here that antagonism of the LPA receptor LPA1 with the small molecule ki16425 reduces the severity of abdominal inflammation and organ damage in the setting of peritoneal endotoxin exposure. Pretreatment of mice with intraperitoneal ki16425 reduces the induction of peritoneal neutrophil chemokine production, liver oxidative stress, liver injury, and cellular apoptosis in visceral organs in the setting of LPS exposure. Mice pretreated with ki16425 are also protected from LPS-induced mortality. Tissue myeloperoxidase activity is not affected by LPA1 antagonism. We have shown that LPA1 is associated with LPS co-receptor CD14, the association is suppressed by ki16425. LPS-induced phosphorylation of PKCδ and p38 MAPK in liver cells and IL-6 production in Raw264 cells are likewise blunted by LPA1 antagonism. These studies indicate that the small molecule inhibitor of LPA1, ki16425, suppresses cytokine responses and inflammation in a peritoneal sepsis model by blunting downstream signaling through the LPA1-CD14-TLR4 receptor complex. This anti-inflammatory effect may represent a therapeutic strategy for the treatment of systemic inflammatory responses to infection of the abdominal cavity.

Id: 61 Effect of Altitude and Cigarette Smoke Exposure on Bronchoalveolar Lavage Fluid Cellularity

M.K. Ishaq, C. Cross, H. Youness, L. Booth, E. Duggan, L. Howard, B.A. Petrone- Holtslander, A. Awab. Pulmonary and Critical Care Medicine, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma. D. Hutchings. Cherokee Nation Distributors, Catoosa, OK, Catoosa, Oklahoma. V. White, D. Self, D.M. Kupfer, D. Burian. Civil Aerospace Medical Institute, federal Aviation Administration, Oklahoma City, OK, Oklahoma City, Oklahoma. J.P. Metcalf. Oklahoma Medical Research foundation, Oklahoma City, OK., Oklahoma City, Oklahoma.

Rationale: Occupational exposure to passive smoke presents a significant health risk to workers. Never smoking, healthy flight attendants exposed to cigarette smoke (CS) on commercial aircrafts in the United States prior to the ban on smoking had significant declines in spirometry and diffusion capacity. Thirteen percent of those examined had declines that would be classified as mild chronic obstructive pulmonary disease (COPD) by American Thoracic Society (ATS) criteria. COPD is associated with, and likely caused by, chronic inflammation due to environmental irritants including CS. We have previously shown that exposure at cabin altitude causes systemic inflammation. We propose that the source of that inflammation is the lung, and that inflammation is enhanced by cigarette smoke exposure.

Methods: We recruited two groups of individuals, non-smoking and light-smoking individuals (£1 pack per day) both with normal lung function. Subjects are matched against age, gender, ethnicity, and smoking status. Each subject undergoes 2 separate sessions at least 30 days apart in a hypobaric altitude chamber. Subjects are exposed to either ground level pressure or a simulated standard cabin altitude of 8,000 feet for 5.5 hours in random order. Vital signs, pulse oximetry, alveolar gases, and blood samples are obtained before, during, and after the session. Within 1 hour following their session, subjects undergo bronchoscopy for bronchoalveolar lavage (BAL) and brushing. Results 227 subjects were initially screened from an original pool of recruitment consisting of patients undergoing pulmonary function testing at the Oklahoma City VA. Only 21 were enrollable, of whom 6 had smoking and non-smoking matches and only 1 consented. Modification of the research protocol allowed a second source of recruitment from the local campus. 112 subjects have been screened, with 97 recruited and 25 subjects enrolled. Of these, a total of 7 non-smoking and 5 smoking subjects have completed both testing phases including BAL. Preliminary results show that smokers desaturated to an average of 93% with non-smokers desaturating to 95% with ascent to 8,000 feet. Results from BAL fluid (BALf) cell counts revealed a significant increase in total cells recovered from smokers vs non-smokers in both ground or simulated ascent runs (avg. ground 11.32 vs. 4.32 x 106 cells; avg. Flight 12.55 vs. 3.81 x 106 cells, p<0.05 all comparisons). Though there was a 10% increase with ascent in smokers and a 12% decrease in cells recovered with ascent in non-smokers, these differences did not reach statistical significance. Cell concentration in BALf was also increased in smokers vs. non-smokers with either ground or simulated ascent (avg. ground 2.30 vs. 0.78 x 105 cells/ml; avg flight 2.52 vs. 0.72 x 105 cells/ml, p<0.05 all comparisons). Again, though there was an 8% decrease in cell concentration with ascent in non-smokers and a 10% increase in smokers, this did not reach statistical significance. Over 70% of the increase in cell numbers were due to alveolar macrophages, though an increase in other cell types, including neutrophils and lymphocytes was seen in the smoking cohort.

Conclusions: Exposure to cabin altitude causes a decrease in oxygen saturation in normal and nonsmoking subjects. Increased BALf cellularity in light smokers also occurred, and there a trend toward enhancement of this effect with altitude. Whether these findings are due to an increase in BALf markers of inflammation including cytokine levels will be determined in further studies.

Id: 62 Fty720 S-Phosphonate Does Not Activate Grk2-Mediated S1Pr1 Phosphorylation and Ubiquitiation

L. Wang, S. Dudek. Univ of Illinois at Chicago, Chicago, Illinois. R. Bittman. The City University of New York, New York, New York.

Rationale: Modulation of pulmonary vascular barrier function is an important clinical goal in acute lung injury (ALI). We previously demonstrated that fTY720 S-phosphonate (Tysiponate/Tys), an analog of sphingosine 1- phosphate (S1P) and fTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in the LPS- and bleomycin-induced models of mouse ALI. Moreover, Tys preserves expression of the barrier promoting S1P1 receptor (S1PR1), whereas S1P and fTY720 induce its ubiquitination and degradation. In this report, we further characterize the mechanism of preservation of S1PR1 expression by Tys in cultured human pulmonary endothelial cells (EC).

Methods/Results: P-fTY720, but not Tys, significantly increased association of the G protein-coupled receptor regulatory kinase, GRK2, with S1PR1 by co-immunoprecipitation and co-immunostaining, as well as increased the phosphoserine status of S1PR1. Pharmacologic inhibition of the ubiquitin-activating enzyme E1 by PYR41 significantly inhibited S1PR1 degradation induced by p-fTY720 and S1P. Although association of S1PR1 and the E3 ligase WWP2 reportedly is important for S1PR1 degradation, neither p-fTY nor Tys alters this association. Furthermore, pharmacological inhibition of PKA, PKG, PKC, GSK3, PI3K, ERK, Src, or c-Abl activity, as well as inhibition of calcium flux, all fail to inhibit p-fTY720-induced S1PR1 degradation.

Conclusion: Unlike other S1PR1 agonists, Tys fails to activate the GRK2-mediated ubiquitination pathway and thus preserves S1PR1 expression. These results provide additional mechanistic insights into the barrier-regulatory effects of this potential ALI therapy.

Id: 63 Inhibition of the Actin Related Protein 2/3 Complex Decreases Pulmonary Endothelial Barrier Function and Recovery

B.N. Brinley. Pulmonary and Critical Care, University of Illinois Hospital and Health Science System, Chicago, Illinois. B.N. Brinley. Internal Medicine, Mercy Hospital and Medical Center, Chicago, Illinois.

RATIONALE: Regulation of theThe pulmonary endothelial barrier between the lung vascular and interstitial spaces is disrupted during the development of sepsis and acute lung injury (ALI). Barrier function is determined by actin cytoskeletal structure and rearrangements which in turn alter cell membrane and junctional structures. The actin related protein 2/3 complex (Arp 2/3) is a key regulator of branched actin polymerization particularly at the cell periphery. In this study we investigate the role of Arp 2/3 in pulmonary endothelial cell (EC) actin dynamics and barrier function.

METHODS: In vitro studies were performed using cultured human pulmonary artery endothelial cells (HPAEC). EC barrier function was measured under barrier enhancing (sphingosine-1-phosphate, S1P 1μM) and disruptive (thrombin 1U/ml) conditions by transendothelial electrical resistance (TER) in the presence or absence of a specific Arp 2/3 inhibitor (CK-666 50 μM). Inter-endothelial gaps were assessed by measuring monolayer permeability to FITC conjugated avidin of cells grown on biotinylated gelatin in the presence or absence of CK-666 (50 μM x 1 hr) after treatment with S1P (1μM x 15 min), thrombin (1U/ml x 15 min) or vehicle. Actin polymerization was assessed by the ratio of filamentous (f) to globular (G) actin in cell lysates.

RESULTS: Arp 2/3 inhibition significantly decreased baseline EC resistance, measured by TER, when compared to vehicle (-27% +/- 4.8%, p=0.04). This effect was rapid (∼15 min) and sustained (>2 hrs). Interestingly, following treatment with S1P (1μM) there was no significant difference in the increase in TER between cells pre-treated with Arp 2/3 inhibitor (Ck- 666 50 μM x 1 hr) or vehicle, (41% +/- 9% vs 44% +/- 4%) respectively. Similarly, Arp 2/3 inhibition (Ck-666 50 μM x 1 hr) did not alter the absolute drop in resistance induced by thrombin (1U/ml) but did significantly delay the recovery of resistance to baseline (97 +/- 12 min vs 193 +/- 24 min, p=0.04). In preliminary studies of EC gap formation, Arp 2/3 inhibition (Ck-666 50 μM x 1 hr) increased thrombin induced paracellular permeability to FITC-Avidin by 14%. Actin polymerization following S1P treatment (1μM x 15 min) revealed a 12% decrease in actin (f/G) ratio in cells pretreated with Arp 2/3 inhibitor (Ck-666 50 μM x 1 hr).

CONCLUSION: These results highlight the importance of Arp 2/3- mediated actin polymerization in the determination of pulmonary endothelial barrier function. In particular, the Arp 2/3 complex may play a critical role in EC gap closure and recovery of barrier integrity.

Id: 64 the Central Role of Nuclear Factor (Erythroid-Derived 2)-Like 2 in Ventilatorinduced Lung Injury

T. Wang, T. Jiang, T. Zhou, H. Quijada, J.B. Mascarenhas, V. Ramamoorthi Elangovan, L. Hecker, D.D. Zhang, J.G. Garcia. University of Arizona, Tucson, Arizona.

BACKGROUND AND RATIONALE: Ventilator-induced lung injury (VILI) is an acute lung injury produced by mechanical ventilation with high tidal volumes in patients in the intensive care unit (ICU). VILI is a common entity with an unacceptable mortality rate (∼20%). As no current therapies exist for VILI, novel therapeutic targets for VILI and mechanical stress-induced tissue injury are needed. Nuclear factor (erythroid-derived 2)-like 2, also known as NfE2L2 or Nrf2, is a transcription factor that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation. In this study, we defined the central role of Nrf2 in VILI and mechanical stress-induced endothelial cell pathobiology.

METHODS AND RESULTS: first, we defined Nrf2 as a sensory marker for mechanical stress-induced human lung endothelial cell injury. Antioxidant response element (ARE) luciferase plasmids were transfected into both endothelial cells and murine lung tissues. Both EC exposed to 18% cyclic stretch (18% CS) to produce excessive mechanical stress and mimic VILI, and mice exposed to high tidal volume ventilation (40 ml/kg), resulted in ARE activation, the genomic site of Nrf2 binding. We next confirmed Nrf2 signaling is essential for gene expression dysregulation by 18% CS. Nrf2 silencing RNAs (siRNA) to reduce Nrf2 protein expression, resulted in significantly amelioration of 18% CS-induced gene expression, including signaling pathways of “cytokine-cytokine interaction”, “adhesion and diapedesis of lymphocytes”, and “PPAR signaling pathway”. Finally, we validated that Nrf2 is a novel and effective therapeutic target for VILI in pre-clinical models of VILI. The Nrf2 activator, sulforaphane (Sf, 11.5 mg/kg), significantly attenuated VILI (40 ml/kg, 4 hrs)-induced lung inflammation, including BAL protein leakage and white blood cell infiltration.

CONCLUSION: These novel findings characterize Nrf2 as a sensor of mechanical stress produced by high tidal volume mechanical ventilation, and as a central mediator of mechanical stress-induced gene expression dysregulation. Nrf2 may represent a novel therapeutic target in VILI.

Id: 65 Mice with Sickle Cell Disease Are Less Susceptible to Ventilator-Induced Lung Injury but Not Lps-Induced Lung Injury

J.R. Sysol. Pharmacology, University of Illinois College of Medicine at Chicago, Chicago, Illinois. J.R. Sysol, S. Sammani, E. Letsiou, S. Singla, S.M. Dudek, R.F. Machado. Medicine, University of Illinois College of Medicine at Chicago, Chicago, Illinois.

Rationale: Sickle cell anemia, the most common and severe form of sickle cell disease (SCD), is an autosomal recessive disorder affecting more than 300,000 individuals worldwide. Patients with this disease have an abnormal form of hemoglobin (HbS) within their erythrocytes due to a single amino acid substitution in the b chain of Hb. Mutant HbS has reduced solubility when deoxygenated, leading to polymerization and aggregation of sickle erythrocytes in the microvasculature. A major complication in SCD is acute chest syndrome (ACS), an acute lung injury (ALI) syndrome of multiple etiologies that can lead to respiratory failure and need for mechanical ventilation in close to 15% of patients. Although ACS is the leading cause of mechanical ventilation in patients with SCD, the role of ventilator-induced lung injury (VILI) in this patient population is unknown. In this study, we compared established in vivo models of lipopolysaccharide (LPS)-induced lung injury and VILI in mice with sickle cell disease (Townes strain).

Methods: for VILI studies, sickle mice and control littermates (age 10-12wks) were exposed to high tidal volume mechanical ventilation (HVt=40mL/kg) for 4h. For LPS-induced lung injury studies, mice were injected intratracheally with LPS (1mg/kg) and studied after 16h. Bronchoalveolar lavage fluid (BALf), plasma, and lung tissues for histological analysis were collected from all mice. Total cell counts, neutrophil counts, and protein levels were analyzed in BALf, and cytokines (TNf-α, KC, and MIP-2) were measured in both BALf and plasma.

Results: following VILI, sickle mice showed significantly less total cells (p<0.05), neutrophils (p<0.01), and protein (p<0.05) in BALf compared to controls. Induction of neutrophil chemo-attractants KC and MIP-2 were also attenuated in BALf from sickle mice exposed to VILI versus controls (53% reduction, p<0.05 and 64% reduction, p<0.05, respectively). Plasma KC levels were also significantly lower in VILI sickle mice (p<0.01), and histological analyses of lung tissue showed reduced inflammation and alveolar damage. In contrast to VILI, sickle mice were not protected from LPSinduced lung injury. Sickle mice had elevated TNf-α levels in BALf following LPS challenge compared to controls (p<0.05), with no significant changes observed in BALf protein, neutrophil counts, or other cytokine levels. The extent of lung injury was similar between sickle and control mice after LPS challenge.

Conclusion: These studies demonstrate that sickle mice are protected from VILI but not LPS-induced lung injury. The diminished induction of neutrophil chemo-attractants and chemotaxis in sickle mice during VILI may functionally explain this protection. These studies warrant future exploration into the differential mechanisms of lung injury in these models and may lead to improved management strategies for SCD patients in the intensive care unit.

ID: 66 Anti-Inflammatory Effects Of C-Abl Inhibition In Lps-Induced ALI

A.N. Rizzo. Pharmacology, UIC College of Medicine, Chicago, Illinois. A.N. Rizzo, E. Letsiou, S. Sammani, M.E. Brown, S.M. Dudek. Medicine, University of Illinois College of Medicine, Chicago, Illinois. J.G. Garcia. University of Arizon, Tuscon, Arizona.

Rationale: Acute lung injury (ALI) occurs due to inflammation-induced disruption of the pulmonary endothelial cell (EC) barrier, which causes protein-rich fluid and inflammatory cells to flood the airspaces. A therapeutic goal is to identify agents that both dampen injurious inflammation and increase EC barrier integrity. Recently published work indicates that the fDA-approved Abl family kinase inhibitor imatinib attenuates EC barrier dysfunction in murine sepsis. Additionally, imatinib decreases NfκB activity and downstream pro-inflammatory signaling in lymphocytes. In the present study, we explored the effects of imatinib on LPS-induced NfκB activation and downstream neutrophil recruitment using cell culture and murine models of LPS-induced ALI. Additionally, we utilized selective Abl family kinase inhibitors (nilotinib and GNf-2) and c-Abl and Arg siRNA to determine the specific role of these kinases in LPS-induced ALI.

Methods: Human pulmonary artery ECs were treated with imatinib (40μM, 0- 60min) and challenged with LPS (1μg/mL, 0-3hrs). Phosphorylated NfκB and downstream EC activation (VCAM-1, IL- 6, IL-8) were assessed by western blots (WB) and ELISAs. NfκB localization was determined using immunofluorescence (If) microscopy. Nilotinib (5μM), GNf-2 (15μM), and c-Abl or Arg specific siRNA were used in complementary in vitro assays. C57BL6 mice received imatinib (75mg/kg, IP) or vehicle 4 hours after intratracheal LPS (1mg/kg, 18hrs) and inflammation was assessed by lung NfκB expression and bronchoalveolar lavage (BAL) cytokine levels and neutrophil counts. Complementary intravital two-photon microscopy studies assessed pulmonary neutrophil recruitment in real time.

Results: Imatinib inhibits LPS-induced NFκB phosphorylation (54%, p=0.03) and nuclear translocation in ECs as well as downstream EC activation as measured by the release of inflammatory cytokines IL-8 (60%, p<0.001) and IL-6 (93%, p<0.001) and expression of cellular adhesion molecules (VCAM-1). The more specific inhibitors nilotinib and GNf-2 blocked LPS-induced VCAM-1 expression more potently than imatinib. Furthermore, silencing c-Abl, but not Arg, attenuated LPS-induced VCAM-1 expression (33%, p=0.04) and IL-8 release (48%, p=0.048). In murine ALI, imatinib decreased LPS-induced lung NFκB expression, BAL levels of TNfα (88%, p=0.001) and pulmonary neutrophils recruitment (43%, p=0.02).

Conclusions: Imatinib exhibits potent antiinflammatory effects in lung EC including inhibition of LPS-induced NFκB activation, VCAM-1 expression, inflammatory cytokine release (IL-6, IL-8, TNfα), and ultimately neutrophil recruitment. These effects are mimicked with specific Abl family kinase inhibitors as well as selective c-Abl silencing. Thus, the fDA approved Abl kinase inhibitors (imatinib, nilotinib, dasatinib) may be potential therapeutic options for patients with ALI and other inflammatory lung diseases.

Id: 67 Parkin Deficiency Protects against Lps-Induced Acute Lung Injury

E. Letsiou, S. Sammani, S.M. Dudek. University of Illinois at Chicago, Chicago, Illinois.

Introduction: Acute lung injury (ALI) and its more severe form, the Acute Respiratory Distress Syndrome (ARDS), are serious complications of inflammatory conditions and are associated with an unacceptable mortality of up to 40%. ALI is characterized by increased alveolo-capillary permeability, excessive accumulation of neutrophils in the lungs, and pulmonary edema. We have recently demonstrated that imatinib, a tyrosine kinase inhibitor, protects against LPS-induced pulmonary leak and inflammation, by targeting c-Abl kinase. In the present study, we investigate the role of a novel c-Abl substrate, parkin, in regulating ALI. Parkin (PARK2) is an E3 ubiquitin ligase with multifunctional roles in mitophagy and inflammation; however, its role in ALI is almost completely unexplored.

Methods: C57BL/6 (WT) and parkin deficient (PARK2 KO) male mice (8-12 wks, n=5) were subjected to LPS (intratracheally, 1 mg/kg) or PBS (controls), and allowed to recover prior to harvest 18 hours later. Leakage of proteins into the alveolar space was assessed by measuring the protein levels in the bronchoalveolar lavage (BAL). To assess lung inflammation, BAL total and neutrophil cell counts were performed. BAL myeloperoxidase (MPO) activity was determined in BAL as a marker of neutrophil activation. BAL was also assessed for cytokine levels (IL-6, TNf-a, and IL-1b). Parkin deficiency in the PARK2 KO mice was confirmed by immunoblotting analysis of lung tissue lysates.

Results: Control PARK2 KO mice display a normal lung phenotype. However, PARK2 KO mice exhibited less ALI after LPS compared to WT mice. In PARK2 KO, BAL protein levels were reduced by 23.6% (p=0.0046) compared to WT mice. LPS induced a large influx of inflammatory cells into the lungs of WT mice which was attenuated in the PARK2 KO; BAL total cell and neutrophil counts in PARK2 KO mice were substantially decreased by 44.7% (p=0.039), and by 45.7% (p=0.046) respectively, compared to WT. BAL MPO activity levels were also significantly lower in PARK2 KO by 52.1 % (p=0.03). Interestingly, BAL cytokine levels did not differ between the two strains after LPS.

Conclusion: Our studies demonstrate that parkin deficiency protects against LPS-induced lung permeability and inflammatory cell infiltration. These studies identify parkin as a novel mediator involved in ALI pathogenesis that may potentially represent a therapeutic target for treatment of ALI/ARDS.

Id: 68 Maladaptive Apoptosis Responses to Cigarette Smoke in Human Bronchial Epithelial Cells from Individuals with Copd

G.E. Holt, L. Kuenzi, M. Salathe. Medicine/Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Miami, Miami, florida.

Objective: Cigarette smoke induces apoptosis in human bronchial epithelial (HBE) cells however the rate of apoptosis is higher in smokers with COPD than in those without due to unclear mechanisms. We believe HBE cells from healthy smokers adapt to the chronicity of smoke exposure by attenuating the smoke mediated induction of apoptosis. We hypothesize that HBE cells from individuals with COPD fail to adopt these protective measures and thus have unfettered rates of smoke-mediated apoptosis ultimately leading to parenchymal cell loss and clinical disease.

Methods: We use primary, fully differentiated HBE cells grown at the air-liquid interface and expose them to cigarette smoke from a smoke robot. Endpoints measured are activation of the intrinsic pathway of apoptosis and cell death. HBE cells are derived from non-smokers, healthy smokers and smokers with COPD.

Results: HBE cells exposed to cigarette smoke exhibit DNA strand fragmentation, activation of the pro-apoptotic protein caspase 3 and inactivation of the anti-apoptotic protein, PARP-1 consistent with initiation of apoptosis. HBE cells from healthy smokers downregulate the pro-apoptotic protein caspase-3 and upregulate the anti-apoptosis protein XIAP in comparison to smokers with COPD and non-smokers. Smokers with and without COPD both upregulate the antiapoptosis protein PARP-1 in comparison to non-smokers. Evaluation of smoke-mediated cell death revealed HBE cells from healthy smokers were more resistant to cell death than HBE cells from the other cohorts.

Conclusions: Healthy smokers adapt to the chronicity of smoke exposure by altering the expression of apoptotic proteins to attenuate smoke mediated cell death. This adaptive response does not occur to the same extent in smokers with COPD and this may explain why these individuals contract disease. A sound understanding of these adaptive mechanisms may allow formulation of strategies to prevent disease in at risk individuals and in those for whom disease progresses despite avoidance of smoke.

Id: 69 Role of Voltage-Gated Na+ Channels in Hypoxia-Induced Pulmonary Vasoconstriciton

Y. Gu, Q. Gu, H. Tang. Department of Medicine, University of Arizona, Tucson, Arizona. D.R. Fraidenburg. Department of Medicine, University of Illinois, Chicago, Illinois. A. Makino. Department of Physiology, University of Arizona, Tucson, Arizona. J.J. Yuan. Department of Medicine and Physiology, University of Arizona, Tucson, Arizona.

Rational: Pulmonary arterial hypertension (PAH) is a rare but progressive and fatal disease characterized by increased pulmonary vascular resistance (PVR). Sustained pulmonary vasoconstriction and pulmonary vascular remodeling are the major causes of increase PVR in patients with PAH. Our previous studies demonstrated that human PASMC express TTX-sensitive voltage-gated Na+ channels. However, their functions in pulmonary vasoconstriction remain unresolved.

Methods and Results: In the current study, we have utilized the isolated, perfused, and ventilated mouse lung preparation to determine the roles of voltage-gated Na+ channels in modulating HPV. We analyzed the hypoxia-induced response in isolated lungs from mice. Ventilation of lungs from mice with 1% O2 provoked a vasoconstrictor response and reached to the plateau within 4 min. Perfusion with tetrodotoxin (TTX), an inhibitor of votage-gated Na+ channels, slightly but significantly decrease hypoxia-induced pulmonary vasoconstriction. Normoxic vascular ton were not affected by MTT perfusion and there is no difference of vasoconstriction induced by high K+ before and after MTT perfusion.

Conclusion: Our study has demonstrated that perfusion with TTX inhibit the acute hypoxia-induced pulmonary vasoconstriction. These results implicated that voltage-gated Na+ channels play roles in acute hypoxic pulmonary vasoconstriction but are not involved in high K+ induced response. Thus, voltage-gated Na+ channels may present a target for a molecular intervention to control pulmonary hemodynamics.

Id: 70 Hemin-Induced Acute Lung Injury is Mediated by Gvpla2 in Mice

S. Sammani, E. Letsiou, A. Rizzo, J. Sysol, L. Meliton, R. Machado, S. Dudek. UIC, Chicago, Illinois.

Background: Acute chest syndrome (ACS) is a major complication and a chief cause of morbidity and mortality in sickle cell disease (SCD) patients. Recent studies have revealed that hemin, a hemolysis byproduct and inflammatory agent, induces intravascular hemolysis, autoamplification of extracellular hemin, and acute chest syndrome in sickle cell mice (Ghosh et al, JCI, 2013). Hemin-induced ACS is a novel preclinical model of acute lung injury (ALI) that is characterized by lung infiltration of inflammatory cells and pulmonary endothelial cell (EC) barrier disruption, leading to lung edema. Previous studies by our group and others have identified Group V phospholipase A2 (gVPLA2) as an important mediator of ALI. Inhibition of gVPLA2 attenuates LPS-induced pulmonary EC permeability in vitro as well as ALI induced by LPS or mechanical ventilation (VILI) in vivo.

Goals and Methods: We hypothesized that the genetic absence of gVPLA2 in gVPLA2 knockout (KO) mice would attenuate hemin-induced ALI compared to wild-type (WT) mice. To explore the effects of gVPLA2 on the pulmonary barrier function in vivo, 8-weekold gVPLA2 KO and WT (C57bl/6) (N=3-4) mice were challenged with hemin (70 μmol/kg) or saline, ∼200μl volume, IV injection via jugular vein. Mice were harvested 18 hours later. Bronchoalveolar (BAL) fluid was collected for total BAL protein concentrations as well as BAL inflammatory cells total and differential cell counts. Lung tissue samples were used for histological evaluation and western blot.

Results: BAL protein levels and BAL inflammatory cell counts were reduced significantly in hemin-challenged gVPLA2 KO compared to hemin-challenged WT mice (P£0.05). These results indicate that gVPLA2 knockout mice are protected in the novel hemin-induced ALI mice model.

Conclusion: GVPLA2 plays an essential role in hemin-induced lung inflammation, and its inhibition represents a therapeutic intervention for ACS in sickle cell patients.

Id: 71 Therapeutic Benefits of Young, but Not Old, Adipose-Derived Mesenchymal Stem Cells in a Chronic Mouse Model of Bleomycin-Induced Pulmonary Fibrosis

J. Tashiro, D.J. Gerth, S.J. Elliot, M.K. Glassberg. Medicine, University of Miami Miller School of Medicine, Miami, florida.

Rationale: The fact that pulmonary inflammatory lesions and bleomycin (BLM)-induced fibrosis spontaneously resolve in young mice, while remaining irreversible in aged mice, suggests that impairment of pulmonary regeneration and repair is associated with aging. Since mesenchymal stem cells (MSCs) may promote repair following injury, we postulated that differences in MSCs from aged mice may underlie post-injury fibrosis in aging. The potential for young-donor MSCs to inhibit BLM-induced pulmonary fibrosis in aged male mice (>22 months) has not been studied.

Methods: Adipose-derived MSCs (ASCs) from young (4-month) and old (22-month) male mice were infused 1-day following intratracheal BLM administration. Lung fibrosis was evaluated using Ashcroft score, collagen content, and αv-integrin mRNA expression. Additional endpoints of inflammation and cellular damage included matrix metalloproteinase (MMP)-2 activity, oxidative stress, and markers of apoptosis.

Results: At 21-day sacrifice, aged BLM mice demonstrated lung fibrosis. Lung tissue from aged BLM mice receiving young ASCs exhibited decreased fibrosis, MMP-2 activity, oxidative stress, and markers of apoptosis vs. BLM controls. Lung mRNA expression of TNfα was also decreased in aged BLM mice receiving young-donor ASCs vs. controls. In contrast, old-donor ASC treatment in aged BLM mice did not reduce fibrosis and related markers. Compared to olddonor ASCs, young-donor ASCs had decreased mRNA expression of MMP-2, insulin-like growth factor receptor, and AKT activation.

Conclusions: These results show that the BLM-induced pulmonary fibrosis in aged mice could be blocked by young-donor ASCs and that the mechanisms may include changes in collagen turnover and inflammation.

Id: 72 Aspergillus Tracheobronchitis: A Successful Treatment of An Unusual Infection

M.U. Khawar. Internal Medicine, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma. M.K. Ishaq, K. Jones. Pulmonary & Critical Care Medicine, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma.

INTRODUCTION: Isolated invasive Aspergillus tracheobronchitis (ATB) is an uncommon clinical form of invasive pulmonary Aspergillosis (IPA) in which Aspergillus infection is limited entirely or predominantly to the tracheobronchial tree. Only a few cases of isolated invasive ATB associated with systemic lupus erythematosus (SLE) have been described in the literature. We report a patient with recently diagnosed SLE, who was found to have isolated invasive ATB and treated successfully with Voriconazole.

CASE PRESENTATION: A 44-year-old woman diagnosed with SLE four months prior was evaluated for chronic dry cough of more than two months duration. Her past medical history included a recent diagnosis of CMV pneumonitis for which she was taking a therapeutic dose of Valganciclovir. Initial physical examination including the examination of chest was unremarkable. Chest imaging with an X-ray and computed tomography did not show any airway or lung parenchymal abnormalities. A bronchoscopic examination performed later showed multiple small sized nodules with superficial ulceration in the trachea and bronchi. Endobronchial biopsies of these nodules were performed and histopathological examination disclosed mucosal accumulation of the chronic inflammatory cells. Special fungal staining further showed septate fungal hyphae compatible with Aspergillus species. Cultures of sputum, bronchial aspirate, and biopsy specimens all grew Aspergillus fumigatus, confirming the diagnosis of isolated invasive ATB. Our patient was started on oral Voriconzaole and a follow up bronchoscopic examination at three months showed no gross evidence of residual Aspergillus infection.

DISCUSSION: Infections with Aspergillus species have been described only rarely in patients with SLE. Invasive aspergillosis more commonly occurs in patients with hematological and other malignancies (often following bone marrow transplantation), AIDS, immunosuppression from chronic corticosteroid therapy, or after solid organ transplantation. Its nonspecific clinical presentation includes fever and respiratory symptoms (cough, wheezing or stridor), and initial radiographic examination lacks sensitivity for early diagnosis. Blood cultures are usually negative, and positive sputum cultures are often considered to represent contamination or colonization. In patients with a high index of suspicion, bronchoscopy with cytology and cultures from bronchoalveolar lavage have been shown to be >90% specific. Voriconazole is currently the first-line agent for the treatment of invasive aspergillosis. Isolated invasive ATB may be an early stage of Aspergillus invasion and it may progress to more extensive and invasive infection if early diagnosis is not established. Given the nonspecific and variable clinical presentation, clinicians should be aware of this rare entity in select patient population so that early antifungal therapy can be initiated to halt the progression to more invasive disease.

Id: 73 Pulmonary Embolism in Neurofibromatosis

W. Wong. Medicine, Hennepin County Medical Center, Shoreview, Minnesota.

A 58-year-old man with a history of neurofibromatosis type 1 and seizure disorder was brought to the emergency room after being found down at his apartment for an unknown period of time. The patient was unresponsive but hemodynamically stable. External survey was significant for a scalp laceration on the left parietal skull. The patient was immediately intubated for airway protection. CT of the head revealed a large left subdural hematoma. He was taken to the operating room for an emergent craniotomy. He was subsequently admitted to the surgical intensive care unit for further management. CT of the chest showed that patient has a right lower lobe obstructing pulmonary embolism. Ultrasound of bilateral lower extremities was negative for a deep vein thrombosis. PT and INR were within normal limits. Hypercoagulability panel was sent and was negative. Due to bleeding risk from subdural hematoma, patient was not a candidate for anticoagulation. He gradually improved and was discharged following sessions of physical and occupational therapy. The etiology of his presentation remained unclear. However, it is likely that patient lost consciousness secondary to a hypoxic event from the obstructing pulmonary embolism. Here we present a unique case of pulmonary embolism of unknown etiology in a patient with neurofibromatosis. Upon literature review, pulmonary neurofibromatosis is a common finding in patients who have mucosal neurofibromatosis. This is indeed the case for our patient, who not only has >70% of body surface area affected by neurofibromatosis, but also has mucosal neurofibromata. Pulmonary neurofibromatosis could erupt later on in life, or increase in number with age, thus predisposing patients to thromboembolism in the pulmonary vasculature. Our presentation aims at increasing clinical awareness of this possible link, so that timely therapeutic interventions would be provided. In particular, anticoagulation should be initiated for an optimal outcome. Mucosal neurofibromata.

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Figure 1

Mucosal neurofibromata

Id: 74 Death Due to Dysautonomia in a Patient with Fulminant Guillain-Barre Syndrome Mimicking Brain Death

D. Prasanna, N. Madan, S. Kapoor. Jacobi Medical Center, Bronx, New York.

Introduction: Guillain-Barre Syndrome (GBS) is characterized by acute areflexic paralysis and albumino-cytological dissociation on CSF. Fulminant GBS mimicking clinical brain death has been described in the literature and has overall poor prognosis. We describe, to the best of our knowledge, third reported case of death due to dysautonomia in a patient with fulminant GBS mimicking brain death.

Case: A healthy 44 year old male with no significant medical history presented to the Emergency department with acute respiratory failure requiring mechanical ventilation in the setting of vomiting, diarrhea and fever of 5 days duration. His neurological exam was consistent with severe flaccid quadraparesis and complete areflexia in all four extremities, anisocoria but intact extraocular movements. Computerized tomography scan of the head was unremarkable. On Day 2 of ICU stay, his neurological exam became consistent with clinical brain death as he developed coma, bilaterally fixed and dilated pupils and loss of extraocular movements. He was not triggering the ventilator. Initial Lumbar Puncture was non-confirmatory but repeat showed albumino-cytological dissociation. Electromyographic study showed severe demyelinating polyneuropathy. Electro-encephalographic study demonstrated intact cortical function with patient in persistently awake state. His ICU course was complicated by episodes of severe dysautonomia, extremes of blood pressure and symptomatic bradycardia requiring insertion of temporary pacemaker. He received one course of plasmapharesis and two courses of IV immunoglobulin with no clinical recovery. He underwent tracheostomy and gastrostomy tube placement and died about 6 weeks later after he developed sudden unexplained asystolic cardiac arrest related to his severe dysautonomia.

Discussion: fulminant GBS mimicking brain death is a rare occurrence with only about 20 cases been reported in the literature. Diagnosis is based on the clinical exam (quadraparesis and areflexia), CSf findings of albumino-cytological dissociation, typical electrophysiological findings and normal brain imaging. Serious and fatal autonomic dysfunction, like arrhythmias and extreme hypertension or hypotension, has been well described in patients with GBS. Three to eight percent of patients with GBS die from complications like sepsis, acute respiratory distress syndrome, pulmonary embolism, or in rare cases, unexplained cardiac arrest, related to dysautonomia. In the largest case series of 13 patients with fulminant GBS by Vargas et al, of the 3 deaths, 2 died from cardiac arrest due to dysautonomia and one died due to large anterior wall myocardial infarction. GBS mimicking brain death has a poor recovery rate and a high mortality, particularly, related to dysautonomia. Our patient died of unexplained asystolic cardiac arrest, most likely due to dysautonomia. Dysautonomia should be regarded as a poor prognostic sign in fulminant GBS and a marker of mortality in this population.

Id: 75 a Unique Presentation and Management of Hypercapnic Respiratory Failure in Overlap Syndrome

D. Lin, A.R. Abreu. Division of Pulmonary, Allergy, Critical Care, Sleep Medicine, UHealth Sleep Medicine Program, University of Miami - Miller School of Medicine, Miami, florida.

Introduction: Obstructive sleep apnea (OSA) is associated with a variety of metabolic and cardiovascular consequences. We present an unusual case of overlap syndrome requiring increased ventilator support during Positive Airway Pressure (PAP) therapy. Report of Case A 54 year-old male with a history of COPD, overweight, idiopathic bilateral pleural fibrosis, systemic hypertension presented with dyspnea, hypersomnia and witnessed apneas during sleep. The diagnostic overnight polysomnography revealed an Apnea-Hypopnea Index (AHI) of 13.9 and Respiratory Disturbance Index (RDI) of 28.9 per hour sleep and the arterial blood gas (ABG) during the daytime while awake showed evidence of chronic hypercapnia with a pH of 7.34 and a PaCO2 of 67mmHg. Subsequently, an optimal PAP titration was obtained with Bi-level PAP with Inspiratory Airway Pressure (IPAP) and Expiratory Airway Pressure (EPAP) of 15 and 10 cmH20 on room air, respectively. During an admission for acute hypercapnic, hypoxemic respiratory failure secondary to COPD exacerbation, his PaCO2 increased to 100mmHg and he was found to have pulmonary hypertension with a mean pulmonary artery pressure of 31 mmHg and a normal pulmonary capillary wedge pressure during a right heart catheterization. After this hospitalization he was discharged home on oxygen supplementation at 3 liters per minute, 24 hours per day. The pulmonary function test after recovery subsequently showed both severe obstructive and restrictive changes with forced expiratory volume of 0.7 liter per second, total lung capacity of 36% and diffusion capacity of 27% of predicted, without bronchodilator response. His arterial blood gas with a pH of 7.29 and a PaCO2 of 77 mmHg suggested acute on chronic respiratory acidosis. Patient refused to be re-admitted and chose to be treated as an outpatient with close clinical observation. An overnight polysomnography for PAP re-titration could not be performed. Over a period of 6 weeks, multiple ABGs were performed and the pressure support of his Bi-level PAP device was gradually increased to 10cmH2O until his PaCO2 decreased to 68 mmHg and his pH improved to 7.33 with resolution of acute respiratory acidosis. The EPAP remained unchanged at 10 cmH2O with a residual AHI below 5 per hour of sleep.

Conclusion: This is an unusual case of overlap syndrome with development of worsening ventilation and oxygenation after an episode of COPD exacerbation with the need for sequential ABGs to appropriately adjust the patient's noninvasive mechanical ventilator support in an outpatient setting avoiding hospital re-admission. Future studies should be performed to evaluate the benefit from an attended overnight polysomnography with capnometry monitoring, in patients with overlap syndrome, with the goal to optimize their therapy with noninvasive mechanical support without the need for invasive procedures.

Id: 76 Metastatic Glioblastoma – a Deceptive Killer

S. Naqvi, K. Allen. Pulmonary & Critical Care Medicine, University of Oklahoma Health Sciences Center, Edmond, Oklahoma. M. Khawar. Internal Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.

Introduction: Glioblastoma, a highly aggressive tumor of astrocytic cell origin accounts for 17.1% of all adult primary brain tumors. Extra cranial metastasis has been rarely reported in the literature & occurs in only 0.2% of all GBM patients. Lung metastasis is a prognostic factor of extremely poor outcome and may create a diagnostic dilemma. Case Report A 41 year old otherwise healthy male presented with 2 day history of acute onset occipital headache with nausea, vomiting and blurred vision. CT head revealed a mass, followed by an MRI showing 3.8 X 3.3 X 3.7cm enhancing lesion in the parietal-occipital region. As a part of cancer work up, PETCT showed a 3.2 X 4.4cm RUL lung mass (SUV 34.2) concerning for primary lung malignancy; no other extra cranial uptake was noticed. Differential included metastatic lung tumor vs primary brain malignancy with a separate pathological pulmonary process. Resection of the brain mass was consistent with Glioblastoma (WHO grade IV). Bronchoscopy with TBB and BAL was negative for malignant cells. Patient received one cycle of chemo and radiation and was admitted few days later with chest pain. Imaging revealed increasing size of RUL lung mass with hilar and pleural extension; there was also associated pleural effusion and rim enhancing lesions in the liver. FNA of the liver lesion was consistent with highly malignant, pleomorphic cells which stained negative for glial fibrillary acidic protein (GfAP). Serial analysis of pleural fluid revealed malignant cells, positive for GfAP & morphologically similar to occipital lesion. Palliative care was offered to the patient. Discussion first reported in 1928, extra cranial metastasis is a unique and rare manifestation of GBM. Metastatic disease at presentation can be hard to differentiate from a separate malignant process. With growing literature pointing towards aggressive metastatic nature of GBM, clinicians should be aware & attention should be paid to immunoreactivity along with other clinical details. When diagnosed, therapeutic options are few with life expectancy spanning only few months. We will also like to emphasize the importance of serial analysis of pleural fluid when a malignant disease is suspected; with positive yield approaching 80% on analysis of three separate specimens.

Conclusion: To our knowledge, there are only few cases of extra cranial GBM reported, with majority occurring month to years after treatment of the primary lesion. Extra cranial spread at presentation represents grave prognosis.

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Id: 77 a Critical Central Airway Obstruction Managed with Adjuvent Cardio-Pulmonary Bypass

H. Bhardwaj, H. Youness, B. Brown. Pulmonary & Critical Care Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.

Introduction: Central airway obstruction is one of the most serious emergencies faced by the physicians. Near total central airway obstruction can result in critical hypoxia and fatal cardiopulmonary arrest. Although, various interventional procedures areavailable to manage central airway obstruction; in extreme situations, use of adjuvant cardiopulmonary bypass(CPB) should be considered to maintain oxygenation during the procedure.

Case Report: A 58 year old male with history of squamous cell esophageal cancer presented with worsening dyspnea and stridor. He was in moderate respiratory distress with tachypnea at 26 breaths/min. Other vital signs & cardio-pulmonary examination were normal. ABG showed a Ph. of 7.29, PaCO2 at 64 mmHg, & PaO2 at 65 mmHg. CT-scan of the chest revealed significant distal tracheal stenosis 2 cm above the carina. Flexible bronchoscopy showed a critical ‘near total’ obstruction of the tracheal lumen by external compression and intraluminal invasion of tumor. Unfortunately, the procedure had to be aborted before the airway stenting due to significant hypoxia during the procedure. At this point, it was decided to attempt the stent placement in the operating room with adjuvant venovenous femoro-femoral CPB. After the initiation of CPB, suspension laryngoscopy was performed with the induction of the general anesthesia to secure airway access. Flexible bronchoscope was extended through the mouth and a guidewire was placed beyond the obstruction. A 18x60 mm silicon covered metal stent (AlveolusTM) was deployed over the wire resulting in resolution of the tracheal obstruction and patient's respiratory distress & stridor. Patient was successfully taken of the CPB. Discussion first introduced in 1990, airway stenting is a safe and effective intervention used in the maintenance of the airway patency in various central airway obstructions. Although certain types of airway stents (e.g. self-expanding metal stents) can be placed using flexible bronchoscope, the procedure can be very tricky in patients with near total occlusion of the central airways. Any airway manipulation in these patients can result in complete obstruction resulting in loss of ventilation and fatal hypoxia. This necessitates the use of adjuvent CPB to maintain the adequate oxygenation during the procedure in some extreme cases. There are two methods of CPB depending on the route of the returning oxygenated blood: veno-venous or veno-arterial and anyone of these methods can be used during complex airway procedures.

Conclusion: In extreme central airway obstructions, adjuvant CPB should be considered to maintain oxygenation. CPB can both provide adequate oxygenation during airway intervention and allow extra time to reestablish airway patency even if the ventilation is completely impossible.

Id: 78 An Unusual Cause of Recurrent Pleural Effusion

M.U. Khawar, M.A. Farooq. Internal Medicine, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma. H. Youness. Pulmonary & Critical Care Medicine, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma.

INTRODUCTION: Pancreatico-pleural fistula (PPf) is a rare condition in which pancreatic enzymes drain directly in to the pleural cavity, most commonly from an enlarging pseudocyst. We describe a patient with recurrent pleural effusion, who later was found to have PPF.

CASE PRESENTATION: A 63-year-old male with a past medical history of alcohol abuse and pancreatitis presented with dyspnea due to left sided pleural effusion. Pleural fluid analysis revealed exudative effusion. CT-Abdomen showed numerous pancreatic pseudocysts with fluid around the pancreas. Patient was discharged after symptomatic improvement of pancreatitis and pleural effusion. Patient's pleural effusion and symptoms recurred after a month and a chest tube had to be placed. Repeat pleural fluid analysis yielded blood tinged fluid with chemical analysis suggestive of an exudate and high amylase level of 15252 U/L. CTChest/ Abdomen was repeated showing multiple pancreatic pseudocysts with a paraesophageal fistulous tract extending to the mediastinum and associated with bilateral pleural effusion and a pericardial effusion. MRCP showed pancreatic pseudocysts with a fistula extending into the mediastinum, normal sized common bile duct and proximal pancreatic duct. Since the fistula was thought to originate from the distal pancreas, ERCP was not done. Patient was treated with a conservative management including NPO status, parenteral nutrition and Octreotide. His chest tube drainage was around 300-400 cc/day and decreased over four weeks. Repeat CT-Chest/Abdomen showed improvement in pleural effusion, pancreatic pseudocysts and resolution of the paraesophageal tract. Chest tube was removed thereafter.

DISCUSSION: PPf is a rare condition that occurs in 0.4% of patient presenting with pancreatitis and 4.5% of patients with pancreatic pseudocyst. It requires a high index of suspicion in patients with pleural effusion, history of pancreatitis and alcohol abuse. Rapidly re-accumulating pleural effusion with elevated amylase in pleural fluid is a clue to the diagnosis. MRCP or ERCP can well demonstrate the pancreatic pathology and fistulous tract. Initial treatment focuses on control of pleural effusion along with bowel rest and Octreotide infusion. Spontaneous closure is achieved in almost half of the cases over 2-3 weeks. If failed to resolve, endoscopic stenting or surgical intervention is required in carefully selected patients.

ID: 79 Myofibroblast Differentiation Requires Signaling Via Mtor Complexes 1 And 2

K.T. Ferguson. Medicine, University of Wisconsin, Madison, Wisconsin, Sherris, RestorGenex, Buffalo Grove, Illinois. E.E. Torr, K. Bernau, N.K. Sandbo. Allergy, Pulmonary, & Critical Care Medicine, University of Wisconsin, Madison, Wisconsin.

Rationale: Idiopathic pulmonary fibrosis is a progressive and deadly disorder with very few therapeutic options. Signaling via the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway has been implicated in myofibroblast differentiation, but in vivo work with these compounds has had mixed results. In these studies, we investigated the potential antifibrotic effect of the inhibitor RestorGenex 529 (8-(1-hydroxyethyl)-2-methoxy-3-(4-methoxybenzyloxy)-benzo[c]chromen-6-one) (RES-529), which disrupts the formation of mTOR complex-1 (mTORC1) and mTOR Complex-2 (mTORC2), on myofibroblast differentiation and function.

Methods: Primary cultures of human lung fibroblasts (HLf) were utilized to study the effects of RES-529 on TGf-b dependent signals, myofibroblast differentiation, and fibroblast function. Analogs of RES-529 were also investigated for possible more potent inhibition of myofibroblast differentiation and function.

Results: TGf-beta-induced phosphorylation of the downstream targets of mTORC1, p70S6 kinase and 4E-BP1 were dose-dependently inhibited by RES-529, with a maximal effect occurring at the 10 micromolar concentration. Additionally, phosphorylation of Akt at the S473 site, a reported downstream target of mTORC2, was dosedependently inhibited by RES-529. This was associated with inhibition of TGf-beta-induced myofibroblast differentiation by RES-529 with the same dose-dependency. Expression of fibronectin and collagen were similarly decreased by RES-529 at these concentrations. In contrast, there was no effect of RES-529 on canonical TGf-betainduced smad signaling, as assessed by receptor-associated smad 2/3 phosphorylation. Likewise, assessment of smad-dependent gene expression via smad-binding element driven luciferase assay showed no effect on TGf-betainduced SBE expression. Likewise, there was no observed anti-proliferative effect of RES-529 in HLF.

Conclusions: TGf-beta-induced myofibroblast differentiation requires signaling via both mTORC1 and mTORC2, as assessed by the novel dual TOR complex inhibitor RES-529. Disruption of this signaling pathway completely inhibited matrix protein expression, without effecting canonical smad-signaling or proliferation of HLF. These properties make inhibition of mTORC1/mTORC2 with RES-529 a potentially promising approach to inhibit the development of pulmonary fibrosis in vivo.

Id: 80 the Crucial Role of Pecam1 Induced by Simvastatin for Endothelial Barrier Function

W. Chen, M. Habeck, X. Ni, J.R. Jacobson. Medicine, Univ of Illinois at Chicago, Chicago, Illinois.

Rational: Platelet endothelial cell adhesion molecule (PECAM) plays an important role neutrophil adhesion and rolling respondent to inflammatory stimulus, endothelial cellular junction intact. Statin is HMGCoenzyme reductase inhibitor and has an outstanding anti-inflammatory effect and endothelial barrier functional enhancement effect. Previous studies indicated that atorvastatin-mediated PECAM-1 expression increase contributed to endothelial barrier functional enhancement effect, but the mechanism has not well characterized.

Methods/Results: In this study, our results confirmed that simvastatin had similar PECAM-1 induction effect in human pulmonary artery endothelial cells (HPAECs) at mRNA and protein level. At same time, simvastatin-mediated PECAM-1 induction enhanced endothelial barrier function by ECIS and monolayer HPAECs permeability measurement. Silence of PECAM-1 diminished endothelial barrier protective effect of simvastatin. Promoter activity assay showed that simvastatin-directed target sequence located in promoter 788-688 nucleotides region. Transcriptional factor binding sequence assay showed that GATA(R) and C/EBPb potentially bind these target sequence of PECAM-1 promoter. Deletion of GATA(R) and C/EBPb target sequence from PECAM-1 promoter showed that simvastatin- induced PECAM-1 promoter activity significantly decreased at condition of deletion of C/EBPb binding sequence but not for GATA(R) binding sequence. Microarray and western blotting study showed that simvastatin induced increase of C/EBPb as time dependent manner and significantly increase at 24 hours and reach to maximum at 48 hours. Cytosolic and nuclear fraction protein assay showed that 16 hours simvastatin treatment of HPAECs significantly increase C/EBPb in both of cytosolic and nucleusin HPAECs. Moreover, silence of C/EBPb diminished simvastatinmediated induction effect of PECAM-1 in HPAECs.

Conclusion: Simvastatin-mediated PECAM-1 induction plays an important role in endothelial barrier enhancement effect. Simvastatin induced PECAM-1 expression in HPAECs through induction of C/EBPb signal pathway.

Id: 81 Baseline Serum Interferon Beta/Alpha Ratio Predicts Response to Tumor Necrosis Factor Alpha Inhibitoin in Rheumatoid Arthritis

T. Wampler Muskardin, J.M. Dorschner, M.A. Jensen, T.B. Niewold. Mayo Clinic, Rochester, Minnesota P. Vashisht, University of Nebraska Medical Center, Omaha, Nebraska. R. Aggrawal, B.S. Chrabot. Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois. M. Kern, P.K. Gregersen. Center for Genomics and Human Genetics, feinstein Institute Medical Research, North Shore LIJ Health System, Manhasset, New York. S. Bridges. University of Alabama at Birmingham, Birmingham, Alabama.

Background: A major unmet goal in the treatment of rheumatoid arthritis (RA) is to define parameters of the abnormal immune response that allow for more rational selection of therapy. Prognostic serum markers would greatly expedite the process of achieving control over disease activity and allow us to avoid exposing patients to medication to which they will not respond. Biologic agents are costly, and having knowledge of which patients will respond would also allow for more efficient use of these medications. In this study, we evaluate baseline circulating type I interferon activity as a predictor of treatment response to TNf-alpha inhibition in RA.

Methods: We studied sera from a test set of 32 RA patients from the ABCoN Consortium and a validation set of 92 RA patients from the TETRAD registry. The test set included those with a good response or no response to TNf-alpha inhibition at 14 weeks by EULAR criteria. The validation set included subjects with good, moderate, or no response at 12 weeks by EULAR criteria. Total serum type I IFN activity, IFN-α and IFN-b activity were measured using a functional reporter cell assay.

Results: In the test set, an increased ratio of IFN-b to IFN-α (IFN b/α ratio) in pre-treatment serum associated with lack of response to TNf-α inhibition (p=0.013). A receiver-operator curve supported a ratio of 1.3 as the optimal cut-off. In the validation set, IFN b/α ratio >1.3 was a strong discriminator of non-response vs. either moderate or good response (OR=3.32, 95% CI 1.290-8.524). Anti-CCP antibody titer and mechanism of action of TNf-α inhibitor did not influence this relationship. In meta-analysis, pre-treatment IFN b/α ratio >1.3 was strongly associated with non-response to TNf-alpha inhibition (p=0.004).

Conclusion: The data demonstrate that pre-treatment circulating type I IFN activity can be predictive of therapeutic response to TNf-α inhibition in RA. The ratio of IFN b/α at baseline was the best predictive marker, suggesting that type I IFN subtype plays an important role in the therapeutic response. This test could be relevant in other conditions treated with TNf-alpha inhibitors, including inflammatory bowel disease and others.

Id: 82 Single Cell Gene Expression in Human Lupus Monocytes Reveals a Differential Impact of Interferon Signaling between Monocyte Subsets

Z. Jin, M.A. Jensen, J.M. Dorschner, D.M. Vsetecka, S. Amin, A. Makol, F.C. Ernste, T. Osborn, K.G. Moder, V. Chowdhary, T.B. Niewold. Division of Rheumatology and Department of Immunology, Mayo Clinic, Rochester, Minnesota.

Background/Purpose: Type I interferon (IFN) is a primary pathogenic factor in human systemic lupus erythematosus (SLE). IFN gene expression signatures have been observed in whole blood and mixed peripheral blood mononuclear cell populations in SLE, but the significance of this IFN signaling in immune cell subsets is still incompletely understood. We examined gene expression in individual SLE patient monocytes to explore the impact of increased IFN-induced transcription in single cells.

Methods: CD14++CD16- classical monocytes and CD14dimCD16+ non classical monocytes from SLE patients were purified by magnetic separation. The fluidigm C1 System was used for single cell capture and target gene pre-amplification, and equal numbers of classical and non-classical monocytes were studied. rtPCR was used to quantify expression of 87 monocyte-related genes, 17 of which were IFN-induced genes. An individual cell IFN score was generated based upon the expression of the 17 IFN-induced genes.

Results: Monocytes from the same SLE patient blood sample demonstrated varying levels of IFN-induced gene expression. In classical monocytes, high IFN score correlated with CD32a, IL1B, and IL8 expression. In non-classical monocytes, high IFN score was correlated with a larger number of inflammatory mediators, including cytokines such as IL12, IL23, and IL15; the immune receptors CD36, CD32a, CD80, and TLR7; and inflammatory signaling genes such as RELA, STAT2, IRAK1, IRAK4, and MyD88. CD16 transcripts were detected in a small group of classical monocytes, despite a lack of surface CD16 expression, suggesting that these cells may be transitioning from classical to non-classical subgroup. In these cells, CD16 expression was positively correlated with IFN score (p=0.019).

Conclusion: This study revealed striking diversity in the IFN responses of individual monocytes, and supports the idea that IFN signaling has distinct effects upon classical and non-classical monocytes, as the IFN signature correlated with a diverse panel of inflammatory mediators in the non-classical subset. IFN may contribute to the transition from classical to non-classical subtype in SLE. Single cell studies can reveal effects of IFN on single immune cells and uncommon populations such as non-classical monocytes, which may be masked in whole blood or mixed cell populations.

Id: 83 a Novel Model of Allergen-Specific Immunotherapy for House Dust Mite-Induced Asthma

S.J. Bracken, A.J. Adami, A. Mills, L.A. Guernsey, K. Parham, R.S. Thrall. Uconn Health Center, Unionville, Connecticut. E. Rafti, A.P. Matson, C.M. Schramm. Connecticut Children's Medical Center, Hartford, Connecticut.

BACKGROUND: Allergic asthma is a chronic lung disorder that affects 300 million people worldwide. Annual healthcare expenditures for asthma exceed $50 billion, yet current pharmacologic agents for asthma are primarily directed at symptom relief and have limited curative potential and poor prospects for reducing disease prevalence. Allergen-specific immunotherapy (AIT) is the only etiologic treatment for atopic disorders and has been shown to reduce symptoms and improve quality of life in asthma patients. Although the mechanisms by which AIT exerts its effects are actively being investigated, most studies have been conducted in humans and thus have been unable to evaluate the immunologic and physiologic changes occurring within the lungs. Our limited understanding of the mechanisms underlying the effects of AIT has been a barrier to the development of evidence-based clinical practice guidelines and universally-effective AIT for asthma. The goal of this study was to develop a clinically-relevant murine model to investigate the specific effects of AIT on allergic airway inflammation.

METHODS: IL-10gfp mice were intranasally administered house dust mite extract (HDM) over an 11 week period to induce allergic airway disease (AAD). Biweekly, subcutaneous AIT with a standardized HDM extract was initiated following development of early AAD (week 2) and continued for the remaining 9 weeks (18 total injections) at either a low dose (10 AU/ml) or high dose (200 AU/ml). Control animals received equal volumes of vehicle (normal saline phenol) in a time-matched manner. Upon sacrifice, cell differentials were assessed in bronchoalveolar lavage (BAL). Flow cytometric analysis was performed on hilar lymph node (HLN), lung tissue, and blood. Total serum IgE levels were assessed via ELISA. H&E and PAS-stained lung tissue sections were blindly scored for degree of inflammation and mucus production.

RESULTS: Both low and high-dose AIT significantly reduced total leukocyte counts (55% reduction; p <0.05) as well as frequency (45% reduction) and number (75% reduction) of eosinophils in the BAL relative to control animals. AIT also reduced frequency and number of lung Th2 cells (gated as CD4+ST2+ T cells), the main orchestrators of allergic inflammation, in a dose-dependent manner (p<0.05; high-dose AIT vs. control). While IgE levels were comparably elevated across groups before initiation of AIT, low- and high-dose AIT prevented further increases in IgE (73% and 35% reduction, respectively) relative to control animals. Total inflammation scores were significantly decreased by low- and high-dose AIT (p<0.05), with low-dose AIT having a drastic effect on number and size of inflammatory clusters (p<0.001 vs. control) and high-dose AIT more greatly affecting mucus production. Surprisingly, neither lownor high-dose AIT affected levels of local (HLN, lung) or systemic (blood) foxp3+ regulatory T cells, which are often elevated in the blood of patients receiving long-term AIT. In addition, AIT did not affect levels of IL-10+ alveolar macrophages, which we have shown to increase with tolerance to HDM. Additional studies are currently ongoing to evaluate AIT's potential to improve pulmonary function.

CONCLUSION: Herein, we describe the first clinically-relevant murine model of subcutaneous AIT for treatment of HDM-induced asthma. Results from this study demonstrate that AIT attenuates severity of AAD by affecting multiple parameters of allergic inflammation. Further evaluation of this model may yield valuable insight into the clinical mechanisms of AIT, which may in turn be applied to develop safer, more effective AIT protocols.

Funding: R01AI043573, f30HL122018

Id: 84 Exposure to House Dust Mite is Associated with the Appearance of Pro-Regulatory Gut Microorganisms and Increases in Pulmonary Regulatory T Cells

A.J. Adami, S.J. Bracken, L.A. Guernsey, R.S. Thrall. Immunology, University of Connecticut Health Center, West Hartford, Connecticut. J. Graf. Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut. C.M. Schramm. Pediatrics, Connecticut Childrens Medical Center, Hartford, Connecticut.

Introduction: Allergic asthma is a major global cause of morbidity, impacting over 300 million individuals. Asthma is characterized by an inappropriate immune response to harmless allergens such as house dust mite (HDM). Determining how the immune system loses tolerance to allergens is critical for development of curative asthma therapies. Much work by our laboratory and others has suggested that regulatory T cells (Tregs) play a critical role in the immunological disturbances behind asthma. A growing body of evidence points to the microbiome as a key player in Treg induction, and multiple studies have suggested that disruption of the microbiome may predispose individuals to developing asthma.

Methods: To explore the role of the microbiome in immune regulation, allergic airway disease (AAD) was induced in C57BL/6 mice (n=5-9 mice per group) by daily intranasal challenge with HDM extract for up to 11 weeks. Controls received intranasal saline. Fecal pellets were collected weekly during the exposure period. DNA was extracted from fecal pellets and the V4 region of the 16S rRNA gene was amplified. Amplified DNA was sequenced on an Illumina MiSeq and processed using QIIME software and the GreenGenes reference database. At 2, 5, and 11 weeks, groups of mice were sacrificed and cells were isolated from the lungs, broncho-alveolar lavage (BAL), and lung-draining (hilar) lymph node (HLN) for flow cytometric analysis and BAL cellular differentials. Comparisons were made using one- and two-way ANOVA and student's t-test.

Results: HDM induced AAD with classic signs of asthma between 2-5 weeks of exposure, including BAL eosinophilia and increased cellularity in the BAL and lungs. By 11 weeks of exposure, tolerance and disease suppression developed (i.e. resolution of BAL eosinophilia). This phenotype was associated with an increase in the proportion of Tregs in the HLN relative to control and acute HDM-exposed animals. Exposure to HDM was associated with distinct changes in the gut microbiome. After 5 weeks of HDM exposure, organisms of the genus Blautia increased from zero to between 5 and 10 percent of all organisms present in HDM-exposed mice while remaining undetectable in controls (p < 0.0001). A similar pattern was noted for genus Bacteroides, which increased to between 5 and 20 percent of all organisms by 11 weeks in HDM-exposed mice while remaining below 2 percent in controls (p < 0.0001). Intriguingly, members of both groups of organisms have been shown to influence immune regulation through the induction and maintenance of Tregs.

Conclusions: Development and suppression of HDM-induced AAD is associated with changes in the gut microbiome, including the appearance of organisms associated with immune regulation. These changes coincide with increases in pulmonary Tregs and reduction in disease markers, including BAL eosinophilia. These data demonstrate a correlation of an altered microbiome with the development of tolerance and suggest that the microbiome may play a role in the regulation of the immune response to inhaled allergens. This work was supported by NIAID (R01 AI-043573 to RST) and NHLBI (f30 HL-126324 to AJA).

Id: 85 T Cell-Mediated Systemic Recruitment of Neutrophils and Monocytes after Intranasal Staphylococcal Enterotoxin a Exposure

J. Svedova, A.T. Vella. Immunology, University of Connecticut Health Center, Farmington, Connecticut.

Staphylococcus aureus (S. aureus), a common pathogen colonizing the human nose and skin, is one of the most important causes of life-threatening infections. The high morbidity and mortality of S. aureus infections are ascribed to various virulence factors, including superantigens. Superantigens, such as staphylococcal enterotoxin A (SEA), mediate food poisoning and toxic shock syndrome and are also implicated in a number of pulmonary diseases and even sepsis. Their potency is embedded in their ability to directly bind to MHC II on antigen presenting cells and then crosslink with specific TCR Vb chains on T cells (e.g. Vb3 for SEA). The subsequent oligoclonal expansion and activation of T cells unleashes a massive immune response. Although SEA has been studied for decades, the exact mechanism of its action is not completely understood. We previously showed that intranasal administration of SEA in mice leads to a T-cell mediated recruitment of neutrophils and monocytes to the lungs. We hypothesized that the local migration of these innate cells to the lungs would be coincident with a systemic immune response. The aim of this study was to understand the early systemic responses to intranasal SEA exposure, and in particular, to illuminate the mechanism of how SEA-activated T cells orchestrate migration and activation of neutrophils and monocytes. Intranasal instillation of SEA in mice caused a rapid influx of neutrophils and monocytes to peripheral blood, followed by their recruitment to spleen and draining and non-draining lymph nodes. This migration was dependent on T cells since TCR bδ-/- mice were unable to recruit these cells unless they received an adoptive T cell transfer. Confocal microscopy imaging showed that the recruited neutrophils localized to the T cell zone within the lymph nodes. Utilizing qPCR and multiplex assays, an increased expression of several known neutrophil and monocyte chemotactic factors, including CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL7 was detected in the lymph node tissue as early as 1 h after SEA exposure. By sorting specific cell populations we found that these chemokines were differentially expressed. Interestingly, CXCL1 and CXCL2 were mostly produced by dendritic cells while CCL3 and CCL4 were made by SEAactivated Vb3+ T cells. Our future studies will focus on illuminating the mechanism of chemokine release by T cells and dendritic cells and on describing the consequences of neutrophil and monocyte recruitment to lymph nodes. Understanding how SEA-activated T cells affect the migration and function of these innate cells is not only important for designing appropriate countermeasures against SEA-impacted diseases but it will also enhance our knowledge of inflammatory mechanisms.

Id: 86 a Commensal Bacteria-Derived Lipid, Deficient in the Serum of Multiple Sclerosis Patients, Causes Attenuation of Experimental Autoimmune Encephalomyelitis and An Increase in Cd39+ Tregs

E.J. Anstadt, M. Fujiwara, R.B. Clark. Immunology, University of Connecticut Health Center, Farmington, Connecticut. F. Nichols. Oral and Health Diagnostic Sciences, University of Connecticut Health Center, Farmington, Connecticut. R. Nemati. Chemistry, University of Connecticut, Storrs, Connecticut.

The role of the microbiome in multiple sclerosis (MS) remains unknown. We have identified a Toll-like receptor 2 agonist produced by bacteria of the phylum Bacteroidetes (composing 30% of the normal gastrointestinal flora), called Lipid 654 (L654). We found that L654 can be recovered in healthy human serum and that levels are significantly lower in patients with MS. To determine the relevance of this finding, we administered L654 to mice that were actively immunized to induce experimental autoimmune encephalomyelitis (EAE) and found significant attenuation of disease. The mechanism underlying the attenuation induced by L654 is not yet known. CD39, the ratelimiting ectonucleotidase responsible for the conversion of adenosine triphosphate to adenosine, has been implicated as a critical suppressive mechanism of foxP3+ T regulatory cells (Tregs) in both EAE and MS. In MS, circulating CD39+ Tregs are decreased and also impaired in their ability to control Th17 proliferation. Intriguingly, CD39+ Tregs are increased during periods of remission in MS patients. The purpose of this study was to 1) determine if L654 could attenuate clinical disease severity in a transfer model of EAE to better test the effect of L654 on activated, encephalitogenic T cells and 2) determine the role of CD39+ Tregs in the disease inhibition caused by L654 administration. An adoptive transfer model of EAE in SJL/J mice was utilized. Mice received transfer of PLP-specific activated T cells and EAE developed 8-11 days later. A 1 ug dose of L654 was administered to mice at day 0 or at day 0 and 7. Mice were observed for disease severity or spinal cords were harvested for cellular analysis. L654 administration significantly attenuated clinical disease severity in the transfer model of EAE (p < 0.0001). Cellular analysis of mononuclear cells in the spinal cord at day 18 showed a significant decrease in total cell number (p = 0.02) as well as a titrating decrease in percentage of IL-17 producing, IFNγ producing and IL-17/IFNγ double-producing CD4+ T cells in mice administered L654. At day 10, cellular analysis showed a decrease in total mononuclear cell number and an increase in percentage of CD39+ Tregs in mice administered L654. Therefore, L654 is of significant interest as a potential treatment for MS, as it may increase CD39+ Tregs, known to be deficient in MS patients, and potentially suppress clinical disease. Future directions will determine if the suppression induced by L654 is dependent on CD39+ Tregs, and what effect L654 administration has on spontaneous relapses in EAE.

Id: 87 Inhibition of Ezh2 Simultaneously Decreases Pro-Inflammatory and Enhances Antiinflammatory Microglial Phenotypes

E. Koellhoffer, R. Ritzel, L. McCullough. Neuroscience, University of Connecticut Health Center, Farmington, Connecticut. J. Grenier. Immunology, University of Connecticut Health Center, Farmington, Connecticut.

The aging central nervous system (CNS) is characterized by a chronic baseline elevation in inflammation which contributes to the pathogenicity of many neurological diseases. Understanding the source of this inflammation is critical as manipulations may allow for the reversal of these age-related changes, thereby reducing the risk of CNS disease. This has led to a focus on the role of microglia, the resident innate immune cell of the brain, in CNS inflammation. Microglia possess the ability to adapt to the environment by altering their phenotype between a resting (M0) state to either an activated pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. In the setting of aging, microglia have a more predominant M1 phenotype at baseline. However, the molecular mechanisms underlying these changes with age remain largely unknown. Recent work has shown that Jumonji Domain Containing 3 (Jmjd3), a histone demethylase, is essential for M2 polarization. By demethylating histone H3 lysine 27 trimethylation (H3K27me3) to a monomethylation mark (H3K27me1), Jmjd3 alters the epigenetic landscape of target promoters from a transcriptionally repressed state to a transcriptionally active state. This in turn allows for expression of target genes to occur. In contrast, Enhancer of Zeste Homologue 2 (Ezh2) functions in an opposite manner by selectively writing the H3K27me3 mark and repressing transcription of target genes. We hypothesized that inhibition of Ezh2 may tilt the balance between Jmjd3 and Ezh2 respective activities in favor of higher Jmjd3 activity, thereby enhancing polarization toward an anti-inflammatory and neuroprotective M2 phenotype. Mixed glial cultures were isolated from P0.5-P2 C57Bl/6 mice and cultured for 10-14 days before microglia were isolated. Primary microglia were treated with GSK343 (an inhibitor of Ezh2) for 1hr before being stimulated with LPS+IFNγ or IL-4. After 4hr or 24hr RNA was isolated for qRT-PCR analysis. In our model of primary microglia, pre-treatment with GSK343 significantly diminished LPS+IFNγ- induced IL6 and IL1B expression at both 4hr and 24hr (p<0.05). Additionally, Ezh2 inhibition rescued the expression of M2-associated genes ARG1, CD206, and IRf4 at 24hr (p<0.05) which were otherwise significantly down-regulated in the presence of LPS+IFNγ. In line with our hypothesis, inhibition of Ezh2 also resulted in an augmented IL-4- induced upregulation of ARG1, CD206, and IRf4 at 4hr (p<0.05). Our data implies that there is epigenetic regulation of M1/M2 microglia polarization and that Ezh2 has a role in this regulation. Pre-treatment with an inhibitor of Ezh2 limits LPS+IFNγ-induced microglial M1 polarization and restores M2-associated gene expression to baseline. This suggests that altering the balance between Ezh2 and Jmjd3 allows for reprograming of both M1- and M2-associated genes.

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