Life Sciences Commons^™

Genetic Determinants Of Cerebral Edema In Severe Traumatic Brain Injury: A Pilot Study Of The Role Of Cacna1 And Aqp4 Gene Mutations, Raphael A. Carandang, Susanne Muehlschlegel, Wiley R. Hall, Cynthia Ouillette, Robert H. Brown Jr.

Dr Robert Brown

Cerebral edema is the one of the most significant predictors of poor outcome after traumatic brain injury. It is still unclear what the pathophysiological and cellular mechanisms and predictors of post-traumatic edema are. The exponential growth in genetic information has opened an avenue for investigation in traumatic brain injury and implicated specific genes in the pathophysiology of post-traumatic injury edema. Two examples are the Aquaporin-4 and CACNA1 genes, which respectively encode water and calcium channels. The Aquaporin-4 gene on chromosome 18q11.2-12.1 encodes the Aquaporin-4 protein (AQP4) water channel. AQP4 is one of the bidirectional high capacity water channels that is …

Homologous Recombination Mediates Functional Recovery Of Dysferlin Deficiency Following Aav5 Gene Transfer, William E. Grose, K. Reed Clark, Danielle Griffin, Vinod Malik, Kimberly M. Shontz, Chrystal L. Montgomery, Sarah Lewis, Robert H. Brown Jr., Paul M. L. Janssen, Jerry R. Mendell, Louise R. Rodino-Klapac

Dr Robert Brown

The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique …

Adrenoceptor Function And Expression In Bladder Urothelium And Lamina Propria, Christian Moro, Lotti Tajouri, Russ Chess-Williams

Christian Moro

Microrna Signature In Alcoholic Liver Disease, Shashi Bala, Gyongyi Szabo

Gyongyi Szabo

Alcoholic liver disease (ALD) is a major global health problem. Chronic alcohol use results in inflammation and fatty liver, and in some cases, it leads to fibrosis and cirrhosis or hepatocellular carcinoma. Increased proinflammatory cytokines, particularly TNF alpha, play a central role in the pathogenesis of ALD. TNF alpha is tightly regulated at transcriptional and posttranscriptional levels. Recently, microRNAs (miRNAs) have been shown to modulate gene functions. The role of miRNAs in ALD is getting attention, and recent studies suggest that alcohol modulates miRNAs. Recently, we showed that alcohol induces miR-155 expression both in vitro (RAW 264.7 macrophage) and in …

Human Monoclonal Antibody Hcv1 Effectively Prevents And Treats Hcv Infection In Chimpanzees, Trevor J. Morin, Teresa J. Broering, Brett A. Leav, Barbara M. Blair, Kirk J. Rowley, Elisabeth N. Boucher, Yang Wang, Peter S. Cheslock, Michael Knauber, David B. Olsen, Steve W. Ludmerer, Gyongyi Szabo, Robert W. Finberg, Robert H. Purcell, Robert E. Lanford, Donna M. Ambrosino, Deborah C. Molrine, Gregory J. Babcock

Gyongyi Szabo

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, …

Il-1 Receptor Antagonist Ameliorates Inflammasome-Dependent Alcoholic Steatohepatitis In Mice, Jan Petrasek, Shashi Bala, Timea Csak, Dora Lippai, Karen Kodys, Victoria Menashy, Matthew Barrieau, So-Yun Min, Evelyn A. Kurt-Jones, Gyongyi Szabo

Gyongyi Szabo

Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1beta. IL-1beta, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1beta maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra-treated mice as well as 3 mouse models deficient in regulators of IL-1beta activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1beta signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1beta was due to upregulation of Casp-1 activity and inflammasome activation. The …

Plasma Microrna Profiles Distinguish Lethal Injury In Acetaminophen Toxicity: A Research Study, Jeanine Ward, Shashi Bala, Jan Petrasek, Gyongyi Szabo

Gyongyi Szabo

AIM: To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice. METHODS: Using plasma from APAP poisoned mice, either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed, we screened commercially available murine microRNA libraries (SABiosciences, Qiagen Sciences, MD) to evaluate for unique miRNA profiles between these two dosing parameters. RESULTS: We distinguished numerous, unique plasma miRNAs both up- and downregulated in lethally compared to sublethally dosed mice. Of note, many of the greatest up- and downregulated miRNAs, namely 574-5 p, 466 g, 466 f-3p, 375, 29 c, and 148 a, have …

Lipopolysaccharide Induces And Activates The Nalp3 Inflammasome In The Liver, Michal Ganz, Timea Csak, Bharath D. Nath, Gyongyi Szabo

Gyongyi Szabo

AIM: To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide (LPS)-induced stimulation in the liver. METHODS: Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5 mug/g bodyweight LPS and sacrificed 2, 4, 6, 18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase (ALT) levels. To determine if LPS stimulation in the liver led to activation of the inflammasome, real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome. Enzyme-linked immunosorbent assays were used to determine the protein …

Dissecting The Workforce And Workplace For Clinical Endocrinology, And The Work Of Endocrinologists Early In Their Careers, Claude Desjardins, Mark A. Bach, Anne R. Cappola, Ellen W. Seely, Ronald G. Ehrenberg

Ronald G. Ehrenberg

[Excerpt] No national mechanism is in place for an informed, penetrating, and systematic assessment of the physician workforce such as that achieved by the National Science Foundation (NSF) for the periodic evaluation of the nation’s scientists and engineers. Likewise, knowledge of the workforce for clinical research is enigmatic and fragmentary despite the serial recommendations of “blue-ribbon” panels to establish a protocol for the recurrent assessment of clinical investigators early in their careers. Failure to adopt a national system for producing timely, high-quality data on the professional activities of physicians limits the application of improvement tools for advancing clinical investigation and …

Self-Reported Colorectal Cancer Screening Of Medicare Beneficiaries In Family Medicine Vs. Internal Medicine Practices In The United States: A Cross-Sectional Study, Angela (Young) Higgins, Anna R. B. Doubeni, Karon L. Phillips, Adeyinka O. Laiyemo, Becky A. Briesacher, Jennifer Tjia, Chyke A. Doubeni

Jennifer Tjia

BACKGROUND: The benefit of screening for decreasing the risk of death from colorectal cancer (CRC) has been shown, yet many patients in primary care are still not undergoing screening according to guidelines. There are known variations in delivery of preventive health care services among primary care physicians. This study compared self-reported CRC screening rates and patient awareness of the need for CRC screening of patients receiving care from family medicine (FPs) vs. internal medicine (internists) physicians.

METHODS: Nationally representative sample of non-institutionalized beneficiaries who received medical care from FPs or internists in 2006 (using Medicare Current Beneficiary Survey). The main …

Increased Microrna-155 Expression In The Serum And Peripheral Monocytes In Chronic Hcv Infection, Shashi Bala, Yaphet Tilahun, Odette Taha, Hawau Alao, Karen Kodys, Donna Catalano, Gyongyi Szabo

Gyongyi Szabo

BACKGROUND: Hepatitis C Virus (HCV), a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA) regulate inflammation (miR-155, -146a and -125b) as well as hepatocyte function (miR-122). METHODS: Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection. RESULTS: We found that monocytes from chronic HCV …

Consumption Of High Ω-3 Fatty Acid Diet Suppressed Prostate Tumorigenesis In C3(1) Tag Mice, Juliana A. Akinsete, Gabriela Ion, Theodore R. Witte, W. Elaine Hardman

Elaine Hardman Ph.D.

Prostate cancer incidence and mortality are high in the Western world and high ω-6/ω-3 PUFA in the Western diet may be a contributing factor. We investigated whether changing from a diet that approximates ω-6 fat content of the Western diet to a high ω-3 fat diet at adulthood might reduce prostate cancer risk. Female SV 129 mice that had consumed a high ω-6 diet containing corn oil for 2 weeks were bred with homozygous C3(1)Tag transgenic male mice. All male offspring were weaned to the corn oil diet (CO) until postpuberty when half of the male offspring were transferred to …

Global Analysis Of Gene Expression Changes During Retinoic Acid-Induced Growth Arrest And Differentiation Of Melanoma: Comparison To Differentially Expressed Genes In Melanocytes Vs Melanoma, Mary H. Estler, Goran Boskovic, James Denvir, Sarah Miles, Donald A. Primerano, Richard M. Niles

Donald A. Primerano

BACKGROUND: The incidence of malignant melanoma has significantly increased over the last decade. Some of these malignancies are susceptible to the growth inhibitory and pro-differentiating effects of all-trans-retinoic acid (RA). The molecular changes responsible for the biological activity of RA in melanoma are not well understood. RESULTS: In an analysis of sequential global gene expression changes during a 4-48 h RA treatment of B16 mouse melanoma cells, we found that RA increased the expression of 757 genes and decreased the expression of 737 genes. We also compared the gene expression profile (no RA treatment) between non-malignant melan-a mouse melanocytes and …

Global Analysis Of Gene Expression Changes During Retinoic Acid-Induced Growth Arrest And Differentiation Of Melanoma: Comparison To Differentially Expressed Genes In Melanocytes Vs Melanoma, Mary H. Estler, Goran Boskovic, James Denvir, Sarah Miles, Donald A. Primerano, Richard M. Niles

Richard M. Niles

BACKGROUND: The incidence of malignant melanoma has significantly increased over the last decade. Some of these malignancies are susceptible to the growth inhibitory and pro-differentiating effects of all-trans-retinoic acid (RA). The molecular changes responsible for the biological activity of RA in melanoma are not well understood. RESULTS: In an analysis of sequential global gene expression changes during a 4-48 h RA treatment of B16 mouse melanoma cells, we found that RA increased the expression of 757 genes and decreased the expression of 737 genes. We also compared the gene expression profile (no RA treatment) between non-malignant melan-a mouse melanocytes and …

Expression And Function Of Hypoxia Inducible Factor-1 Alpha In Human Melanoma Under Non-Hypoxic Conditions, Caroline N. Mills, Sandeep S. Joshi, Richard M. Niles

Richard M. Niles

Background Hypoxia inducible factor-1 alpha (HIF-1α) protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP), vertical growth phase (VGP) and metastatic (MET) melanomas. Results HIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part …

Retinoic Acid Decreases Atf-2 Phosphorylation And Sensitizes Melanoma Cells To Taxol-Mediated Growth Inhibition, Ying Huang, Jennifer Minigh, Sarah Miles, Richard N. Niles

Richard M. Niles

Cutaneous melanoma is often resistant to chemo- and radiotherapy. This resistance has recently been demonstrated to be due, at least in part, to high activating transcription factor 2 (ATF-2) activity in these tumors. In concordance with these reports, we found that B16 mouse melanoma cells had higher levels of ATF-2 than immortalized, but non-malignant mouse melanocytes. In addition, the melanoma cells had a much higher amount of phosphorylated (active) ATF-2 than the immortalized melanocytes. In the course of determining how retinoic acid (RA) stimulates activating protein-1 (AP-1) activity in B16 melanoma, we discovered that this retinoid decreased the phosphorylation of …

Mid-Gestation Angiogenic Biomarker Levels Are Increased In Women At High Risk For Preeclampsia, Tiffany A. Moore Simas, Sybil L. Crawford, Susie Bathgate, Jing Yang, Laura Robidoux, Melissa J. Moore, Sharon E. Maynard

Sybil L. Crawford

Background: Pre-pregnancy hypertension and diabetes mellitus, multiple gestations, prior preeclampsia, are significant risk factors for preeclampsia. Whether altered maternal levels of angiogenic factors contribute to increased preeclampsia risk in these conditions is unknown. Our objective was to compare maternal serum angiogenic biomarker levels in women with major risk factors for preeclampsia and healthy controls. Methods: Women presenting for prenatal care were enrolled if they had one of the following preeclampsia risk factors: pre-pregnancy hypertension and/or diabetes mellitus, nulliparity with pre-pregnancy BMI>30, multiple gestations, or prior preeclampsia. Healthy control pregnancies without these risk factors were enrolled for comparison. Maternal serum …

Serum Microrna-122 And Mir-155 As Biomarkers Of Liver Injury And Inflammation In Models Of Acute And Chronic Liver Disease, Shashi Bala, Jan Petrasek, Shiv Mundkur, Donna Catalano, Jeanine Ward, Ivan Levin, Hawau Alao, Karen Kodys, Gyongyi Szabo

Gyongyi Szabo

Background: MicroRNAs (miRs) are small non-coding molecules that regulate gene expression. MiRs expression levels change not only in diseased tissues but also in circulation. Further, miRs are stable in frozen samples that make them attractive for biomarker discovery. Recent reports suggest altered expression of circulating miRNAs in various diseases. MiR-122 is highly abundant in hepatocytes where it regulates different metabolic pathways while miR-155 is a central regulator of inflammation. The aim of this study was to evaluate circulating miRNAs as potential markers of hepatocyte damage and inflammation in liver diseases. Methods: Serum/plasma and liver samples were collected from C57/BL6 mice …

Increased Oxidative Capacity Of Circulating Polymorphonuclear Neutrophils (Pmns) In Non-Diabetic Nash Patients, Timea Csak, Karen Kodys, Angela Dolganiuc, Dora Lippai, Michal Ganz, Christopher Marshall, Gyongyi Szabo

Gyongyi Szabo

Background: Inflammation and oxidative stress are key factors in the pathogenesis of non-alcoholic steatohepatitis (NASH). Polymorphonuclear neutrophils are capable to produce significant amounts of reactive oxygen species (ROS) via the NADPH oxidase complex. Increased hepatic neutrophil infiltration has been described in steatohepatitis. We aimed to investigate the in vitro ROS generation by neutrophils of NASH patients and the hepatic NADPH oxidase activity in murine steatohepatitis. Material and methods: PMNs were isolated from peripheral blood of NASH patients (n=16) and healthy controls (n=16). In vitro ROS production was measured by luminol chemiluminescence after phorbol myristate acetate (PMA) or opsonized zymosan stimulation. …

Caspase-1-Dependent, Il-1ss-Mediated Alcoholic Steatohepatitis Is Ameliorated By Il-1 Receptor Antagonist Treatment In Mice, Jan Petrasek, Shashi Bala, Dora Lippai, Karen Kodys, Victoria Menashy, Matthew Barrieau, Evelyn A. Kurt-Jones, Gyongyi Szabo

Gyongyi Szabo

Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of pro-inflammatory cytokines, including interleukin (IL)-1β. IL-1β, Type-I IL-1 receptor (IL1R1) and IL-1 receptor antagonist (IL-1Ra) are all part of the IL-1 superfamily that play a role in inflammation. IL-1β maturation is dependent on Caspase-1. Using wild-type (WT), Caspase-1-, IL-1R1- and IL-1Ra deficient mice fed with Lieber-DeCarli alcohol or control diet, we have identified that signaling mediated by the active IL-1β was required for development of alcohol-induced steatosis, inflammation and injury. Increased IL-1β was due to upregulation of Caspase-1 activity and inflammatory activation. The pathogenic role of IL-1 signaling in …

Cd81/Cd9 Tetraspanins Aid Plasmacytoid Dendritic Cells In Recognition Of Hcv-Infected Cells And Induction Of Ifnα, Shuye Zhang, Karen Kodys, Gyongyi Szabo

Gyongyi Szabo

Recognition of hepatitis C virus (HCV)-infected hepatocyes and interferon (IFN) induction are critical in antiviral immune response. We hypothesized that cell-cell contact between pDCs and HCV-infected cells was required for IFNα induction via involvement of cell surface molecules. Co-culture of human peripheral blood mononuclear cells (PBMCs) with genotype 1a full length HCV genomic replicon cells (FL) or genotype 2a JFH-1 virus infected hepatoma cells (JFH-1), not with uninfected hepatoma cells (Huh7.5), induced IFNα production. Depletion of pDCs from PBMCs attenuated IFNα release and purified pDCs produced high levels of IFNα after co-culture with FL replicons or JFH-1 infected cells. IFNα …

Inhibition Of Tlr8- And Tlr4-Induced Type I Ifn Induction By Alcohol Is Different From Its Effects On Inflammatory Cytokine Production In Monocytes, Maoyin Pang, Shashi Bala, Karen Kodys, Donna Catalano, Gyongyi Szabo

Gyongyi Szabo

BACKGROUND: Prolonged alcohol consumption is a significant co-factor in the progression of chronic viral infections including hepatitis C and HIV, which are both single-stranded RNA viruses. Toll like receptor 8 (TLR8), a pattern recognition receptor expressed in monocytes, senses viral single stranded RNA as a danger signal and leads to the induction of Type I interferon (IFN) as well as the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF alpha). Lipopolysaccharide (LPS), a Toll like receptor 4 (TLR4) ligand, was shown to affect inflammatory cell activation after alcohol consumption and in HIV and HCV infections. Here we hypothesized that alcohol exposure …

Phase 2 Clinical Trial Of A Recombinant Adeno-Associated Viral Vector Expressing Α1-Antitrypsin: Interim Results, Terence R. Flotte, Bruce C. Trapnell, Margaret Humphries, Brenna Carey, Roberto Calcedo, Farshid Rouhani, Martha Campbell-Thompson, Anthony T. Yachnis, Robert A. Sandhaus, Noel G. Mcelvaney, Christian Mueller, Louis M. Messina, James M. Wilson, Mark L. Brantly, David R. Knop, Guo-Jie Ye, Jeffrey D. Chulay

Christian Mueller

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with …

Induction Of Group Ivc Phospholipase A2 In Allergic Asthma: Transcriptional Regulation By Tnf-Α In Bronchoepithelial Cells, Justin S. Bickford, Kimberly J. Newsom, John-David Herlihy, Christian Mueller, Benjamin Keeler, Xiaolei Qiu, Jewell N. Walters, Nan Su, Shannon M. Wallet, Terence R. Flotte, Harry S. Nick

Christian Mueller

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus, Aspergillus fumigatus (Af), in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC phospholipase A2 (cPLA2γ or PLA2G4C). Our results infer that Af extract can induce cPLA2γ levels directly in eosinophils while induction in lung epithelial cells is most likely a consequence of TNF-α secretion by …

Sustained Transgene Expression Despite T Lymphocyte Responses In A Clinical Trial Of Raav1-Aat Gene Therapy, Mark L. Brantly, Jeffrey D. Chulay, Lili Wang, Christian Mueller, Margaret Humphries, L. Terry Spencer, Farshid Rouhani, Thomas J. Conlon, Roberto Calcedo, Michael R. Betts, Carolyn Spencer, Barry J. Bryne, James M. Wilson, Terence R. Flotte

Christian Mueller

Alpha-1 antitrypsin (AAT) deficiency is well-suited as a target for human gene transfer. We performed a phase 1, open-label, dose-escalation clinical trial of a recombinant adeno-associated virus (rAAV) vector expressing normal (M) AAT packaged into serotype 1 AAV capsids delivered by i.m. injection. Nine AAT-deficient subjects were enrolled sequentially in cohorts of 3 each at doses of 6.9 x 10(12), 2.2 x 10(13), and 6.0 x 10(13) vector genome particles per patient. Four subjects receiving AAT protein augmentation discontinued therapy 28 or 56 days before vector administration. Vector administration was well tolerated, with only mild local reactions and 1 unrelated …

Sustained Mirna-Mediated Knockdown Of Mutant Aat With Simultaneous Augmentation Of Wild-Type Aat Has Minimal Effect On Global Liver Mirna Profiles, Christian Mueller, Qiushi Tang, Alisha Gruntman, Keith S. Blomenkamp, Jeffrey H. Teckman, Lina Song, Phillip D. Zamore, Terence R. Flotte

Christian Mueller

Alpha-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently …