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Sample records for coxsackie b-adenovirus receptor-pseudotyped

  1. Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus?

    PubMed Central

    Granio, Ophélia; Porcherot, Marine; Corjon, Stéphanie; Kitidee, Kuntida; Henning, Petra; Eljaafari, Assia; Cimarelli, Andrea; Lindholm, Leif; Miossec, Pierre; Boulanger, Pierre; Hong, Saw-See

    2009-01-01

    Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BVCAR) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BVCAR to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BVCAR-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BVCARGFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BVCAR-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BVCAR to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BVCAR-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BVCAR in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BVCAR-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BVCAR-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BVCAR-Ad5GFP complex. Various situations in vitro or ex vivo in which our BVCAR-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed. PMID:19357170

  2. Myocardial imaging. Coxsackie myocarditis

    SciTech Connect

    Wells, R.G.; Ruskin, J.A.; Sty, J.R.

    1986-09-01

    A 3-week-old male neonate with heart failure associated with Coxsackie virus infection was imaged with Tc-99m PYP and TI-201. The abnormal imaging pattern suggested myocardial infarction. Autopsy findings indicated that the cause was myocardial necrosis secondary to an acute inflammatory process. Causes of abnormal myocardial uptake of Tc-99m PYP in pediatrics include infarction, myocarditis, cardiomyopathy, bacterial endocarditis, and trauma. Myocardial imaging cannot provide a specific cause diagnosis. Causes of myocardial infarction in pediatrics are listed in Table 1.

  3. Coxsackie A virus-associated herpetiform angina.

    PubMed

    Zavate, O; Avram, G; Pavlov, E; Burlea-Iriciuc, A; Ivan, A; Cotor, F

    1984-01-01

    Virological investigations were performed in 25 herpangina cases that occurred in a large urban centre in the summer season of 1982. Twelve Coxsackie A virus strains (10 Coxsackie A5 strains, one Coxsackie A4 and one Coxsackie A6 strain) could be isolated from 9 (36.0%) of the patients. PMID:6324449

  4. Model generation of viral channel forming 2B protein bundles from polio and coxsackie viruses

    E-print Network

    Watts, Anthony

    Model generation of viral channel forming 2B protein bundles from polio and coxsackie viruses by enteroviruses such as polio and coxsackie viruses with two transmembrane domains. The protein is found to make this has on the in vivo activity of 2B. Keywords: 2B, polio virus, coxsackie virus, membrane proteins

  5. ZEB1 limits adenoviral infectability by transcriptionally repressing the Coxsackie virus and Adenovirus Receptor

    E-print Network

    Lacher, Markus D; Shiina, Marisa; Chang, Peter; Keller, Debora; Tiirikainen, Maarit I; Korn, W Michael

    2011-01-01

    Molecular Cancer 2011, 10:91 http://www.molecular-cancer.com/content/10/1/91 Page 16 of 16 44. Ginzinger DG: GeneMolecular Cancer 2011, 10:91 http://www.molecular-cancer.com/content/10/1/91 Background The coxsackie virus and adenovirus receptor (CAR), encoded by the CXADR gene,

  6. Medical and Surgical Treatment in Pediatric Orbital Myositis Associated with Coxsackie Virus

    PubMed Central

    Gil, Pedro; Gil, João; Paiva, Catarina; Castela, Guilherme; Castela, Rui

    2015-01-01

    Purpose. To report a case of orbital myositis associated with Coxsackie virus and its medical and surgical approach. Methods. Complete ophthalmological examination and imaging and analytical investigation were performed. Results. A 6-year-old male presented with subacute painless binocular horizontal diplopia. Examination revealed bilateral best-corrected visual acuity (BCVA) of 20/20 and right eye 45-prism-dioptre (PD) esotropia in near and distance fixations, with no motility restrictions. Serologic screening was positive for Coxsackie virus acute infection and computerized tomography (CT) suggested right eye medial rectus orbital myositis. An oral corticosteroid 1.0?mg/kg/day regimen was started. A new CT after two months showed symmetrical lesions in both medial rectus muscles. Corticosteroids were increased to 1.5?mg/kg/day. After imagiological resolution on the 4th month, alternating 45?PD esotropia persisted. Bilateral 7?mm medial rectus recession was performed after 1 year without spontaneous recovery. At 1-year follow-up, the patient is orthophoric with 200?? stereopsis and bilateral 20/20 BCVA. Conclusions. To our knowledge, this is the first reported case of orbital myositis associated with Coxsackie virus. This is also the first reported case of isolated strabismus surgery after orbital myositis in pediatric age, highlighting the favourable aesthetic and functional outcomes even in cases of late ocular motility disorders. PMID:26550508

  7. ACQUISITION OF ANTIBODIES TO VARIOUS COXSACKIE AND ECHO VIRUSES AND HEPATITIS A VIRUS IN AGRICULTURAL COMMUNAL SETTLEMENTS IN ISRAEL

    EPA Science Inventory

    A seroepidemiological study was conducted to measure the antibody prevalence for eight different enteric viruses. These include seven 'classical' enteroviruses, ie, Coxsackie virus types A9, B1, B3, B4 and three ECHO virus types 4,7, and 9, as well as hepatitis A virus (HAV), rec...

  8. DEVELOPMENT OF A RAPID, QUANTITATIVE METHOD FOR THE DETECTION OF INFECTIVE COXSACKIE AND ECHO VIRUSES IN DRINKING WATER

    EPA Science Inventory

    The objectives of this research are to improve on the current analytical methods for quantitative detection of infective coxsackie and echo viruses in drinking water. The specific objectives of this research are to: (1) Improve the sensitivity and specificity of IMS-PCR for in...

  9. Isoform-specific regulation and localization of the coxsackie and adenovirus receptor in human airway epithelia.

    PubMed

    Excoffon, Katherine J D A; Gansemer, Nicholas D; Mobily, Matthew E; Karp, Philip H; Parekh, Kalpaj R; Zabner, Joseph

    2010-01-01

    Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR(Ex7) and CAR(Ex8)). We found low-level expression of the CAR(Ex8) isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR(Ex7), CAR(Ex8) localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR(Ex7), CAR(Ex8) differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR(Ex8) providing a potential mechanism for the apical localization of CAR(Ex8) in airway epithelial. In summary, apical localization of CAR(Ex8) may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins. PMID:20361046

  10. Recombination between Poliovirus and Coxsackie A Viruses of Species C: A Model of Viral Genetic Plasticity and Emergence

    PubMed Central

    Combelas, Nicolas; Holmblat, Barbara; Joffret, Marie-Line; Colbère-Garapin, Florence; Delpeyroux, Francis

    2011-01-01

    Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV), an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C), in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs. PMID:21994791

  11. Enhanced therapeutic efficacy of an adenovirus-PEI-bile-acid complex in tumors with low coxsackie and adenovirus receptor expression.

    PubMed

    Lee, Cho-Hee; Kasala, Dayananda; Na, Youjin; Lee, Min Sang; Kim, Sung Wan; Jeong, Ji Hoon; Yun, Chae-Ok

    2014-07-01

    Adenovirus (Ad) is a potential vehicle for cancer gene therapy. However, cells that express low levels of the coxsackie and adenovirus receptor (CAR) demonstrate poor Ad infection efficiency. We developed a bile acid-conjugated poly(ethyleneimine) (DA3)-coated Ad complex (Ad/DA3) to enhance Ad transduction efficiency. The size distribution and zeta potential of Ad/DA3 increased to 324 ± 3.08 nm and 10.13 ± 0.21 mV, respectively, compared with those of naked Ad (108 ± 2.26 nm and -17.7 ± 1.5 mV). The transduction efficiency of Ad/DA3 increased in a DA3 polymer concentration-dependent manner. Enhanced gene transfer by Ad/DA3 was more evident in CAR-moderate and CAR-negative cancer cells. Competition assays with a CAR-specific antibody revealed that internalization of Ad/DA3 was not mediated primarily by CAR but involved clathrin-, caveolae-, and macropinocytosis-mediated endocytosis. Cancer cell death was significantly increased when oncolytic Ad and DA3 were complexed (RdB-KOX/DA3) compared to that of naked oncolytic Ad and was inversely proportional to CAR levels. Importantly, RdB-KOX/DA3 significantly enhanced apoptosis, reduced angiogenesis, reduced proliferation, and increased active viral replication in human tumor xenografts compared to that of naked Ad. These results demonstrate that a hybrid vector system can increase the efficacy of oncolytic Ad virotherapy, particularly in CAR-limited tumors. PMID:24731708

  12. Coxsackie Virus A16 Infection of Placenta with Massive Perivillous Fibrin Deposition Leading to Intrauterine Fetal Demise at 36 Weeks Gestation.

    PubMed

    Yu, Weiming; Tellier, Raymond; Wright, James R

    2015-01-01

    Massive perivillous fibrin deposition (MPFD) is an uncommon placental disorder, associated with significant fetal morbidity, mortality, and recurrence; its etiology is unknown. We describe a 31-year-old mother, diagnosed with Coxsackievirus infection and hand-foot-and-mouth disease at 35 weeks gestation. Ultrasound at 35 weeks revealed a normal fetus and placenta. One week later, the mother experienced decreased fetal movement and ultrasound demonstrated intrauterine demise. The autopsy showed mild, acute pericarditis and hypoxic-ischemic encephalopathy. Placenta examination showed MPFD involving 80% of the parenchyma. Molecular viral analysis and serotyping showed Coxsackie A16 virus. The mother had an uneventful pregnancy 15 months later. Coxsackievirus infections in pregnant mothers are often asymptomatic. Transplacental Coxsackievirus infection is very rare but is associated with spontaneous abortion, intrauterine demise, or serious neonatal morbidity. Mild, nonspecific histologic changes have been reported in the placenta. To our knowledge, this is the first report of MPFD associated with Coxsackievirus infection. PMID:25826430

  13. Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

    SciTech Connect

    Vigl, Benjamin; Zgraggen, Claudia; Rehman, Nadia; Banziger-Tobler, Nadia E.; Detmar, Michael; Halin, Cornelia

    2009-01-15

    Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.

  14. [CANCER RESEARCH 63, 847853, February 15, 2003] Modulation of Coxsackie-Adenovirus Receptor Expression for Increased Adenoviral

    E-print Network

    Hemminki, Akseli

    Wang, Ronald D. Alvarez, Gene P. Siegal, and David T. Curiel Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center [A. H., A. K., B. L., M. W., G. P 35294, and Cancer Gene Therapy Group, Rational Drug Development Program, Department of Oncology

  15. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells

    PubMed Central

    Lenman, Annasara; Liaci, A. Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S.; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-01-01

    Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy. PMID:25674795

  16. CAR chasing: canine adenovirus vectors-all bite and no bark?

    PubMed

    Kremer, Eric J

    2004-02-01

    This review deals primarily with canine adenovirus serotype 2 (CAV-2) vectors and gives a simplified overview of how the various domains of virology, cellular and molecular biology, as well as immunology, come into play when trying to understand and ameliorate adenovirus (Ad)-mediated gene transfer. The generation of early region 1 (E1)-deleted (DeltaE1) CAV-2 vectors, the lack of pre-existing humoral immunity, trafficking, the use of the coxsackie B adenovirus receptor (CAR), the surprising neuronal tropism, and the ability to migrate via axons to afferent regions of the central and peripheral nervous system, are described. Due to these intrinsic properties, CAV-2 vectors may be powerful tools for the study of the pathophysiology and potential treatment of neurodegenerative diseases like lysosomal storage disorders, Parkinson's, Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and others. Other potential uses include anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, pain therapy, cancer therapy (e.g. K9 CRAds), and gene transfer to other somatic tissues. PMID:14978757

  17. Emphysematous gastritis in a patient with coxsackie B3 myocarditis and cardiogenic shock requiring veno-arterial extra-corporeal membrane oxygenation

    PubMed Central

    Ashfaq, Awais; Chapital, Alyssa B.

    2015-01-01

    Introduction Emphysematous gastritis is a rare condition in which gas accumulates in the stomach lining usually due to an infectious source. Case presentation We present a 16 year old female with viral myocarditis and cardiogenic shock transferred to our hospital on extracorporeal membrane oxygenation (ECMO) who developed emphysematous gastritis. After listing the patient for heart transplant, patient underwent Bi-VAD placement requiring veno-venous ECMO support. Subsequently, she developed worsening abdominal distention. CT of abdomen/pelvis showed the stomach to be diffusely edematous, thick-walled, containing intramural gas collections, consistent with emphysematous gastritis. Patient underwent nonoperative management and two weeks later had complete resolution of the gastritis. Unfortunately, her overall condition deteriorated in the subsequent days and support was withdrawn. Discussion Management of emphysematous gastritis usually revolves around supportive care, broad spectrum antibiotics and bowel rest. Our patients’ gastritis resolved with non-operative management, albeit, she succumbed to multiorgan failure due to other causes. Conclusion We believe, this is a unique case of a veno-arterial ECMO causing emphysematous gastritis. PMID:26263451

  18. Hand, foot, and mouth disease on the soles (image)

    MedlinePLUS

    Hand, foot, and mouth disease is cause by a coxsackie virus. It produces mouth ulcers and small blisters (vesicles) on the hands and feet. The vesicles often have a reddish border with a white or lighter colored area in ...

  19. THE WORLD WITHIN REACH Dean's List of Distinguished Students

    E-print Network

    Alexandrova, Ivana

    Algozzine, Michael Coxsackie NY Ali, Nicole Sayville NY Ali-Pearson, Tyana Bronx NY Allegretta, Robert, Will Spring Hill FL Allison, Karl Bay Shore NY Alloush, Yunis Schenectady NY Almenas, Alexis Bronx NY Almes

  20. Detection by immunofluorescence of possible viral implications in "idiopathic" peripheral facial paralysis.

    PubMed

    P?un, L; Pârvu, C; Ceau?u, E M

    1985-01-01

    The presence of viral antigens was detected by the indirect immunofluorescence technique in exfoliated cells occurring in the pharyngeal exudate of 18 out of 29 patients with peripheral facial paralysis. The most frequently encountered antigens were: Coxsackie A and B virus (33.3%), adenovirus (16.7%), and the association Coxsackie B virus + adenovirus (16.7%). The possibility that some of the so-called "idiopathic" peripheral facial paralyses may have a viral etiology is discussed. PMID:3004020

  1. Realms of the Viruses Online

    ERIC Educational Resources Information Center

    Liu, Dennis

    2007-01-01

    Viruses have evolved strategies for infecting all taxa, but most viruses are highly specific about their cellular host. In humans, viruses cause diverse diseases, from chronic but benign warts, to acute and deadly hemorrhagic fever. Viruses have entertaining names like Zucchini Yellow Mosaic, Semliki Forest, Coxsackie, and the original terminator,…

  2. EFFECT OF PARTICULATES ON DISINFECTION OF ENTEROVIRUSES IN WATER BY CHLORINE DIOXIDE

    EPA Science Inventory

    The inactivation kinetics of ClO2 on two enteroviruses, poliovirus 1 (Mahoney) and coxsackie virus A9, and an enteric indicator of fecal pollution, Escherichia coli, were examined in laboratory studies. In addition, the disinfecting ability of ClO2 as affected by particulates (bo...

  3. IDENTIFICATION AND DETECTION OF WATER-BORNE VIRUSES BY IMMUNOENZYMATIC METHODS

    EPA Science Inventory

    A quantitative enzyme-linked immunosorbent assay (ELISA) was used for identification of viruses selected as representative water-borne viruses: poliovirus 1, echovirus 6, coxsackievirus A9, and coxsackie B viruses. Partially purified viral antigens or virus-specific antibodies we...

  4. JOURNAL OF VIROLOGY, Jan. 1990, p. 185-194 0022-538X/90/010185-10$02.00/0

    E-print Network

    Kirkegaard, Karla

    of poliovirus (29, 44) and for picoma- viruses such as coxsackie B3 virus (20) and hepatitis A virus (6 that full-length cDNA replicas ofRNA viral genomes can initiate authentic viral infections either as DNA and characterize mutations in RNA genomes on the DNA level. Viral mutants whose phenotypic defects can

  5. ORIGINAL ARTICLE A heparan sulfate-targeted conditionally replicative

    E-print Network

    Hemminki, Akseli

    Therapy Group, Rational Drug Design Program and Haartman Institute, University of Helsinki, Helsinki in gene therapy, is the coxsackie-adenovirus receptor (CAR). CAR is expressed variably and often at low pathway-dependent CRAd, Ad5.pK7-D24, with a polylysine motif in the fiber C-terminus, enabling CAR

  6. Analysis of Type 1 Diabetes with Wavelets and Other Mathematical Tools

    E-print Network

    Tefera, Akalu

    a coxsackie virus, which are polio-related viruses that cause upper respiratory infections. After fighting off the virus, the body's immune system turns on the beta cells in the pancreas. However, you cannot catch to regulate the amount of glucose in the blood. Insulin is a hormone that helps the body move the glucose

  7. Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

    PubMed Central

    Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan

    2002-01-01

    AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381

  8. Collection and Testing of Respiratory Samples

    ClinicalTrials.gov

    2012-05-22

    QIAGEN ResPlex II Advanced Panel; Influenza A; Influenza B; Respiratory Syncytial Virus Infections; Infection Due to Human Parainfluenza Virus 1; Parainfluenza Type 2; Parainfluenza Type 3; Parainfluenza Type 4; Human Metapneumovirus A/B; Rhinovirus; Coxsackie Virus/Echovirus; Adenovirus Types B/C/E; Coronavirus Subtypes 229E; Coronavirus Subtype NL63; Coronavirus Subtype OC43; Coronavirus Subtype HKU1; Human Bocavirus; Artus Influenza A/B RT-PCR Test; Influenza A, Influenza B,

  9. Isolation of polioviruses and other enteroviruses in south Greece between 1994 and 1998.

    PubMed

    Siafakas, N; Georgopoulou, A; Markoulatos, P; Spyrou, N

    2000-01-01

    During the five-year period between 1994 and 1998, a total of 217 clinical samples were assessed for the isolation of enteroviruses at the Enterovirus Reference Centre for South Greece. Fourteen enterovirus strains belonging to different serotypes were isolated. These field strains were detected by cell culture in appropriate cell lines. They were subsequently identified by neutralizing antibodies with the LBM (Lim-Benyesh Melnick) mixed antisera pools up to 1995 and RIVM (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) pools from 1996 onwards. The isolated viruses included two strains of poliovirus type 2 Sabin-like, three strains of poliovirus type 1 non-Sabin-like, one Coxsackie B2 (CBV2) strain, one Coxsackie B5 (CBV5) strain, one Echo 5 (ECV5) strain, one Echo 7 (ECV7) strain, three Coxsackie A16 (CAV16) strains, and two currently enteroviral strains unidentified by RIVM pools. Reverse transcription-polymerase chain reaction (RT-PCR) using poliovirus-specific primers or poliovirus type-specific primers and enterovirus specific primers from the highly conserved 5'-UTR, the latter followed by RFLP, was also applied in 6 clinical isolates (3 strains of poliovirus type 1 non-Sabin-like, 1 polio type 2 Sabin-like, and 2 non-identified by RIVM pools enteroviruses). The advantages and the drawbacks of these assays against the conventional ones are discussed here. The isolations and the subsequent identification of the strains were carried out from fecal samples of clinical cases that included hand-foot-and-mouth disease, meningitis, and acute flaccid paralysis. The reappearance of non-Sabin-like poliovirus strains in Greece in 1996 after 14 years is considered to have an important medical and clinical value. PMID:10906768

  10. The role of some viruses in acute pneumonias in adults.

    PubMed

    Câmpeanu, A; Nicolaescu, V; M?gureanu, E; Grobnicu, M; Niculescu, I; Dinulescu, M; Zili?teanu, E; Cre?escu, L

    1977-01-01

    Virological and bacteriological investigations were performed in 85 patients with acute pneumonias and virus isolation or serological evidence of virus infection were obtained in 37.6% of the cases. Influenza A2 and B viruses were incriminated in 14.1% of the patients; parainfluenza viruses in 7% and adenoviruses in 17.2% of the cases. Coxsackie virus was isolated from one patient's blood, and poliovirus 3 was recovered in 3 cases. In 5 cases associated virus infections were detected. PMID:194393

  11. [Severe acute pancreatitis and infection by influenza A (H1N1) virus in a child: case report].

    PubMed

    Rodríguez Schulz, Diego; Martínez, Agustina; Guzmán, María Belén; Robledo, Hugo; Capocasa, Patricia; Martínez, Luz; Garnero, Analía

    2015-08-01

    Acute pancreatitis is an inflammatory disease of the pancreas, characterized by abdominal pain and high level of pancreatic enzymes. Pancreatitis is the most common disease of pancreas in children and adults. For the diagnosis we need 2 of 3 characteristics: abdominal pain characteristic of acute pancreatitis, amylase and/or lipase 3 times higher than the normal upper limit and characteristic findings in images. The etiologies are multiple: trauma, metabolic disease and infections: mixovirus, HIV, measles, coxsackie, hepatitis B, C, cytomegalovirus, varicella, herpes simplex. Three cases of PA associated with H1N1 Influenza virus were reported, only one in a child with uncomplicated features. PMID:26172021

  12. Mollolide A, a diterpenoid with a new 1,10:2,3-disecograyanane skeleton from the roots of Rhododendron molle.

    PubMed

    Li, Yong; Liu, Yun-Bao; Zhang, Jian-Jun; Li, Yu-Huan; Jiang, Jian-Dong; Yu, Shi-Shan; Ma, Shuang-Gang; Qu, Jing; Lv, Hai-Ning

    2013-06-21

    Mollolide A (1), a diterpenoid featuring a new 1,10:2,3-disecograyanane skeleton, was isolated from the roots of Rhododendron molle. Its structure was elucidated through extensive MS, IR, and NMR spectroscopy analyses. The absolute configuration was determined by single-crystal X-ray diffraction of its p-bromobenzoate derivative (1b). Compound 1 exhibits a significant analgesic effect at a dose of 20 mg/kg and antiviral activity against the Coxsackie B3 virus with an IC50 value of 27.7 ?M. PMID:23724861

  13. Psoriasis Herpeticum due to Varicella Zoster Virus: A Kaposi's Varicelliform Eruption in Erythrodermic Psoriasis.

    PubMed

    Garg, Geeta; Thami, Gurvinder P

    2012-05-01

    Kaposi's varicelliform eruption (KVE) or eczema herpeticum is characterized by disseminated papulovesicular eruption caused by a number of viruses like Herpes simplex virus I and II, Coxsackie virus, and Vaccinia and Small pox viruses in patients with pre-existing skin disease. The occurrence of KVE with psoriasis has been reported recently as a new entity psoriasis herpeticum. The rare causation of psoriasis herpeticum due to Varicella zoster virus in a patient with underlying psoriasis is being reported for the first time. PMID:22707775

  14. Volatile organic compounds generated by cultures of bacteria and viruses associated with respiratory infections.

    PubMed

    Abd El Qader, Amir; Lieberman, David; Shemer Avni, Yonat; Svobodin, Natali; Lazarovitch, Tsilia; Sagi, Orli; Zeiri, Yehuda

    2015-12-01

    Respiratory infections (RI) can be viral or bacterial in origin. In either case, the invasion of the pathogen results in production and release of various volatile organic compounds (VOCs). The present study examines the VOCs released from cultures of five viruses (influenza A, influenza B, adenovirus, respiratory syncitial virus and parainfluenza 1 virus), three bacteria (Moraxella catarrhalis, Haemophilus influenzae and Legionella pneumophila) and Mycoplasma pneumoniae isolated colonies. Our results demonstrate the involvement of inflammation-induced VOCs. Two significant VOCs were identified as associated with infectious bacterial activity, heptane and methylcyclohexane. These two VOCs have been linked in previous studies to oxidative stress effects. In order to distinguish between bacterial and viral positive cultures, we performed principal component analysis including peak identity (retention time) and VOC concentration (i.e. area under the peak) revealing 1-hexanol and 1-heptadecene to be good predictors. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26033043

  15. PARTICIPATION OF BICARBONATE IN RNA AND PROTEIN SYNTHESES AS INDICATED BY VIRUS PROPAGATION IN HUMAN CELLS

    PubMed Central

    Chang, R. Shihman

    1959-01-01

    The propagation of a strain of Coxsackie virus, group B type 1, in human cell cultures depleted of bicarbonate has been studied. Under the described experimental conditions, bicarbonate depletion suppresses the propagation of this virus. This suppressive effect may be reversed by the addition of the following compounds to the bicarbonate-depleted cultures: (a) bicarbonate; (b) adenosine, guanosine, cytidine, and uridine; (c) adenylic, guanylic, cytidylic, and uridylic acids; (d) enzymatically degraded RNA prepared from yeasts or human embryo, or (e) RNA. The following compounds are unable to reverse the suppressive effect of bicarbonate depletion: (a) adenine, guanine, cytosine, and uracil, with or without ribose; (b) adenine, guanine, cytosine, and thymine; (c) deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine; (d) deoxyadenylic, deoxyguanylic, deoxycytidylic, and thymidylic acids; (e) enzymatically degraded DNA, or (f) DNA. The same general results as with the Coxsackie virus have been obtained with a strain of poliovirus and vaccinia virus. The failure of bicarbonate depletion to suppress completely the propagation of the poliovirus under the described condition constitutes a major difference. The significance of these findings is discussed. PMID:13620851

  16. [Studies on anti-EV71 virus activity of traditional Chinese medicine and its clinical application in treatment of HFMD].

    PubMed

    Xue, Bailin; Yao, Zhihong; Yu, Rongmin

    2011-12-01

    Hand-foot-and-mouth disease is caused by intestinal virus infection. The viruses coxsackie A16 (CA16) and enterovirus 71 (EV71) are the main pathogens. Between them, the virus EV71 is more dangerous and easier to cause serious complications, which leads to death or disability. Currently, there are no effective antiviral drugs to treat EV71 infection. Therefore, developing an effective drug against EV71 virus activity is significant. It has a huge potency of screening the anti-EV71 components and developing the new drugs from the abundant traditional Chinese medicines (TCMs). Meanwhile, since hand-foot-and-mouth disease spread in Shanghai in 1981, a growing number of reports on TCMs treatment in clinic have been published. In addition, most of treatments with various ways are effective, which play a positive role on improving clinic treatment and controlling diseases. Moreover, special clinic advantages and features of TCMs were obviously shown. PMID:22393752

  17. Glucosylated caffeoylquinic acid derivatives from the flower buds of Lonicera japonica

    PubMed Central

    Yu, Yang; Jiang, Zhibo; Song, Weixia; Yang, Yongchun; Li, Yuhuan; Jiang, Jiandong; Shi, Jiangong

    2015-01-01

    Three new glucosylated caffeoylquinic acid isomers (1–3), along with six known compounds, have been isolated from an aqueous extract of the flower buds of Lonicera japonica. Structures of the new compounds were determined by spectroscopic and chemical methods as (?)-4-O-(4-O-?-d-glucopyranosylcaffeoyl)quinic acid (1), (?)-3-O-(4-O-?-d-glucopyranosylcaffeoyl)quinic acid (2), and (?)-5-O-(4-O-?-d-glucopyranosylcaffeoyl)quinic acid (3), respectively. In the preliminary in vitro assays, two known compounds methyl caffeate and 2?-O-methyladenosine showed inhibitory activity against Coxsackie virus B3 with IC50 values of 3.70 ?mol/L and 6.41 ?mol/L and SI values of 7.8 and 12.1, respectively. PMID:26579448

  18. The Molecular Interaction of CAR and JAML Recruits the Central Cell Signal Transducer PI3K

    SciTech Connect

    Verdino, Petra; Witherden, Deborah A.; Havran, Wendy L.; Wilson, Ian A.

    2010-11-15

    Coxsackie and adenovirus receptor (CAR) is the primary cellular receptor for group B coxsackieviruses and most adenovirus serotypes and plays a crucial role in adenoviral gene therapy. Recent discovery of the interaction between junctional adhesion molecule-like protein (JAML) and CAR uncovered important functional roles in immunity, inflammation, and tissue homeostasis. Crystal structures of JAML ectodomain (2.2 angstroms) and its complex with CAR (2.8 angstroms) reveal an unusual immunoglobulin-domain assembly for JAML and a charged interface that confers high specificity. Biochemical and mutagenesis studies illustrate how CAR-mediated clustering of JAML recruits phosphoinositide 3-kinase (P13K) to a JAML intracellular sequence motif as delineated for the {alpha}{beta} T cell costimulatory receptor CD28. Thus, CAR and JAML are cell signaling receptors of the immune system with implications for asthma, cancer, and chronic nonhealing wounds.

  19. Detection of bioagents using a shear horizontal surface acoustic wave biosensor

    DOEpatents

    Larson, Richard S; Hjelle, Brian; Hall, Pam R; Brown, David C; Bisoffi, Marco; Brozik, Susan M; Branch, Darren W; Edwards, Thayne L; Wheeler, David

    2014-04-29

    A biosensor combining the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325 MHz with the specificity provided by antibodies and other ligands for the detection of viral agents. In a preferred embodiment, a lithium tantalate based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against Coxsackie virus B4 or the negative-stranded category A bioagent Sin Nombre virus (SNV). Rapid detection of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, and the sensor's selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1 The biosensor was able to delect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS).

  20. Development of a new cell culture-based method and optimized protocol for the detection of enteric viruses.

    PubMed

    Lee, Jae Ho; Lee, Gyu-Cheol; Kim, Jong Ik; Yi, Hyun Ah; Lee, Chan Hee

    2013-07-01

    The development of rapid and effective methods to detect water- and food-borne enteric viruses is important for the prevention and control of mass infection. This study represents an attempt to develop a reliable cell culture-based detection system and optimize an effective and rapid protocol for the assaying of environmental samples for the presence of infectious enteric viruses. Six enteric viruses were used in this study: poliovirus, Coxsackie virus A9, Coxsackie virus B5, human rotavirus G1, hepatitis A virus, and adenovirus type 41. Among the cell lines from humans (A549, HeLa, HEK293, and HFF) and other primates (Vero, BS-C-1, FRhK-4, BGMK, and MA104), a cytopathic effect (CPE) analysis indicated that the MA104 cell line was the most optimal for use in the detection of infectious enteric viruses. Both the sensitivity and specificity of virus detection in MA104 cells were similar to or higher than those in standard BGMK cells. Next, a method was developed for the determination of the infectiousness of enteric viruses using the colorimetric thiazolyl blue (MTT) assay. This assay utilizes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to yield % values based on colorimetric results. These results were compared with those from a conventional CPE-based TCID(50) assay, revealing no statistically significant difference between the two methods. The MTT% values in MA104 cells were comparable to those in BGMK cells. This MA104 cell-based MTT assay could substitute for the classical BGMK cell-based CPE assay for infectious enteric viruses. PMID:23578702

  1. Detection of Viral Pathogens by Reverse Transcriptase PCR and of Microbial Indicators by Standard Methods in the Canals of the Florida Keys

    PubMed Central

    Griffin, Dale W.; Gibson, Charles J.; Lipp, Erin K.; Riley, Kelley; Paul, John H.; Rose, Joan B.

    1999-01-01

    In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, enterococci, coliphages, F-specific (F+) RNA coliphages, Giardia lamblia, Cryptosporidium parvum, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1,410, E. coli organisms from <1 to 130, Clostridium spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F+ phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks. PMID:10473424

  2. Rethinking Molecular Mimicry in Rheumatic Heart Disease and Autoimmune Myocarditis: Laminin, Collagen IV, CAR, and B1AR as Initial Targets of Disease

    PubMed Central

    Root-Bernstein, Robert

    2014-01-01

    Rationale: Molecular mimicry theory (MMT) suggests that epitope mimicry between pathogens and human proteins can activate autoimmune disease. Group A streptococci (GAS) mimics human cardiac myosin in rheumatic heart disease (RHD) and coxsackie viruses (CX) mimic actin in autoimmune myocarditis (AM). But myosin and actin are immunologically inaccessible and unlikely initial targets. Extracellular cardiac proteins that mimic GAS and CX would be more likely. Objectives: To determine whether extracellular cardiac proteins such as coxsackie and adenovirus receptor (CAR), beta 1 adrenergic receptor (B1AR), CD55/DAF, laminin, and collagen IV mimic GAS, CX, and/or cardiac myosin or actin. Methods: BLAST 2.0 and LALIGN searches of the UniProt protein database were employed to identify potential molecular mimics. Quantitative enzyme-linked immunosorbent assay was used to measure antibody cross-reactivity. Measurements: Similarities were considered to be significant if a sequence contained at least 5 identical amino acids in 10. Antibodies were considered to be cross-reactive if the binding constant had a Kd less than 10-9 M. Main results: Group A streptococci mimics laminin, CAR, and myosin. CX mimics actin and collagen IV and B1AR. The similarity search results are mirrored by antibody cross-reactivities. Additionally, antibodies against laminin recognize antibodies against collagen IV; antibodies against actin recognize antibodies against myosin, and antibodies against GAS recognize antibodies against CX. Thus, there is both mimicry of extracellular proteins and antigenic complementarity between GAS-CX in RHD/AM. Conclusion: Rheumatic heart disease/AM may be due to combined infections of GAS with CX localized at cardiomyocytes that may produce a synergistic, hyperinflammatory response that cross-reacts with laminin, collagen IV, CAR, and/or B1AR. Epitope drift shifts the immune response to myosin and actin after cardiomyocytes become damaged. PMID:25191648

  3. GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases.

    PubMed

    Roberts, Jane L; Tavallai, Mehrad; Nourbakhsh, Aida; Fidanza, Abigail; Cruz-Luna, Tanya; Smith, Elizabeth; Siembida, Paul; Plamondon, Pascale; Cycon, Kelly A; Doern, Christopher D; Booth, Laurence; Dent, Paul

    2015-10-01

    Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged 'rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the 'Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. 'Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of 'Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. PMID:25858032

  4. Viruses associated with influenza-like-illnesses in Papua New Guinea, 2010.

    PubMed

    Kono, Jacinta; Jonduo, Marinjho H; Omena, Matthew; Siba, Peter M; Horwood, Paul F

    2014-05-01

    Influenza-like-illness can be caused by a wide range of respiratory viruses. The etiology of influenza-like-illness in developing countries such as Papua New Guinea is poorly understood. The etiological agents associated with influenza-like-illness were investigated retrospectively for 300 nasopharyngeal swabs received by the Papua New Guinea National Influenza Centre in 2010. Real-time PCR/RT-PCR methods were used for the detection of 13 respiratory viruses. Patients with influenza-like-illness were identified according to the World Health Organization case definition: sudden onset of fever (>38°C), with cough and/or sore throat, in the absence of other diagnoses. At least one viral respiratory pathogen was detected in 66.3% of the samples tested. Rhinoviruses (17.0%), influenza A (16.7%), and influenza B (12.7%) were the pathogens detected most frequently. Children <5 years of age presented with a significantly higher rate of at least one viral pathogen and a significantly higher rate of co-infections with multiple viruses, when compared to all other patients >5 years of age. Influenza B, adenovirus, and respiratory syncytial virus were all detected at significantly higher rates in children <5 years of age. This study confirmed that multiple respiratory viruses are circulating and contributing to the presentation of influenza-like-illness in Papua New Guinea. PMID:24136362

  5. Vascular endothelial growth factor signalling in endothelial cell survival: A role for NF{kappa}B

    SciTech Connect

    Grosjean, Jennifer . E-mail: Jennifer.grosjean@imperial.ac.uk; Kiriakidis, Serafim; Reilly, Kerri; Feldmann, Marc; Paleolog, Ewa

    2006-02-17

    Angiogenesis is the development of blood capillaries from pre-existing vessels. Vascular endothelial growth factor (VEGF) is a key regulator of vessel growth and regression, and acts as an endothelial survival factor by protecting endothelial cells from apoptosis. Many genes involved in cell proliferation and apoptosis are regulated by the nuclear factor kappa B (NF{kappa}B) transcription factor family. This study aimed to address the hypothesis that VEGF-mediated survival effects on endothelium involve NF{kappa}B. Using an NF{kappa}B-luciferase reporter adenovirus, we observed activation of NF{kappa}B following VEGF treatment of human umbilical vein endothelial cells. This was confirmed using electrophoretic mobility shift assay and found to involve nuclear translocation of NF{kappa}B sub-unit p65. However, NF{kappa}B activation occurred without degradation of inhibitory I{kappa}B proteins (I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon}). Instead, tyrosine phosphorylation of I{kappa}B{alpha} was observed following VEGF treatment, suggesting NF{kappa}B activation was mediated by degradation-independent dissociation of I{kappa}B{alpha} from NF{kappa}B. Adenovirus-mediated over-expression of either native I{kappa}B{alpha}, or of I{kappa}B{alpha} in which tyrosine residue 42 was mutated to phenylalanine, inhibited induction of NF{kappa}B-dependent luciferase activity in response to VEGF. Furthermore, VEGF-induced upregulation of mRNA for the anti-apoptotic protein Bcl-2 and cell survival following serum withdrawal was reduced following I{kappa}B{alpha} over-expression. This study highlights that different molecular mechanisms of NF{kappa}B activation may be involved downstream of stimuli which activate the endothelial lining of blood vessels.

  6. Improving gene transfer in human renal carcinoma cells: Utilization of adenovirus vectors containing chimeric type 5 and type 35 fiber proteins.

    PubMed

    Acharya, Bishnu; Terao, Shuji; Suzuki, Toru; Naoe, Michio; Hamada, Katsuyuki; Mizuguchi, Hiroyuki; Gotoh, Akinobu

    2010-05-01

    The transduction efficacy of adenovirus serotype 5 (Ad5) vector in human renal carcinoma cells is generally low due to the down-regulated expression of Coxsackie and adenovirus receptor (CAR) in target cells. By contrast, the infectivity of adenovirus serotype 35 vectors depends on the binding rate to CD46 receptor, independent of CAR. In this study, we examined whether an adenovirus vector containing chimeric type 5 and type 35 fiber proteins (Ad5/F35) increases transduction efficiency compared to Ad5 vector in human renal carcinoma cells in vitro. The expression of CAR was much lower in the human renal carcinoma cells than in control HEK293 cells. By contrast, the expression of CD46 was similar and perhaps at a higher level in the human renal carcinoma cells than in the HEK293 cells. The transduction efficacy of Ad5/F35 vector was dramatically higher compared to that of Ad5 in human renal carcinoma cells, and was correlated to the expression of CD46. Thus, Ad5/35 vector may be useful for the development of novel gene therapy approaches to renal cell carcinoma. PMID:22993573

  7. Pseudotyping the adenovirus serotype 5 capsid with both the fibre and penton of serotype 35 enhances vascular smooth muscle cell transduction.

    PubMed

    Parker, A L; White, K M; Lavery, C A; Custers, J; Waddington, S N; Baker, A H

    2013-12-01

    Ex vivo gene therapy during coronary artery bypass grafting (CABG) holds great potential to prevent excessive smooth muscle cell (SMC) proliferation, neointima formation and graft failure. The most successful preclinical strategies to date have utilised vectors based on the species C adenovirus, Ad5, which engages the Coxsackie and Adenovirus receptor (CAR) as its primary attachment receptor. Profiling receptors on human SMCs demonstrated the absence of CAR but substantial expression of the species B receptor CD46. We performed transduction experiments using Ad5 and the CD46-utilising adenovirus Ad35, and found Ad35 significantly more efficient at transducing SMCs. To evaluate whether transduction could be further augmented, we evaluated chimeric CD46-utilising Ad5/Ad35 vectors comprising the Ad5 capsid pseudotyped with the Ad35 fibre alone (Ad5/F35) or in combination with the Ad35 penton (Ad5/F35/P35). In human smooth muscle cells (hSMCs), Ad5/F35/P35 mediated significantly higher levels of transduction than either parental vector or Ad5/F35. Ex vivo transduction experiments using mouse aortas from CD46 transgenics demonstrated that Ad5/F35/P35 was significantly more efficient at transducing SMCs than the other vectors tested. Finally, ex vivo transduction and immunofluorescent colocalisation experiments using human tissue from CABG procedures confirmed the preclinical potential of Ad5/F35/P35 as an efficient vector for vascular transduction during CABG. PMID:24005577

  8. Polyanion inhibitors of human immunodeficiency virus and other viruses. 5. Telomerized anionic surfactants derived from amino acids.

    PubMed

    Leydet, A; Barragan, V; Boyer, B; Montéro, J L; Roque, J P; Witvrouw, M; Este, J; Snoeck, R; Andrei, G; De Clercq, E

    1997-01-31

    omega-Acryloyl anionic surfactants, whose polar heads are derived from amino acids, have been telomerized to prepare polyanions of a predetermined molecular weight. The main goal of this study was to verify whether the antiviral activity is influenced by the degree of polymerization of the polyanions. The oligomeric polyanions were evaluated for their activity against human immunodeficiency virus (HIV-1 or HIV-2) and various other RNA and DNA viruses. With regard to their anti-HIV activity, a minimum number of anionic groups was necessary to achieve an inhibitory effect. Moreover, to be active the overall conformation of the polyanion must be such that the anionic groups are located on the external site of the molecule. With some of the polyanions, a 50% inhibition concentration (IC50) as low as 1 microgram/ mL, or even 0.1 microgram/mL, was noted against HIV-1 in CEM-4 and MT-4 cells, respectively. The most potent polyanions also proved active against human cytomegalovirus and herpex simplex virus at concentrations of 5-10 and 20-40 micrograms/mL, respectively. No activity was observed against any of the other viruses tested (i.e., vesicular stomatitis, Sindbis, Semliki forest, parainfluenza, Junin, Tacaribe, Coxsackie, polio, reo, and vaccinia). No toxicity for the host cells was observed at concentrations up to 200 micrograms/mL. PMID:9022800

  9. Inactivation of coxsackieviruses B3 and B5 in water by chlorine.

    PubMed Central

    Jensen, H; Thomas, K; Sharp, D G

    1980-01-01

    The inactivation rates of coxsackievirus B3 (CB3) and B5 (CB5) by chlorine in dilute buffer at pH 6 were very nearly the same and about half that of poliovirus (Mahoney) under similar conditions. Purified CB3, like the poliovirus, aggregated in the acid range but not at pH 7 and above. Purified CB5 aggregated rapidly at all pH values; still, the graph of log surviving infectivity versus time was a straight line. No chlorine inactivation data were obtained with dispersed CB5, for it could be dispersed only by addition of diethylaminoethyl dextran, which would react with the chlorine. Addition of 0.1 M NaCl to the buffer at pH 6 did not influence the aggregation of CB5 or the rate of chlorine action on either of the coxsackie-viruses, but at pH 10 it increased the disinfection activity of OCl- for both viruses roughly 20-fold. Cesium chloride had a similar but smaller effect. KCl was the most active of the three in this respect, making the inactivating effect of OCl- at pH 10 about equal to that of HOCl at pH 6. Images PMID:6252839

  10. Borna disease virus nucleoprotein inhibits type I interferon induction through the interferon regulatory factor 7 pathway

    SciTech Connect

    Song, Wuqi; Department of Microbiology, Harbin Medical University ; Kao, Wenping; Department of Microbiology, Harbin Medical University ; Zhai, Aixia; Qian, Jun; Li, Yujun; Zhang, Qingmeng; Zhao, Hong; Hu, Yunlong; Li, Hui; Zhang, Fengmin; The Key Laboratory of Pathogenic Biology, Heilongjiang Higher Education Institutions; Department of Microbiology, Harbin Medical University

    2013-09-06

    Highlights: •IRF7 nuclear localisation was inhibited by BDV persistently infected. •BDV N protein resistant to IFN induction both in BDV infected OL cell and N protein plasmid transfected OL cell. •BDV N protein is related to the inhibition of IRF7 nuclear localisation. -- Abstract: The expression of type I interferon (IFN) is one of the most potent innate defences against viral infection in higher vertebrates. Borna disease virus (BDV) establishes persistent, noncytolytic infections in animals and in cultured cells. Early studies have shown that the BDV phosphoprotein can inhibit the activation of type I IFN through the TBK1–IRF3 pathway. The function of the BDV nucleoprotein in the inhibition of IFN activity is not yet clear. In this study, we demonstrated IRF7 activation and increased IFN-?/? expression in a BDV-persistently infected human oligodendroglia cell line following RNA interference-mediated BDV nucleoprotein silencing. Furthermore, we showed that BDV nucleoprotein prevented the nuclear localisation of IRF7 and inhibited endogenous IFN induction by poly(I:C), coxsackie virus B3 and IFN-?. Our findings provide evidence for a previously undescribed mechanism by which the BDV nucleoprotein inhibits type I IFN expression by interfering with the IRF7 pathway.

  11. RGD-modifided oncolytic adenovirus exhibited potent cytotoxic effect on CAR-negative bladder cancer-initiating cells

    PubMed Central

    Yang, Y; Xu, H; Shen, J; Yang, Y; Wu, S; Xiao, J; Xu, Y; Liu, X-Y; Chu, L

    2015-01-01

    Cancer-initiating cell (CIC) is critical in cancer development, maintenance and recurrence. The reverse expression pattern of coxsackie and adenovirus receptor (CAR) and ?? integrin in bladder cancer decreases the infection efficiency of adenovirus. We constructed Arg-Gly-Asp (RGD)-modified oncolytic adenovirus, carrying EGFP or TNF-related apoptosis-inducing ligand (TRAIL) gene (OncoAd.RGD-hTERT-EGFP/TRAIL), and applied them to CAR-negative bladder cancer T24 cells and cancer-initiating T24 sphere cells. OncoAd.RGD-hTERT-EGFP had enhanced infection ability and cytotoxic effect on T24 cells and T24 sphere cells, but little cytoxicity on normal urothelial SV-HUC-1 cells compared with the unmodified virus OncoAd.hTERT-EGFP. Notably, OncoAd.RGD-hTERT-TRAIL induced apoptosis in T24 cells and T24 sphere cells. Furthermore, it completely inhibited xenograft initiation established by the oncolytic adenovirus-pretreated T24 sphere cells, and significantly suppressed tumor growth by intratumoral injection. These results provided a promising therapeutic strategy for CAR-negative bladder cancer through targeting CICs. PMID:25973680

  12. Viruses in idiopathic inflammatory myopathies: absence of candidate viral genomes in muscle.

    PubMed

    Leff, R L; Love, L A; Miller, F W; Greenberg, S J; Klein, E A; Dalakas, M C; Plotz, P H

    1992-05-16

    There is indirect evidence that various viruses have aetiological roles in the idiopathic inflammatory myopathies. By means of a sensitive and specific method based on the polymerase chain reaction (PCR), we sought direct evidence for the presence in affected muscle of nucleic acid sequences from Coxsackie virus, mumps virus, encephalomyocarditis virus, adenovirus, human T-lymphotropic virus types I and II, and human immunodeficiency virus. RNA was extracted from muscle biopsy samples obtained from 44 patients with idiopathic inflammatory myopathies a mean of 45 (range 0-216) months after disease onset. All the subjects were older than 16 years at disease onset. The integrity of the mRNA extracted was confirmed by the successful PCR amplification of insulin receptor mRNA in all samples. The PCR method was able to detect between 1 and 20 molecules of added viral nucleic acid for the picornaviruses sought. No detectable virus sequences were found, however, in any of the patients' muscle samples or in samples from 13 controls. We tested for retroviral DNA in 22 samples (17 patients, 5 controls) that met our criterion for adequate DNA extraction (detectable beta-actin DNA by PCR); again no virus sequences were found. Persistence in muscle of these or closely related viruses is unlikely to be a continuing stimulus for disease in the idiopathic inflammatory myopathies. PMID:1349938

  13. Echovirus 22 is an atypical enterovirus.

    PubMed Central

    Coller, B A; Chapman, N M; Beck, M A; Pallansch, M A; Gauntt, C J; Tracy, S M

    1990-01-01

    Although echovirus 22 (EV22) is classified as an enterovirus in the family Picornaviridae, it is atypical of the enterovirus paradigm, typified by the polioviruses and the coxsackie B viruses. cDNA reverse transcribed from coxsackievirus B3 (CVB3) RNA does not hybridize to genomic RNA of EV22, and conversely, cDNA made to EV22 does not hybridize to CVB3 genomic RNA or to molecular clones of CVB3 or poliovirus type 1. EV22 cDNA does not hybridize to viral RNA of encephalomyocarditis virus or to a molecular clone of Theiler's murine encephalomyelitis virus, members of the cardiovirus genus. The genomic RNA of EV22 cannot be detected by the polymerase chain reaction using generic enteroviral primers. EV22 does not shut off host cell protein synthesis, and the RNA of EV22 is efficiently translated in vitro in rabbit reticulocyte lysates. Murine enterovirus-immune T cells recognize and proliferate against EV22 as an antigen in vitro, demonstrating that EV22 shares an epitope(s) common to enteroviruses but not found among other picornaviruses. Images PMID:2159539

  14. Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development, Function or Stress Response

    PubMed Central

    Schell, Christoph; Kretz, Oliver; Bregenzer, Andreas; Rogg, Manuel; Helmstädter, Martin; Lisewski, Ulrike; Gotthardt, Michael; Tharaux, Pierre-Louis; Huber, Tobias B.; Grahammer, Florian

    2015-01-01

    The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive Cxadr knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, Cxadr is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of Cxadr for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific Cxadr knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of Cxadr on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of Cxadr. PMID:26076477

  15. Efficient targeting of adenoviral vectors to integrin positive vascular cells utilizing a CAR-cyclic RGD linker protein.

    PubMed

    Krom, Y D; Gras, J C E; Frants, R R; Havekes, L M; van Berkel, T J; Biessen, E A L; van Dijk, K Willems

    2005-12-16

    Vascular smooth muscle (VSMC) and endothelial cells (EC) are particularly resistant to infection by type 5 adenovirus (Ad) vectors. To overcome this limitation and target Ad vectors to ubiquitously expressed alpha(V)beta(3/5) integrins, we have generated a linker protein consisting of the extracellular domain of the coxsackie adenovirus receptor (CAR) connected via avidin to a biotinylated cyclic (c) RGD peptide. After optimization of CAR to cRGD and to Ad coupling, infection of mouse heart endothelial cells (H5V) could be augmented significantly, as demonstrated by 600-fold increased transgene expression levels. In EOMAs, a hemangioendothelioma-derived cell line, the fraction of infected cells was enhanced 4- to 6-fold. Furthermore, the fraction of infected primary mouse VSMC was increased from virtually 0% to 25%. Finally, in human umbilical vein endothelial cells, the number of GFP positive cells was enhanced from 2% to 75%. In conclusion, CAR-cRGD is a versatile and highly efficient construct to target Ad vectors to both transformed and primary VSMC and EC. PMID:16259946

  16. Enhanced Prostate Cancer Gene Transfer and Therapy Using a Novel Serotype Chimera Cancer Terminator Virus (Ad.5/3-CTV)

    PubMed Central

    AZAB, BELAL M.; DASH, RUPESH; DAS, SWADESH K.; BHUTIA, SUJIT K.; SARKAR, SIDDIK; SHEN, XUE-NING; QUINN, BRIDGET A.; DENT, PAUL; DMITRIEV, IGOR P.; WANG, XIANG-YANG; CURIEL, DAVID T.; PELLECCHIA, MAURIZIO; REED, JOHN C.; SARKAR, DEVANAND; FISHER, PAUL B.

    2015-01-01

    Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting. PMID:23868767

  17. Rapid and highly sensitive detection of Enterovirus 71 by using nanogold-enhanced electrochemical impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Hsing-Yuan; Tseng, Shing-Hua; Cheng, Tsai-Mu; Chu, Hsueh-Liang; Lu, Yu-Ning; Wang, Fang-Yu; Tsai, Li-Yun; Shieh, Juo-Yu; Yang, Jyh-Yuan; Juan, Chien-Chang; Tu, Lung-Chen; Chang, Chia-Ching

    2013-07-01

    Enterovirus 71 (EV71) infection is an emerging infectious disease causing neurological complications and/or death within two to three days after the development of fever and rash. A low viral titre in clinical specimens makes the detection of EV71 difficult. Conventional approaches for detecting EV71 are time consuming, poorly sensitive, or complicated, and cannot be used effectively for clinical diagnosis. Furthermore, EV71 and Coxsackie virus A16 (CA16) may cross react in conventional assays. Therefore, a rapid, highly sensitive, specific, and user-friendly test is needed. We developed an EV71-specific nanogold-modified working electrode for electrochemical impedance spectroscopy in the detection of EV71. Our results show that EV71 can be distinguished from CA16, Herpes simplex virus, and lysozyme, with the modified nanogold electrode being able to detect EV71 in concentrations as low as 1 copy number/50 ?l reaction volume, and the duration between sample preparation and detection being 11 min. This detection platform may have the potential for use in point-of-care diagnostics.

  18. Antiviral and antitumor activities of the lectin extracted from Aspidistra elatior.

    PubMed

    Xu, Xiao-Chao; Zhang, Zhong-Wei; Chen, Yang-Er; Yuan, Ming; Yuan, Shu; Bao, Jin-Ku

    2015-01-01

    Lectins, a group of highly diverse proteins of non-immune origin and are ubiquitously distributed in plants, animals and fungi, have multiple significant biological functions, such as anti-fungal, anti-viral and, most notably, anti-tumor activities. A lectin was purified from the rhizomes of Aspidistra elatior Blume, named A. elatior lectin (AEL). In vitro experiments showed that the minimum inhibitory concentrations of AEL against the vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus were all the same at about 4 ?g/mL. However, AEL was ineffective against the Sindbis virus and reovirus-1. AEL also showed significant in vitro antiproliferative activity towards Bre-04, Lu-04, HepG2, and Pro-01 tumor cell lines by increasing the proportion of their sub-G1 phase. However, AEL failed to restrict the proliferation of the HeLa cell line. Western blotting indicated that AEL induced the upregulation of cell cycle-related proteins p53 and p21. The molecular basis and species-specific effectiveness of the anti-proliferative and anti-viral potential of AEL are discussed. PMID:25854839

  19. A vesicular stomatitis virus glycoprotein epitope-incorporated oncolytic adenovirus overcomes CAR-dependency and shows markedly enhanced cancer cell killing and suppression of tumor growth.

    PubMed

    Yoon, A-Rum; Hong, Jinwoo; Yun, Chae-Ok

    2015-10-27

    Utility of traditional oncolytic adenovirus (Ad) has been limited due to low expression of coxsackie and adenovirus receptor (CAR) in cancer cells which results in poor infectivity of Ads. Here with an aim of improving the efficiency of Ad's entry to the cell, we generated a novel tropism-expanded oncolytic Ad which contains the epitope of vesicular stomatitis virus glycoprotein (VSVG) at the HI-loop of Ad fiber. We generated 9 variants of oncolytic Ads with varying linkers and partial deletion to the fiber. Only one VSVG epitope-incorporated variant, RdB-1L-VSVG, which contains 1 linker and no deletion to fiber, was produced efficiently. Production of 3-dimensionaly stable fiber in RdB-1L-VSVG was confirmed by immunoblot analysis. RdB-1L-VSVG shows a remarkable improvement in cytotoxicity and total viral yield in cancer cells. RdB-1L-VSVG demonstrates enhanced cytotoxicity in cancer cells with subdued CAR-expression as it can be internalized by an alternate pathway. Competition assays with a CAR-specific antibody (Ab) or VSVG receptor, phosphatidyl serine (PS), reveals that cell internalization of RdB-1L-VSVG is mediated by both CAR and PS. Furthermore, treatment with RdB-1L-VSVG significantly enhanced anti-tumor effect in vivo. These studies demonstrate that the strategy to expand oncolytic Ad tropism may significantly improve therapeutic profile for cancer treatment. PMID:26430798

  20. Paleobiogeography of the Onondaga Limestone in southeastern New York: A second shelf to basin ramp

    SciTech Connect

    Lindemann, R.H. . Dept. of Geology); Feldman, H.R. . Biology Dept.)

    1993-03-01

    As a logical consequence of outcrop pattern and density, research on the Middle Devonian Onondaga Limestone has concentrated on the formation in an essentially east-west transect between the mid-Hudson Valley and Buffalo. Paleogeographic analysis reveals a symmetrical pattern, with the axis of the Appalachian Basin in central New York and the Edgecliff reefs flourishing in the eastern and western shelf regions. The subsurface reef trend of western New York and Pennsylvania is consistent with this model and is suggestive of a parallel reef trend in the east. However, recent examination of the formation in the southeastern part of the state has led to a pronounced reinterpretation of Onondaga paleobiogeography in that area. The authors have determined that there was a shallow carbonate shelf in the Helderberg-Coxsackie areas, a thick accumulation of shelf-margin bryozoan bafflestone between Leeds and Saugerties, and an even thicker accumulation of sparse to packed biocalcisiltites deposited on a carbonate ramp dipping southward into the Port Jervis area. It appears that this ramp occupied the northern margin of a structural base depocenter, located to the east of and not directly related to the topographic basin of central New York, which was centered in the Tristates vicinity from the Late Silurian until the early Middle Devonian.

  1. Enhanced prostate cancer gene transfer and therapy using a novel serotype chimera cancer terminator virus (Ad.5/3-CTV).

    PubMed

    Azab, Belal M; Dash, Rupesh; Das, Swadesh K; Bhutia, Sujit K; Sarkar, Siddik; Shen, Xue-Ning; Quinn, Bridget A; Dent, Paul; Dmitriev, Igor P; Wang, Xiang-Yang; Curiel, David T; Pellecchia, Maurizio; Reed, John C; Sarkar, Devanand; Fisher, Paul B

    2014-01-01

    Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting. PMID:23868767

  2. Species D Adenoviruses as Oncolytics Against B Cell Cancers

    PubMed Central

    Chen, Christopher Y.; Senac, Julien S.; Weaver, Eric A.; May, Shannon M.; Jelinek, Diane F.; Greipp, Philip; Witzig, Thomas; Barry, Michael A.

    2011-01-01

    Purpose Oncolytic viruses are self-amplifying anti-cancer agents that make use of the natural ability of viruses to kill cells. Adenovirus serotype 5 (Ad5) has been extensively tested against solid cancers, but less so against B cell cancers since these cells do not generally express the coxsackie and adenoviral receptor (CAR). To determine if other adenoviruses might have better potency, we “mined” the adenovirus virome of 55 serotypes for viruses that could kill B cell cancers. Experimental Design 15 adenoviruses selected to represent Ad species B, C, D, E, and F were tested in vitro against cell lines and primary patient B cell cancers for their ability to infect, replicate in, and kill these cells. Select viruses were also tested against B cell cancer xenografts in immunodeficient mice. Results Species D adenoviruses mediated most robust killing against a range of B cell cancer cell lines, against primary patient marginal zone lymphoma cells, and against primary patient CD138+ myeloma cells in vitro. When injected into xenografts in vivo, single treatment with select species D viruses Ad26 and Ad45 delayed lymphoma growth. Conclusions Relatively unstudied species D adenoviruses have a unique ability to infect and replicate in B cell cancers as compared to other adenovirus species. These data suggest these viruses have unique biology in B cells and support translation of novel species D adenoviruses as oncolytics against B cell cancers. PMID:21890454

  3. Photochemical studies and nanomolar photodynamic activities of phthalocyanines functionalized with 1,4,7-trioxanonyl moieties at their non-peripheral positions.

    PubMed

    Sobotta, Lukasz; Wierzchowski, Marcin; Mierzwicki, Michal; Gdaniec, Zofia; Mielcarek, Jadwiga; Persoons, Leentje; Goslinski, Tomasz; Balzarini, Jan

    2016-02-01

    Manganese(III), cobalt(II), copper(II), magnesium(II), zinc(II) and metal-free phthalocyanines, possessing 1,4,7-trioxanonyl substituents, at their non-peripheral positions, were subjected to photochemical, photodynamic and biological activity studies. Demetallated phthalocyanine and its metallated d-block analogues, with copper(II), cobalt(II), manganese(III) chloride, were found to be less efficient singlet oxygen generators in comparison to the zinc(II) analogue and zinc(II) phthalocyanine reference. Irradiation of several phthalocyanines for short time periods resulted in a substantially increased cytostatic activity against both suspension (leukemic/lymphoma at 85nM) and solid (cervix carcinoma at 72nM and melanoma at 81nM) tumour cell lines (up to 200-fold). Noteworthy is that enveloped viruses, such as for herpesvirus and influenza A virus, but not, non-enveloped virus strains, such as Coxsackie B4 virus and reovirus-1, exposed to irradiation in the presence of the phthalocyanines, markedly lost their infectivity potential. PMID:26638008

  4. Cytokine and Chemokine Production by Human Pancreatic Islets Upon Enterovirus Infection

    PubMed Central

    Schulte, Barbara M.; Lanke, Kjerstin H.W.; Piganelli, Jon D.; Kers-Rebel, Esther D.; Bottino, Rita; Trucco, Massimo; Huijbens, Richard J.F.; Radstake, Timothy R.D.J.; Engelse, Marten A.; de Koning, Eelco J.P.; Galama, Jochem M.; Adema, Gosse J.; van Kuppeveld, Frank J.M.

    2012-01-01

    Enteroviruses of the human enterovirus B species (HEV-Bs) (e.g., coxsackie B viruses [CVBs] and echoviruses) have been implicated as environmental factors that trigger/accelerate type 1 diabetes, but the underlying mechanism remains elusive. The aim of this study was to gain insight into the cytokines and chemokines that are produced by human pancreatic islets upon infection with CVBs. To this end, we studied the response of human islets of Langerhans upon mock or CVB3 infection. Using quantitative PCR, we showed that upon CVB3 infection, transcription of interferon (IFN), IFN-stimulated genes, and inflammatory genes was induced. Analysis of secreted cytokines and chemokines by Luminex technology confirmed production and secretion of proinflammatory cytokines (e.g., interleukin [IL]-6 and tumor necrosis factor-?) as well as various chemotactic proteins, such as IFN-?–induced protein 10, macrophage inflammatory protein (MIP)-1?, MIP-1?, and IL-8. Infection with other HEV-Bs induced similar responses, yet their extent depended on replication efficiency. Ultra violet–inactivated CVB3 did not induce any response, suggesting that virus replication is a prerequisite for antiviral responses. Our data represent the first comprehensive overview of inflammatory mediators that are secreted by human islets of Langerhans upon CVB infection and may shed light on the role of enteroviruses in type 1 diabetes pathogenesis. PMID:22596052

  5. EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

    PubMed Central

    Grünwald, Geoffrey K; Vetter, Alexandra; Klutz, Kathrin; Willhauck, Michael J; Schwenk, Nathalie; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Zach, Christian; Wagner, Ernst; Göke, Burkhard; Holm, Per S; Ogris, Manfred; Spitzweg, Christine

    2013-01-01

    We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by 123I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. 124I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of 131I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy. PMID:24193032

  6. CD47-retargeted oncolytic adenovirus armed with melanoma differentiation-associated gene-7/interleukin-24 suppresses in vivo leukemia cell growth.

    PubMed

    Li, Gongchu; Wu, Hu; Cui, Lianzhen; Gao, Yajun; Chen, Lei; Li, Xin; Liang, Tianxiang; Yang, Xinyan; Cheng, Jianhong; Luo, Jingjing

    2015-12-22

    Our previous studies have suggested that harboring a soluble coxsackie-adenovirus receptor-ligand (sCAR-ligand) fusion protein expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting to serotype 5 adenovirus infection. We report here a novel oncolytic adenovirus vector redirected to CD47+ leukemia cells though carrying a sCAR-4N1 expression cassette in the viral genome, forming Ad.4N1, in which 4N1 represents the C-terminal CD47-binding domain of thrombospondin-1. The infection and cytotoxicity of Ad.4N1 in leukemia cells were determined to be mediated by the 4N1-CD47 interaction. Ad.4N1 was further engineered to harbor a gene encoding melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), forming Ad.4N1-IL24, which replicated dramatically faster than Ad.4N1, and elicited significantly enhanced antileukemia effect in vitro and in a HL60/Luc xenograft mouse model. Our data suggest that Ad.4N1 could act as a novel oncolytic adenovirus vector for CD47+ leukemia targeting gene transfer, and Ad.4N1 harboring anticancer genes may provide novel antileukemia agents. PMID:26554307

  7. Viral serologies in patients with chronic fatigue and chronic fatigue syndrome.

    PubMed

    Buchwald, D; Ashley, R L; Pearlman, T; Kith, P; Komaroff, A L

    1996-09-01

    Chronic fatigue syndrome (CFS) is an illness characterized by disabling fatigue associated with complaints of fevers, sore throat, myalgia, lymphadenopathy, sleep disturbances, neurocognitive difficulties, and depression. A striking feature of CFS is its sudden onset following an acute, presumably viral, illness and the subsequent recurrent "flu-like" symptoms. It has been speculated that both CFS and debilitating chronic fatigue (CF) that does not meet strict criteria for CFS may be the direct or indirect result of viral infections. We therefore tested 548 chronically fatigued patients who underwent a comprehensive medical and psychiatric evaluation for antibodies to 13 viruses. Our objectives were to compare the seroprevalence and/or geometric mean titer (GMT) of antibodies to herpes simplex virus 1 and 2, rubella, adenovirus, human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, and Cox-sackie B virus, types 1-6 in patients with CF to healthy control subjects. Other goals were to determine if greater rates of seropositivity or higher GMTs occurred among subsets of patients with CFS, fibromyalgia, psychiatric disorders, a self-reported illness onset with a viral syndrome, and a documented temperature > 37 degrees C on physical examination. Differences in the seroprevalence or GMTs of antibodies to 13 viruses were not consistently found in those with CF compared with control subjects, or in any subsets of patients including those with CFS, an acute onset of illness, or a documented fever. These particular viral serologies were not useful in evaluating patients presenting with CF. PMID:8890037

  8. Hydrophobic pocket targeting probes for enteroviruses

    NASA Astrophysics Data System (ADS)

    Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

    2015-10-01

    Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells.Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to le

  9. Multimerization of Adenovirus Serotype 3 Fiber Knob Domains Is Required for Efficient Binding of Virus to Desmoglein 2 and Subsequent Opening of Epithelial Junctions?

    PubMed Central

    Wang, Hongjie; Li, ZongYi; Yumul, Roma; Lara, Stephanie; Hemminki, Akseli; Fender, Pascal; Lieber, André

    2011-01-01

    Recently, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population (H. Wang et al., Nat. Med. 17:96–104, 2011). In this study, we have attempted to delineate structural details of the Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We found that the DSG2-interacting domain(s) within Ad3 is formed by several fiber knob domains that have to be in the spatial constellation that is present in viral particles. Based on this finding, we generated a small recombinant, self-dimerizing protein containing the Ad3 fiber knob (Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blocked Ad3 infection. We demonstrated by confocal immunofluorescence and transmission electron microscopy analyses that Ad3-K/S/Kn, through its binding to DSG2, triggered the transient opening of intercellular junctions in epithelial cells. The pretreatment of epithelial cells with Ad3-K/S/Kn resulted in increased access to receptors that are localized in or masked by epithelial junctions, e.g., CAR or Her2/neu. Ad3-K/S/Kn treatment released CAR from tight junctions and thus increased the transduction of epithelial cells by a serotype Ad5-based vector. Furthermore, the pretreatment of Her2/neu-positive breast cancer cells with Ad3-K/S/Kn increased the killing of cancer cells by the Her2/neu-targeting monoclonal antibody trastuzumab (Herceptin). This study widens our understanding of how Ads achieve high avidity to their receptors and the infection of epithelial tissue. The small recombinant protein Ad3-K/S/Kn has practical implications for the therapy of epithelial cancer and gene/drug delivery to normal epithelial tissues. PMID:21525338

  10. pH-sensitive oncolytic adenovirus hybrid targeting acidic tumor microenvironment and angiogenesis

    PubMed Central

    Choi, Joung-Woo; Jung, Soo-Jung; Kasala, Dayananda; Hwang, June Kyu; Hu, Jun; Bae, You Han; Yun, Chae-Ok

    2015-01-01

    Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH 6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH 6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH 7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX. PMID:25575865

  11. CD123 targeting oncolytic adenoviruses suppress acute myeloid leukemia cell proliferation in vitro and in vivo

    PubMed Central

    Li, G; Li, X; Wu, H; Yang, X; Zhang, Y; Chen, L; Wu, X; Cui, L; Wu, L; Luo, J; Liu, X Y

    2014-01-01

    We report here a novel strategy to redirect oncolytic adenoviruses to CD123 by carry a soluble coxsackie-adenovirus receptor (sCAR)-IL3 expression cassette in the viral genome to form Ad.IL3, which sustainably infected acute myeloid leukemia (AML) cells through CD123. Ad.IL3 was further engineered to harbor gene encoding manganese superoxide dismutase (MnSOD) or mannose-binding plant lectin Pinellia pedatisecta agglutinin (PPA), forming Ad.IL3-MnSOD and Ad.IL3-PPA. As compared with Ad.IL3 or Ad.sp-E1A control, Ad.IL3-MnSOD and Ad.IL3-PPA significantly suppressed in vitro proliferation of HL60 and KG-1 cells. Elevated apoptosis was detected in HL60 and KG-1 cells treated with either Ad.IL3-MnSOD or Ad.IL3-PPA. The caspase-9–caspase-7 pathway was determined to be activated by Ad.IL3-MnSOD as well as by Ad.IL3-PPA in HL60 cells. In an HL60/Luc xenograft nonobese diabetic/severe-combined immunodeficiency mice model, Ad.IL3-MnSOD and Ad.IL3-PPA suppressed cancer cell growth as compared with Ad.IL3. A significant difference of cancer cell burden was detected between Ad.IL3 and Ad.IL3-PPA groups at day 9 after treatment. Furthermore, Ad.IL3-MnSOD significantly prolonged mouse survival as compared with Ad.sp-E1A. These findings demonstrated that Ad.IL3-gene could serve as a novel agent for AML therapy. Harboring sCAR-ligand expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting serotype 5 adenovirus infection. PMID:24658372

  12. The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

    PubMed

    Danielsson, Angelika; Dzojic, Helena; Rashkova, Victoria; Cheng, Wing-Shing; Essand, Magnus

    2011-01-01

    The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected. PMID:21379379

  13. Antiviral triterpenoids from the medicinal plant Schefflera heptaphylla.

    PubMed

    Li, Yaolan; Jiang, Renwang; Ooi, Linda S M; But, Paul P H; Ooi, Vincent E C

    2007-05-01

    Schefflera heptaphylla (L.) Frodin is a principal ingredient of an herbal tea formulation widely used for the treatment of common cold in southern China. An extract of the long leafstalk of the compound leaf of S. heptaphylla exhibited the most potent antiviral activity against respiratory syncytial virus (RSV). Further antiviral-guided fractionation and isolation of the leafstalk extract of S. heptaphylla led to obtain two highly active pure triterpenoids, namely 3alpha-hydroxylup-20(29)-ene-23,28-dioic acid and 3-epi-betulinic acid 3-O-sulfate, together with an inactive saponin, 3alpha-hydroxylup-20(29)-ene-23,28-dioic acid 28-O-alpha-l-rhamnopyranosyl-(1-->4)-O-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranoside. An antiviral assay using a cytopathic effect (CPE) reduction method showed that the two triterpenoids possessed broader antiviral activity against respiratory syncytial virus (RSV) with a similar 50% inhibition concentration (IC(50)) value of 6.25 microg/mL, influenza A (H1N1) virus with IC(50) values of 25 and 31.3 microg/mL, Coxsackie B3 (Cox B3) virus with IC(50) values of 12.5 and 20 microg/mL and herpes simplex virus type 1 (HSV-1) with IC(50) values of 18.8 and 25 microg/mL, respectively, whereas the saponin did not have antiviral activity against these four viruses at a concentration of 100 microg/mL. PMID:17357972

  14. CAR-dependent and CAR-independent pathways of adenovirus vector–mediated gene transfer and expression in human fibroblasts

    PubMed Central

    Hidaka, Chisa; Milano, Eric; Leopold, Philip L.; Bergelson, Jeffrey M.; Hackett, Neil R.; Finberg, Robert W.; Wickham, Thomas J.; Kovesdi, Imre; Roelvink, Peter; Crystal, Ronald G.

    1999-01-01

    Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by Ad?RGD?gal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) ?V integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)?gal (bearing seven lysines on the end of fiber) (b) AdF(RGD)?gal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK ?gal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors. PMID:10021467

  15. Strategies to enhance transductional efficiency of adenoviral-based gene transfer to primary human fibroblasts and keratinocytes as a platform in dermal wounds

    PubMed Central

    Stoff, Alexander; Rivera, Angel A.; Banerjee, N. S.; Mathis, J. Michael; Espinosa-de-los-Monteros, Antonio; Le, Long P.; De la Torre, Jorge I.; Vasconez, Luis O.; Broker, Thomas R.; Richter, Dirk F.; Stoff-Khalili, Mariam A.; Curiel, David T.

    2007-01-01

    Genetically modified keratinocytes and fibroblasts are suitable for delivery of therapeutic genes capable of modifying the wound healing process. However, efficient gene delivery is a prerequisite for successful gene therapy of wounds. Whereas adenoviral vectors (Ads) exhibit superior levels of in vivo gene transfer, their transductional efficiency to cells resident within wounds may nonetheless be suboptimal, due to deficiency of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR). We explored CAR-independent transduction to fibroblasts and keratinocytes using a panel of CAR-independent fiber-modified Ads to determine enhancement of infectivity. These fiber-modified adenoviral vectors included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7). To evaluate whether transduction efficiencies of the fiber-modified adenoviral vectors correlated with the expression of their putative receptors on keratinocytes and fibroblasts, we analyzed the mRNA levels of CAR, ?? integrin, syndecan-1, and glypican-1 using quantitative polymerase chain reaction. Analysis of luciferase and green fluorescent protein transgene expression showed superior transduction efficiency of Ad5.pK7 in keratinocytes and Ad5.RGD.pK7 in fibroblasts. mRNA expression of ?? integrin, syndecan-1 and glypican-1 was significantly higher in primary fibroblasts than CAR. In keratinocytes, syndecan-1 expression was significantly higher than all the other receptors tested. Significant infectivity enhancement was achieved in keratinocytes and fibroblasts using fiber-modified adenoviral vectors. These strategies to enhance infectivity may help to achieve higher clinical efficacy of wound gene therapy. PMID:17014674

  16. Increased adenovirus Type 5 mediated transgene expression due to RhoB down-regulation.

    PubMed

    Majhen, Dragomira; Stojanovi?, Nikolina; Vuki?, Dunja; Pichon, Chantal; Leduc, Chloé; Osmak, Maja; Ambriovi?-Ristov, Andreja

    2014-01-01

    Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking. PMID:24466204

  17. Regulation of the Heme Oxygenase-1/carbon monoxide system by hydrogen sulfide in murine coxsackievirus B3-induced myocarditis.

    PubMed

    Zhang, S; Wu, T; Xia, T; Rong, X; Wu, T; Chu, M; Wu, R

    2015-01-01

    To explore the impact of hydrogen sulfide (H2S) on the heme oxygenase—1 (HO—1)/carbon monoxide (CO) system in coxsackie virus B3 (CVB3)—induced myocarditis. A total of 80 Balb/c mice were divided randomly into four groups designated N, C, P and S. Group N served as the negative control while groups C, P, and S were infected with CVB3 to induce myocarditis. Group P was additionally treated with DL—propargylglycine (PAG) to inhibit the generation of H2S while Group S was treated with NaHS, an H2S donor. Ten days after infection, heart sections were scored for histopathology. We also measured carboxyhemoglobin (COHb) levels in the blood and HO—1 expression by immunohistochemistry. 1. Each CVB3—infected group (C, P, and S) exhibited increased pathology, COHb levels, and HO—1 expression compared to uninfected controls. 2. Regarding histopathology, the score of group P was worse, while that of group S was better, than that of group C. 3. The P group COHb level was lower than group C, while the S group COHb level was higher than group C. 4. Positive HO—1 expression was seen in group C with reduced expression in group P and increased expression in group S. 5. A positive correlation was observed between the COHb concentration and HO—1expression; alternatively, a negative correlation was found between the histopathologic scores and both the concentration of COHb and the expression level of HO—1. Modulation of H2S can play a regulatory role in the pathogenesis of VMC by impacting the HO—1/CO pathway. PMID:26025406

  18. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    PubMed Central

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3?-to-5? unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities. PMID:26218680

  19. Prevalence of human enteroviruses among apparently healthy nursery school children in Accra

    PubMed Central

    Attoh, Juliana; Obodai, Evangeline; Adiku, Theophilus; Odoom, John Kofi

    2014-01-01

    Introduction Human enteroviruses are common in children causing asymptomatic infections ranging from mild to severe illnesses. In Ghana, information on the prevalence of non-polio enterovirus causing acute flaccid paralysis is available but data on surveillance of these viruses in school children is scanty. Here, the prevalence of human enteroviruses among apparently healthy children in selected school in Accra was studied. Methods Stool samples from 273 apparently healthy children less than eight years of age in 9 selected nursery schools were collected between December 2010 and March 2011and processed for human enteroviruses on L20B, RD and Hep-2 cell lines. Positive Isolates were characterized by microneutralisation assay with antisera pools from RIVM, the Netherlands according to standard methods recommended by WHO. Results Of the 273 samples processed, 66 (24.2%) non-polio enteroviruses were isolated. More growth was seen on Hep-2C (46%) only than RD (18%) only and on both cell lines (34%). No growth was seen on L20B even after blind passage. Excretion of non-polio enteroviruses was found in all the schools with majority in BD school. Serotyping of the isolates yielded predominantly Coxsackie B viruses followed by echoviruses 13 and 7. More than half of the isolates could not be typed by the antisera pools. Conclusion The study detected 13 different serotypes of non-polio enteroviruses in circulation but no poliovirus was found. BD school was found to have the highest prevalence of NPEV. Complete identification through molecular methods is essential to establish the full range of NPEVs in circulation in these schools. PMID:25400833

  20. Hepatoma targeting peptide conjugated bio-reducible polymer complexed with oncolytic adenovirus for cancer gene therapy.

    PubMed

    Choi, Joung-Woo; Kim, Hyun Ah; Nam, Kihoon; Na, Youjin; Yun, Chae-Ok; Kim, SungWan

    2015-12-28

    Despite adenovirus (Ad) vector's numerous advantages for cancer gene therapy, such as high ability of endosomal escape, efficient nuclear entry mechanism, and high transduction, and therapeutic efficacy, tumor specific targeting and antiviral immune response still remain as a critical challenge in clinical setting. To overcome these obstacles and achieve cancer-specific targeting, we constructed tumor targeting bioreducible polymer, an arginine grafted bio-reducible polymer (ABP)-PEG-HCBP1, by conjugating PEGylated ABP with HCBP1 peptides which has high affinity and selectivity towards hepatoma. The ABP-PEG-HCBP1-conjugated replication incompetent GFP-expressing ad, (Ad/GFP)-ABP-PEG-HCBP1, showed a hepatoma cancer specific uptake and transduction compared to either naked Ad/GFP or Ad/GFP-ABP. Competition assays demonstrated that Ad/GFP-ABP-PEG-HCBP1-mediated transduction was specifically inhibited by HCBP1 peptide rather than coxsackie and adenovirus receptor specific antibody. In addition, ABP-PEG-HCBP1 can protect biological activity of Ad against serum, and considerably reduced both innate and adaptive immune response against Ad. shMet-expressing oncolytic Ad (oAd; RdB/shMet) complexed with ABP-PEG-HCBP1 delivered oAd efficiently into hepatoma cancer cells. The oAd/ABP-PEG-HCBP1 demonstrated enhanced cancer cell killing efficacy in comparison to oAd/ABP complex. Furthermore, Huh7 and HT1080 cancer cells treated with oAd/shMet-ABP-PEG-HCBP1 complex had significantly decreased Met and VEGF expression in hepatoma cancer, but not in non-hepatoma cancer. In sum, these results suggest that HCBP1-conjugated bioreducible polymer could be used to deliver oncolytic Ad safely and efficiently to treat hepatoma. PMID:26437261

  1. “Bath salts” induced severe reversible cardiomyopathy

    PubMed Central

    Sivagnanam, Kamesh; Chaudari, Dhara; Lopez, Pablo; Sutherland, Michael E; Ramu, Vijay K.

    2013-01-01

    Patient: Male, 27 Final Diagnosis: Bath salt induced cardiomyopathy Symptoms: Agitation • fever • pedal edema Medication: Intravenous nor-epinephrine for less than 6 hours Clinical Procedure: — Specialty: Internal medicine • cardiology Objective: Unusual clinical course Background: “Bath salts” is the street name for a group of recently identified and increasingly abused stimulant synthetic cathinones that are associated with multiple systemic effects. We present a case of a patient who developed reversible dilated cardiomyopathy secondary to their use. Case Report: A 27 year old male with no past medical history was brought to emergency department with agitation. He had been inhaling and intravenously injecting “bath salts”, containing a mephedrone/Methylenedioxypyrovalerone (MDPV) combination. On presentation, he was tachycardic, hypotensive and febrile. His initial labs showed an elevated white count, creatinine and creatinine phosphokinase levels. His erythrocyte sedimentation rate; C-reactive protein; urinalysis; urine drug screen; Human Immunodeficiency Virus, hepatitis, coxsackie, and influenza serology were normal. EKG showed sinus tachycardia. An echocardiogram was done which showed dilated cardiomyopathy with an ejection fraction (EF) of 15–20% and global hypokinesia. A left heart catheterization was done and was negative for coronary artery disease. At a 20 week follow up, he had stopped abusing bath salts and was asymptomatic. A repeat echocardiogram showed an EF of 52%. Cocnlusions: Bath salts (MDPV, mephedrone) are synthetic cathinones with amphetamine/cocaine like properties with potential cardiotoxic effects. Cardiovascular manifestations reported include tachycardia, hypertension, myocardial infarction, arrhythmias and cardiac arrest. “Bath salts” can also cause severe reversible dilated cardiomyopathy. Prior to diagnosis, other causes of cardiomyopathy including ischemic, infectious, familial, immunological, metabolic and cytotoxic may need to be ruled out; as was done in our patient. PMID:23919103

  2. [Occurrence and isolation of human enteroviruses from the air of waste removal and disposal plants].

    PubMed

    Pfirrmann, A; vanden Bossche, G

    1994-08-01

    Aerosols from waste treatment plants were examined with regard to the presence of airborne viruses. For the purpose of a comparative evaluation, two different collecting devices consisting of an electroprecipitator and a special-impinger apparatus were used for extraction and collection of viruses from air samples. The collected suspensions were concentrated and fractionated by means of hydroextraction in combination with a differential centrifugation procedure. After solubilisation of the sedimented material with the anionic detergent, sodium-dodecylsulfate, and following ultrasonic treatment, viral infectivity could be demonstrated in 12 out of 36 examined specimens, after inoculation on BGM cells. The highest virus isolation rates were obtained with the electroprecipitator. Based on the results of investigations of biological, physicochemical as well as antigenic characteristics, the isolated strains revealed to belong to the family of Picornaviridae. According to the results of additional characterization assays, the isolates were identified as Coxsackie-B and ECHO-viruses. The linkage between the occurrence of these viruses and a possible risk of infection for humans remains to be elucidated by further epidemiological studies. However, the results of the present work indicate that, besides of an increased dust and germ concentration in such facilities, there is substantial evidence of increased viral contamination as well. Enteroviruses are generally considered as indicator viruses revealing the presence of viral contaminants in tap water and sewage. As human enteroviruses can be regularly isolated from such aerosols, the detection of these viruses in air samples may also be an appropriate criterion to estimate the amount to which virus concentrations may build up within waste treatment plants. PMID:7802896

  3. Safety Profiles and Antitumor Efficacy of Oncolytic Adenovirus Coated with Bioreducible Polymer in the Treatment of a CAR Negative Tumor Model

    PubMed Central

    2015-01-01

    Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy. PMID:25400213

  4. The antiviral effect of jiadifenoic acids C against coxsackievirus B3

    PubMed Central

    Ge, Miao; Wang, Huiqiang; Zhang, Guijie; Yu, Shishan; Li, Yuhuan

    2014-01-01

    Coxsackievirus B type 3 (CVB3) is one of the major causative pathogens associated with viral meningitis and myocarditis, which are widespread in the human population and especially prevalent in neonates and children. These infections can result in dilated cardiomyopathy (DCM) and other severe clinical complications. There are no vaccines or drugs approved for the prevention or therapy of CVB3-induced diseases. During screening for anti-CVB3 candidates in our previous studies, we found that jiadifenoic acids C exhibited strong antiviral activities against CVB3 as well as other strains of Coxsackie B viruses (CVBs). The present studies were carried out to evaluate the antiviral activities of jiadifenoic acids C. Results showed that jiadifenoic acids C could reduce CVB3 RNA and proteins synthesis in a dose-dependent manner. Jiadifenoic acids C also had a similar antiviral effect on the pleconaril-resistant variant of CVB3. We further examined the impact of jiadifenoic acids C on the synthesis of viral structural and non-structural proteins, finding that jiadifenoic acids C could reduce VP1 and 3D protein production. A time-course study with Vero cells showed that jiadifenoic acids C displayed significant antiviral activities at 0–6 h after CVB3 inoculation, indicating that jiadifenoic acids C functioned at an early step of CVB3 replication. However, jiadifenoic acids C had no prophylactic effect against CVB3. Taken together, we show that jiadifenoic acids C exhibit strong antiviral activities against all strains of CVB, including the pleconaril-resistant variant. Our study could provide a significant lead for anti-CVB3 drug development.

  5. Incorporation of Porcine Adenovirus 4 Fiber Protein Enhances Infectivity of Adenovirus Vector on Dendritic Cells: Implications for Immune-Mediated Cancer Therapy

    PubMed Central

    Wilkinson-Ryan, Ivy; Kim, Julius; Kim, Sojung; Ak, Ferhat; Dodson, Lindzy; Colonna, Marco; Powell, Matthew; Mutch, David; Spitzer, Dirk; Hansen, Ted; Goedegebuure, Simon P.; Curiel, David; Hawkins, William

    2015-01-01

    One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-?) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses. PMID:25933160

  6. Replication-competent adenoviruses with the type 35-derived fiber-knob region achieve reactive oxygen species-dependent cytotoxicity and produce greater toxicity than those with the type 5-derived region in pancreatic carcinoma.

    PubMed

    Yamauchi, Suguru; Kawamura, Kiyoko; Okamoto, Shinya; Morinaga, Takao; Jiang, Yuanyuan; Shingyoji, Masato; Sekine, Ikuo; Kubo, Shuji; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

    2015-12-01

    Pancreatic carcinoma is relatively resistant to chemotherapy and cell death induced by replication of adenoviruses (Ad) can be one of the therapeutic options. Transduction efficacy of conventional type 5 Ad (Ad5) is however low and the cytotoxic mechanism by replication-competent Ad was not well understood. We constructed replication-competent Ad5 of which the E1A promoter region was replaced with a transcriptional regulatory region of the midkine, the survivin or the cyclooxygenase-2 gene, all of which were expressed at a high level in human tumors. We also prepared replication-competent Ad5 that were activated with the same region but had the type 35 Ad-derived fiber-knob region (AdF35) to convert the major cellular receptor for Ad infection from the coxsackie adenovirus receptor to CD46 molecules. Replication-competent AdF35 that were activated with the exogenous region produced cytotoxic effects on human pancreatic carcinoma cells greater than the corresponding Ad5 bearing with the same regulatory region. Cells infected with the AdF35 showed cytopathic effects and increased sub-G1 fractions. Caspase-9, less significantly caspase-8 and poly (ADP-ribose) polymerase, but not caspase-3 was cleaved and expression of molecules involved in autophagy and caspase-independent cell death pathways remained unchanged. Nevertheless, H2A histone family member X molecules were phosphorylated, and N-acetyl-L-cystein, an inhibitor for reactive oxygen species, suppressed the AdF35-mediated cytotoxicity. These data indicated a novel mechanism of Ad-mediated cell death and suggest a possible clinical application of the fiber-knob modified Ad. PMID:26373551

  7. Mechanisms of trophoblast-virus interaction.

    PubMed

    Parry, S; Holder, J; Strauss, J F

    1997-12-15

    The human placenta serves as a barrier to the transmission of some viruses, but allows others to reach the fetal circulation. The resistance and permissiveness of the placenta to viral transmission appears to be determined in large part by the placental trophoblast. In order to define the mechanisms by which human trophoblast cells influence vertical transmission of viruses, we have studied the interaction of replication-deficient recombinant viral vectors with transformed human choriocarcinoma (BeWo) cells and primary trophoblast cell cultures. Recombinant adenovirus vectors efficiently transduce BeWo cells and primary trophoblast cells. However, as BeWo cells differentiate after treatment with cAMP analogs, they become relatively resistant to adenovirus-mediated transduction due to diminished uptake of the virus particles. This differentiation-dependent loss of adenovirus transduction may be related to the down-regulation of the coxsackie adenovirus receptor, which we have detected in undifferentiated trophoblast cells. Recombinant herpes simplex virus vectors also transduce undifferentiated BeWo cells and isolated cytotrophoblast cells, but transduction by herpes simplex virus vectors declines with cAMP-induced trophoblast differentiation, apparently due to reduced viral uptake. In contrast, cAMP treatment augments trophoblast transduction by recombinant adeno-associated virus, a member of the parvovirus family. This augmentation appears to be due to increased virus uptake by a yet to be determined mechanism. The understanding of the molecular basis of these interactions between recombinant viral vectors and trophoblast cells may yield strategies to prevent vertical transmission of viruses as well as to create opportunities to modify trophoblast function through gene transfer using viral vectors. PMID:9501288

  8. Enhanced gene transfer to rabbit jugular veins by an adenovirus containing a cyclic RGD motif in the HI loop of the fiber knob.

    PubMed

    Hay, C M; De Leon, H; Jafari, J D; Jakubczak, J L; Mech, C A; Hallenbeck, P L; Powell, S K; Liau, G; Stevenson, S C

    2001-01-01

    Gene therapy using recombinant adenoviral vectors represents a promising therapeutic tool to prevent vein graft stenosis, the main complication of coronary artery bypass grafting. However, the low transduction efficiency of vascular smooth muscle cells and endothelial cells (EC) is a potential limitation, presumably due to the low levels of functional adenovirus receptor (coxsackie:adenovirus receptor; CAR). Designing vectors specifically targeted to alpha(v) integrins is a strategy that might overcome the poor expression of CAR in vascular smooth muscle cells and EC. RGD, a receptor-binding motif that can interact with alpha(v) integrins, was inserted into the HI loop and at the C-terminus of the adenoviral fiber protein in two separate adenovirus vectors encoding a beta-galactosidase reporter gene. Av1nBgCRGD (C-terminus) and Av1nBgHIRGD (HI loop) were evaluated in EC in culture and in jugular vein organ culture. Transduction of primary rat and rabbit EC with Av1nBgHIRGD was significantly more efficient when compared to Av1nBgCRGD or Av1nBg. Transduction of mouse, rat and rabbit jugular veins in organ culture using Av1nBg showed that adenovirus-mediated gene expression was greatest in rabbit jugular veins compared to rat and mouse veins. Av1nBgHIRGD augmented gene expression approximately four-fold in rabbit jugular veins when compared to Av1nBg. Histochemical analysis showed that numerous EC but few smooth muscle cells were transduced at all vector concentrations. A substantial number of adventitial fibroblasts were transduced only at the highest vector concentrations of Av1nBgHIRGD. These findings demonstrate that integrin-targeted vectors allow for enhanced gene delivery to veins and strengthen the viability of adenoviral-mediated gene transfer of therapeutic transgenes to human veins prior to vein grafting. PMID:11455202

  9. Viremic HIV controllers exhibit high plasmacytoid dendritic cell\\reactive opsonophagocytic IgG antibody responses against HIV-1 p24 associated with greater antibody isotype diversification

    PubMed Central

    Tjiam, M. Christian; Taylor, James P. A.; Morshidi, Mazmah A.; Sariputra, Lucy; Burrows, Sally; Martin, Jeffrey N.; Deeks, Steven G.; Tan, Dino B.A.; Lee, Silvia; Fernandez, Sonia; French, Martyn A.

    2015-01-01

    Identifying the mechanisms of natural control of HIV-1 infection could lead to novel approaches to prevent or cure HIV infection. Several studies have associated natural control of HIV-1 infection with IgG antibodies against HIV-1 Gag proteins (e.g. p24) and/or production of IgG2 antibodies against HIV-1 proteins. These antibodies likely exert their effect by activating anti-viral effector cell responses rather than virus neutralization. We hypothesized that an opsonophagocytic IgG antibody response against HIV-1 p24 that activates plasmacytoid dendritic cells (pDCs) through Fc?RIIa would be associated with control of HIV and that this would be enhanced by antibody isotype diversification. Using the Gen2.2 pDC cell line, we demonstrated that pDC-reactive opsonophagocytic IgG antibody responses against HIV-1 p24 were higher in HIV controllers (HIV RNA <2000 copies/mL) than non-controllers (HIV RNA >10,000 copies/mL) particularly in controllers with low but detectable viremia (HIV RNA 75–2000 copies/mL). Opsonophagocytic antibody responses correlated with plasma levels of IgG1 and IgG2 anti-HIV-1 p24 and notably, correlated inversely with plasma HIV RNA levels in viremic HIV patients. Phagocytosis of these antibodies was mediated via Fc?RIIa. Isotype diversification (towards IgG2) was greatest in HIV controllers and depletion of IgG2 from immunoglobulin preparations indicated that IgG2 antibodies to HIV-1 p24 do not enhance phagocytosis, suggesting that they enhance other aspects of antibody function, such as antigen opsonization. Our findings emulate those for pDC-reactive opsonophagocytic antibody responses against coxsackie, picorna and influenza viruses and demonstrate a previously undefined immune correlate of HIV-1 control that may be relevant to HIV vaccine development. PMID:25911748

  10. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    PubMed Central

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  11. Developing an Effective Gene Therapy for Prostate Cancer: New Technologies with Potential to Translate from the Laboratory into the Clinic

    PubMed Central

    Dash, Rupesh; Azab, Belal; Shen, Xue-Ning; Sokhi, Upneet K.; Sarkar, Siddik; Su, Zhao-zhong; Wang, Xiang-Yang; Claudio, Pier Paolo; Dent, Paul; Dmitriev, Igor P.; Curiel, David T.; Grant, Steven; Sarkar, Devanand; Fisher, Paul B.

    2015-01-01

    Prostate cancer is the second leading cause of cancer-related deaths in men in the U.S. At present, no single or combination therapy has shown efficacy in decreasing disease progression in patients with metastatic disease. A potentially viable approach for treating late-stage prostate cancer is gene therapy. Adenoviruses (Ad) are the most commonly used mode of gene delivery, but progress using this vector has been hampered by concerns over the safety and practicality of viruses including conditionally replicating Ads (CRAds), particularly for intravenous delivery, and the inefficiency of non-viral transfection techniques. Major challenges for effective gene therapy using Ads are the limited infectivity of regular Ad serotype 5 (Ad5) and the inability to specifically deliver the therapeutic directly into diseased tissue without trapping in the liver or elimination by the immune system. The shortcoming in using Ad5 is mostly attributed to a reduction in Coxsackie-adenovirus receptors (CAR) on the surface of cancer cells, which can be mitigated by generating tropism-modified Ads permitting CAR-independent infection of tumor cells. The limitations of systemic gene delivery can now be overcome by using a novel targeted-delivery approach such as ultrasound (US) contrast agents (microbubbles) to deliver effective therapeutic reagents, Ads, or recombinant proteins, combined with ultrasound-targeted microbubble destruction (UTMD), to develop a site-specific therapy in immune competent transgenic mouse models. These unique strategies for enhancing the efficacy of gene therapy provide a direct path to translation from the laboratory into the clinic for developing an effective gene therapy of prostate cancer. PMID:21276410

  12. Characterization of infectivity of knob-modified adenoviral vectors in glioma.

    PubMed

    Paul, C P L; Everts, M; Glasgow, J N; Dent, P; Fisher, P B; Ulasov, I V; Lesniak, M S; Stoff-Khalili, M A; Roth, J C; Preuss, M A; Dirven, C M F; Lamfers, M L M; Siegal, G P; Zhu, Z B; Curiel, D T

    2008-05-01

    Malignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors,particularly vectors derived from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping,the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types,including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or"xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype"fiber knob domains, including those derived from murine,ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46(an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma. PMID:18756624

  13. Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the Apogossypol derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells

    PubMed Central

    Azab, Belal; Dash, Rupesh; Das, Swadesh K.; Bhutia, Sujit K.; Shen, Xue-Ning; Quinn, Bridget A.; Sarkar, Siddik; Wang, Xiang-Yang; Hedvat, Michael; Dmitriev, Igor P.; Curiel, David T.; Grant, Steven; Dent, Paul; Reed, John C.; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B.

    2011-01-01

    Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients. PMID:21780116

  14. RNASEK is required for internalization of diverse acid-dependent viruses

    PubMed Central

    Hackett, Brent A.; Yasunaga, Ari; Panda, Debasis; Tartell, Michael A.; Hopkins, Kaycie C.; Hensley, Scott E.; Cherry, Sara

    2015-01-01

    Viruses must gain entry into cells to establish infection. In general, viruses enter either at the plasma membrane or from intracellular endosomal compartments. Viruses that use endosomal pathways are dependent on the cellular factors that control this process; however, these genes have proven to be essential for endogenous cargo uptake, and thus are of limited value for therapeutic intervention. The identification of genes that are selectively required for viral uptake would make appealing drug targets, as their inhibition would block an early step in the life cycle of diverse viruses. At this time, we lack pan-antiviral therapeutics, in part because of our lack of knowledge of such cellular factors. RNAi screening has begun to reveal previously unknown genes that play roles in viral infection. We identified dRNASEK in two genome-wide RNAi screens performed in Drosophila cells against West Nile and Rift Valley Fever viruses. Here we found that ribonuclease kappa (RNASEK) is essential for the infection of human cells by divergent and unrelated positive- and negative-strand-enveloped viruses from the Flaviviridae, Togaviridae, Bunyaviridae, and Orthomyxoviridae families that all enter cells from endosomal compartments. In contrast, RNASEK was dispensable for viruses, including parainfluenza virus 5 and Coxsackie B virus, that enter at the plasma membrane. RNASEK is dispensable for attachment but is required for uptake of these acid-dependent viruses. Furthermore, this requirement appears specific, as general endocytic uptake of transferrin is unaffected in RNASEK-depleted cells. Therefore, RNASEK is a potential host cell Achilles’ heel for viral infection. PMID:26056282

  15. Gene delivery into malignant glioma by infectivity-enhanced adenovirus: in vivo versus in vitro models.

    PubMed

    Van Houdt, Winan J; Wu, Hongju; Glasgow, Joel N; Lamfers, Martine L; Dirven, Clemens M; Gillespie, G Yancey; Curiel, David T; Haviv, Yosef S

    2007-07-01

    Adenoviral (Ad) vectors demonstrate several attributes of potential utility for glioma gene therapy. Although Ad infection is limited in vitro by low expression levels of the coxsackie-adenoviral receptor (CAR), in vivo studies have shown the efficacy of Ad vectors as gene delivery vectors. To evaluate the in vivo utility of CAR-independent, infectivity-enhanced Ad vectors, we employed genetically modified Ad vectors in several experimental models of human gliomas. We used three capsid-modified Ad vectors: (1) a chimeric Ad vector with a human Ad backbone and a fiber knob of a canine Ad, (2) an Ad vector with a polylysine motif incorporated into the fiber gene, and (3) a double-modified Ad vector incorporating both an RGD4C peptide and the polylysine motif. These three modified Ad vectors target, respectively, the putative membrane receptor(s) of the canine Ad vector, heparan sulfate proteoglycans (HSPGs), and both integrins and HSPGs. Our in vitro studies indicated that these retargeting strategies all enhanced CAR-independent infectivity in both established and primary low-passage glioma cells. Enhancement of in vitro gene delivery by the capsid-modified vectors correlated inversely with the levels of cellular CAR expression. However, in vivo in orthotopic human glioma xenografts, the unmodified Ad vector was not inferior relative to the capsid-modified Ad vector. Although genetic strategies to circumvent CAR deficiency in glioma cells could reproducibly expand the cellular entry mechanisms of Ad vectors in cultured and primary glioma cells, these approaches were insufficient to confer in vivo significant infectivity enhancement over unmodified Ad vectors. Other factors, probably the extracellular matrix, stromal cells, and the three-dimensional tumor architecture, clearly play important roles in vivo and interfere with Ad-based gene delivery into glioma tumors. PMID:17522331

  16. Characterization of Enterovirus Isolates from Patients with Heart Muscle Disease in a Selenium-Deficient Area of China

    PubMed Central

    Peng, Tianqing; Li, Yanwen; Yang, Yingzhen; Niu, Cunlong; Morgan-Capner, Peter; Archard, Leonard C.; Zhang, Hongyi

    2000-01-01

    An association of enterovirus infection with endemic cardiomyopathy (Keshan disease [KD]) and outbreaks of myocarditis in selenium-deficient rural areas of southwestern China has been established. Enteroviruses have been isolated from patients with KD or during outbreaks of myocarditis in last two decades. Six of these isolates grew readily in cell lines (Vero or HEp-2) and were investigated by a novel molecular typing method apart from serotyping and pathogenicity. A neutralization assay identified two isolates from KD as coxsackievirus serotype B2 (CVB2) and two isolates from myocarditis as coxsackievirus serotype B6 (CVB6) but failed to type the remaining two isolates, also from myocarditis. Direct nucleotide sequencing of reverse transcription-PCR products amplified from the 5? nontranslated region (5?NTR) of these viruses confirmed that they belong to a phylogenetic cluster consisting of coxsackie B-like viruses, including some echovirus serotypes. Sequence analysis of the coding region for viral capsid protein VP1 showed that two isolates serotyped as CVB2 have the highest amino acid sequence homology with CVB2 and that the remaining four isolates, two CVB6 and the two unknown serotypes, are most closely related to the sequence of CVB6. Sequences among these isolates varied from 82.3 to 99% in the 5?NTR and from 69 to 99% in VP1, indicating no cross contamination. The pathogenicity of these viruses in adult and suckling mice was assessed. None caused pathologic changes in the hearts of adult MF-1 or SWR mice, although pancreatitis was evident. However, the four CVB6-like viruses caused death in suckling mice, similar to a virulent coxsackievirus group B3 laboratory strain. In conclusion, the sequence data confirm that coxsackievirus group B serotypes are predominant in the region in which KD is endemic and may be the etiological agents in outbreaks of myocarditis. VP1 genotyping of enteroviruses is accurate and reliable. Animal experiments indicate that isolates may differ in pathogenicity. PMID:11015360

  17. Differential Susceptibility and Response of Primary Human Myeloid BDCA1+ Dendritic Cells to Infection with Different Enteroviruses

    PubMed Central

    Schulte, Barbara M.; Kers-Rebel, Esther D.; Prosser, Amy C.; Galama, Jochem M. D.; van Kuppeveld, Frank J. M.; Adema, Gosse J.

    2013-01-01

    Coxsackie B viruses (CVBs) and echoviruses (EVs) form the Human Enterovirus-B (HEV-B) species within the family Picornaviridae. HEV-B infections are widespread and generally cause mild disease; however, severe infections occur and HEV-B are associated with various chronic diseases such as cardiomyopathy and type 1 diabetes. Dendritic cells (DCs) are the professional antigen-presenting cells of our immune system and initiate and control immune responses to invading pathogens, yet also maintain tolerance to self-antigens. We previously reported that EVs, but not CVBs, can productively infect in vitro generated monocyte-derived DCs. The interactions between HEV-B and human myeloid DCs (mDCs) freshly isolated from blood, however, remain unknown. Here, we studied the susceptibility and responses of BDCA1+ mDC to HEV-B species and found that these mDC are susceptible to EV, but not CVB infection. Productive EV7 infection resulted in massive, rapid cell death without DC activation. Contrary, EV1 infection, which resulted in lower virus input at the same MOI, resulted in DC activation as observed by production of type I interferon-stimulated genes (ISGs), upregulation of co-stimulatory and co-inhibitory molecules (CD80, CD86, PDL1) and production of IL-6 and TNF-?, with a relative moderate decrease in cell viability. EV1-induced ISG expression depended on virus replication. CVB infection did not affect DC viability and resulted in poor induction of ISGs and CD80 induction in part of the donors. These data show for the first time the interaction between HEV-B species and BDCA1+ mDCs isolated freshly from blood. Our data indicate that different HEV-B species can influence DC homeostasis in various ways, possibly contributing to HEV-B associated pathology. PMID:23638101

  18. Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse

    PubMed Central

    Wu, Chia-Ying; Lin, Yi-Wen; Kuo, Chia-Ho; Liu, Wan-Hsin; Tai, Hsiu-Fen; Pan, Chien-Hung; Chen, Yung-Tsung; Hsiao, Pei-Wen; Chan, Chi-Hsien; Chang, Ching-Chuan; Liu, Chung-Cheng; Chow, Yen-Hung; Chen, Juine-Ruey

    2015-01-01

    Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 107 TCID50/mL 10 days after infection when using an MOI of 10?4. The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system. PMID:26287531

  19. Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model.

    PubMed

    Jung, Soo-Jung; Kasala, Dayananda; Choi, Joung-Woo; Lee, Soo-Hwan; Hwang, June Kyu; Kim, Sung Wan; Yun, Chae-Ok

    2015-01-12

    Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy. PMID:25400213

  20. pH-sensitive oncolytic adenovirus hybrid targeting acidic tumor microenvironment and angiogenesis.

    PubMed

    Choi, Joung-Woo; Jung, Soo-Jung; Kasala, Dayananda; Hwang, June Kyu; Hu, Jun; Bae, You Han; Yun, Chae-Ok

    2015-05-10

    Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX. PMID:25575865

  1. Hydrophobic pocket targeting probes for enteroviruses.

    PubMed

    Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

    2015-10-15

    Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells. PMID:26440968

  2. The Low-Cost Compound Lignosulfonic Acid (LA) Exhibits Broad-Spectrum Anti-HIV and Anti-HSV Activity and Has Potential for Microbicidal Applications

    PubMed Central

    D’huys, Thomas; Petrova, Mariya I.; Lebeer, Sarah; Snoeck, Robert; Andrei, Graciela; Schols, Dominique

    2015-01-01

    Objectives Lignosulfonic acid (LA), a low-cost lignin-derived polyanionic macromolecule, was extensively studied for its anti-HIV and anti-HSV activity in various cellular assays, its mechanism of viral inhibition and safety profile as potential microbicide. Results LA demonstrated potent inhibitory activity of HIV replication against a wide range of R5 and X4 HIV strains and prevented the uptake of HIV by bystander CD4+ T cells from persistently infected T cells in vitro (IC50: 0.07 – 0.34 ?M). LA also inhibited HSV-2 replication in vitro in different cell types (IC50: 0.42 – 1.1 ?M) and in rodents in vivo. Furthermore, LA neutralized the HIV-1 and HSV-2 DC-SIGN-mediated viral transfer to CD4+ T cells (IC50: ?1 ?M). In addition, dual HIV-1/HSV-2 infection in T cells was potently blocked by LA (IC50: 0.71 ?M). No antiviral activity was observed against the non-enveloped viruses Coxsackie type B4 and Reovirus type 1. LA is defined as a HIV entry inhibitor since it interfered with gp120 binding to the cell surface of T cells. Pretreatment of PBMCs with LA neither increased expression levels of cellular activation markers (CD69, CD25 and HLA-DR), nor enhanced HIV-1 replication. Furthermore, we found that LA had non-antagonistic effects with acyclovir, PRO2000 or LabyA1 (combination index (CI): 0.46 – 1.03) in its anti-HSV-2 activity and synergized with tenofovir (CI: 0.59) in its anti-HIV-1 activity. To identify mechanisms of LA resistance, we generated in vitro a mutant HIV-1 NL4.3LAresistant virus, which acquired seven mutations in the HIV-1 envelope glycoproteins: S160N, V170N, Q280H and R389T in gp120 and K77Q, N113D and H132Y in gp41. Additionally, HIV-1 NL4.3LAresistant virus showed cross-resistance with feglymycin, enfuvirtide, PRO2000 and mAb b12, four well-described HIV binding/fusion inhibitors. Importantly, LA did not affect the growth of vaginal Lactobacilli strains. Conclusion Overall, these data highlight LA as a potential and unique low-cost microbicide displaying broad anti-HIV and anti-HSV activity. PMID:26132818

  3. Formalin-Inactivated EV71 Vaccine Candidate Induced Cross-Neutralizing Antibody against Subgenotypes B1, B4, B5 and C4A in Adult Volunteers

    PubMed Central

    Chang, Jui-Yuan; Jiang, Renee; Hsieh, Yi-Chin; Tsao, Amanda; Wu, Chien-Long; Huang, Ju-Lan; Fung, Chang-Phone; Hsieh, Szu-Min; Wang, Ya-Fang; Wang, Jen-Ren; Hu, Mei-Hua; Chiang, Jen-Ron; Su, Ih-Jen; Chong, Pele Choi-Sing

    2013-01-01

    Background Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac) at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16. Methods Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses. Results The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had <8 pre-vaccination neutralization titers (Nt) against the B4 vaccine strain. After the first EV71vac immunization, 95% of vaccinees have >4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (?20% of participants) against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8) against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16. Conclusion EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials. Trial Registration ClinicalTrials.gov __NCT01268787 PMID:24278177

  4. Chemoprevention gene therapy (CGT) of pancreatic cancer using perillyl alcohol and a novel chimeric serotype cancer terminator virus.

    PubMed

    Sarkar, S; Azab, B; Quinn, B A; Shen, X; Dent, P; Klibanov, A L; Emdad, L; Das, S K; Sarkar, D; Fisher, P B

    2014-01-01

    Conditionally replication competent adenoviruses (Ads) that selectively replicate in cancer cells and simultaneously express a therapeutic cytokine, such as melanoma differentiation associated gene- 7/Interleukin-24 (mda-7/IL-24), a Cancer Terminator Virus (CTV-M7), hold potential for treating human cancers. To enhance the efficacy of the CTV-M7, we generated a chimeric Ad.5 and Ad.3 modified fiber bipartite CTV (Ad.5/3-CTV-M7) that can infect tumor cells in a Coxsackie Adenovirus receptor (CAR) independent manner, while retaining high infectivity in cancer cells containing high CAR. Although mda-7/IL-24 displays broad-spectrum anticancer properties, pancreatic ductal adenocarcinoma (PDAC) cells display an intrinsic resistance to mda-7/IL-24-mediated killing due to an mda-7/IL-24 mRNA translational block. However, using a chemoprevention gene therapy (CGT) approach with perillyl alcohol (POH) and a replication incompetent Ad to deliver mda-7/IL-24 (Ad.mda-7) there is enhanced conversion of mda-7/IL-24 mRNA into protein resulting in pancreatic cancer cell death in vitro and in vivo in nude mice containing human PDAC xenografts. This combination synergistically induces mda-7/IL-24-mediated cancer-specific apoptosis by inhibiting anti-apoptotic Bcl-xL and Bcl-2 protein expression and inducing an endoplasmic reticulum (ER) stress response through induction of BiP/GRP-78, which is most evident in chimeric-modified non-replicating Ad.5/3- mda-7- and CTV-M7-infected PDAC cells. Moreover, Ad.5/3-CTV-M7 in combination with POH sensitizes therapy-resistant MIA PaCa-2 cell lines over-expressing either Bcl-2 or Bcl-xL to mda-7/IL-24-mediated apoptosis. Ad.5/3-CTV-M7 plus POH also exerts a significant antitumor 'bystander' effect in vivo suppressing both primary and distant site tumor growth, confirming therapeutic utility of Ad.5/3-CTV-M7 plus POH in PDAC treatment, where all other current treatment strategies in clinical settings show minimal efficacy. PMID:24236457

  5. The HDAC Inhibitors Scriptaid and LBH589 Combined with the Oncolytic Virus Delta24-RGD Exert Enhanced Anti-Tumor Efficacy in Patient-Derived Glioblastoma Cells

    PubMed Central

    Berghauser Pont, Lotte M.E.; Kleijn, Anne; Kloezeman, Jenneke J.; van den Bossche, Wouter; Kaufmann, Johanna K.; de Vrij, Jeroen; Leenstra, Sieger; Dirven, Clemens M.F.; Lamfers, Martine L.M.

    2015-01-01

    Background A phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastoma-pathway and enters cells via ?v?3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi. Methods SAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and ?v?3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness. Results Scriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both drugs moderately increased ?v?3 integrin levels and viral infection in responding but not in non-responding GSCs. LBH589 moderately increased late viral gene expression, however, virus titration revealed diminished viral progeny production by both HDACi, Scriptaid augmented caspase-3/7 activity, LC3B conversion, p62 and phospho-p70S6K consumption, as well as LDH levels. LBH589 increased LDH and phospho-p70S6K consumption. Responsiveness correlated with expression of various Rb-pathway genes and integrins. Combination treatments induced limited toxicity to human astrocytes. Conclusion LBH589 and Scriptaid combined with Delta24-RGD revealed synergistic anti-tumor activity in a subset of GSCs. Both HDACi moderately augmented viral infection and late gene expression, but slightly reduced progeny production. The drugs differentially activated multiple cell death pathways. The limited toxicity on astrocytes supports further evaluation of the proposed combination therapies. PMID:25993039

  6. Assessment of the microbial removal capabilities of riverbank filtration

    NASA Astrophysics Data System (ADS)

    Partinoudi, V.; Collins, M.; Margolin, A.; Brannaka, L.

    2003-04-01

    Riverbank filtrate includes both groundwater and river water that has percolated through the banks or bed of a river to an extraction well. One of the primary objectives of this study was to assess the microbial removal capabilities of riverbank filtration (RBF) independent of any groundwater dilution, i.e. a worse case scenario. A total of five sites were chosen: the Pembroke Waterworks (NH), the Milford State Fish Hatchery (NH), Jackson (NH) (where an infiltration gallery exists), Louisville Water Company (KY), and Cedar Rapids (IA). This study has been monitoring total coliforms, E.coli and aerobic spore forming bacteria amongst other water quality parameters over the past twelve months. Male specific (MS2) and somatic coliphage viruses were also monitored intensively for two weeks, using a single agar overlay and a two-step enrichment method, in December 2002 in Louisville, KY and in Cedar Rapids, IA. This intensive coliphage monitoring was followed by the collection of samples for special analysis of enteric viruses (Adenovirus type 40 and 41, Astrovirus, Poliovirus, Coxsackie virus, Rotavirus and Echovirus). The virus samples were analyzed using the ICC-nPCR method, due to its high specificity and sensitivity. Typical river water total coliforms, E.coli and aerobic spore forming bacteria concentrations ranged between 43-145000 CFU/100mL, 0-24192 CFU/100mL and 83-1997 CFU/100mL, respectively. All three of these microbial concentrations were below detection limits (<1CFU/100mL) in the riverbank filtration extraction well water, even after eliminating the “dilution” effects with groundwater. The male specific and the somatic coliphages ranged between 328-491 PFU/25mL and 3-21 PFU/25mL, respectively, in the river water. The concentration of the male specific coliphages was reduced by as much as 77% by the riverbank passage whereas the concentrations of the somatic coliphages were reduced by 100%. In summary the sites evaluated in this study indicated the conservative effectiveness of RBF in removing bacteria and virus indicators. Any groundwater dilution with the RBF extract should contribute to even lower microbial concentrations.

  7. Probing the structure and function of biopolymer-carbon nanotube hybrids with molecular dynamics

    NASA Astrophysics Data System (ADS)

    Johnson, Robert R.

    2009-12-01

    Nanoscience deals with the characterization and manipulation of matter on the atomic/molecular size scale in order to deepen our understanding of condensed matter and develop revolutionary technology. Meeting the demands of the rapidly advancing nanotechnological frontier requires novel, multifunctional nanoscale materials. Among the most promising nanomaterials to fulfill this need are biopolymer-carbon nanotube hybrids (Bio-CNT). Bio-CNT consists of a single-walled carbon nanotube (CNT) coated with a self-assembled layer of biopolymers such as DNA or protein. Experiments have demonstrated that these nanomaterials possess a wide range of technologically useful properties with applications in nanoelectronics, medicine, homeland security, environmental safety and microbiology. However, a fundamental understanding of the self-assembly mechanics, structure and energetics of Bio-CNT is lacking. The objective of this thesis is to address this deficiency through molecular dynamics (MD) simulation, which provides an atomic-scale window into the behavior of this unique nanomaterial. MD shows that Bio-CNT composed of single-stranded DNA (ssDNA) self-assembles via the formation of high affinity contacts between DNA bases and the CNT sidewall. Calculation of the base-CNT binding free energy by thermodynamic integration reveals that these contacts result from the attractive pi--pi stacking interaction. Binding affinities follow the trend G > A > T > C. MD reveals that long ssDNA sequences are driven into a helical wrapping about CNT with a sub-10 nm pitch by electrostatic and torsional interactions in the backbone. A large-scale replica exchange molecular dynamics simulation reveals that ssDNA-CNT hybrids are disordered. At room temperature, ssDNA can reside in several low-energy conformations that contain a sequence-specific arrangement of bases detached from CNT surface. MD demonstrates that protein-CNT hybrids composed of the Coxsackie-adenovirus receptor are biologically active and function as a nanobiosensor with specific recognition of Knob proteins from the adenovirus capsid. Simulation also shows that the rigid CNT damps structural fluctuations in bound proteins, which may have important ramifications for biosensing devices composed of protein-CNT hybrids. These results expand current knowledge of Bio-CNT and demonstrate the effectiveness of MD for investigations of nanobiomolecular systems.