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Sample records for hsv thymidine kinase

  1. Glucose transport and apoptosis after gene therapy with HSV thymidine kinase

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    Haberkorn, U.; Henze, M. [Heidelberg Univ. (Germany). Abt. fuer Klinische Nuklearmedizin; Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Altmann, A.; Morr, I.; Jiang Shiming [Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Kamencic, H.; Traut, U.; Metz, J.; Kinscherf, R. [Dept. of Anatomy and Cell Biology III, Univ. of Heidelberg (Germany)

    2001-11-01

    The relation between tumour metabolism and induction of apoptosis by gene therapy was investigated in a rat Morris hepatoma (MH3924A) model expressing the HSV thymidine kinase (HSVtk) gene. In vivo the amount of glucose transporter (GLUT1 and GLUT3 isoforms) expressing cells was determined in tumours of untreated and treated animals using immunohistochemistry. In vitro uptake studies with 2-fluoro-2-deoxy-D-glucose (FDG), 3-O-methylglucose and thymidine (TdR) and a TUNEL (TdT-mediated dUTP nick end labelling) assay for the assessment of apoptosis were done immediately and 24 h after treatment of the recombinant cells with different doses of ganciclovir (GCV). Immunohistochemistry revealed a significant increase in GLUT1 in treated tumours which showed enhanced transport activity for FDG. In vitro the FDG and 3-O-methylglucose uptake increased to 186% when compared with that of the non-treated cells immediately after incubation with GCV. However, 24 h later the FDG uptake had declined to its normal level, whereas the accumulation of 3-O-methylglucose remained elevated. The uptake of TdR, which was determined simultaneously, decreased in the acid-insoluble fraction of the cells to 27% and 11%, respectively, immediately and 24 h after therapy, while in the acid-soluble fraction it increased to 229% and to 167%, respectively. Employing the TUNEL technique, 25% of cells were found to be apoptotic 24 h after the termination of GCV treatment. Inhibition of glucose transport by cytochalasin B or competition with deoxyglucose resulted in a 78% (cytochalasin B) and 88% (deoxyglucose) decrease in FDG uptake in the recombinant hepatoma cells and in an increase in the apoptotic cell fraction. It is concluded that inhibition of enhanced glucose transport in GCV-treated cells increased apoptosis. Therefore, enhanced glucose transport seems to represent a stress reaction of tumour cells dedicated for the prevention of cell death. (orig.)

  2. PET imaging of oncolytic VSV expressing the mutant HSV-1 thymidine kinase transgene in a preclinical HCC rat model.

    Science.gov (United States)

    Muñoz-Álvarez, Kim A; Altomonte, Jennifer; Laitinen, Iina; Ziegler, Sibylle; Steiger, Katja; Esposito, Irene; Schmid, Roland M; Ebert, Oliver

    2015-04-01

    Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk-treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

  3. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression

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    Andreas Jacobs

    1999-06-01

    Full Text Available Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp and HSV-1-tk genes was generated (tkgfp gene and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS. TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil (FIAU, in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT], and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.

  4. Cytotoxicity and cellular uptake of pyrimidine nucleosides for imaging herpes simplex type-1 thymidine kinase (HSV-1 TK) expression in mammalian cells

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    Morin, Kevin W.; Duan Weili; Xu Lihua; Zhou Aihua; Moharram, Sameh; Knaus, Edward E.; McEwan, Alexander J.B.; Wiebe, Leonard I. E-mail: leonard.wiebe@ualberta.ca

    2004-07-01

    In vivo transfer of the herpes simplex virus type-1 thymidine kinase (HSV-1 TK) gene, with subsequent administration of antiviral drugs such as ganciclovir, has emerged as a promising gene therapy protocol for treating proliferative disorders. The in vitro cytotoxicities (IC{sub 50}) for two series of 5-iodo- and (E)-5-(2-iodovinyl)-substituted 2'-deoxy- and 2'-deoxy-2'-fluoro-pyrimidine nucleosides ranged from millimolar to low nanomolar concentrations in mammalian tumor cell lines (KBALB; R-970-5; 143B; EMT-6) and their counterparts engineered to express HSV-1 TK (KBALB-STK; 143B-LTK). Their HSV-1 TK selectivity indices ranged from one (nonselective) to one million (highly selective) based on cytotoxicity, with FIRU being the least toxic to all cell lines, and FIAU being most toxic. HSV-1 TK selectivity, based on uptake, ranged from 10 to 140, with IVDU being most selective for HSV-1 TK expressing cells, followed by IVFRU, FIRU, FIAU, IVFAU and finally IUDR. Phosphorylation of [{sup 125}I]FIAU led to incorporation of the radiolabel into nucleic acids, whereas IVFRU and FIRU radioactivity was trapped primarily in the nucleotide pool. These data indicate that cytotoxicity does not depend on initial metabolic trapping (e.g., phosphorylation), but on elaboration of the mononucleotides to more cytotoxic anabolites. Lipophilicities and nucleoside transport rates of the six nucleosides tested were within narrow ranges. This supports the premise that cellular biochemistry, and not cellular bioavailability, is responsible for the observed broad range of cytotoxicity and trapping. In vivo biodistribution studies with 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyribouridine (FIRU), 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyarabinouridine (FIAU) and (E)-5-(2-[{sup 125}I]iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) demonstrate selective accumulation of all three radiotracers in HSV-1 TK-expressing KBABK-STK tumors, compared to their very low

  5. Intravenous Administration Is an Effective and Safe Route for Cancer Gene Therapy Using the Bifidobacterium-Mediated Recombinant HSV-1 Thymidine Kinase and Ganciclovir

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    Huicong Zhou

    2016-06-01

    Full Text Available The herpes simplex virus thymidine kinase/ganciclovir (HSV TK/GCV system is one of the best studied cancer suicide gene therapy systems. Our previous study showed that caspase 3 expression was upregulated and bladder tumor growth was significantly reduced in rats treated with a combination of Bifidobacterium (BF and HSV TK/GCV (BF-rTK/GCV. However, it was raised whether the BF-mediated recombinant thymidine kinase combined with ganciclovir (BF-rTK/GCV was safe to administer via venous for cancer gene therapy. To answer this question, the antitumor effects of BF-rTK/GCV were mainly evaluated in a xenograft nude mouse model bearing MKN-45 gastric tumor cells. The immune response, including analysis of cytokine profiles, was analyzed to evaluate the safety of intramuscular and intravenous injection of BF-rTK in BALB/c mice. The results suggested that gastric tumor growth was significantly inhibited in vivo by BF-rTK/GCV. However, the BF-rTK/GCV had no effect on mouse body weight, indicating that the treatment was safe for the host. The results of cytokine profile analysis indicated that intravenous injection of a low dose of BF-rTK resulted in a weaker cytokine response than that obtained with intramuscular injection. Furthermore, immunohistochemical analysis showed that intravenous administration did not affect the expression of immune-associated TLR2 and TLR4. Finally, the BF-rTK/GCV inhibited vascular endothelial growth factor (VEGF expression in mouse model, which is helpful for inhibiting of tumor angiogenesis. That meant intravenous administration of BF-rTK/GCV was an effective and safe way for cancer gene therapy.

  6. Intravenous Administration Is an Effective and Safe Route for Cancer Gene Therapy Using the Bifidobacterium-Mediated Recombinant HSV-1 Thymidine Kinase and Ganciclovir.

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    Zhou, Huicong; He, Zhiliang; Wang, Changdong; Xie, Tingting; Liu, Lin; Liu, Chuanyang; Song, Fangzhou; Ma, Yongping

    2016-06-06

    The herpes simplex virus thymidine kinase/ganciclovir (HSV TK/GCV) system is one of the best studied cancer suicide gene therapy systems. Our previous study showed that caspase 3 expression was upregulated and bladder tumor growth was significantly reduced in rats treated with a combination of Bifidobacterium (BF) and HSV TK/GCV (BF-rTK/GCV). However, it was raised whether the BF-mediated recombinant thymidine kinase combined with ganciclovir (BF-rTK/GCV) was safe to administer via venous for cancer gene therapy. To answer this question, the antitumor effects of BF-rTK/GCV were mainly evaluated in a xenograft nude mouse model bearing MKN-45 gastric tumor cells. The immune response, including analysis of cytokine profiles, was analyzed to evaluate the safety of intramuscular and intravenous injection of BF-rTK in BALB/c mice. The results suggested that gastric tumor growth was significantly inhibited in vivo by BF-rTK/GCV. However, the BF-rTK/GCV had no effect on mouse body weight, indicating that the treatment was safe for the host. The results of cytokine profile analysis indicated that intravenous injection of a low dose of BF-rTK resulted in a weaker cytokine response than that obtained with intramuscular injection. Furthermore, immunohistochemical analysis showed that intravenous administration did not affect the expression of immune-associated TLR2 and TLR4. Finally, the BF-rTK/GCV inhibited vascular endothelial growth factor (VEGF) expression in mouse model, which is helpful for inhibiting of tumor angiogenesis. That meant intravenous administration of BF-rTK/GCV was an effective and safe way for cancer gene therapy.

  7. Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

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    Najjar, Amer M; Manuri, Pallavi R; Olivares, Simon; Flores, Leo; Mi, Tiejuan; Huls, Helen; Shpall, Elizabeth J; Champlin, Richard E; Turkman, Nashaat; Paolillo, Vincenzo; Roszik, Jason; Rabinovich, Brian; Lee, Dean A; Alauddin, Mian; Gelovani, Juri; Cooper, Laurence J N

    2016-12-01

    We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19(+) artificial antigen-presenting cells. After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19(+) targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[(18)F]fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil ([(18)F]FEAU). This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

  8. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  9. Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene

    NARCIS (Netherlands)

    Chalmers, D; Ferrand, C; Apperley, JF; Melo, JV; Ebeling, S; Newton, [No Value; Duperrier, A; Hagenbeek, A; Garrett, E; Tiberghien, P; Garin, M

    Introduction of the Herpes simplex virus thymidine kinase (HSV-tk) gene into target cells renders them susceptible to killing by ganciclovir (GCV). We are studying the use of HSV-tk-transduced T lymphocytes in the context of hematopoietic stem cell transplantation. We have previously shown, in vitro

  10. Enzymatic Regulation of Cytosolic Thymidine Kinase 1 and Mitochondrial Thymidine Kinase 2

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2010-01-01

    . Apart from the complex de novo synthesis of dTTP through UDP reduction, dTTP is provided through salvage of thymidine catalyzed by the thymidine kinases, the cytosolic and cell cycle regulated TK1 and the mitochondrial and constitutively expressed TK2. The complex enzymatic regulation of TK1 and TK2...

  11. Treatment of malignant gliomas with a replicating adenoviral vector expressing herpes simplex virus-thymidine kinase

    NARCIS (Netherlands)

    D. Nanda (Dharminderkoemar); R. Vogels; M. Havenga; C.J.J. Avezaat (Cees); A. Bout; P.S. Smitt

    2001-01-01

    textabstractWe evaluated the interaction between oncolytic, replication-competent adenoviral vectors and the herpes simplex virus-1 thymidine kinase (HSV1-tk) gene/ganciclovir (GCV) suicide system for the treatment of malignant gliomas. We constructed a panel of

  12. Role of Genotypic Analysis of the Thymidine Kinase Gene of Herpes Simplex Virus for Determination of Neurovirulence and Resistance to Acyclovir

    OpenAIRE

    Lee, N. Y.; Tang, Y.-W.; Espy, M. J.; Kolbert, C. P.; Rys, P. N.; Mitchell, P. S.; Day, S. P.; Henry, S. L.; Persing, D. H.; Smith, T. F.

    1999-01-01

    Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) have been associated with resistance to acyclovir (ACY) and possible recognition of neurotropic strains. We sequenced a 335-bp segment of the TK gene to determine the frequency of mutations in HSV strains recovered from dermal, genital, and cerebrospinal fluid (CSF) specimens (n = 200; 102 HSV type 1 [HSV-1] 98 HSV-2 strains). Four polymorphic sites were detected in HSV-1 strains; C513T, A528G, C575T, and C672T. Among t...

  13. Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase

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    Haberkorn, Uwe; Khazaie, Khashayarsha; Morr, Iris; Altmann, Annette; Mueller, Markus; Kaick, Gerhard van

    1998-05-01

    Assessment of suicide enzyme activity would have considerable impact on the planning and the individualization of suicide gene therapy of malignant tumors. This may be done by determining the pharmacokinetics of specific substrates. We generated ganciclovir (GCV)-sensitive human mammary carcinoma cell lines after transfection with a retroviral vector bearing the herpes simplex virus thymidine kinase (HSV-tk) gene. Thereafter, uptake measurements and HPLC analyses were performed up to 48 h in an HSV-tk-expressing cell line and in a wild-type cell line using tritiated GCV. HSV-tk-expressing cells showed higher GCV uptake and phosphorylation than control cells, whereas in wild-type MCF7 cells no phosphorylated GCV was detected. In bystander experiments the total GCV uptake was related to the amount of HSV-tk-expressing cells. Furthermore, the uptake of GCV correlated closely with the growth inhibition (r=0.92). Therefore, the accumulation of specific substrates may serve as an indicator of the HSV-tk activity and of therapy outcome. Inhibition and competition experiments demonstrated slow transport of GCV by the nucleoside carriers. The slow uptake and low affinity to HSV-tk indicate that GCV is not an ideal substrate for the nucleoside transport systems or for HSV-tk. This may be the limiting factor for therapy success, necessitating the search for better substrates of HSV-tk.

  14. Mutation Study of Two Thymidine Kinases 

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Munch-Petersen, Birgitte; Eklund, Hans

    The deoxyribonucleoside kinase, thymidine kinase 1 (TK1), is a key enzyme in the salvage pathway, where it catalyzes the first of three phosphate transfers from ATP to thymidine. Besides from their importance in DNA metabolism deoxyribonucleoside kinases are also important for activation of antic...... not phosphorylate the anticancer analog 1-β-D-arabinofuranosylcytosine (AraC), however. The HuTK1 mutant has been crystallized, and azidothymidine monophosphate has been modelled into the active site.......The deoxyribonucleoside kinase, thymidine kinase 1 (TK1), is a key enzyme in the salvage pathway, where it catalyzes the first of three phosphate transfers from ATP to thymidine. Besides from their importance in DNA metabolism deoxyribonucleoside kinases are also important for activation...... of anticancer and antiviral nucleoside pro-drugs. Humans have four different deoxyribonucleoside kinases, the two cytosolic: TK1 and deoxycytidine kinase (dCK) and the two mitochondrial: thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK). In insects, a single gene encodes a multi substrate kinase...

  15. Prodrugs of herpes simplex thymidine kinase inhibitors.

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    Yanachkova, Milka; Xu, Wei-Chu; Dvoskin, Sofya; Dix, Edward J; Yanachkov, Ivan B; Focher, Federico; Savi, Lida; Sanchez, M Dulfary; Foster, Timothy P; Wright, George E

    2015-04-01

    Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo. © The Author(s) 2015.

  16. Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice

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    Gong Jianping

    2010-12-01

    Full Text Available Abstract Objective The purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK suicide gene system on mice hepatoma. Methods Forty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1 PBS; (2 HSV-TK (3 HSV-TK+ ultrasound (HSV-TK+US; (4 HSV-TK+ultrasound+microbubbles (HSV-TK+US+MB. The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups. Results The TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p Conclusion Ultrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer.

  17. Pasteurella multocida thymidine kinase 1 efficiently activates pyrimidine nucleoside analogs.

    Science.gov (United States)

    Clausen, A R; Al Meani, S A L; Piskur, J

    2010-06-01

    In the Pasteurella multocida genome only one putative deoxyribonucleoside kinase encoding gene, for thymidine kinase 1 (PmTK1), was identified. The PmTK1 gene was sub-cloned into Escherichia coli KY895 and it sensitized the host towards 2',2'-difluoro-deoxycytidine (gemcitabine, dFdC), 3'-azido-thymidine (AZT) and 5-fluoro-deoxyuridine (5F-dU). PmTK1 was over-expressed and purified with two different tags. Apparently, deoxyuridine (dU), and not thymidine (dT), is the preferred substrate. We suggest that PmTK1s could be employed as a species-specific activator of uracil-based nucleoside antibiotics.

  18. Tomato thymidine kinase is subject to inefficient TTP feedback regulation

    DEFF Research Database (Denmark)

    Larsen, Nicolai Balle; Munch-Petersen, Birgitte; Piskur, Jure

    2014-01-01

    A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood–brain barrier. Transduction with toTK1 has been found...

  19. Structures of thymidine kinase 1 of human and mycoplasma origin

    DEFF Research Database (Denmark)

    Welin, Martin; Kosinska, Urszula; Mikkelsen, Nils-Egil

    2004-01-01

    Cytosolic thymidine kinase, TK1, is a well-known cell cycle regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs as AZT. We have now determined the first structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in com...... differs fundamentally from the structures of the other deoxyribonucleoside kinases indicating a different evolutionary origin....

  20. A mathematical model of human thymidine kinase 2 activity

    DEFF Research Database (Denmark)

    Radivoyevitch, Tom; Munch-Petersen, Birgitte; Wang, Liya

    2011-01-01

    _ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative cooperati......_ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative...

  1. A pseudoreceptor modelling study of the varicella-zoster virus and human thymidine kinase binding sites

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    Greenidge, Paulette A.; Merz, Alfred; Folkers, Gerd

    1995-12-01

    A representative range of pyrimidine nucleoside analogues that are known to inhibit herpes simplex virus (HSV) replication have been used to construct receptor binding site models for the varicella-zoster virus (VZV), thymidine kinase (TK) and human TK1. Given a set of interacting ligands, superimposed in such a manner as to define a pharmacophore, the pseudoreceptor modelling technique Yak provides a means of building binding site models of macromolecules for which no three-dimensional experimental structures are available. Once the models have been evaluated by their ability to reproduce experimental binding data [Vedani et al., J. Am. Chem. Soc., 117 (1995) 4987], they can be used for predictive purposes. Calculated and experimental values of relative binding affinity are compared. Our models suggest that the substitution of one residue may be sufficient to determine ligand subtype affinity.

  2. Thymidine kinase 1 regulatory fine-tuning through tetramer formation

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Clausen, Anders R.; Andersson, Karl-Magnus

    2013-01-01

    Abstract: Thymidine kinase 1 (TK1) provides a crucial precursor, deoxythymidine monophosphate, for nucleic acid synthesis, and the activity of TK1 increases by up to 200-fold during the S-phase of cell division in humans. An important part of the regulatory checkpoints is the ATP and enzyme...... in higher vertebrates and was especially pronounced in mammalian and bird TK1s. We suggest that the dimer form is the original form and that the tetramer originated in the animal lineage after the split of Dictyostelium and the lineages leading to invertebrates and vertebrates. The efficient switching...

  3. Vesicular Stomatitis Virus G Glycoprotein and ATRA Enhanced Bystander Killing of Chemoresistant Leukemic Cells by Herpes Simplex Virus Thymidine Kinase/Ganciclovir.

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    Hu, Chenxi; Chen, Zheng; Zhao, Wenjun; Wei, Lirong; Zheng, Yanwen; He, Chao; Zeng, Yan; Yin, Bin

    2014-02-01

    Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

  4. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

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    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon; (UIC); (MXPL-G)

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

  5. In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, C.-H. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Liu, R.-S. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); National Yang-Ming University Medical School and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Wang, H.-E. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Hwang, J.-J. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Deng, W.-P. [Institute of Biomedical Material, Taipei Medical University, Taipei 112, Taiwan (China); Chen, J.-C. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Chen, F.-D. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China) and Institute of Radiological Sciences, Central Taiwan University of Science and Technology Taichung 112, Taiwan (China)]. E-mail: d49220009@ym.edu.tw

    2006-07-15

    The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [{sup 131}I] FIAU and [{sup 3}H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by

  6. Methodology and problems of protein-ligand docking: case study of dihydroorotate dehydrogenase, thymidine kinase, and phosphodiesterase 4.

    Science.gov (United States)

    Pospisil, Pavel; Kuoni, Thomas; Scapozza, Leonardo; Folkers, Gerd

    2002-01-01

    The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear-cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.

  7. A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing.

    Science.gov (United States)

    McSorley, Theresa; Ort, Stephan; Monnerjahn, Christian; Konrad, Manfred

    2014-02-01

    The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Comparative active‐site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    National Research Council Canada - National Science Library

    Skovgaard, Tine; Uhlin, Ulla; Munch‐Petersen, Birgitte

    2012-01-01

    The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1...

  9. Design of Thymidine Analogues Targeting Thymidilate Kinase of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Luc Calvin Owono Owono

    2013-01-01

    Full Text Available We design here new nanomolar antituberculotics, inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt, by means of structure-based molecular design. 3D models of TMPKmt-inhibitor complexes have been prepared from the crystal structure of TMPKmt cocrystallized with the natural substrate deoxythymidine monophosphate (dTMP (1GSI for a training set of 15 thymidine analogues (TMDs with known activity to prepare a QSAR model of interaction establishing a correlation between the free energy of complexation and the biological activity. Subsequent validation of the predictability of the model has been performed with a 3D QSAR pharmacophore generation. The structural information derived from the model served to design new subnanomolar thymidine analogues. From molecular modeling investigations, the agreement between free energy of complexation (ΔΔGcom and Ki values explains 94% of the TMPKmt inhibition (pKi=-0.2924ΔΔGcom+3.234;R2=0.94 by variation of the computed ΔΔGcom and 92% for the pharmacophore (PH4 model (pKi=1.0206×pKipred-0.0832,  R2=0.92. The analysis of contributions from active site residues suggested substitution at the 5-position of pyrimidine ring and various groups at the 5′-position of the ribose. The best inhibitor reached a predicted Ki of 0.155 nM. The computational approach through the combined use of molecular modeling and PH4 pharmacophore is helpful in targeted drug design, providing valuable information for the synthesis and prediction of activity of novel antituberculotic agents.

  10. Thymidine kinase 1 deficient cells show increased survival rate after UV-induced DNA damage

    DEFF Research Database (Denmark)

    Skovgaard, T; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    2010-01-01

    Balanced deoxynucleotide pools are known to be important for correct DNA repair, and deficiency for some of the central enzymes in deoxynucleotide metabolism can cause imbalanced pools, which in turn can lead to mutagenesis and cell death. Here we show that cells deficient for the thymidine salvage...... enzyme thymidine kinase 1 (TK1) are more resistant to UV-induced DNA damage than TK1 positive cells although they have thymidine triphosphate (dTTP) levels of only half the size of control cells. Our results suggest that higher thymidine levels in the TK- cells caused by defect thymidine salvage to d...

  11. Integrated homology modelling and X-ray study of herpes simplex virus I thymidine kinase: a case study.

    Science.gov (United States)

    Folkers, G; Alber, F; Amrhein, I; Behrends, H; Bohner, T; Gerber, S; Kuonen, O; Scapozza, L

    1997-01-01

    Knowledge-based homology modelling together with site-directed mutagenesis, epitope and conformational mapping is an approach to predict the structures of proteins and for the rational design of new drugs. In this study we present how this procedure has been applied to model the structure of herpes simplex virus type 1 thymidine kinase (HSV1 TK, HSV1 ATP-thymidine-5'-phosphotransferase, EC 2.7.1.21). We have used, and evaluated, several secondary structure prediction methods, such as the classical one based on Chou and Fastman algorithm, neural networks using the Kabsch and Sander classification, and the PRISM method. We have validated the algorithms by applying them to the porcine adenylate kinase (ADK), whose three-dimensional structure is known and that has been used for the alignment of the TKs as well. The resulting first model of HSV1-TK consisted of the first beta-strand connected to the phosphate binding loop and its subsequent alpha-helix, the fourth beta-strand connected to the conserved FDRH sequence and two alpha-helix with basic amino acids. The 3D structure was built using the X-ray structure of ADK as template and following the general procedure for homology modelling. We extended the model by means of COMPOSER, an automatic process for protein modelling. Site-directed mutagenesis was used to experimentally verify the predicted active-site model of HSV1-TK. The data measured in our lab and by others support the suggestion that the FDRH motif is part of the active site and plays an important role in the phosphorylation of substrates. The structure of HSV1 TK, recently solved in collaboration with Prof. G. Schulz at 2.7 A resolution, includes 284 of 343 residues of the N-terminal truncated TK. The secondary structures could be clearly assigned and fitted to the density. The comparison between crystallographically determined structure and the model shows that nearly 70% of the HSV1 TK structure has been correctly modelled by the described integrated

  12. Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice.

    Science.gov (United States)

    Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu; Hsu, Sheng-Min; Chen, Shun-Hua

    2017-02-15

    Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised

  13. Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major.

    Directory of Open Access Journals (Sweden)

    Jennifer Timm

    2015-05-01

    Full Text Available Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd forming thymidine monophosphate (dTMP. L. major Type II TK (LmTK has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s(-1 and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.

  14. Human telomerase reverse-transcriptase promoter-controlled and herpes simplex virus thymidine kinase-armed adenoviruses for renal cell carcinoma treatment

    Directory of Open Access Journals (Sweden)

    Tian DW

    2013-04-01

    Full Text Available Dawei Tian,1–4 Yan Sun,3 Yang Yang,2,3 Mingde Lei,3 Na Ding,3 Ruifa Han2,31Tianjin Medical University, Tianjin, People's Republic of China; 2Department of Urinary Surgery, 3Tianjin Institute of Urology, Second Hospital of Tianjin Medical University, Tianjin, People's Republic of China; 4Tianjin Nankai Hospital, Tianjin, People's Republic of ChinaAbstract: New treatment strategies are required for renal cell carcinoma (RCC due to its relative insensitivity to conventional radio- and chemotherapies. The promising strategy of tumor inhibition using human telomerase reverse transcriptase (hTERT-controlled herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV in the hTERT promoter-driven HSV-TK/GCV suicide gene system was investigated. Tumor volume, weight, relative proliferation rate, and cell-apoptosis levels were examined in mice injected with adenovirus (Ad-hTERT-HSV-TK and GCV. Increased cell death occurred following treatment with Ads carrying hTERT-HSV-TK/GCV or cytomegalovirus promoter-controlled (CMV-HSV-TK/GCV for human RCC 786-0 and fibroblast MRC-5 cells. In mice, Ad-hTERT-HSV-TK/GCV more specifically inhibited tumor and RCC xenograft growth than Ad-CMV-HSV-TK/GCV (P < 0.05. Furthermore, Ad-hTERT-HSV-TK/GCV did not significantly damage normal fibroblasts or organ systems (heart, lung, liver, brain, kidney, and spleen. Thus, Ad-hTERT-HSV-TK/GCV is an effective RCC inhibitor in human cells in vitro and in vivo mouse models, indicating potential usefulness in RCC-targeted gene therapy.Keywords: hTERT promoter, HSV-TK/GCV, renal cell carcinoma, adenovirus

  15. Role of Genotypic Analysis of the Thymidine Kinase Gene of Herpes Simplex Virus for Determination of Neurovirulence and Resistance to Acyclovir

    Science.gov (United States)

    Lee, N. Y.; Tang, Y.-W.; Espy, M. J.; Kolbert, C. P.; Rys, P. N.; Mitchell, P. S.; Day, S. P.; Henry, S. L.; Persing, D. H.; Smith, T. F.

    1999-01-01

    Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) have been associated with resistance to acyclovir (ACY) and possible recognition of neurotropic strains. We sequenced a 335-bp segment of the TK gene to determine the frequency of mutations in HSV strains recovered from dermal, genital, and cerebrospinal fluid (CSF) specimens (n = 200; 102 HSV type 1 [HSV-1] 98 HSV-2 strains). Four polymorphic sites were detected in HSV-1 strains; C513T, A528G, C575T, and C672T. Among the polymorphisms, only C575T resulted in a change of amino acid sequence (residue 192, Ala→Val). For HSV-2 strains, only one polymorphism (G420T) which resulted in an amino acid substitution (residue 139, Leu→Phe) was detected. Phenotypic determination of resistance to ACY by a plaque reduction assay of 48 HSV isolates was not correlated with the sequence results of 11 strains in that 7 of these with genotypic polymorphisms were susceptible to the drug in vitro. In addition, of 32 ACY-resistant HSV strains, 28 (87.5%) had no polymorphisms detected in the 335-bp amplicon of the TK gene. There was no statistical difference in the frequency of polymorphisms according to the source of the specimens. We conclude that the detection of nucleic acid polymorphisms in a previously implicated 335-bp segment of the TK gene cannot be interpreted as indicative of either ACY resistance or neurotropism of HSV strains from dermal, genital, and CSF sources. PMID:10488172

  16. In vivo PET imaging with {sup 18}F-FHBG of hepatoma cancer gene therapy using herpes simplex virus thymidine kinase and ganciclovir

    Energy Technology Data Exchange (ETDEWEB)

    Lee, TaeSup; Kim, JunYoup; Moon, ByungSeok; Kang, JooHyun; Song, Inho; Kwon, HeeChung; Kim, KyungMin; Cheon, GiJeong; Choi, ChangWoon; Lim, SangMoo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-07-01

    Monitoring gene expression in vivo to evaluate the gene therapy efficacy is a critical issue for scientists and physicians. Non-invasive nuclear imaging can offer information regarding the level of gene expression and its location when an appropriate reporter gene is constructed in the therapeutic gene therapy. Herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) is the most common reporter gene and is used in cancer gene therapy by activating relatively nontoxic compounds, such as acyclovir or ganciclovir (GCV), to induce cell death. In this study, we investigate the feasibility of monitoring cancer gene therapy using retroviral vector transduced HSV1-tk and GCV, in vitro cellular uptake and in vivo animal studies, including biodistribution and small animal positron emission tomography (PET) imaging, were performed in HSV1-tk and luciferase (Luc)-transduced MCA-TK/Luc and enhanced green fluorescent protein (eGFP)-transduced MCA-eGFP hepatoma cell lines.

  17. Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase.

    Science.gov (United States)

    Tieng, Vannary; Cherpin, Ophelie; Gutzwiller, Eveline; Zambon, Alexander C; Delgado, Christophe; Salmon, Patrick; Dubois-Dauphin, Michel; Krause, Karl-Heinz

    2016-01-01

    Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK). We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC) were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells.

  18. Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase

    Directory of Open Access Journals (Sweden)

    Vannary Tieng

    2016-01-01

    Full Text Available Pluripotent stem cell (PSC-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK. We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells.

  19. Thymidine kinase enzyme selective imaging radiopharmaceutical. {sup 99m}Tc(CO){sub 3}-Ganciclovir

    Energy Technology Data Exchange (ETDEWEB)

    Gedik, B.; Teksoez, S.; Ichedef, C.; Kilcar, A.Y.; Medine, E.I.; Ucar, E. [Ege Univ., Bornova, Izmir (Turkey). Dept. of Nuclear Applications

    2013-03-01

    The aim of this study is to radiolabel Ganciclovir, known as having selective antiviral properties against thymidine kinase, with technetium tricarbonylcore ({sup 99m}Tc(CO){sub 3}{sup +}) and to investigate the biological behavior of this complex in vitro and in vivo. Commercially provided Ganciclovir (GCV) was radiolabeled with {sup 99m}Tc(CO){sub 3}{sup +}. Initially, optimum radiolabeling conditions were determined by analyzing factors such as temperature, pH and time. Quality control of the radiolabeled compound was performed. The radiolabeling yield was found to be 97%. The {sup 99m}Tc(CO){sub 3}-GCV complex also displayed good in vitro stability during the 24 h period. In vitro cell uptake studies showed that the {sup 99m}Tc(CO){sub 3}-GCV complex is highly uptaken in A-549, PC-3, HeLa cell lines according to the control group {sup 99m}Tc(I)-tricarbonyl core. The knowledge gained from in vivo and in vitro studies of {sup 99m}Tc(CO){sub 3}-GCV could contribute to the development of a new HSV1-tk gene imaging agent. (orig.)

  20. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5′ of a deoxyribonucleoside. This salvage pathway is well characterized....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.......7.1.21)‐like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T‐DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a...

  1. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Anders Ranegaard Clausen, Anders Ranegaard; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.......7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a...

  2. Tanshinone IIA increases the bystander effect of herpes simplex virus thymidine kinase/ganciclovir gene therapy via enhanced gap junctional intercellular communication.

    Directory of Open Access Journals (Sweden)

    Jianyong Xiao

    Full Text Available The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV. This effect is reported to be mediated by gap junctional intercellular communication (GJIC, and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA, a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.

  3. Tanshinone IIA increases the bystander effect of herpes simplex virus thymidine kinase/ganciclovir gene therapy via enhanced gap junctional intercellular communication.

    Science.gov (United States)

    Xiao, Jianyong; Zhang, Guangxian; Qiu, Pengxiang; Liu, Xijuan; Wu, Yingya; Du, Biaoyan; Li, Jiefen; Zhou, Jing; Li, Jingjing; Tan, Yuhui

    2013-01-01

    The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.

  4. Synthesis and Biological Evaluation of a New Acyclic Pyrimidine Derivative as a Probe for Imaging Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression

    Directory of Open Access Journals (Sweden)

    Simon M. Ametamey

    2013-07-01

    Full Text Available With the idea of finding a more selective radiotracer for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk gene expression by means of positron emission tomography (PET, a novel [18F]fluorine radiolabeled pyrimidine with 4-hydroxy-3-(hydroxymethylbutyl side chain at N-1 (HHB-5-[18F]FEP was prepared and evaluated as a potential PET probe. Unlabeled reference compound, HHB-5-FEP, was synthesized via a five-step reaction sequence starting from 5-(2-acetoxyethyl-4-methoxypyrimidin-2-one. The radiosynthesis of HHB-[18F]-FEP was accomplished by nucleophilic radiofluorination of a tosylate precursor using [18F]fluoride-cryptate complex in 45% ± 4 (n = 4 radiochemical yields and high purity (>99%. The biological evaluation indicated the feasibility of using HHB-5-[18F]FEP as a PET radiotracer for monitoring HSV1-tk expression in vivo.

  5. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different...... ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket...... surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3...

  6. Characterization of Oligomeric and Kinetic Properties of Tomato Thymidine Kinase 1

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Larsen, Nicolai Balle; Andersson, Karl-Magnus

    2011-01-01

    The gene encoding thymidine kinase 1 from tomato (toTK1) has in combination with azidothymidine (AZT) recently been proposed as a powerful suicide gene for anticancer gene therapy. The toTK1/AZT combination has been demonstrated to have several advantages for the treatment of glioblastomas because...

  7. Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Sherley, J.L.; Kelly, T.J.

    1988-01-05

    The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity.

  8. Targeting of herpes simplex virus 1 thymidine kinase gene sequences into the OCT4 locus of human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Wu Ou

    Full Text Available The in vitro differentiation of human induced pluripotent stem cells (hiPSC to generate specific types of cells is inefficient, and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC, we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK suicide gene at the endogenous OCT4 (POU5F1 locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only, we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus, we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences, flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed, three contained the HSV1-TK transgene at the OCT4 locus, but they were not sensitive to GCV. The other six clones were GCV-sensitive, but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days, indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed.

  9. A human osteosarcoma cell line expressing herpes simplex type-1 thymidine kinase: studies with radiolabeled (E)-5-(2-iodovinyl)-2'-fluoro-2'-deoxyuridine

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Kevin W. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); Duan Weili [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); Knaus, Edward E. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); McEwan, Alexander J.B. [Department of Radiology and Diagnostic Imaging, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, T6G 2N8 (Canada); Wiebe, Leonard I. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada)]. E-mail: leonard.wiebe@ualberta.ca

    2005-07-01

    Introduction: (E)-5-(2-Iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) is a pyrimidine nucleoside analogue that accumulates selectively in murine cells expressing herpes simplex type-1 thymidine kinase (HSV-1 TK). The uptake of [{sup 125}I]IVFRU in human 143B osteosarcoma cells transduced with a retroviral vector bearing the HSV-1 TK gene (143B-LTK cells) is now reported. Methods: HSV-1 TK gene expression in 143B-LTK cells was confirmed by Western blotting and reverse transcriptase (RT)-PCR. Cell and subcellular uptake of [{sup 125}I]IVFRU was determined in cell culture, and whole body biodistribution after intravenous injection of [{sup 125}I]IVFRU was determined using nude mice bearing implanted 143B or 143B-LTK tumors. Results: Although IVFRU was less toxic to the human cell line expressing HSV-1 TK (143B-LTK) than ganciclovir, both IVFRU and ganciclovir were not toxic to the cell line not expressing HSV-1 TK (143B). When cells were exposed to [{sup 125}I]IVFRU in vitro, only the 143B-LTK cells accumulated radioactivity. The acid-soluble fraction from 143B-LTK cell lysates contained 8-fold greater activity than the acid-insoluble fraction after an 8-h exposure to [{sup 125}I]IVFRU. Biodistribution of [{sup 125}I]IVFRU in nude mice bearing subcutaneous 143B and 143B-LTK tumors revealed widespread distribution of the nucleoside in vivo but with specific localization in 143B-LTK tumors. Conclusion: The underlying biochemical process of metabolic entrapment of IVFRU in human osteosarcoma cells expressing HSV-1 TK is responsible for selective localization in these cells. The differences in subcellular distribution into the nucleic acid fraction, and in cytotoxicity, reflect the importance of cell type and lineage as determinants of the performance of gene imaging radiopharmaceuticals.

  10. Role of Equilibrative Nucleobase Transporter 1/SLC43A3 as a Ganciclovir Transporter in the Induction of Cytotoxic Effect of Ganciclovir in a Suicide Gene Therapy with Herpes Simplex Virus Thymidine Kinase.

    Science.gov (United States)

    Furukawa, Junji; Inoue, Katsuhisa; Ohta, Kinya; Yasujima, Tomoya; Mimura, Yoshihisa; Yuasa, Hiroaki

    2017-01-01

    A suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV) has been under development as a tumor-targeted therapy; however, the mechanism of cellular GCV uptake, which is prerequisite in the therapy, has not been clarified. In an attempt to resolve this situation and gain information to optimize HSV-TK/GCV system for cancer therapy, we found that human equilibrative nucleobase transporter 1 (ENBT1) can transport GCV with a Michaelis constant of 2.75 mM in Madin-Darby canine kidney II (MDCKII) cells stably transfected with this transporter. In subsequent experiments using green fluorescent protein (GFP)-tagged ENBT1 (GFP-ENBT1) and HSV-TK, the uptake of GCV (30 μM), which was minimal in MDCKII cells and unchanged by their transfection with HSV-TK alone, was increased extensively by their transfection with GFP-ENBT1, together with HSV-TK. Accordingly, cytotoxicity, which was assessed by the WST-8 cell viability assay after the treatment of those cells with GCV (30 μM) for 72 hours, was induced in those transfected with GFP-ENBT1, together with HSV-TK but not in those transfected with HSV-TK alone. These results suggest that ENBT1 could facilitate GCV uptake and thereby enhance cytotoxicity in HSV-TK/GCV system. We also identified Helacyton gartleri (HeLa) and HepG2 as cancer cell lines that are rich with ENBT1 and A549, HCT-15 and MCF-7 as those poor with ENBT1. Accordingly, the HSV-TK/GCV system was effective in inducing cytotoxicity in the former but not in the latter. Thus, ENBT1 was found to be a GCV transporter that could enhance the performance of HSV-TK/GCV suicide gene therapy. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  11. Serum thymidine kinase in vitamin B/sub 12/ deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Hagberg, H.; Gronowitz, S.; Killander, A.; Kaellander, C. (University Hospital, Uppsala, Sweden)

    1984-01-01

    In DNA synthesis deoxythymidine kinase (TK) catalyses the conversion of deoxythymidine to deoxythymidine monophosphate (dTMP) via the 'salvage pathway'. Serum deoxythymidine kinase (S=TK) was measured in this study in 75 patients with vitamin B/sub 12/ deficiency by a new, very sensitive method, using /sup 125/I-deoxyuridine as substrate. Elevated S-TK levels were found in those patients who had developed haemolysis and anaemia and the more advanced the disease the higher the S-TK value. Thus there was a highly significant correlation between S-TK, haemoglobin level and lactic dehydrogenase activity. These findings are consistent with the theory that elevated levels of S-TK are due to release from unstable proliferating tissue.

  12. Deoxyribonucleoside kinases in two aquatic bacteria with high specificity for thymidine and deoxyadenosine

    DEFF Research Database (Denmark)

    Tinta, Tinkara; Christiansen, Louise Slot; Konrad, Anke

    2012-01-01

    Deoxyribonucleoside kinases (dNKs) are essential in the mammalian cell but their 'importance' in bacteria, especially aquatic ones, is less clear. We studied two aquatic bacteria, Gram-negative Flavobacterium psychrophilum JIP02/86 and Polaribacter sp. MED152, for their ability to salvage...... deoxyribonucleosides (dNs). Both had a Gram-positive-type thymidine kinase (TK1), which could phosphorylate thymidine, and one non-TK1 dNK, which could efficiently phosphorylate deoxyadenosine and slightly also deoxycytosine. Surprisingly, the four tested dNKs could not phosphorylate deoxyguanosine, and apparently......, these two bacteria are missing this activity. When tens of available aquatic bacteria genomes were examined for the presence of dNKs, a majority had at least a TK1-like gene, but several lacked any dNKs. Apparently, among aquatic bacteria, the role of the dN salvage varies....

  13. Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

    OpenAIRE

    Lu, Qiaosheng; Hwang, Ying T.; Hwang, Charles B. C.

    2002-01-01

    To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominately frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion ...

  14. Bystander killing of malignant cells via the delivery of engineered thymidine-active deoxycytidine kinase for suicide gene therapy of cancer.

    Science.gov (United States)

    Neschadim, A; Wang, J C M; Lavie, A; Medin, J A

    2012-05-01

    Activity and specificity of chemotherapeutic agents against solid tumors can be augmented via the targeted or localized delivery of 'suicide' genes. Selective activation of specific prodrugs in cells expressing the 'suicide' gene drives their elimination by apoptosis, while also enabling the killing of adjacent bystander cells. Strong bystander effects can compensate for poor 'suicide' gene delivery, and depend on the prodrugs used and mechanisms for the acquisition of activated drug by the bystander population, such as the presence of gap junctional intercellular communications. Although a number of 'suicide' gene therapies for cancer have been developed and characterized, such as herpes simplex virus-derived thymidine kinase (HSV-tk)-based activation of ganciclovir, their limited success highlights the need for the development of more robust approaches. Limiting activation kinetics and evolution of chemoresistance are major obstacles. Here we describe 'suicide' gene therapy of cancer based on the lentivirus-mediated delivery of a thymidine-active human deoxycytidine kinase variant. This enzyme possesses substrate plasticity that enables it to activate a multitude of prodrugs, some with distinct mechanisms of action. We evaluated the magnitude and mechanisms of bystander effects induced by different prodrugs, and show that when used in combination, they can synergistically enhance the bystander effect while avoiding off-target toxicity.

  15. Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

    Science.gov (United States)

    Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

    2013-11-01

    Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

  16. Preclinical and therapeutic utility of HVJ liposomes as a gene transfer vector for hepatocellular carcinoma using herpes simplex virus thymidine kinase.

    Science.gov (United States)

    Hasegawa, H; Shimada, M; Yonemitsu, Y; Utsunomiya, T; Gion, T; Kaneda, Y; Sugimachi, K

    2001-04-01

    Although gene therapy has been suggested to be a novel strategy to treat hepatocellular carcinoma (HCC), no study showing the clinical feasibility of vectors to treat HCC has been reported. In this preclinical study, we show evidence indicating that hemagglutinating virus of Japan (HVJ) liposomes are a feasible vector to treat HCC in a clinical setting using ganciclovir (GCV) and herpes simplex virus thymidine kinase (HSV-tk), which is driven by the cytomegalovirus immediate early enhancer/promoter (plasmid pcDNA3/HSV-tk). In in vitro experiments, almost complete tumor cell regression was achieved with the optimal GCV concentration (100 microg/mL) and more than 1/3 regression was seen even with a 20% transduction ratio using HuH7 HCC cells stably transformed by HSV-tk. HVJ liposomes showed a 19.7% (mean) transduction rate of the lacZ gene in a relatively large mass of more than 300 mm3 in vivo, which is a clinically detectable size, implanted into SCID mice. Moreover, a single HSV-tk injection of HVJ liposomes followed by GCV treatment inhibited tumor growth at least within a week, and repeat administration was more effective. Furthermore, subcutaneous injection of an HVJ liposomes vehicle induced no apparent inflammatory response in C3H/HeN mice, whereas lacZ gene transfection resulted in inflammatory pathology, suggesting a lower immunogenicity of the HVJ envelope protein than those of bacteria-derived plasmid DNA or the beta-galactosidase gene product. From these findings, we conclude that HVJ liposomes are a clinically safe and effective gene transfer vector to treat HCC.

  17. Radiochemotherapy of hepatocarcinoma via lentivirus-mediated transfer of human sodium iodide symporter gene and herpes simplex virus thymidine kinase gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen Libo, E-mail: libochen888@hotmail.com [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China); Guo Guoying [Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Liu Tianjing; Guo Lihe [Division of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Zhu Ruisen [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China)

    2011-07-15

    Herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system has been widely used as a traditional gene therapy modality, and the sodium/iodide symporter gene (NIS) has been found to be a novel therapeutic gene. Since the therapeutic effects of radioiodine therapy or prodrug chemotherapy on cancers following NIS or HSV-TK gene transfer need to be enhanced, this study was designed to investigate the feasibility of radiochemotherapy for hepatocarcinoma via coexpression of NIS gene and HSV-TK gene. Methods: HepG2 cells were stably transfected with NIS, TK and GFP gene via recombinant lentiviral vector and named HepG2/NTG. Gene expression was examined by reverse transcriptase polymerase chain reaction, fluorescence imaging and iodide uptake. The therapeutic effects were assessed by MTT assay and clonogenic assay. Results: HepG2/NTG cells concentrated {sup 125}I{sup -} up to 76-fold higher than the wild-type cells within 20 min, and the efflux happened with a T{sub 1/2eff} of less than 10 min. The iodide uptake in HepG2/NTG cells was specifically inhibited by sodium perchlorate. Dose-dependent toxicity to HepG2/NTG cells by either GCV or {sup 131}I was revealed by clonogenic assay and MTT assay, respectively. The survival rate of HepG2/NTG cells decreased to 49.7%{+-}2.5%, 43.4%{+-}2.8% and 8.6%{+-}1.2% after exposure to {sup 131}I, GCV and combined therapy, respectively. Conclusion: We demonstrate that radiochemotherapy of hepatocarcinoma via lentiviral-mediated coexpression of NIS gene and HSV-TK gene leads to stronger killing effect than single treatment, and in vivo studies are needed to verify these findings.

  18. Imaging of Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression with Radiolabeled 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in Liver by Hydrodynamic-based Procedure

    Energy Technology Data Exchange (ETDEWEB)

    Song, In Ho; Lee, Tae Sup; Kang, Joo Hyun; Lee, Yong Jin; Kim, Kwang Il; An, Gwang Il; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-10-15

    Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynamic injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU.

  19. Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Win-Ping; Lai, Wen-Fu [Graduate Institute of Biomedical Materials, Taipei Medical University, Taipei (Taiwan); Yang, Wen K.; Yang, Den-Mei [Institute of Biological Science, Academic Sinica, Taipei (Taiwan); Liu, Ren-Shyan [Department of Nuclear Medicine and National PET Cyclotron Center, Veterans General Hospital, Taipei (Taiwan); Hwang, Jeng-Jong; Wang, Hsin-Ell [Institute of Radiological Science, National Yang-Ming University, 155, Sec. 2, Lih-Nong Street, 112, Pei-tou, Taipei (Taiwan); Fu, Ying-Kai [Institute of Nuclear Energy, Atomic Energy Council, Taoyuan (Taiwan)

    2004-01-01

    An experimental cancer gene therapy model was employed to develop a non-invasive imaging procedure using radiolabelled 2'-fluoro-2'-deoxy-5-iodo-1-{beta}-d-arabinofuranosyluracil (FIAU) as an enzyme substrate for monitoring retroviral vector-mediated herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) transgene expression. Iodine-131 labelled FIAU was prepared by a no-carrier-added (n.c.a.) synthesis process and lyophilised to give ''hot kits''. The labelling yield was over 95%, with a radiochemical purity of more than 98%. The stability of [{sup 131}I]FIAU in the form of lyophilised powder (the hot kit) was much better than that in the normal saline solution. The shelf life of the final [{sup 131}I]FIAU hot kit product is as long as 4 weeks. Cellular uptake of [{sup 131}I]FIAU after different periods of storage was investigated in vitro with HSV1-tk-retroviral vector transduced NG4TL4-STK and parental non-transduced NG4TL4 murine sarcoma cell lines over an 8-h incubation period. The NG4TL4-STK cells accumulated more radioactivity than NG4TL4 cells in all conditions, and accumulation increased with time up to 8 h. The kinetic profile of the cellular uptake of n.c.a. [{sup 131}I]FIAU formulated from the lyophilised hot kit or from the stock solution was qualitatively similar. For animal model cancer gene therapy studies, FVB/N mice were inoculated subcutaneously with the HSV1-tk(+) and tk(-) sarcoma cells into the flank to produce tumours. Biodistribution studies showed that tumour/blood ratios were 2, 3.5, 8.2 and 386.8 at 1, 4, 8 and 24 h post injection, respectively, for the HSV1-tk(+) tumours, and 0.5, 0.5, 0.7 and 5.4, respectively, for the HSV1-tk(-) tumours. Radiotracer clearance from blood was completed in 24 h and was bi-exponential. A significant difference in radioactivity accumulation was revealed among the HSV1-tk(+) tumours, the tk(-) tumours and other tissues. At 24 h p.i., higher activity retention was observed

  20. Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans-Splicing Induces Selective Killing of HIV-Producing Cells

    Directory of Open Access Journals (Sweden)

    Carin K. Ingemarsdotter

    2017-06-01

    Full Text Available Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

  1. Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation.

    Science.gov (United States)

    Knecht, Wolfgang; Petersen, Gitte Ebert; Munch-Petersen, Birgitte; Piskur, Jure

    2002-01-25

    In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group. Copyright 2002 Academic Press.

  2. Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK Expression with [124I]FIAU and PET

    Directory of Open Access Journals (Sweden)

    Trevor Hackman

    2002-01-01

    Full Text Available Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CDherpes simplex virus type 1 thymidine kinase (HSV1-tk fusion gene (CD/TK with 5-fluorocytosine (5FC, ganciclovir (GCV, and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil and positron emission tomography (PET for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells. The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

  3. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Egeblad, Louise; Munch-Petersen, Birgitte

    2015-01-01

    Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been...... tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated...... for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene...

  4. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.

    Science.gov (United States)

    Tabin, C J; Hoffmann, J W; Goff, S P; Weinberg, R A

    1982-01-01

    We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors. Images PMID:6180306

  5. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  6. Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: possibility for a rapid human cell mutation assay

    Energy Technology Data Exchange (ETDEWEB)

    Skopek, T.R.; Liber, H.L.; Penman, B.W.; Thilly, W.G.

    1978-09-29

    A thymidine kinase heterozygote designated H2BT has been isolated from the human lymphoblast line HH4. Significant increase in the trifluorothymidine-resistant fraction was observed in the new cell line following treatment with the mutagens ICR-191 and butylmethansulfonate. Phenotypic expression was complete forty-eight hours after treatment.

  7. Secretion of thymidine kinase to increase the effectivity of suicide gene therapy results in the loss of enzymatic activity

    NARCIS (Netherlands)

    Beerens, A. M. J.; Rots, M. G.; Bermudez, B.; De Vries, E. F. J.; Haisma, H. J.

    2008-01-01

    Low efficiency of gene transfer is one of the major limitations of gene therapy. A solution to this problem may be transmission; by modification of the transgene, the gene product can be secreted and internalized by the surrounding cells. Cancer gene therapy using the herpes simplex thymidine kinase

  8. Development and trial of a bovine herpesvirus 1-thymidine kinase deletion virus as a vaccine.

    Science.gov (United States)

    Smith, G A; Young, P L; Rodwell, B J; Kelly, M A; Storie, G J; Farrah, C A; Mattick, J S

    1994-03-01

    An Australian bovine herpesvirus 1 (BHV1) isolate with a defined (427 base pair) deletion in the protein coding region of the thymidine kinase gene was obtained by standard marker rescue procedures. After selection in the presence of the nucleotide analogue 5'-iodo-deoxy-uridine the virus was analysed by hybridisation with three differential oligonucleotide probes, restriction endonuclease profile studies and DNA sequence analysis. The virus elicited an immune response in recipient animals after either intramuscular or intravenous administration and produced no significant deleterious side-effects when administered at a dose sufficient to stimulate the host immune response. The safety and immunogenicity of the recombinant BHV1 virus 39B1 were similar to those reported for other registered BHV1 vaccines and the virus would appear to be suitable for the production of a vaccine seed lot and more exhaustive field trials as a prelude to commercial vaccine production and registration.

  9. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic...... properties are found in cancer cells. Investigation of these factors offers possibilities to understand the molecular background for TKl expression including to clarify general regulation patterns. It also gives valuable information for constructing new nucleoside analogs for the therapy of cancer and virus...... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  10. Use of Thymidine Kinase Recombinant Adenovirus and Ganciclovir Mediated Mouse Liver Preconditioning for Hepatocyte Xenotransplantation.

    Science.gov (United States)

    Moreno, Daniel; Neri, Leire; Vicente, Eva; Vales, Africa; Aldabe, Rafael

    2017-01-01

    Hepatocyte transplantation is the best approach to maintain and propagate differentiated hepatocytes from different species. Host liver has to be adapted for transplanted hepatocytes productive engraftment and proliferation being required a chronic liver injury to eliminate host hepatocytes and provide a proliferative advantage to the transplanted hepatocytes. Most valuable mouse models for xenograft hepatocyte transplantation are based on genetically modified animals to cause a chronic liver damage and to limit host hepatocyte regeneration potential. We present a methodology that generates a chronic liver damage and can be applied to any host mouse strain and animal species based on the inoculation of a recombinant adenovirus to express herpes simplex thymidine kinase in host hepatocytes sensitizing them to ganciclovir treatment. This causes a prolonged liver damage that allows hepatocyte transplantation and generation of regenerative nodules in recipient mouse liver integrated by transplanted cells and host sinusoidal. Obtained chimeric animals maintain functional chimeric nodules for several weeks, ready to be used in any study.

  11. Clinical significance of thymidine kinase in Egyptian children with acute lymphoblastic leukemia

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    Adel A Hagag

    2015-01-01

    Full Text Available Background: Acute lymphoblastic leukemia (ALL is the most common childhood malignancy, representing one-third of pediatric cancers. Thymidine kinase-1 (TK-1 is expressed in proliferating cells so elevated TK-1 indicates active tumor growth. Objective: To study the clinical significance of TK-1 in children with ALL. Patients and Methods: This study was carried out on 40 children with newly diagnosed ALL who were admitted to Oncology Unit, Pediatric department, Tanta University (26 males and 14 females with their ages ranged from 4 to 10 years and 30 healthy children of matched age and sex as a control group. For all patients the following were done: Complete blood picture, bone marrow examination, immunophenotyping and TK-1 serum levels. Results: Mean TK-1 level was significantly higher in patients at diagnosis than controls and significantly higher in patients with unfavorable outcome than patients with favorable outcome. Mean TK-1 level was significantly higher in patients in relapse than patients in remission and controls. No significant differences in mean TK-1 level between patients in remission and controls. There were statistically significant differences in disease free survival and overall survival between patients with favorable and unfavorable outcome. Conclusion: From this study we concluded that TK is a helpful marker in diagnosis and follow-up of patients with ALL. Recommendations: Thymidine kinase-1 should be routinely assessed at diagnosis and during follow-up in ALL patients for better diagnostic and prognostic assessment and should be taken in consideration in designing future therapeutic strategies based on patients-specific risk factors.

  12. Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

    Directory of Open Access Journals (Sweden)

    Hanan Sharif

    2012-06-01

    Full Text Available Abstract Background Thymidine kinase 1 (TK1 is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies. Results Canine TK1 phosphorylated the thymidine (dThd analog 3'-azido-thymidine (AZT as efficiently as it did dThd, whereas AZT phosphorylation by human TK1 was less efficient than that of dThd. Dog TK1 was also more thermostable and pH tolerant than the human enzyme. Oligomeric forms were observed with both enzymes in addition to the tetrameric and dimeric forms. Cellular TK1 was predominantly seen in dimeric and tetrameric forms, in the case of both dog TK1 from MDCK cells and human TK1 from CEM cells. Active serum TK1 was found mainly in a high molecular weight form, and treatment with a reducing agent shifted the high molecular weight complex to lower molecular weight forms with reduced total activity. Western blot analysis demonstrated a polypeptide of 26 kDa (dog and 25 kDa (human for cellular and serum TK1. There was no direct correlation between serum TK1 activity and protein level. It appears that a substantial fraction of serum TK1 is not enzymatically active. Conclusions These results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. The results presented here provide important information for the future development and use of serum TK1 as a diagnostic biomarker in human and veterinary medicine.

  13. Structure of vaccinia virus thymidine kinase in complex with dTTP: insights for drug design

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    Balzarini Jan

    2006-10-01

    Full Text Available Abstract Background Development of countermeasures to bioterrorist threats such as those posed by the smallpox virus (variola, include vaccination and drug development. Selective activation of nucleoside analogues by virus-encoded thymidine (dThd kinases (TK represents one of the most successful strategies for antiviral chemotherapy as demonstrated for anti-herpes drugs. Vaccinia virus TK is a close orthologue of variola TK but also shares a relatively high sequence identity to human type 2 TK (hTK, thus achieving drug selectivity relative to the host enzyme is challenging. Results In order to identify any differences compared to hTK that may be exploitable in drug design, we have determined the crystal structure of VVTK, in complex with thymidine 5'-triphosphate (dTTP. Although most of the active site residues are conserved between hTK and VVTK, we observe a difference in conformation of residues Asp-43 and Arg-45. The equivalent residues in hTK hydrogen bond to dTTP, whereas in subunit D of VVTK, Asp-43 and Arg-45 adopt a different conformation preventing interaction with this nucleotide. Asp-43 and Arg-45 are present in a flexible loop, which is disordered in subunits A, B and C. The observed difference in conformation and flexibility may also explain the ability of VVTK to phosphorylate (South-methanocarbathymine whereas, in contrast, no substrate activity with hTK is reported for this compound. Conclusion The difference in conformation for Asp-43 and Arg-45 could thus be used in drug design to generate VVTK/Variola TK-selective nucleoside analogue substrates and/or inhibitors that have lower affinity for hTK.

  14. 5-[{sup 18}F]Fluoroalkyl pyrimidine nucleosides: probes for positron emission tomography imaging of herpes simplex virus type 1 thymidine kinase gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Chacko, Ann-Marie [Institute for Environmental Medicine, Targeted Therapeutics Program, University of Pennsylvania, Philadelphia, PA 19104 (United States); Blankemeyer, Eric; Lieberman, Brian P.; Qu, Wenchao [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Kung, Hank F. [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104 (United States)], E-mail: kunghf@gmail.com

    2009-01-15

    Introduction: The preliminary in vivo evaluation of novel 5-[{sup 18}F]fluoroalkyl-2'-deoxyuridines ([{sup 18}F]FPrDU, [{sup 18}F]FBuDU, [{sup 18}F]FPeDU; [{sup 18}F]1a-c, respectively) and 2'-fluoro-2'-deoxy-5-[{sup 18}F]fluoroalkyl-1-{beta}-D-arabinofuranosyl uracils ([{sup 18}F]FFPrAU, [{sup 18}F]FFBuAU, [{sup 18}F]FFPeAU; [{sup 18}F]1d-f, respectively) as probes for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is described. Methods: [{sup 18}F]1a-f were successfully synthesized by a rapid and efficient two-step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [{sup 18}F]F{sup -}. For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1-tk (RG2TK+) and wild-type cells (RG2). Results: Biodistribution studies at 2 h pi revealed that the uptake of [{sup 18}F]1a-b and [{sup 18}F]1d-e in RG2TK+ tumors was not significantly different from control tumors. However, [{sup 18}F]1c and [{sup 18}F]1f had an average 1.6- and 1.7-fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [{sup 18}F]1c and [{sup 18}F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [{sup 18}F]1c and [{sup 18}F]1f showed higher initial uptake (3.5- and 1.4-fold, respectively) in RG2TK+ tumors than in control tumors, with continued washout of activity from both tumors over time. Conclusions: Biological evaluations suggest that [{sup 18}F]1c and [{sup 18}F]1f may have limited potential for imaging HSV1-tk gene expression due to fast washout of activity from the blood, thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk-expressing tumors.

  15. Investigation on Cell Proliferation with a New Antibody against Thymidine Kinase 1

    Directory of Open Access Journals (Sweden)

    Naining Wang

    2001-01-01

    Full Text Available The cytosolic thymidine kinase 1 (TK1 is one of the enzymes involved in DNA replication. Based on biochemical studies, TK1 is activated at late G1 of cell cycle, and its activity correlates with the cell proliferation. We have developed a polyclonal anti‐TK1 antibody against a synthetic peptide from the C‐terminus of human TK1. Using this antibody, here we demonstrate the exclusive location of TK1 in the cytoplasm of cells. Cell cycle dependent TK1 expression was studied by simultaneous fluorescence staining for TK1 and bromodeoxyuridine, by using elutriated cells, and by quantitation of the amount TK1 in relation to the cellular DNA content. TK1, which was strongly expressed in the cells in S+G2 period, raised at late G1 and decreased during mitosis. The amount of TK1 increased three folds from late G1 to G2. TK1 positive cells were demonstrated in areas of proliferation activity of various normal and malignant tissues. The new anti‐TK1 antibody works in archival specimens and is a specific marker of cell proliferation.

  16. Loss of arylformamidase with reduced thymidine kinase expression leads to impaired glucose tolerance

    Directory of Open Access Journals (Sweden)

    Alison J. Hugill

    2015-11-01

    Full Text Available Tryptophan metabolites have been linked in observational studies with type 2 diabetes, cognitive disorders, inflammation and immune system regulation. A rate-limiting enzyme in tryptophan conversion is arylformamidase (Afmid, and a double knockout of this gene and thymidine kinase (Tk has been reported to cause renal failure and abnormal immune system regulation. In order to further investigate possible links between abnormal tryptophan catabolism and diabetes and to examine the effect of single Afmid knockout, we have carried out metabolic phenotyping of an exon 2 Afmid gene knockout. These mice exhibit impaired glucose tolerance, although their insulin sensitivity is unchanged in comparison to wild-type animals. This phenotype results from a defect in glucose stimulated insulin secretion and these mice show reduced islet mass with age. No evidence of a renal phenotype was found, suggesting that this published phenotype resulted from loss of Tk expression in the double knockout. However, despite specifically removing only exon 2 of Afmid in our experiments we also observed some reduction of Tk expression, possibly due to a regulatory element in this region. In summary, our findings support a link between abnormal tryptophan metabolism and diabetes and highlight beta cell function for further mechanistic analysis.

  17. Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus

    Energy Technology Data Exchange (ETDEWEB)

    Upton, C.; McFadden, G.

    1986-12-01

    The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK Gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 187 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively.

  18. Thymidine Kinase Suicide Gene-mediated Ganciclovir Ablation of Autologous Gene-modified Rhesus Hematopoiesis

    Science.gov (United States)

    Barese, Cecilia N; Krouse, Allen E; Metzger, Mark E; King, Connor A; Traversari, Catia; Marini, Frank C; Donahue, Robert E; Dunbar, Cynthia E

    2012-01-01

    Despite the genotoxic complications encountered in clinical gene therapy trials for primary immunodeficiency diseases targeting hematopoietic cells with integrating vectors; this strategy holds promise for the cure of several monogenic blood, metabolic and neurodegenerative diseases. In this study, we asked whether the inclusion of a suicide gene in a standard retrovirus vector would allow elimination of vector-containing stem and progenitor cells and their progeny in vivo following transplantation, using our rhesus macaque transplantation model. Following stable engraftment with autologous CD34+ cells transduced with a retrovirus vector encoding a highly sensitive modified Herpes simplex virus thymidine kinase SR39, the administration of the antiviral prodrug ganciclovir (GCV) was effective in completely eliminating vector-containing cells in all hematopoietic lineages in vivo. The sustained absence of vector-containing cells over time, without additional GCV administration, suggests that the ablation of TkSR39 GCV-sensitive cells occurred in the most primitive hematopoietic long-term repopulating stem or progenitor cell compartment. These results are a proof-of-concept that the inclusion of a suicide gene in integrating vectors, in addition to a therapeutic gene, can provide a mechanism for later elimination of vector-containing cells, thereby increasing the safety of gene transfer. PMID:22910293

  19. Antagonism of HSV-tk transfection and ganciclovir treatment on chemotherapeutic drug sensitivity

    NARCIS (Netherlands)

    Van Dillen, IJ; Mulder, NH; Meijer, C; Dam, WA; Kamstra, E; De Vries, L; Meersma, GJ; Van Der Zee, AGJ; De Vries, EFJ; Vaalburg, W; Hospers, GAP

    Our study focused on the influence of herpes simplex virus thymidine kinase (HSV-tk) expression and ganciclovir (GCV) treatment on the sensitivity of C6 glioma cells to frequently used chemotherapeutic drugs, i.e. adriamycin (ADR), cisplatin (CDDP), 5-fluorouracil (5-FU), and methotrexate (MTX).

  20. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    Directory of Open Access Journals (Sweden)

    Louise Slot Christiansen

    2015-06-01

    Full Text Available Nucleoside analogues (NA are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1 is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT.We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.

  1. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    Energy Technology Data Exchange (ETDEWEB)

    Slot Christiansen, Louise [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Egeblad, Louise [Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Munch-Petersen, Birgitte [Department of Science, Systems and Models, Roskilde University, Roskilde 4000 (Denmark); Piškur, Jure [Department of Biology, Lund University, Lund 22362 (Sweden); Knecht, Wolfgang, E-mail: Louise.Slot_Christiansen@biol.lu.se [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden)

    2015-06-08

    Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.

  2. Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation.

    Science.gov (United States)

    Amacher, D E; Paillet, S C; Turner, G N; Ray, V A; Salsburg, D S

    1980-08-01

    The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFTR mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample loge t test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.

  3. Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

    Science.gov (United States)

    Khan, Zahidul; Knecht, Wolfgang; Willer, Mette; Rozpedowska, Elzbieta; Kristoffersen, Peter; Clausen, Anders Ranegaard; Munch-Petersen, Birgitte; Almqvist, Per M; Gojkovic, Zoran; Piskur, Jure; Ekström, Tomas J

    2010-06-01

    The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas.

  4. Identification of Fhit as a post-transcriptional effector of Thymidine Kinase 1 expression.

    Science.gov (United States)

    Kiss, Daniel L; Waters, Catherine E; Ouda, Iman M; Saldivar, Joshua C; Karras, Jenna R; Amin, Zaynab A; Mahrous, Seham; Druck, Teresa; Bundschuh, Ralf A; Schoenberg, Daniel R; Huebner, Kay

    2017-03-01

    FHIT is a genome caretaker gene that is silenced in >50% of cancers. Loss of Fhit protein expression promotes accumulation of DNA damage, affects apoptosis and epithelial-mesenchymal transition, though molecular mechanisms underlying these alterations have not been fully elucidated. Initiation of genome instability directly follows Fhit loss and the associated reduced Thymidine Kinase 1 (TK1) protein expression. The effects on TK1 of Fhit knockdown and Fhit induction in the current study confirmed the role of Fhit in regulating TK1 expression. Changes in Fhit expression did not impact TK1 protein turnover or transcription from the TK1 promoter, nor steady-state levels of TK1 mRNA or turnover. Polysome profile analysis showed that up-regulated Fhit expression resulted in decreased TK1 RNA in non-translating messenger ribonucleoproteins and increased ribosome density on TK1 mRNA. Fhit does not bind RNA but its expression increased luciferase expression from a transgene bearing the TK1 5'-UTR. Fhit has been reported to act as a scavenger decapping enzyme, and a similar result with a mutant (H96) that binds but does not cleave nucleoside 5',5'-triphosphates suggests the impact on TK1 translation is due to its ability to modulate the intracellular level of cap-like molecules. Consistent with this, cells expressing Fhit mutants with reduced activity toward cap-like dinucleotides exhibit DNA damage resulting from TK1 deficiency, whereas cells expressing wild-type Fhit or the H96N mutant do not. The results have implications for the mechanism by which Fhit regulates TK1 mRNA, and more broadly, for its modulation of multiple functions as tumor suppressor/genome caretaker. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene: detailed immunological function following add-back after haplo-identical transplantation.

    Science.gov (United States)

    Hashimoto, Hisayoshi; Kitano, Shigehisa; Yamagata, Shizuka; Miyagi Maeshima, Akiko; Ueda, Ryosuke; Ito, Ayumu; Tada, Kohei; Fuji, Shigeo; Yamashita, Takuya; Tomura, Daisuke; Nukaya, Ikuei; Mineno, Junichi; Fukuda, Takahiro; Mori, Shinichiro; Takaue, Yoichi; Heike, Yuji

    2015-12-01

    Haplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. We investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vβ repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. A comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8(+) non-TK cells were also depleted in response to ganciclovir administration. The TCR Vβ-chain repertoire of both TK cells and non-TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non-TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. We demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  6. Monitoring HSV-TK/ganciclovir cancer suicide gene therapy using CdTe/CdS core/shell quantum dots

    NARCIS (Netherlands)

    Shao, D.; Zeng, Q.; Fan, Z.; Li, J.; Zhang, M.; Zhang, Y.; Li, O.; Chen, L.; Kong, X.; Zhang, H.

    2012-01-01

    To be able to label a gene and monitor its migration are key important approaches for the clinical application of cancer suicide gene therapy. Photonic nanomaterials are introduced in this work. One of the most promised suicide genes - herpes simplex virus thymidine kinase (HSV-TK) gene - is

  7. Improved long-term expression from helper virus-free HSV-1 vectors packaged using combinations of mutated HSV-1 proteins that include the UL13 protein kinase and specific components of the VP16 transcriptional complex

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    Geller Alfred I

    2009-06-01

    Full Text Available Abstract Background Herpes Simplex Virus (HSV-1 gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (~99% of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase, or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days. These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex. Results A HSV-1 vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. For supporting long-term expression, the preferred combination of mutated HSV-1 proteins was mutated UL13, UL46, and UL47. Vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for 3 months, the longest time examined. Conclusion Vector particles containing this combination of mutated HSV-1 proteins improve recombinant gene expression. Implications of these results for strategies to further improve

  8. Improved long-term expression from helper virus-free HSV-1 vectors packaged using combinations of mutated HSV-1 proteins that include the UL13 protein kinase and specific components of the VP16 transcriptional complex.

    Science.gov (United States)

    Liu, Meng; Wang, Xiaodan; Geller, Alfred I

    2009-06-16

    Herpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (approximately 99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase), or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex. A HSV-1 vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. For supporting long-term expression, the preferred combination of mutated HSV-1 proteins was mutated UL13, UL46, and UL47. Vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for 3 months, the longest time examined. Vector particles containing this combination of mutated HSV-1 proteins improve recombinant gene expression. Implications of these results for strategies to further improve long-term expression are discussed

  9. F-18-FEAU as a radiotracer for herpes simplex virus thymidine kinase gene expression : in-vitro comparison with other PET tracers

    NARCIS (Netherlands)

    Buursma, AR; Rutgers, [No Value; Hospers, GAP; Mulder, NH; Vaalburg, W; de Vries, EFJ

    Objective The herpes simplex virus thymidine kinase (HSVtk) gene has frequently been applied as a reporter gene for monitoring transgene expression in animal models. In clinical gene therapy protocols, however, extremely low expression levels of the transferred gene are generally observed.

  10. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

    DEFF Research Database (Denmark)

    Dou, Q P; Zhao, S; Levin, A H

    1994-01-01

    By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we....... (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide...

  11. Evaluation of F-18-labeled 5-iodocytidine ({sup 18}F-FIAC) as a new potential positron emission tomography probe for herpes simplex virus type 1 thymidine kinase imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Pei-Chia; Wu, Chun-Yi; Chang, Wen-Yi; Chang, Wei-Ting [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China); Alauddin, Mian [Department of Experimental Diagnostic Imaging, MD Anderson Cancer Center, University of Texas, TX, 77054 (United States); Liu, Ren-Shan [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China); Department of Nuclear Medicine, Faculty of Medicine, National Yang-Ming University, Taipei, 11221, Taiwan (China); Department of Nuclear Medicine and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, 11217, Taiwan (China); Lin, Wuu-Jyh [Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, 32546, Taiwan (China); Chen, Fu-Du [College of Health and Leisure Science, TransWorld University, Yunlin, 64063, Taiwan (China); Chen, Chuan-Lin, E-mail: clchen2@ym.edu.tw [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China); Wang, Hsin-Ell, E-mail: hewang@ym.edu.tw [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China)

    2011-10-15

    Objective: Herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene in combination with radiolabeled nucleoside substrates is the most widely used reporter system. This study characterized 1-(2'-deoxy-2'-[{sup 18}F]fluoro-{beta}-D-arabinofuranosyl)-5-iodocytosine ({sup 18}F-FIAC) as a new potential positron emission tomography (PET) probe for HSV1-tk gene imaging and compared it with 2'-deoxy-2'-[{sup 18}F]fluoro-5-iodo-1-{beta}-D-arabinofuranosyluracil ({sup 18}F-FIAU) and 2'-deoxy-2'-[{sup 18}F]fluoro-5-ethyl-1-{beta}-D-arabinofuranosyluracil({sup 18}F-FEAU) (thymidine analogues) in an NG4TL4-WT/STK sarcoma-bearing mouse model. Methods: A cellular uptake assay, biodistribution study, radioactive metabolites assay and microPET imaging of NG4TL4-WT/STK tumor-bearing mice post administration of {sup 18}F-FIAC, {sup 18}F-FIAU and {sup 18}F-FEAU were conducted to characterize the biological properties of these tracers. Results: Highly specific uptake of {sup 18}F-FIAC, {sup 18}F-FIAU and {sup 18}F-FEAU in tk-transfected [tk(+)] cells was observed. The tk(+)-to-tk(-) cellular uptake ratio after a 2-h incubation was 66.6{+-}25.1, 76.3{+-}18.2 and 247.2{+-}37.2, respectively. In biodistribution studies, {sup 18}F-FIAC showed significant tk(+) tumor specificity (12.6; expressed as the tk(+)-to-tk(-) tumor uptake ratio at 2 h postinjection) comparable with {sup 18}F-FIAU (15.8) but lower than {sup 18}F-FEAU (48.0). The results of microPET imaging also revealed the highly specific accumulation of these three radioprobes in the NG4TL4-tk(+) tumor. Conclusion: Our findings suggested that the cytidine analogue {sup 18}F-FIAC is a new potential PET probe for the imaging of HSV1-tk gene expression. {sup 18}F-FIAC may be regarded as the prodrug of {sup 18}F-FIAU in vivo.

  12. Influence of p53 status on the HSV-Tk/GCV-induced bystander effect in a panel of human ovarian carcinoma cell lines

    NARCIS (Netherlands)

    van Dillen, IJ; Mulder, NH; Sluiter, WJ; Meijer, C; de Jong, S; Loncarek, J; Mesnil, M; de Vries, EFJ; Vaalburg, W; Hospers, GAP

    2005-01-01

    The p53 protein is mutated in 50% of all human tumors and plays a key role in apoptosis, cell cycle, and the expression of several genes. We investigated if p53 mutations influence the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV)-induced bystander effect (BE). Additionally, we

  13. Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models.

    Science.gov (United States)

    Kang, N-H; Hwang, K-A; Yi, B-R; Lee, H J; Jeung, E-B; Kim, S U; Choi, K-C

    2012-06-01

    As human amniotic fluid-derived stem cells (hAFSCs) are capable of multiple lineage differentiation, extensive self-renewal and tumor targeting, they may be valuable for clinical anticancer therapies. In this study, we used hAFSCs as vehicles for targeted delivery of therapeutic suicide genes to breast cancer cells. hAFSCs were engineered to produce AF2.CD-TK cells in order to express two suicide genes encoding bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) that convert non-toxic prodrugs, 5-fluorocytosine (5-FC) and mono-phosphorylate ganciclovir (GCV-MP), into cytotoxic metabolites, 5-fluorouracil (5-FU) and triphosphate ganciclovir (GCV-TP), respectively. In cell viability test in vitro, AF2.CD-TK cells inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the 5-FC or GCV prodrugs, or a combination of these two reagents. When the mixture of 5-FC and GCV was treated together, an additive cytotoxic effect was observed in the cell viability. In animal experiments using female BALB/c nude mouse xenografts, which developed by injecting MDA-MB-231 cells, treatment with AF2.CD-TK cells in the presence of 5-FC and GCV significantly reduced tumor volume and weight to the same extent seen in the mice treated with 5-FU. Histopathological and fluorescent staining assays further showed that AF2.CD-TK cells were located exactly at the site of tumor formation. Furthermore, breast tissues treated with AF2.CD-TK cells and two prodrugs maintained their normal structures (for example, the epidermis and reticular layers) while breast tissue structures in 5-FU-treated mice were almost destroyed by the potent cytotoxicity of the drug. Taken together, these results indicate that AF2.CD-TK cells can serve as excellent vehicles in a novel therapeutic cell-based gene-directed prodrug system to selectively target breast malignancies.

  14. The thymidine kinase-1 as a potential tumor marker: structure, function, activity in normal and malignant tissues

    Directory of Open Access Journals (Sweden)

    N. S. Sergeeva

    2017-01-01

    Full Text Available In the review the role of the thymidine kinase (TK to ensure the replication of DNA de novo and spare (salvage the way in health and activate alternate ways in carcinogenesis is described. The structure of cytoplasmic TK (TК-1, also called fetal, and the level of regulation of its activity in the cells and their change during the cell cycle is described. Considering the data about the absence of TK-1 in resting (G0 cells, TK-1 is positioned as a marker of proliferating cells, which activity is recorded from late G1 phase, peaking in S-phase, it is stored in the G2 and mitosis, quickly decreasing to undetectable levels in the early G1 phase. Data on the expression TK-1 (as compared with Ki-67 and PCNA (proliferating cell nuclear antigen in tumor tissues (colorectal, breast, cervical, lung, renal, prostate and ovarian cancer, as well as some benign and precancerous pathological processes in relation to the clinical and diagnostic features of these processes are systemized. These data suggest that the proliferative index studies on TK-1 (antibody to the domain HRA-210 should be used together with Ki-67 and PCNA, for a more complete assessment of the proliferative status of malignant tumors and pre-cancerous and benign conditions, with the aim of prognosis of the tumor process and treatment planning.

  15. An Adenovirus Vector Containing the Suicide Gene Thymidine Kinase for a Broad Application in Cancer Gene Therapy

    Directory of Open Access Journals (Sweden)

    Magalhães GS

    2002-01-01

    Full Text Available Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK gene, followed by administration of the antiviral drug ganciclovir (GCV. The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK driven by three strong promoters (P CMV IE, SV40 and EN1 and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

  16. Oncolytic adenoviruses armed with thymidine kinase can be traced by PET imaging and show potent antitumoural effects by ganciclovir dosing.

    Directory of Open Access Journals (Sweden)

    Daniel Abate-Daga

    Full Text Available Replication-competent adenoviruses armed with thymidine kinase (TK combine the concepts of virotherapy and suicide gene therapy. Moreover TK-activity can be detected by noninvasive positron emission-computed tomography (PET imaging, what could potentially facilitate virus monitoring in vivo. Here, we report the generation of a novel oncolytic adenovirus that incorporates the Tat8-TK gene under the control of the Major Late Promoter in a highly selective backbone thus providing selectivity by targeting the retinoblastoma pathway. The selective oncolytic TK virus, termed ICOVIR5-TK-L, showed reduced potency compared to a non-selective counterpart. However the combination of ICOVIR5-TK-L with ganciclovir (GCV induced a potent antitumoural effect similar to that of wild type adenovirus in a preclinical model of pancreatic cancer. Although the treatment with GCV provoked a reduction in the viral yield, both in vitro and in vivo, a two-cycle treatment of virus and GCV resulted in an enhanced antitumoral response that correlated with high TK-activity, based on microPET measurements. Thus, TK-expressing oncolytic adenoviruses can be traced by PET imaging providing real time information on the activity of the virus and its antitumoral potency can be optimized by GCV dosing.

  17. Usage of adenovirus expressing thymidine kinase mediated hepatocellular damage for enabling mouse liver repopulation with allogenic or xenogenic hepatocytes.

    Directory of Open Access Journals (Sweden)

    Daniel Moreno

    Full Text Available It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/- γc(-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk and two consecutive doses of ganciclovir (GCV. We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

  18. Cloning of the koi herpesvirus (KHV gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

    Directory of Open Access Journals (Sweden)

    Gilad Oren

    2005-03-01

    Full Text Available Abstract Background Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV. Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. Results A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV and the channel catfish virus (CCV. The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. Conclusion The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

  19. Clinical effect of recombinant adenovirus containing thymidine kinase suicide gene in preventing postoperative recurrence of hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    MENG Jian

    2017-10-01

    Full Text Available ObjectiveTo investigate the clinical effect of surgical operation combined with gene therapy in preventing postoperative recurrence of hepatocellular carcinoma (HCC. MethodsA total of 102 patients with single HCC (TNM stage 1-2, tumor diameter <10 cm who were admitted to Beijing YouAn Hospital, Capital Medical University, from July 2006 to February 2013 were enrolled, and among these patients, 60 underwent the gene therapy with recombinant adenovirus containing thymidine kinase suicide gene (ADV-TK before and after surgery (gene group and 42 underwent surgical resection alone (surgery group. The patients were followed up regularly after surgery to observe postoperative recurrence. The t-test was used for comparison of continuous data between groups, and the chi-square test was used for comparison of categorical data between groups. The log-rank test was used for the analysis of cumulative recurrence rate, and univariate and multivariate Cox regression analyses were used to identify influencing factors for recurrence rate. ResultsThe 1-, 3-, and 5-year recurrence rates of tumor were 13.8%, 33.7%, and 47.7% in the gene group and 18.5%, 53.2%, and 69.2% in the surgery group, and there was a significant difference between the two groups (χ2=2643,P=0.041. The gene group had a significantly higher proportion of patients with pyrexia after surgery than the surgery group, and there were no significant differences in the incidence rates of other complications and length of hospital stay between the two groups. The multivariate analysis showed that gene therapy was an independent influencing factor for cumulative rumulative recurrence rate (odds ratio=2752,95 confidence interval:1164-4251,P=0038. ConclusionGene therapy combined with surgical resection can effectively reduce postoperative recurrence of tumor, and therefore, it holds promise for clinical application.

  20. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael

    2006-01-01

    , while none was found in Nanoarchaeum. The identified TK1s have high identity to Gram-positive bacteria TK1s. The TK1s from archaea, Gram-positive bacteria and eukaryotes share the same common ancestor, while the TK1s from Gram-negative bacteria belong to a less-related subgroup. It seems...

  1. Herpes simplex virus thymidine kinase imaging in mice with (1-(2'-deoxy-2'-[{sup 18}F]fluoro-1-{beta}-D-arabinofuranosyl)-5-iodouracil) and metabolite (1-(2'-deoxy-2'-[{sup 18}F]fluoro-1-{beta}-D-arabinofuranosyl)-5-uracil)

    Energy Technology Data Exchange (ETDEWEB)

    Nimmagadda, Sridhar; Lawhorn-Crews, Jawana M.; Shields, Anthony F. [Wayne State University, Karmanos Cancer Institute, Detroit, MI (United States); Wayne State University, Department of Medicine, Detroit, MI (United States); Mangner, Thomas J. [Wayne State University, Karmanos Cancer Institute, Detroit, MI (United States); Wayne State University, Department of Radiology, Detroit, MI (United States); Haberkorn, Uwe [University of Heidelberg, Department of Nuclear Medicine, Heidelberg (Germany)

    2009-12-15

    FIAU, (1-(2{sup '}-deoxy-2{sup '}-fluoro-1-{beta}-D-arabinofuranosyl)-5-iodouracil) has been used as a substrate for herpes simplex virus thymidine kinases (HSV-TK and HSV-tk, for protein and gene expression, respectively) and other bacterial and viral thymidine kinases for noninvasive imaging applications. Previous studies have reported the formation of a de-iodinated metabolite of {sup 18}F-FIAU. This study reports the dynamic tumor uptake, biodistribution, and metabolite contribution to the activity of {sup 18}F-FIAU seen in HSV-tk gene expressing tumors and compares the distribution properties with its de-iodinated metabolite {sup 18}F-FAU. CD-1 nu/nu mice with subcutaneous MH3924A and MH3924A-stb-tk+ xenografts on opposite flanks were used for the biodistribution and imaging studies. Mice were injected IV with either {sup 18}F-FIAU or {sup 18}F-FAU. Mice underwent dynamic imaging with each tracer for 65 min followed by additional static imaging up to 150 min post-injection for some animals. Animals were sacrificed at 60 or 150 min post-injection. Samples of blood and tissue were collected for biodistribution and metabolite analysis. Regions of interest were drawn over the images obtained from both tumors to calculate the time-activity curves. Biodistribution and imaging studies showed the highest uptake of {sup 18}F-FIAU in the MH3924A-stb-tk+ tumors. Dynamic imaging studies revealed a continuous accumulation of {sup 18}F-FIAU in HSV-TK expressing tumors over 60 min. The mean biodistribution values (SUV {+-} SE) for MH3924A-stb-tk+ were 2.07 {+-} 0.40 and 6.15 {+-} 1.58 and that of MH3924A tumors were 0.19 {+-} 0.07 and 0.47 {+-} 0.06 at 60 and 150 min, respectively. In {sup 18}F-FIAU injected mice, at 60 min nearly 63% of blood activity was present as its metabolite {sup 18}F-FAU. Imaging and biodistribution studies with {sup 18}F-FAU demonstrated no specific accumulation in MH3924A-stb-tk+ tumors and SUVs for both the tumors were similar to those

  2. The antitumor effect of suicide gene therapy using Bifidobacterium infantis-mediated herpes simplex virus thymidine kinase/ganciclovir in a nude mice model of renal cell carcinoma.

    Science.gov (United States)

    Xiao, Xiao; Jin, Ren; Li, Jiang; Bei, Yu; Wei, Tang

    2014-10-01

    To confirm the effectivity of Bifidobacterium infantis-mediated herpes simplex virus thymidine kinase/ganciclovir suicide gene system on the treatment of renal cell carcinoma in nude mice and further explore the mechanisms. A B infantis thymidine kinase (B infantis-TK) suicide gene system was constructed in our previous study. Tumor-bearing nude mice were randomized into 4 groups and injected with normal saline, B infantis, B infantis/pGEX-1λT, and B infantis-TK, respectively, via tail vein, followed by intraperitoneal injection of ganciclovir. The treatment effects were evaluated by the terminal deoxynucleotidyl transferase-mediated deoxynucleotide triphosphate nick end labeling assay, quantitative reverse transcriptase polymerase chain reaction, and Western blotting. Side effects were also recorded. Compared with the other 3 treatments, the treatment with B infantis-TK resulted in a significant effective antitumor activity and stronger apoptotic response. Western blot analysis showed that the expression levels of Rel A and Bcl-xL were significantly lower, whereas those of caspase 3 and Bax were significantly higher in tumor tissues resected from group B infantis-TK, which were consistent with the quantitative reverse transcriptase-polymerase chain reaction results. The B infantis-TK/ganciclovir therapy system exhibits an effective antitumor activity by promoting tumor cell apoptosis through both the intrinsic and the extrinsic apoptotic pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Synthesis and Cellular Uptake of Radioiodine labeled 2{sup '}-deoxyuridine derivatives with HSV1-TK

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Ah; Lee, Kyo Chul; Hong, Su Hee; Kim, Eun Jung; Lee, Jong Chan; An, Gwang Il; Choi, Tae Hyun; Cheon, Gi Jeong; Chun, Kwon Soo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    Several different radiolabeled probes have been developed to image Herpes Simplex Virus Type-1 thyrimidine kinase gene (HSV1-TK) expression. The nucleoside prodrugs under investigation for HSV1-TK imaging generally fall into two main chemical and radioisotope categories: the pyrimidine nucleosides, primarily radioiodinated, and the purin nucleosides, primarily radiofluorinated, and their respective analogues. In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have been recommended as HSV1-TK substrates. Most of these nucleoside derivatives have been developed as prodrugs for tumor proliferation imaging or as anti-viral drugs. For example, 5-fluorouracil (5-FU) and IUdR have been used as tumor agents and acyclovir (ACV), ganciclovir (GCV) and (E)-5-(2-bromovinyl)-2{sup '}- deoxyuridine (BVDU) as an anti-viral agents for virus infection several 5-substituted uracil nucleoside derivatives have been identified to have high sensitivity and selective accumulation in HSV1-TK expressing cells. Of those, IVDU was shown to be rapidly accumulated in HSV1- TK expressing cells in vitro. Imaging of the HSV1-TK reporter gene along with various reporter probes is of current interest. In contrast to the mammalian kinase, which phosphorylates thymidine preferentially, HSV1-TK is less discriminative and phosphorylates a wide range of nucleoside analogues such as acycloguanosines and 2{sup '}-fluoro-2{sup '}-deoxyuridine derivatives that are not phosphorylated efficiently by the native enzyme. More specifically, 5-substituted 2{sup '}-fluoro-2{sup '}-deoxyarabinofuranosyluracil nucleosides are efficiently phosphorylated by HSV- TK. This property, together with the presence of fluorine in the 2{sup '}-arabino-position, endows the 2{sup '}-fluoro-2{sup '}-deoxyuridines with antiviral activity against HSV.

  4. Caspase-3-independent pathways proceeding in bystander effect of HSV-tk/GCV system

    Science.gov (United States)

    Lin, Juqiang; Ma, Yan; Zeng, Shaoqun; Zhang, Zhihong

    2008-02-01

    HSV-tk/GCV system, which is the virus-directed enzyme/prodrug therapy of herpes simplex virus (HSV) thymidine kinase (tk) gene / the anti-viral reagent ganciclovir (GCV), is one of the promising approaches in the rapidly growing area of gene therapy. As gene therapy of cancer such as suicide gene therapy has entered the clinic, another therapy effect which is called 'bystander effect' was reported. Bystander effect can lead to killing of non-transduced tumor cells in the immediate vicinity of GCV-treated HSV-TK-positive cells. Now the magnitude of 'bystander effect' is an essential factor for this anti-tumor approach in vivo. However, the mechanism which HSV-tk/ACV brings "bystander effect" is poorly understood. In this study, we monitor the activation of caspase-3 in HSV-tk/GCV system by a FRET probe CD3, a FRET-based indicator for activity of caspase3, which is composed of an enhanced cyan fluorescent protein, a caspase-sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. Through application of CD3 we have visualized the activation of caspase-3 in tk gene positive human adenoid cystic carcinoma (ACC-M) cells but not in bystander effect of HSV-tk/GCV system induced by GCV. This finding provides needed information for understanding the mechanisms by which suicide gene approaches actually kill cancer cells, and may prove to be helpful for the clinical treatment of cancers.

  5. Combination of MPPa-PDT and HSV1-TK/GCV gene therapy on prostate cancer.

    Science.gov (United States)

    Liang, Liming; Bi, Wenxiang; Chen, Weiwen; Lin, Yani; Tian, Yuanyuan

    2018-02-01

    We combined pyropheophorbide-a methyl ester, photodynamic therapy (MPPa-PDT), 670 ± 10 nm, 4 mW/cm 2 , with herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV1-TK/GCV) to improve the therapeutic effect. We built HSV1-TK expression vector GV230-TK and we observed a bright green fluorescence under fluorescence microscope. It indicated the recombinant plasmid was transfected into PC-3M prostate cancer cells successfully. As the abundant glucose-regulated protein 78 (GRP78) promoter in PC-3M cells can cause active expression of HSV1-TK, cell protein was collected for western blot to determine the expression of HSV1-TK. In CCK-8 assay (n = 6), the cell survival rate of combined treatment group was about 10%, less than that of pure MPPa-PDT group (23%) and pure HSV1-TK/GCV group (35%) (t test, P PDT group (about 22%) and pure HSV1-TK/GCV group (about 19%). The results showed that the combination of the two treatments can effectively improve the cytocidal effect in PC-3M cells.

  6. Effect of p53 activation on cell growth, thymidine kinase-1 activity, and 3'-deoxy-3'fluorothymidine uptake

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, Jeffrey L. E-mail: jschwart@u.washington.edu; Tamura, Yasuko; Jordan, Robert; Grierson, John R.; Krohn, Kenneth A

    2004-05-01

    The use of thymidine (TdR) and thymidine analogs such as 3'-deoxy-3'-fluorothymidine (FLT) as positron emission tomography (PET)-based tracers of tumor proliferation rate is based on the hypothesis that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK{sub 1}) activity, provides an accurate measure of cell proliferation in tumors. Tumor growth is influenced by many factors including the oxygen concentration within tumors and whether tumor cells have been exposed to cytotoxic therapies. The p53 gene plays an important role in regulating growth under both of these conditions. The goal of this study was to investigate the influence of p53 activation on cell growth, TK{sub 1} activity, and FLT uptake. To accomplish this, TK{sub 1} activity, S phase fraction, and the uptake of FLT were determined in plateau-phase and exponentially growing cultures of an isogenic pair of human tumor cell lines in which p53 expression was normal or inactivated by human papilloma virus type 16 E6 expression. Ionizing radiation exposure was used to stimulate p53 activity and to induce alterations in cell cycle progression. We found that exposure of cells to ionizing radiation induced dose-dependent changes in cell cycle progression in both cell lines. The relationship between S phase percentage, TK{sub 1} activity, and FLT uptake were essentially unchanged in the p53-normal cell line. In contrast, TK{sub 1} activity and FLT uptake remained high in the p53-deficient variant even when S phase percentage was low due to a p53-dependent G2 arrest. We conclude that a functional p53 response is required to maintain the normal relationship between TK1 activity and S phase percentage following radiation exposure.

  7. Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

    Energy Technology Data Exchange (ETDEWEB)

    Villarroya, Joan, E-mail: joanvillarroya@gmail.com [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Institut de Recerca l' Hospital de la Santa Creu i Sant Pau, Barcelona (Spain); Lara, Mari-Carmen [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Department of Neurology, Columbia University Medical Center, New York, NY (United States); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII (Spain); Dorado, Beatriz [Department of Neurology, Columbia University Medical Center, New York, NY (United States); Garrido, Marta [Unitat de Biologia Cel.lular i Molecular, IMIM-Hospital del Mar, Barcelona (Spain); Garcia-Arumi, Elena [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII (Spain); Meseguer, Anna [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Hirano, Michio [Department of Neurology, Columbia University Medical Center, New York, NY (United States); Vila, Maya R. [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain)

    2011-04-08

    Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity

  8. PET imaging of HSV1-tk mutants with acquired specificity toward pyrimidine- and acycloguanosine-based radiotracers

    Energy Technology Data Exchange (ETDEWEB)

    Likar, Yury; Dobrenkov, Konstantin; Olszewska, Malgorzata; Shenker, Larissa; Hricak, Hedvig; Ponomarev, Vladimir [Memorial Sloan-Kettering Cancer Center, Molecular Imaging Laboratory, Department of Radiology, New York, NY (United States); Cai, Shangde [Memorial Sloan-Kettering Cancer Center, Radiochemistry/Cyclotron Core Facility, New York, NY (United States)

    2009-08-15

    The aim of this study was to create an alternative mutant of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene with reduced phosphorylation capacity for acycloguanosine derivatives, but not pyrimidine-based compounds that will allow for successful PET imaging. A new mutant of HSV1-tk reporter gene, suitable for PET imaging using pyrimidine-based radiotracers, was developed. The HSV1-tk mutant contains an arginine-to-glutamine substitution at position 176 (HSV1-R176Qtk) of the nucleoside binding region of the enzyme. The mutant-gene product showed favorable enzymatic characteristics toward pyrimidine-based nucleosides, while exhibiting reduced activity with acycloguanosine derivatives. In order to enhance HSV1-R176Qtk reporter activity with pyrimidine-based radiotracers, we introduced the R176Q substitution into the more active HSV1-sr39tk mutant. U87 human glioma cells transduced with the HSV1-R176Qsr39tk double mutant reporter gene showed high {sup 3}H-FEAU pyrimidine nucleoside and low {sup 3}H-penciclovir acycloguanosine analog uptake in vitro. PET imaging also demonstrated high {sup 18}F-FEAU and low {sup 18}F-FHBG accumulation in HSV1-R176Qsr39tk+ xenografts. The feasibility of imaging two independent nucleoside-specific HSV1-tk mutants in the same animal with PET was demonstrated. Two opposite xenografts expressing the HSV1-R176Qsr39tk reporter gene and the previously described acycloguanosine-specific mutant of HSV1-tk, HSV1-A167Ysr39tk reporter gene, were imaged using a short-lived pyrimidine-based {sup 18}F-FEAU and an acycloguanosine-based {sup 18}F-FHBG radiotracer, respectively, administered on 2 consecutive days. We conclude that in combination with acycloguanosine-specific HSV1-A167Ysr39tk reporter gene, a HSV1-tk mutant containing the R176Q substitution could be used for PET imaging of two different cell populations or concurrent molecular biological processes in the same living subject. (orig.)

  9. Gene Therapy with HSV1-sr39TK/GCV Exhibits a Stronger Therapeutic Efficacy Than HSV1-TK/GCV in Rat C6 Glioma Cells

    Directory of Open Access Journals (Sweden)

    Lei-qing Li

    2013-01-01

    Full Text Available Although the combination of herpes simplex virus type 1 (HSV-1 thymidine kinase (TK with ganciclovir (GCV has been shown as a promising suicide gene treatment strategy for glioma, the almost immunodepressive dose of GCV required for its adequate in vivo efficacy has hampered its further clinical application. Therefore, In order to reduce the GCV dose required, we aim to compare the therapeutic efficacy of HSV1-sr39TK, an HSV1-TK mutant with increased GCV prodrug catalytic activity, with wildtype TK in C6 glioma cells. Accordingly, rat C6 glioma cells were first transfected with pCDNA-TK and pCDNA-sr39TK, respectively, and the gene transfection efficacy was verified by immunocytochemistry and western blot analysis. Then the in vivo sensitivity of these transfected C6-TK and C6-sr39TK cells to GCV was determined by 3-(4,5-dimethylthiahiazo-(-z-y1-3,5-di-phenytetrazoliumromide (MTT colorimetric assay and Hoechst-propidium iodide (PI staining. Finally, a subcutaneously C6 xenograft tumor model was established in the nude mice to test the in vitro efficacy of TK/GCV gene therapy. Our results showed that, as compared with wildtype TK, HSV1-sr39TK/GCV demonstrated a stronger therapeutic efficacy against C6 glioma both in vitro and in vivo, which, by reducing the required GCV dose, might warrant its future use in the treatment of glioma under clinical setting.

  10. Micro-PET/CT Monitoring of Herpes Thymidine Kinase Suicide Gene Therapy in a Prostate Cancer Xenograft: The Advantage of a Cell-specific Transcriptional Targeting Approach

    Directory of Open Access Journals (Sweden)

    Mai Johnson

    2005-10-01

    Full Text Available Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase and the cytotoxic gene (herpes simplex thymidine kinase were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen. Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols.

  11. THE THYMIDINE KINASE-1(TK-1 AS A POTENTIAL TUMOR MARKER: SERUM LEVELS IN SERUM OF PATIENTS WITH SOLID AND SYSTEM MALIGNANCE NEOPLASMS

    Directory of Open Access Journals (Sweden)

    N. S. Sergeeva

    2017-01-01

    Full Text Available The review summarizes the results of studies of levels and/or activity in the blood serum of a metabolic marker thymidine kinase-1 (TK-1 of proliferating cells in patients with lymphoproliferative diseases (LPD and malignant neopasms (NM.Comparison of the data in the literature in some cases have been difficult due to the fundamentally different methods of detection the activity or concentration of TK-1, used by authors, even despite the presence of relatively high (but not absolute correlation between these parameters (maximum 0.8.Many clinical and laboratory studies have shown levels of correlation and/or TK-1 activity with clinical stages and different types of LPD and solid MN and can serve as a prognostic factor for overall and recurrence-free survival of patients. When solid MN shown that the activity of TK-1 accurately reflects the proliferative status of tumor.A comparison of the dynamics of TC-1 in the process of chemotherapy and its clinical efficacy, different authors have received fundamentally different results: in some cases the marker reduction was associated with treatment efficacy, and in part of publications they show that the clinically relevant effects of the treatment observed increase in the marker after the first chemotherapy.The entire set of received data demonstrates the relevance of the further development of the algorithm use of TK-1 in oncology practice.

  12. Humanized thymidine kinase-NOG mice can be used to identify drugs that cause animal-specific hepatotoxicity: a case study with furosemide.

    Science.gov (United States)

    Xu, Dan; Michie, Sara A; Zheng, Ming; Takeda, Saori; Wu, Manhong; Peltz, Gary

    2015-07-01

    Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)-NOG mice with humanized livers could prevent this unfortunate outcome (i.e., "rescue" a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Estrogenic Activity of Some Phytoestrogens on Bovine Oxytocin and Thymidine Kinase-ERE Promoter through Estrogen Receptor-α in MDA-MB 231 Cells

    Directory of Open Access Journals (Sweden)

    Ehsan Zayerzadeh

    2014-08-01

    Full Text Available Background: Phytoestrogens, a group of plant-derived polyphenolic compounds have recently come into considerable attention due to the increasing information on their potential adverse effects in human health. Some of phytoestrogens show estrogenic activity that may be carcinogenic for human. In the present study, we investigated the transcriptional effects of variety of phytoestrogens on the bovine oxytocin and the thymidine kinase-ERE promoter by estrogen receptor α in MDA-MB 231 breast cancer cell line. Materials and Methods: Cells were seeded for transfections into 12- well plates at a density of 100000 cells per well were transfected with a total of 3 μg of plasmid DNA using calcium phosphate coprecipitation. Estrogen and some phytoestrogens (naringenin, 8-prenyl-naringenin and 6-( 1, 1 - dimethylallyl naringenin were used for the stimulation of transfected cells. Results: Findings of our study clearly demonstrated the subtype-selective activation of estrogen receptor (ERα and (ERβ by the p hytoestrogen naringenin (activating estrogen receptor β and its substituted forms 8-prenyl-naringenin and 6-( 1, 1 - dimethylallyl naringenin (activating estrogen receptor α , on the ERE-controlled promoter as well as on the oxytocin gene promoter. Conclusion: The study revealed that some p hytoestrogen s show estrogenic activity by classical or non-classical mechanisms as well as exhibit estrogenic activity by undetermined mechanisms in transfected MDA-MB 231 cell line.

  14. Correlations of {sup 18}F-fluorothymidine uptake with pathological tumour size, Ki-67 and thymidine kinase 1 expressions in primary and metastatic lymph node colorectal cancer foci

    Energy Technology Data Exchange (ETDEWEB)

    Nakajo, Masatoyo; Nakajo, Masayuki [Kagoshima University, Department of Radiology, Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Nanpuh Hospital, Department of Radiology, Kagoshima (Japan); Kajiya, Yoriko; Tani, Atsushi [Nanpuh Hospital, Department of Radiology, Kagoshima (Japan); Goto, Yuko; Higashi, Michiyo [Kagoshima University, Department of Human Pathology, Graduate School of Medical and Dental Sciences, Kagoshima, 890-8544 (Japan); Jinguji, Megumi; Fukukura, Yoshihiko [Kagoshima University, Department of Radiology, Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Tanaka, Sadao [Nanpuh Hospital, Department of Pathology, Kagoshima (Japan)

    2014-12-15

    To examine correlations of {sup 18}F-fluorothymidine (FLT) uptake with pathological tumour size and immunohistochemical Ki-67, and thymidine kinase 1 (TK-1) expressions in primary and metastatic node colorectal cancer foci. Thirty primary cancers (PCs) and 37 metastatic nodes (MNs) were included. FLT uptake was assessed by visual scores (non-visible: 0-1 and visible: 2-4), standardized uptake value (SUV), and correlated with size, Ki-67, and TK-1. SUV was measured in visible lesions. FLT heterogeneity was assessed by visual scores (no heterogeneous uptake: 0 and heterogeneous uptake: 1-4). Forty-two lesions were visible. The visible group showed significantly higher values than the non-visible group in size, Ki-67, and TK-1 (each p < 0.05). Size correlated significantly with visual score (PC; ρ = 0.74 and MN; ρ = 0.63), SUVmax (PC; ρ = 0.49, and MN; ρ = 0.76), and SUVmean (PC; ρ = 0.40 and MN; ρ = 0.76) (each p < 0.05). Visual score correlated significantly with size (ρ = 0.86), Ki-67max (ρ = 0.35), Ki-67mean (ρ = 0.38), TK-1max (ρ = 0.35) and TK-1mean (ρ = 0.25) (each p < 0.05). No significant correlations were found between FLT uptake and Ki-67 or TK-1 in 42 visible lesions (each p > 0.05). Heterogeneous FLT uptake was noted in 73 % (22/30) of PCs. FLT uptake correlated with size. Heterogeneous FLT distribution in colorectal cancers may be one of the causes of weak or lack of FLT uptake/Ki-67 or TK-1 correlation. (orig.)

  15. Genetic polymorphism of thymidine kinase (TK) and DNA polymerase (pol) of clinical varicella-zoster virus (VZV) isolates collected over three decades.

    Science.gov (United States)

    Hoffmann, Anja; Döring, Kristin; Seeger, Natalja Tatjana; Bühler, Martina; Schacke, Michael; Krumbholz, Andi; Sauerbrei, Andreas

    2017-10-01

    Genotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. To examine the genotype of the thymidine kinase (TK) and DNA polymerase (pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. The TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. In the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. Non-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Imaging in vivo herpes simplex virus thymidine kinase gene transfer to tumour-bearing rodents using positron emission tomography and [{sup 18}F]FHPG

    Energy Technology Data Exchange (ETDEWEB)

    Hustinx, R.; Shiue, C.Y.; Alavi, A.; Shiue, G.G.; Zhuang, H.; Karp, J.S. [Hospital of the Univ. of Pennsylvania (HUP), Philadelphia (United States). Dept. of Radiology; McDonald, D. [Pennsylvania Univ., Philadelphia, PA (United States). Dept. of Chemistry; Lanuti, M.; Lambright, E. [Dept. of Surgery, University of Pennsylvania, Philadelphia (United States); Eck, S.L. [Dept. of Medicine, University of Pennsylvania, Philadelphia (United States)

    2001-01-01

    Radiolabelled ganciclovir analogues have shown promise as imaging agents to detect herpes simplex virus thymidine kinase (HSVtk) expression. This study evaluated the use of positron emission tomography (PET) imaging with 9-[(3-[{sup 18}F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([{sup 18}F]FHPG) to assess gene transfer into tumours. HSVtk-positive and HSVtk-negative cell lines were first treated in vitro with [{sup 18}F]FHPG. To assess the efficacy of PET in detecting HSVtk expression following in vivo gene transfer, mice were injected intravenously with an adenovirus encoding HSVtk (Ad.HSVtk), a control vector (Ad.Bgl2) or saline. Subcutaneous human glioma xenografts were grown in mice and treated by direct injection of Ad.HSVtk or Ad.Bgl2. Imaging was performed 48 h after transduction. Similar experiments were performed using Fischer rats implanted with syngeneic tumours. The presence of the HSVtk protein was confirmed by immunohistochemistry. Biodistribution studies were also obtained in 14 naive mice. In vitro studies showed high and specific uptake of [{sup 18}F]FHPG in HSVtk-positive cell lines, with an uptake ratio of up to 27:1. PET imaging and direct counting of major organs demonstrated HSVtk-specific tracer retention. In mice, HSVtk-positive tumours retained 3.4% dose/gram as compared to 0.6% for control tumours (P=0.03). They were clearly seen on the PET images as early as 100 min post injection. Similar results were obtained with syngeneic rat tumours. Biodistribution studies demonstrated the rapid distribution and clearance of the tracer in all major organs. Our results demonstrate that PET imaging of HSVtk gene transfer to tumours is feasible and is highly specific for HSVtk expression. (orig.)

  17. The roles of macrophages and nitric oxide in interleukin-3-enhanced HSV-Sr39tk-mediated prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Ching-Fang Yu

    Full Text Available The herpes simplex virus thymidine kinase/ganciclovir (HSV-sr39tk/GCV system is a well-established prodrug system used in cancer gene therapy. However, this system is currently not effective enough to eradicate malignant tumors completely. This study aimed to evaluate whether co-expression of interleukin-3 (IL-3 could enhance the anti-tumor activity of HSV-sr39tk/GCV prodrug gene therapy using a murine TRAMP-C1 prostate tumor model. In vitro results demonstrated that HSV-sr39tk-transfected cells exhibited enhanced sensitivity to the GCV prodrug, which was not affected by co-expression of the mIL-3 gene. However, in vivo studies showed that co-expression of the mIL-3 gene significantly increased the HSV-sr39tk/GCV-induced tumor growth delay and even cured the tumor. The TRAMP-C1-specific immune response of spleen lymphocytes from mice bearing HSV-sr39tk- and IL-3-expressing TRAMP-C1 tumors was measured by ELISA. Results showed that IL-3-activated IL-4-dominant lymphocytes became IFN-γ- dominant lymphocytes after combined HSV-sr39tk/GCV therapy. The efficacy of combined therapies on tumor regression was reduced when macrophages populations were depleted by carrageenan or NO production was inhibited by administration of the iNOS inhibitor, L-NAME. These results suggest that utilizing a bicistronic vector to express HSV-sr39tk and the IL-3 gene induced an enhanced macrophage- or NO-dependent anti-tumor effect.

  18. Suicide gene therapy for hepatocellular carcinoma cells by survivin promoter-driven expression of the herpes simplex virus thymidine kinase gene.

    Science.gov (United States)

    Qu, Lili; Wang, Yanyun; Gong, Lailing; Zhu, Jin; Gong, Rujun; Si, Jin

    2013-04-01

    The aim of this study was to investigate the selective killing effect of the herpes simplex virus-thymidine kinase/ganciclovir (TK/GCV) suicide gene system controlled by the survivin promoter on hepatocellular carcinoma (HCC) cells in vitro. Recombinant plasmid vectors driven by the survivin promoter were constructed. HepG2 HCC and LO2 normal human liver cells were transfected with the recombinant plasmids, green fluorescent protein (GFP)/pSURV, TK/pSURV and TAT-TK/pSURV. GFP expression was detected by fluoroscopy and flow cytometry (FCM). TK gene expression was detected using RT-PCR and western blot analysis. The selective killing effects after GCV application were evaluated by tetrazolium assay, FCM and western blot analysis. Statistical analysis was performed by ANOVA. After transfection with GFP/pSURV, TK/pSURV and TAT-TK/pSURV for 48 h, GFP expression was observed in the HepG2 cells, but not in the L02 cells and TK gene expression was evidently detected by RT-PCR and western blot analysis in the HepG2 cells. Three stably transfected cell lines (HepG2/pSURV, HepG2/TK/pSURV and HepG2/TAT-TK/pSURV) were successfully established. Compared with the HepG2/TK/pSURV group, a significant 'bystander effect' was observed in the HepG2/TAT-TK/pSURV group with the incorporation of unmodifed HepG2 cells at different ratios. Following transfection with TK/pSURV and TAT-TK/pSURV, the growth of HepG2 cells in the presence of GCV was markedly inhibited. This finding was further corroborated by FCM and immunoblot analysis revealed the repressed expression of proliferating cell nuclear antigen (PCNA). Our results showed that the plasmid vectors carrying the TK and TAT-TK fusion protein gene driven by the survivin promoter were successfully constructed and their specific expression in HepG2 cells provided the basis for the targeted gene therapy of HCC.

  19. Bifidobacterial recombinant thymidine kinase-ganciclovir gene therapy system induces FasL and TNFR2 mediated antitumor apoptosis in solid tumors.

    Science.gov (United States)

    Wang, Changdong; Ma, Yongping; Hu, Qiongwen; Xie, Tingting; Wu, Jiayan; Zeng, Fan; Song, Fangzhou

    2016-07-27

    Directly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms. Bifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression. The results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy. BF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control

  20. Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase - Kinetic properties and oligomerization pattern of nine substitution mutants of V106

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Berenstein, Dvora; Munch-Petersen, Birgitte

    2004-01-01

    and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible...

  1. Dual monitoring using {sup 124}I-FIAU and bioluminescence for HSV1-tk suicide gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, T. S.; Kim, J. H.; Kwon, H. C. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)] (and others)

    2007-07-01

    Herpes simplex virus type I thymidine kinase (HSV-tk) is the most common reporter gene and is used in cancer gene therapy with a prodrug nucleoside analog, ganciclovir (GCV). The aim of this study is to evaluate therapeutic efficacy of suicide gene therapy with 2'-fluoro-2'-deoxy-1-D-arabinofuranosyl-5-[{sup 124}I] iodouracil ({sup 124}I - FIAU) and bioluminescence in retrovirally HSV -tk and firefly luciferase transduced hepatoma model. The HSV -tk and firefly luciferase (Luc) was retrovirally transduced and expressed in MCA rat Morris hepatoma cells. Nude mice with subcutaneous tumors, MCA and MCA-TK-Luc, were subjected to GCV treatment (50mg/Kg/d intraperitoneally) for 5 day. PET imaging and biodistribution with ({sup 124}I-FIAU) were performed at before and after initiation of therapy with GCV. Bioluminescent signal was also measured during GCV treatment. Before GCV treatment, no significant difference in tumor volume was found in tumors between MCA and MCA-TK-Luc. After GCV treatment, tumor volume of MCA-TK-Luc markedly reduced compared to that of MCA. In biodistribution study, {sup 124}I-FIAU uptake after GCV therapy significantly decreased compared with pretreatment levels (34.8 13.67 %ID/g vs 7.6 2.59 %ID/g) and bioluminescent signal was also significantly decreased compared with pretreatment levels. In small animal PET imaging, {sup 124}I-FIAU selectively localized in HSV -tk expressing tumor and the therapeutic efficacy of GCV treatment was evaluated by {sup 124}I-FIAU PET imaging. {sup 124}I-FIAU PET and bioluminescence imaging in HSV-tk suicide gene therapy were effective to evaluate the therapeutic response. {sup 124}I-FIAU may serve as an efficient and selective agent for monitoring of transduced HSV1-tk gene expression in vivo in clinical trials.

  2. In vivo image of radioiodinated IVDU and IVFRU in HSV-TK gene tranduced hepatocellular carcinoma bearing buffalo rat

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Sup; Choi, T. H.; Ahn, S. H.; Woo, K. S.; Chung, W. S.; Lee, S. J.; Choi, C. W. [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    2000-07-01

    The extent of gene delivery and expression in gene therapy with suicide genes such as herpes simplex virus thymidine kinase (HSV-tk) is assessed with measurement of selective localization of radioiodinated HSV-tk substrates in HSV-tk expressing tumor. We compared n vitro uptake of {sup 125}I-IVDU, IVFRU and in vivo image of HSV-tk gene tranduced hepatocellular carcinoma model. Using H{sub 2}O{sub 2}(hydrogen peroxide), IVDU and IVFRU was radiolabeled as carrier free form. The uptake of {sup 125}I-IVDU IVFRU was determined with increasing incubation periods in MCA-tk and MCA cell line (1X10{sup 6}cell/flask). The cell harvested and counted after incubation of 15, 30, 60, 120, 240, 480 minutes. For estimating accumulation of radiolabelled IVDU, IVFRU in HSV-tk expressing tumor, MCA-tk cells (1 X 10{sup 6}/100 {mu}l) injected intramuscularly into right thigh of buffalo rats. To determine selective localization of radiolabelled IVDU, IVFRU in HSV-tk expressing hepatocellular carcinoma bearing buffalo rats, MCA-tk cells (1X 10{sup 7} cell/100 {mu}l) were injected subcutaneously into both shoulders of buffalo rats. Established tumor mass implanted into liver of buffalo rats using intra-hepatic tumor injection. Two weeks later, {sup 123}I labelled IVDU, IVFRU(7.4 X 10{sup 7}Bq/200 {mu}l) injected intravenously into tail veins of each buffalo rats. Gamma camera used as revealing localization of {sup 123}I-IVDU, IVFRU in MCA-tk cells grafts rats and in vivo image was taken 2 hrs, 24 hrs after injection. radioiodinated IVDU, IVFRU were radiolabeled with {sup 123}I as labeling yield 70%, {sup 125}I as 84%. Two compounds showed minimal uptake in MCA cell line, but in MCA-tk cell line, increased uptake was observed. The ratio of MCA-tk to MCA was up to 116-fold in {sup 125}I-IVDU, up to 37-fold in {sup 125}I-IVFRU at 480 min. The uptake of IVDU was 4 times higher than IVFRU in MCA-tk cells. Gamma camera images of HSV-tk gene tranduced MCA tumor showed accumulation of {sup 123}I

  3. Serum thymidine kinase 1 activity as a pharmacodynamic marker of cyclin-dependent kinase 4/6 inhibition in patients with early-stage breast cancer receiving neoadjuvant palbociclib.

    Science.gov (United States)

    Bagegni, Nusayba; Thomas, Shana; Liu, Ning; Luo, Jingqin; Hoog, Jeremy; Northfelt, Donald W; Goetz, Matthew P; Forero, Andres; Bergqvist, Mattias; Karen, Jakob; Neumüller, Magnus; Suh, Edward M; Guo, Zhanfang; Vij, Kiran; Sanati, Souzan; Ellis, Matthew; Ma, Cynthia X

    2017-11-21

    Thymidine kinase 1 (TK1) is a cell cycle-regulated enzyme with peak expression in the S phase during DNA synthesis, and it is an attractive biomarker of cell proliferation. Serum TK1 activity has demonstrated prognostic value in patients with early-stage breast cancer. Because cyclin-dependent kinase 4/6 (CDK4/6) inhibitors prevent G1/S transition, we hypothesized that serum TK1 could be a biomarker for CDK4/6 inhibitors. We examined the drug-induced change in serum TK1 as well as its correlation with change in tumor Ki-67 levels in patients enrolled in the NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774). Patients with clinical stage II/III estrogen receptor-positive (ER+)/HER2-negative breast cancer enrolled in the NeoPalAna trial received an initial 4 weeks of anastrozole, followed by palbociclib on cycle 1, day 1 (C1D1) for four 28-day cycles, unless C1D15 tumor Ki-67 was > 10%, in which case patients went off study owing to inadequate response. Surgery occurred following 3-5 weeks of washout from the last dose of palbociclib, except in eight patients who received palbociclib (cycle 5) continuously until surgery. Serum TK1 activity was determined at baseline, C1D1, C1D15, and time of surgery, and we found that it was correlated with tumor Ki-67 and TK1 messenger RNA (mRNA) levels. Despite a significant drop in tumor Ki-67 with anastrozole monotherapy, there was no statistically significant change in TK1 activity. However, a striking reduction in TK1 activity was observed 2 weeks after initiation of palbociclib (C1D15), which then rose significantly with palbociclib washout. At C1D15, TK1 activity was below the detection limit (palbociclib. There was high concordance, at 89.8% (95% CI: 79.2% - 96.2%), between changes in serum TK1 and tumor Ki-67 in the same direction from C1D1 to C1D15 and from C1D15 to surgery time points. The sensitivity and specificity for the tumor Ki-67-based response by palbociclib-induced decrease in serum TK1 were 94

  4. Synthesis of 2'-deoxy-2'-[{sup 18}F]-fluoro-5-ethyl-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]-FEAU) and micro-PET imaging of HSV-tk gene expression in tumor-bearing nude mice

    Energy Technology Data Exchange (ETDEWEB)

    Alauddin, M.M.; Shahinian, A.; Park, R.; Tohme, M.; Fissekis, J.D.; Conti, P.S. [Univ. of Southern California, Los Angeles, CA (United States). PET Imaging Science Center

    2004-07-01

    Herpes simplex virus type-1 thymidine kinase (HSV1-tk) is being used as a suicide gene for gene therapy of cancer. An in vivo method to assess the HSV1-tk enzyme activity after gene transfer is desirable to monitor gene expression as an indicator of gene delivery. Imaging of the HSV1-tk reporter gene along with various reporter probes is of current interest. We originally developed [{sup 18}F]-FHPG and [{sup 18}F]-FHBG for PET imaging of HSV1-tk gene expression and demonstrated that [{sup 18}F]-FHBG is more useful than [{sup 18}F]-FHPG for this purpose. [{sup 124}I]-FIAU has been shown to be a potential PET imaging agent for HSV1-tk gene expression, and is superior to [{sup 18}F]-FHPG and [{sup 18}F]-FHBG. We also demonstrated that radiolabeled FMAU can be used as a marker for HSV-tk gene expression, and is superior to [{sup 18}F]-FHPG and [{sup 18}F]-FHBG. Earlier we reported a synthesis for 2'-deoxy-2'-[{sup 18}F]fluoro-5-methyl-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]-FMAU) and some other 5-substituted nucleosides. We have synthesized now [{sup 18}F]-FEAU, used the tracer for micro-PET imaging of suicide gene expression in tumor-bearing nude mice, and compared the results with earlier studies using [{sup 14}C]-FMAU. (orig.)

  5. Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae

    Science.gov (United States)

    Vernis, Laurence; Piskur, Jure; Diffley, John F. X.

    2003-01-01

    The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse–chase labelling. PMID:14500848

  6. Rapid and liquid-based selection of genetic switches using nucleoside kinase fused with aminoglycoside phosphotransferase.

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    Masahiro Tominaga

    Full Text Available The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON- and negative (OFF- selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3'-phosphotransferase (APH, we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.

  7. Ovarian cancer gene therapy using HPV-16 pseudovirion carrying the HSV-tk gene.

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    Chien-Fu Hung

    Full Text Available Ovarian cancer is the leading cause of death from all gynecological cancers and conventional therapies such as surgery, chemotherapy, and radiotherapy usually fail to control advanced stages of the disease. Thus, there is an urgent need for alternative and innovative therapeutic options. We reason that cancer gene therapy using a vector capable of specifically delivering an enzyme-encoding gene to ovarian cancer cells will allow the cancer cell to metabolize a harmless prodrug into a potent cytotoxin, which will lead to therapeutic effects. In the current study, we explore the use of a human papillomavirus (HPV pseudovirion to deliver a herpes simplex virus thymidine kinase (HSV-tk gene to ovarian tumor cells. We found that the HPV-16 pseudovirion was able to preferentially infect murine and human ovarian tumor cells when administered intraperitoneally. Furthermore, intraperitoneal injection of HPV-16 pseudovirions carrying the HSV-tk gene followed by treatment with ganciclovir led to significant therapeutic anti-tumor effects in murine ovarian cancer-bearing mice. Our data suggest that HPV pseudovirion may serve as a potential delivery vehicle for ovarian cancer gene therapy.

  8. Real-time visualizing and tracing of HSV-TK/GCV suicide gene therapy by near-infrared fluorescent quantum dots.

    Science.gov (United States)

    Shao, Dan; Li, Jing; Xiao, Xuanang; Zhang, Ming; Pan, Yue; Li, Shuo; Wang, Zheng; Zhang, Xin; Zheng, Huilin; Zhang, Xuewen; Chen, Li

    2014-07-23

    Exploring intracellular behavior of suicide gene is significant for improving the efficacy and safety of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-TK/GCV) system in cancer therapy. Molecular imaging represents a powerful tool to understand gene transportation and function dynamics. In this work, we reported a quantum-dot-based technique for revealing the procedure of HSV-TK/GCV suicide gene therapy by constructing covalent linkage between near-infrared fluorescent quantum dots (QDs) and TK gene. This stable QD labeling did not influence either the QDs fluorescence or the biological activity of TK gene. Furthermore, we visualized and dynamically traced the intracellular behavior antitumor effect of TK gene in vitro and in vivo. It is demonstrated that TK gene was shuttled to the nucleus after a-24 h treatment; at that time the single dose of GCV administration exerts the gradually increasing lethal effect until to 72 h. Real-time tracing the formation of hepatocellular carcinoma treated with HSV-TK/GCV suicide gene system in vivo by QD-based NIR fluorescence imaging provides useful insight toward QD-based theranostics in future cancer therapy.

  9. Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI.

    Science.gov (United States)

    Bar-Shir, Amnon; Liu, Guanshu; Greenberg, Marc M; Bulte, Jeff W M; Gilad, Assaf A

    2013-12-01

    In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.

  10. Different strategies for reducing intestinal background radioactivity associated with imaging HSV1-tk expression using established radionucleoside probes

    Science.gov (United States)

    Ruggiero, Alessandro; Brader, Peter; Serganova, Inna; Zanzonico, Pat; Cai, Shangde; Lipman, Neil S.; Hricak, Hedvig; Blasberg, Ronald G.

    2011-01-01

    One limitation of HSV1-tk reporter PET imaging with nucleoside analogues is the high background radioactivity in the intestine. We hypothesized that endogenous expression of thymidine kinase in bacterial flora could phosphorylate and trap such radiotracers, contributing to the high radioactivity levels in the bowel and therefore explored different strategies to increase fecal elimination of radiotracer. Methods Intestinal radioactivity was assessed by in vivo microPET imaging and ex vivo tissue sampling following intravenous injection of 18F-FEAU, 124I-FIAU or 18F-FHBG in a germ-free mouse strain. We also explored the use of an osmotic laxative agent and/or a 100% enzymatically hydrolyzed liquid diet. Results No significant differences in intestinal radioactivity were observed between germ-free and normal mice. 18F-FHBG-derived intestinal radioactivity levels were higher than those of 18F-FEAU and 124I-FIAU; the intestine-to-blood ratio was more than 20-fold higher for 18F-FHBG than for 18F-FEAU and 124I-FIAU. The combination of Peptamen and Nulytely lowered intestinal radioactivity levels and increased (2.2-fold) the HSV1-tk transduced xenograft-to-intestine ratio for 18F-FEAU. Conclusions Intestinal bacteria in germ-free mice do not contribute to the high intestinal levels of radioactivity following injection of radionucleoside analogs. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration. PMID:20128998

  11. Herpes Simplex Virus 1 (HSV-1) and HSV-2 Mediate Species-Specific Modulations of Programmed Necrosis through the Viral Ribonucleotide Reductase Large Subunit R1.

    Science.gov (United States)

    Yu, Xiaoliang; Li, Yun; Chen, Qin; Su, Chenhe; Zhang, Zili; Yang, Chengkui; Hu, Zhilin; Hou, Jue; Zhou, Jinying; Gong, Ling; Jiang, Xuejun; Zheng, Chunfu; He, Sudan

    2015-11-11

    Receptor-interacting protein kinase 3 (RIP3) and its substrate mixed-lineage kinase domain-like protein (MLKL) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defense mechanism. Here, we report that human herpes simplex virus 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus their impacts in mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of an RIP1/RIP3 necrosome. The HSV ribonucleotide reductase large subunit R1 was sufficient to suppress TNF-induced necrosis, and its RIP homotypic interaction motif (RHIM) domain was required to disrupt the RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defense mechanism of programmed necrosis in human cells. This study demonstrated that infection with HSV-1 and HSV-2 blocked TNF-induced necrosis in human cells while these viruses directly activated programmed necrosis in mouse cells. Expression of HSV R1 suppressed TNF-induced necrosis of human cells. The RHIM domain of R1 was essential for its association with human RIP3 and RIP1, leading to disruption of the RIP1/RIP3 complex. This study provides new insights into the species-specific modulation of programmed necrosis by HSV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. STAT3 silencing enhances the efficacy of the HSV.tk suicide gene in gastrointestinal cancer therapy.

    Science.gov (United States)

    Ahn, Ye-Hyeon; Yi, Hwajung; Shin, Ji-Young; Lee, Kang-Duck; Shin, Seung-Pil; Lee, Sang-Jin; Song, Jaewhan; Chun, Kyung-Hee

    2012-04-01

    Aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling has been shown to be associated with uncontrolled cell proliferation and suppression of host-immune surveillance. Conversely, silencing STAT3 can have the dual effects of inhibiting cancer cell proliferation and inducing anti-tumor immune responses. Here, we report on the effects of STAT3 silencing on suicide gene therapy with thymidine kinase (tk). STAT3 silencing by siRNA inhibited the proliferation of AGS human gastric cancer cells through G1 cell cycle arrest, decreased levels of immune-suppressive cytokines, and increased levels of immune-activating cytokines. CT26 mouse colon adenocarcinoma cells, in which STAT3 expression was knocked-down by a STAT3 shRNA-containing lentivirus, grew more slowly in syngenic model Balb/c mice than control CT26 cells. Moreover, we found that STAT3 silencing augmented the efficacy of suicide gene therapy in CT26 cell xenografted mice. When we administrated adenoviruses harboring the herpes simplex virus thymidine kinase gene (Ad5.CMV.HSV.tk) into STAT3-silenced CT26 cell tumors, extensive apoptosis was observed and there was a significant reduction in the size of CT26 cell tumors. STAT3 silencing also enhanced the recruitment and cytotoxic activity of CD3(+)CD8(+) T-cells, and changed the cytokine expression pattern of CT26 cell tumors, reflecting augmentation of anti-cancer immune responses. We conclude that combining suicide gene therapy with STAT3 silencing can result in enhanced anti-cancer effects.

  13. Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic “Emergency Exit” Switch

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    Maciej Sułkowski

    2018-01-01

    Full Text Available Since their invention in 2006, induced Pluripotent Stem (iPS cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification—exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK. Its expression results in specific vulnerability of genetically modified cells to prodrug—ganciclovir (GCV. We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating “emergency exit” switch allowing eradication of transplanted cells in case of their malfunction.

  14. Serum-resistant gene transfer to oral cancer cells by Metafectene and GeneJammer: application to HSV-tk/ganciclovir-mediated cytotoxicity.

    Science.gov (United States)

    Konopka, Krystyna; Fallah, Basma; Monzon-Duller, JoMarie; Overlid, Nathan; Düzgünes, Nejat

    2005-01-01

    Cationic lipids and polyamines have been used as non-viral gene transfer reagents, both in vitro and in vivo. One of the limitations to their use in vivo is the inhibition of gene delivery by serum. We showed previously that, in the absence of serum, relatively high cytotoxicity in oral cancer cell lines could be achieved via transfection of the Herpes Simplex Virus thymidine kinase (HSV-tk) gene followed by treatment with ganciclovir (GCV), despite the low efficiency of transfection (Konopka et al., Gene Ther. Mol. Biol. 8 (2004) 307-318). In this study we evaluated the effect of high concentrations (20-60%) of fetal bovine serum (FBS) on the transfection efficiency of two novel reagents, the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in HSC-3 and H357 human oral squamous cell carcinoma (OSCC) cells. We also examined whether the HSV-tk gene delivered in the presence of FBS (up to 60%, could induce cell death following treatment with GCV. Transfection was optimized using a luciferase-expressing plasmid. Both Metafectene- and GeneJammer-mediated luciferase gene expression in HSC-3 cells was reduced by 40-50% when transfection was performed in the presence of 20-60% FBS. The delivery of the HSV-tk gene by Metafectene in the absence and the presence of 60% FBS, followed by GCV treatment for 9 days, resulted in 95% and 70% cytotoxicity, respectively. With GeneJammer, transfection in 0% and 60% FBS resulted in 90% and 40% cytotoxicity, respectively, after 9 days. In contrast, very low transfection activity and a much higher inhibitory effect of serum were observed in H357 cells. Nevertheless, about 35% GCV-mediated cytotoxicity was observed with H357 cells at both 0% and 60% FBS, using GeneJamer. Thus, Metafectene and GeneJammer can be used in the delivery of genes in biological milieu and in the gene therapy of OSCC in animal models.

  15. HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

    Science.gov (United States)

    Sufiawati, Irna; Tugizov, Sharof M

    2014-01-01

    Herpes simplex virus (HSV) types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD). Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.

  16. HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

    Directory of Open Access Journals (Sweden)

    Irna Sufiawati

    Full Text Available Herpes simplex virus (HSV types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD. Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.

  17. HSV-tk/GCV gene therapy mediated by EBV-LMP1 for EBV-associated cancer

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    Midan Ai

    2008-09-01

    Full Text Available Abstract Background To investigate the feasibility of gene therapy in treating Epstein-Barr virus (EBV-associated cancer by employing the suicide gene, herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV, which uses the signaling pathway through the HIV-long terminal repeat (LTR gene which is expressed from a nuclear factor-κB (NF-κB-binding motif-containing promoter that is regulated by EBV-latent membrane protein 1 (LMP1 via NF-κB. Methods First, we constructed the plasmid pVLTR-tk, which was regulated by EBV-LMP1 via NF-κB, and then investigated the cytotoxic effect of the pVLTR-tk/GCV on cancer cells, using MTT assays, clonogenic assays, flow cytometry, and animal experiments. Results The activation of TK was increased after transfection of the pVLTR-tk into the EBV-LMP1 positive cells. After GCV treatment, the clonogenicity and survival of the cells substantially declined, and a bystander effect was also observed. The LMP1 positive cells exhibited remarkable apoptosis following pVLTR-tk/GCV treatment, and the pVLTR-tk/GCV restrained tumor growth in vivo for EBV-LMP1 positive cancers. Conclusion The pVLTR-tk/GCV suicide gene system may be used as a new gene targeting strategy for EBV-associated cancer.

  18. Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells

    NARCIS (Netherlands)

    Bijnsdorp, I. V.; Azijli, K.; Jansen, E. E.; Wamelink, M. M.; Jakobs, C.; Struys, E. A.; Fukushima, M.; Kruyt, F. A. E.; Peters, G. J.

    2010-01-01

    Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to

  19. Autoradiography study and SPECT imaging of reporter gene HSV1-tk expression in heart

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: LXL730724@hotmail.com; Liu Ying; He Yong; Wu Tao; Zhang Binqing; Gao Zairong; An Rui [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: zhyx1229@163.com

    2010-04-15

    Aim: To demonstrate the feasibility and optimal conditions of imaging herpes simplex virus 1-thymidine kinase (HSV1-tk) gene transferred into hearts with {sup 131}I-2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil ({sup 131}I-FIAU) using autoradiography (ARG) and single photon emission computed tomography (SPECT) in animal models. Methods: HSV1-tk inserted into adenovirus vector (Ad5-tk) and adenovirus (Ad5-null) was prepared. Rats or rabbits were divided into a study group receiving intramyocardial injection of Ad5-tk, and a control group receiving Ad-null injection. In the study group of rats, two sets of experiments, time-course study and dose-dependence study, were performed. In time-course experiments, rats were injected with {sup 131}I-FIAU on Days 1, 2, 3, 5 and 7, after transfection of 1x10{sup 8} pfu Ad5-tk, to study the feasibility and suitable time course for reporter gene imaging. In dose-dependence study, various titers of Ad5-tk (5x10{sup 8}, 1x10{sup 8}, 5x10{sup 7} and 1x10{sup 7} pfu) were used to determine the threshold and optimal viral titer needed for detection of gene expression. The gamma counts of hearts were measured. The rat myocardium was analyzed by ARG and reverse transcriptase-polymerase chain reaction (RT-PCR). SPECT whole-body planar imaging and cardiac tomographic imaging were performed in the rabbit models. Results: From the ARG images, rats injected with Ad5-tk showed significant {sup 131}I-FIAU activity in the anterolateral wall compared with background signals seen in the control Ad5-null rats. In time-course study, the highest radioactivity in the focal myocardium could be seen on Day 1, and then progressively declined with time. In dose-dependence study, the level of {sup 131}I-FIAU accumulation in the transfected myocardium declined with the decrease of Ad viral titers. From the ARG analysis and gamma counting, the threshold viral titer was 5x10{sup 7} pfu, and the optimal Ad titer was 1x10{sup 8} pfu

  20. Cellular influx, efflux, and anabolism of 3-carboranyl thymidine analogs: potential boron delivery agents for neutron capture therapy.

    Science.gov (United States)

    Sjuvarsson, Elena; Damaraju, Vijaya L; Mowles, Delores; Sawyer, Michael B; Tiwari, Rohit; Agarwal, Hitesh K; Khalil, Ahmed; Hasabelnaby, Sherifa; Goudah, Ayman; Nakkula, Robin J; Barth, Rolf F; Cass, Carol E; Eriksson, Staffan; Tjarks, Werner

    2013-11-01

    3-[5-{2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl]thymidine (N5-2OH) is a first generation 3-carboranyl thymidine analog (3CTA) that has been intensively studied as a boron-10 ((10)B) delivery agent for neutron capture therapy (NCT). N5-2OH is an excellent substrate of thymidine kinase 1 and its favorable biodistribution profile in rodents led to successful preclinical NCT of rats bearing intracerebral RG2 glioma. The present study explored cellular influx and efflux mechanisms of N5-2OH, as well as its intracellular anabolism beyond the monophosphate level. N5-2OH entered cultured human CCRF-CEM cells via passive diffusion, whereas the multidrug resistance-associated protein 4 appeared to be a major mediator of N5-2OH monophosphate efflux. N5-2OH was effectively monophosphorylated in cultured murine L929 [thymidine kinase 1 (TK1(+))] cells whereas formation of N5-2OH monophosphate was markedly lower in L929 (TK1(-)) cell variants. Further metabolism to the di- and triphosphate forms was not observed in any of the cell lines. Regardless of monophosphorylation, parental N5-2OH was the major intracellular component in both TK1(+) and TK1(-) cells. Phosphate transfer experiments with enzyme preparations showed that N5-2OH monophosphate, as well as the monophosphate of a second 3-carboranyl thymidine analog [3-[5-(o-carboran-1-yl)pentan-1-yl]thymidine (N5)], were not substrates of thymidine monophosphate kinase. Surprisingly, N5-diphosphate was phosphorylated by nucleoside diphosphate kinase although N5-triphosphate apparently was not a substrate of DNA polymerase. Our results provide valuable information on the cellular metabolism and pharmacokinetic profile of 3-carboranyl thymidine analogs.

  1. Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

    Science.gov (United States)

    Salabert, Anne-Sophie; Vaysse, Laurence; Beaurain, Marie; Alonso, Mathieu; Arribarat, Germain; Lotterie, Jean-Albert; Loubinoux, Isabelle; Tafani, Mathieu; Payoux, Pierre

    2017-01-01

    Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression. A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo. Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft. The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

  2. Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Salabert

    Full Text Available Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression.A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo.Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft.The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

  3. Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model

    Science.gov (United States)

    Beaurain, Marie; Alonso, Mathieu; Arribarat, Germain; Lotterie, Jean-Albert; Loubinoux, Isabelle; Tafani, Mathieu; Payoux, Pierre

    2017-01-01

    Introduction Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression. Methods A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo. Results Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft. Conclusion The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection. PMID:28926581

  4. Dexamethasone inhibits the HSV-tk/ ganciclovir bystander effect in malignant glioma cells

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    Jolois Olivier

    2005-04-01

    Full Text Available Abstract Background HSV-tk/ ganciclovir (GCV gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed. Methods Stable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and BclXL. Results Dexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC, from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade. Conclusion The overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment.

  5. Dexamethasone inhibits the HSV-tk/ ganciclovir bystander effect in malignant glioma cells

    Science.gov (United States)

    Robe, Pierre A; Nguyen-Khac, Minh; Jolois, Olivier; Rogister, Bernard; Merville, Marie-Paule; Bours, Vincent

    2005-01-01

    Background HSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed. Methods Stable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and BclXL. Results Dexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade. Conclusion The overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment. PMID:15804364

  6. Bystander killing effect of tymidine kinase gene-transduced adult bone marrow stromal cells with ganciclovir on malignant glioma cells.

    Science.gov (United States)

    Mori, Kentaro; Iwata, Junko; Miyazaki, Masahiro; Osada, Hideo; Tange, Yuichi; Yamamoto, Takuji; Aiko, Yasuhisa; Tamura, Masaru; Shiroishi, Toshihiko

    2010-01-01

    Transduction of the suicide gene of Herpes simplex virus thymidine kinase (Hsv-tk) into glioma cells or neural stem cells combined with pro-drug ganciclovir (GCV) treatment has been effective to treat experimental glioma in the rat through the bystander effect. Bone marrow stromal cells (MSCs) in the adult bone marrow have tropism for brain tumors and act as tumor stromal cells. Whether adult MSCs expressing Hsv-tk can also act as effector cells of the bystander killing effect on murine glioma cells was investigated. In vitro study of co-culture between 9L/LacZ (9L) glioma cells and Hsv-tk-transduced MSCs (MSCs/tk(+)) followed by GCV administration in the culture medium resulted in apparent nuclear morphological changes in the 9L glioma cells surrounding the MSCs/tk(+). 9L glioma cell survival in the presence of MSCs/tk(+) and GCV treatment was quantitatively measured and showed significant decrease of 9L glioma cell proliferation with higher MSCs/tk(+) ratio and GCV concentration. Intracerebral co-inoculation experiments in Fisher rats used 9L glioma cells and either MSCs/tk(+) or Hsv-tk-non-transduced MSCs (MSCs/tk(-)) followed by intraperitoneal injection of GCV (100 mg/kg, daily for 7 days). The animals co-inoculated with 9L glioma cells and MSCs/tk(+) showed significant retardation of tumor growth and prolongation of survival time compared with the animals with 9L glioma cells and MSCs/tk(-). Quantitative findings were established of the novel effects of adult MSCs/tk(+) as effector cells of the bystander killing effect on glioma cells.

  7. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease

    Directory of Open Access Journals (Sweden)

    Martine Aubert

    2014-01-01

    Full Text Available Following acute infection, herpes simplex virus (HSV establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs for cure of chronic viral infections.

  8. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.

    Science.gov (United States)

    Aubert, Martine; Boyle, Nicole M; Stone, Daniel; Stensland, Laurence; Huang, Meei-Li; Magaret, Amalia S; Galetto, Roman; Rawlings, David J; Scharenberg, Andrew M; Jerome, Keith R

    2014-02-04

    Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3'-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections.Molecular Therapy-Nucleic Acids (2014) 3, e1; doi:10.1038/mtna.2013.75; published online 4 February 2014.

  9. Comparative study of cellular kinetics of reporter probe [{sup 131}I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: lxl730724@hotmail.com; Yin Xiaohua; Wang Ruihua; Liu Ying [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China) and Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: zhyx1229@163.com

    2009-02-15

    Aim: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyluracil) was labeled with {sup 131}I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [{sup 131}I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry. Results: FAU could be labeled with {sup 131}I, and the labeling efficiency and radiochemical purity rates were 53.82{+-}2.05% and 94.85{+-}1.76%, respectively. Time-dependent increase of the accumulation of [{sup 131}I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55{+-}0.37% and 2.09{+-}0.34%, respectively. Greater uptakes of [{sup 131}I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene

  10. A multisubstrate deoxyribonucleoside kinase from plants.

    Science.gov (United States)

    Clausen, Anders Ranegaard; Girandon, Lenart; Knecht, Wolfgang; Survery, Sabeen; Andreasson, Erik; Munch-Petersen, Birgitte; Piskur, Jure

    2008-01-01

    Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene in anti-cancer therapy.

  11. Molecular moment similarity between several nucleoside analogs of thymidine and thymidine. sil@watson.ibm.com.

    Science.gov (United States)

    Silverman, B D; Pitman, M C; Platt, D E

    1999-06-01

    Molecular moment descriptors of the shape and charge distributions of twenty five nucleoside structures have been examined. The structures include thymidine as well as the difluorotoluene nucleoside analog which has been found to pair efficiently with adenine by polymerase catalysis. The remaining twenty three structures have been chosen to be as structurally similar to thymidine and to the difluorotoluene nucleoside analog as possible. The moment descriptors which include a description of the relationship of molecular charge to shape show the difluorotoluene nucleoside to be one of the most proximate molecules to thymidine in the space of the molecular moments. The calculations, therefore, suggest that polymerase specificity might be not only a consequence of molecular steric features alone but also of the molecular electrostatic environment and its registration with molecular shape.

  12. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin...

  13. Photoreaction of 8-methoxypsoralen with thymidine

    Energy Technology Data Exchange (ETDEWEB)

    Shim, S.C.; Kim, Y.Z. (Korea Advanced Inst. of Science and Technology, Seoul (Republic of Korea))

    1983-09-01

    The photoreaction of 8-methoxypsoralen (8-MOP) with thymidine in solid film state yielded two 4',5'-monoadducts (a pair of diastereomers) and three 3,4-monoadducts. The stereochemistry of two 4',5'monoadducts was found to be cis-syn and trans-syn and one 3,4-monoadduct was cis-anti. In addition to these monoadducts, 3,4-, 4',5-biadducts were also formed during the reaction, but the isolation of each isomer of these adducts was not successful; however, the formation of these biadducts was confirmed by UV, IR, TLC and photosplitting experiments.

  14. Engineering HSV-1 vectors for gene therapy.

    Science.gov (United States)

    Goins, William F; Huang, Shaohua; Cohen, Justus B; Glorioso, Joseph C

    2014-01-01

    Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed efficacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

  15. Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Vernis, L.; Piskur, Jure; Diffley, J.F.X.

    2003-01-01

    as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show...

  16. First HSV-1 non primary genital herpes in two patients.

    Science.gov (United States)

    Fouéré, Sébastien; Chaine, Bénédicte; Maylin, Sarah; Minier, Marine; Vallée, Pascale; Scieux, Catherine; Lassau, François; Legoff, Jérôme; Janier, Michel

    2016-05-01

    First HSV-1 genital episodes in HSV-2 infected patients however, had never been demonstrated until the 2 cases we observed. This scarcity could reflect the lower impact of HSV-2 on western populations but questions the existence of cross-protection between viral types. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Natural infection with herpes simplex virus type 1 (HSV-1) induces humoral and T cell responses to the HSV-1 glycoprotein H : L complex

    NARCIS (Netherlands)

    Westra, DF; Verjans, GMGM; Osterhaus, ADME; van Kooij, A; Welling, GW; Scheffer, AJ; The, TH; Welling-Wester, S

    The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)H-t(His):gL complex of HSV-1 were studied in seven HSV-1-seropositive and three HSV-1-seronegative healthy

  18. Synthesis and evaluation of a {sup 99m}Tc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Celen, Sofie [Laboratory for Radiopharmacy, K.U. Leuven, B-3000 Leuven (Belgium); Groot, Tjibbe de [Radiopharmacy, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Balzarini, Jan [Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven (Belgium); Vunckx, Kathleen [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Terwinghe, Christelle [Radiopharmacy, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Vermaelen, Peter [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Van Berckelaer, Lizette [Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven (Belgium); Vanbilloen, Hubert [Radiopharmacy, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Nuyts, Johan [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Mortelmans, Luc [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Verbruggen, Alfons [Laboratory for Radiopharmacy, K.U. Leuven, B-3000 Leuven (Belgium); Bormans, Guy [Laboratory for Radiopharmacy, K.U. Leuven, B-3000 Leuven (Belgium)]. E-mail: guy.bormans@pharm.kuleuven.be

    2007-04-15

    Introduction: Cytosolic thymidine kinase (TK1) catalyzes phosphorylation of thymidine to its monophosphate. TK1 activity is closely related with DNA synthesis, and thymidine analogs derivatized with bulky carboranylalkyl groups at the N-3 position were reported to be good substrates for TK1. Accordingly, we have synthesized {sup 99m}Tc-MAMA-propyl-thymidine and evaluated it as a potential tumor tracer. Methods: The bis(S-trityl)-protected MAMA-propyl-thymidine precursor (3-N-[S-trityl-2-mercaptoethyl]-N-[N'-(S-trityl-2-mercaptoethyl) amidoacetyl] -aminopropyl-thymidine) was prepared in three steps, and its structure was confirmed with {sup 1}H NMR and mass spectrometry. Deprotection of the thiols and labeling with {sup 99m}Tc were done in a two-step, one-pot procedure, yielding {sup 99m}Tc-MAMA-propyl-thymidine, which was analyzed with high-performance liquid chromatography, radio-LC-MS analysis (ESI+) and electrophoresis, and its log P was determined. The biodistribution in normal mice was evaluated, and its biodistribution in a radiation-induced fibrosarcoma (RIF) tumor mouse was compared with that of 3'-deoxy-3'-[{sup 18}F] fluorothymidine [{sup 18}F]FLT. Results: {sup 99m}Tc-MAMA-propyl-thymidine was obtained with a radiochemical yield of 70%. Electrophoresis indicated that the complex is uncharged, and its log P was 1.0. The molecular ion mass of the Tc complex was 589 Da, which is compatible with the hypothesized N{sub 2}S{sub 2}-oxotechnetium structure. Tissue distribution showed fast clearance from plasma primarily by the hepatobiliary pathway. Whole-body planar imaging after injection of {sup 99m}Tc-MAMA-propyl-thymidine in an RIF tumor-bearing mouse showed high uptake in the liver and the intestines. No uptake was observed in the tumor, in contrast to the clear uptake observed for [{sup 18}F] FLT visualized with {mu}PET. Conclusions: Although it has been reported that TK1 accepts large substituents at the N-3 position of the thymine ring

  19. Efficacy of the anti-VZV (anti-HSV3 vaccine in HSV1 and HSV2 recurrent herpes simplex disease: a prospective study

    Directory of Open Access Journals (Sweden)

    Le Goaster J

    2012-07-01

    Full Text Available Jacqueline Le Goaster,1 Sylvie Gonzalo,2 Patrice Bourée,1 Frederic Tangy,3 Anne-Lise Haenni41Department of Tropical Diseases, Centre Hospitalo-Universitaire (CHU, University of Paris XI, Le Kremlin Bicêtre, 2Biomnis Laboratory, Ivry-sur-Seine, 3Retro-Virology, Centre National de Recherche Scientifique (CNRS, Pasteur Institute, Paris; 4Jacques Monod Institute, Centre National de Recherche Scientifique (CNRS, University of Paris VII, Paris, FranceBackground: The aim of this study was to evaluate the possibility of using the anti-varicella zoster virus (anti-VZV, also known as anti-HSV3 vaccine against orobuccal herpes simplex virus type 1 (HSV1 and genital herpes simplex virus type 2 (HSV2. This was suggested by study of the phylogenetic tree of members of the herpes virus family, which showed a close relationship between VZV (HSV3 and the HSV1 and HSV2 herpes viruses.Methods: The present prospective study was conducted from January 2005 through January 2011. Twenty-four patients afflicted with HSV1 and HSV2 herpes recurrences over a period of years, numbering 6–8 and more recurrences per year, agreed to receive the anti-VZV vaccine. They were compared with 26 nonvaccinated patients presenting with herpes simplex diseases 2–5 times a year. All 50 patients were documented with anti-HSV1, anti-HSV2, and anti-VZV antibody serological testing.Results: From 2005 through 2011, for the 24 anti-VZV vaccinated patients, the average number of herpes relapses decreased to 0, correlated with an increased anti-VZV antibody level and clinical recovery of all patients, whereas no improvement was observed for the 26 nonvaccinated herpes patients.Conclusion: Data for the anti-VZV serological antibody levels tested before and after anti-VZV vaccination showed a significant (P < 0.001 increase among vaccinated patients. This suggests defective anti-VZV immune power in these patients. After 6 years of positive results for anti-VZV vaccine, this is a logical and

  20. Cervical HSV-2 infection causes cervical remodeling and increases risk for ascending infection and preterm birth.

    Directory of Open Access Journals (Sweden)

    Devin McGee

    Full Text Available Preterm birth (PTB, or birth before 37 weeks gestation, is the leading cause of neonatal mortality worldwide. Cervical viral infections have been established as risk factors for PTB in women, although the mechanism leading to increased risk is unknown. Using a mouse model of pregnancy, we determined that intra-vaginal HSV2 infection caused increased rates of preterm birth following an intra-vaginal bacterial infection. HSV2 infection resulted in histological changes in the cervix mimicking cervical ripening, including significant collagen remodeling and increased hyaluronic acid synthesis. Viral infection also caused aberrant expression of estrogen and progesterone receptor in the cervical epithelium. Further analysis using human ectocervical cells demonstrated a role for Src kinase in virus-mediated changes in estrogen receptor and hyaluronic acid expression. In conclusion, HSV2 affects proteins involved in tissue hormone responsiveness, causes significant changes reminiscent of premature cervical ripening, and increases risk of preterm birth. Studies such as this improve our chances of identifying clinical interventions in the future.

  1. Genital HSV Shedding among Kenyan Women Initiating Antiretroviral Therapy.

    Directory of Open Access Journals (Sweden)

    Griffins O Manguro

    Full Text Available Genital ulcer disease (GUD prevalence increases in the first month of antiretroviral treatment (ART, followed by a return to baseline prevalence by month 3. Since most GUD is caused by herpes simplex virus type 2 (HSV-2, we hypothesized that genital HSV detection would follow a similar pattern after treatment initiation.We conducted a prospective cohort study of 122 HSV-2 and HIV-1 co-infected women with advanced HIV disease who initiated ART and were followed closely with collection of genital swab specimens for the first three months of treatment.At baseline, the HSV detection rate was 32%, without significant increase in genital HSV detection noted during the first month or the third month of ART. HIV-1 shedding declined during this period; no association was also noted between HSV and HIV-1 shedding during this period.Because other studies have reported increased HSV detection in women initiating ART and we have previously reported an increase in GUD during early ART, it may be prudent to counsel HIV-1 infected women initiating ART that HSV shedding in the genital tract may continue after ART initiation.

  2. HSV neutralization by the microbicidal candidate C5A

    NARCIS (Netherlands)

    de Witte, L.; Bobardt, M.D.; Chatterji, U.; van Loenen, F.B.; Verjans, G.M.G.M.; Geijtenbeek, T.B.H.; Gallay, P.A.

    2011-01-01

    Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1

  3. HSV neutralization by the microbicidal candidate C5A

    NARCIS (Netherlands)

    L. de Witte (Lot); M.D. Bobardt (Michael); U. Chatterji (Udayan); F.B. van Loenen (Freek); G.M.G.M. Verjans (George); T.B.H. Geijtenbeek (Teunis); P.A. Gallay (Philippe)

    2011-01-01

    textabstractGenital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to

  4. [F-18]FHPG positron emission tomography for detection of herpes simplex virus (HSV) in experimental HSV encephalitis

    NARCIS (Netherlands)

    Buursma, AR; de Vries, EFJ; Garssen, J; Kegler, D; van Waarde, A; Schirm, J; Hospers, GAP; Mulder, NH; Vaalburg, W; Klein, HC

    Herpes simplex virus type 1 (HSV-1) is one of the most common causes of sporadic encephalitis. The initial clinical course of HSV encephalitis (HSE) is highly variable, and the infection may be rapidly fatal. For effective treatment with antiviral medication, an early diagnosis of HSE is crucial.

  5. Efficacy of the Herpes Simplex Virus 2 (HSV-2) Glycoprotein D/AS04 Vaccine against Genital HSV-2 and HSV-1 Infection and Disease in the Cotton Rat Sigmodon hispidus Model.

    Science.gov (United States)

    Boukhvalova, Marina; McKay, Jamall; Mbaye, Aissatou; Sanford-Crane, Hannah; Blanco, Jorge C G; Huber, Ashley; Herold, Betsy C

    2015-10-01

    Subunit vaccines based on the herpes simplex virus 2 (HSV-2) glycoprotein D (gD-2) have been the major focus of HSV-2 vaccine development for the past 2 decades. Based on the promising data generated in the guinea pig model, a formulation containing truncated gD-2, aluminum salt, and MPL (gD/AS04) advanced to clinical trials. The results of these trials, however, were unexpected, as the vaccine protected against HSV-1 infection but not against HSV-2. To address this discrepancy, we developed a Depot medroxyprogesterone acetate (DMPA)-treated cotton rat Sigmodon hispidus model of HSV-2 and HSV-1 genital infection. The severity of HSV-1 genital herpes was less than that of HSV-2 genital herpes in cotton rats, and yet the model allowed for comparative evaluation of gD/AS04 immunogenicity and efficacy. Cotton rats were intramuscularly vaccinated using a prime boost strategy with gD/AS04 (Simplirix vaccine) or control vaccine formulation (hepatitis B vaccine FENDrix) and subsequently challenged intravaginally with HSV-2 or HSV-1. The gD/AS04 vaccine was immunogenic in cotton rats and induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies but failed to efficiently protect against HSV-2 disease or to decrease the HSV-2 viral load. However, gD/AS04 significantly reduced vaginal titers of HSV-1 and better protected animals against HSV-1 compared to HSV-2 genital disease. The latter finding is generally consistent with the clinical outcome of the Herpevac trial of Simplirix. Passive transfer of serum from gD/AS04-immunized cotton rats conferred stronger protection against HSV-1 genital disease. These findings suggest the need for alternative vaccine strategies and the identification of new correlates of protection. In spite of the high health burden of genital herpes, there is still no effective intervention against the disease. The significant gap in knowledge on genital herpes pathogenesis has been further highlighted by the recent failure of GSK

  6. Spontaneous and Engineered Compensatory HSV Mutants that Counteract the Host Antiviral PKR Response

    Directory of Open Access Journals (Sweden)

    Kevin A. Cassady

    2009-10-01

    Full Text Available A virulent recombinant HSV lacking the diploid γ134.5 gene (Δγ134.5 have been investigated over the last two decades both for anti-tumor therapy and as vaccine vectors. The first generation vectors, while safe, are incapable of sustained replication in the majority of treated patients. An interferon inducible host antiviral kinase, protein kinase R (PKR, limits late viral protein synthesis and replication of Δγ134.5 viruses. This review describes the development of new Δγ134.5 vectors, through serial passage selection and direct viral genome engineering, which demonstrate selective PKR evasion in targeted cells and improved viral replication without restoring neurovirulence.

  7. A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2.

    Science.gov (United States)

    Lieveld, M; Carregosa, A; Benoy, I; Redzic, N; Berth, M; Vanden Broeck, D

    2017-10-01

    Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Application of shRNA-containing herpes simplex virus type 1 (HSV-1)-based gene therapy for HSV-2-induced genital herpes.

    Science.gov (United States)

    Liu, Zhihong; Xiang, Yang; Wei, Zhun; Yu, Bo; Shao, Yong; Zhang, Jie; Yang, Hong; Li, Manmei; Guan, Ming; Wan, Jun; Zhang, Wei

    2013-11-01

    HSV-1-based vectors have been widely used to achieve targeted delivery of genes into the nervous system. In the current study, we aim to use shRNA-containing HSV-1-based gene delivery system for the therapy of HSV-2 infection. Guinea pigs were infected intravaginally with HSV-2 and scored daily for 100 days for the severity of vaginal disease. HSV-2 shRNA-containing HSV-1 was applied intravaginally daily between 8 and 14 days after HSV-2 challenge. Delivery of HSV-2 shRNA-containing HSV-1 had no effect on the onset of disease and acute virus shedding in animals, but resulted in a significant reduction in both the cumulative recurrent lesion days and the number of days with recurrent disease. Around half of the animals in the HSV-2 shRNA group did not develop recurrent disease 100 days post HSV-2 infection. In conclusion, HSV-2 shRNA-containing HSV-1 particles are effective in reducing the recurrence of genital herpes caused by HSV-2. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Nested PCR for detection of HSV-1 in oral mucosa.

    Science.gov (United States)

    Jalouli, Miranda-Masoumeh; Jalouli, Jamshid; Hasséus, Bengt; Öhman, Jenny; Hirsch, Jan-Michaél; Sand, Lars

    2015-11-01

    It has been estimated that 15%-20% of human tumours are driven by infection and inflammation, and viral infections play an important role in malignant transformation. The evidence that herpes simplex virus type 1 (HSV-1) could be involved in the aetiology of oral cancer varies from weak to persuasive. This study aimed to investigate by nested PCR (NPCR) the prevalence of HSV-1 in samples from normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma (OSCC). We investigated the prevalence of HSV-1 in biopsies obtained from 26 fresh, normal oral mucosa from healthy volunteers as well as 53 oral leukoplakia and 27 OSCC paraffin-embedded samples. DNA was extracted from the specimens and investigated for the presence of HSV-1 by nested polymerase chain reaction (NPCR) and DNA sequencing. HSV-1 was detected in 14 (54%) of the healthy samples, in 19 (36%) of the oral leukoplakia samples, and in 14 (52%) of the OSCC samples. The differences were not statistically significant. We observed a high incidence of HSV-1 in healthy oral mucosa, oral leukoplakia, and OSCC tissues. Thus, no connection between OSCC development and presence of HSV-1 was detected.

  10. Strong Country Level Correlation between Syphilis and HSV-2 Prevalence

    Directory of Open Access Journals (Sweden)

    Chris Richard Kenyon

    2016-01-01

    Full Text Available Background. Syphilis is curable but Herpes Simplex Virus-2 (HSV-2 is not. As a result, the prevalence of syphilis but not HSV-2 may be influenced by the efficacy of national STI screening and treatment capacity. If the prevalence of syphilis and HSV-2 is found to be correlated, then this makes it more likely that something other than differential STI treatment is responsible for variations in the prevalence of both HSV-2 and syphilis. Methods. Simple linear regression was used to evaluate the relationship between national antenatal syphilis prevalence and HSV-2 prevalence in women in two time periods: 1990–1999 and 2008. Adjustments were performed for the laboratory syphilis testing algorithm used and the prevalence of circumcision. Results. The prevalence of syphilis was positively correlated with that of HSV-2 for both time periods (adjusted correlations, 20–24-year-olds: 1990–99: R2=0.54, P<0.001; 2008: R2=0.41, P<0.001 and 40–44-year-olds: 1990–99: R2=0.42, P<0.001; 2008: R2=0.49, P<0.001. Conclusion. The prevalence of syphilis and HSV-2 is positively correlated. This could be due to a common set of risk factors underpinning both STIs.

  11. The impact of HSV for inflammatory arthropathy patients.

    LENUS (Irish Health Repository)

    O'Connor, Mortimer B

    2012-02-01

    Herpes simplex virus type 1 (HSV-1), also known as herpes labialis, is the etiologic agent of vesicular lesions of the oral mucosa commonly referred to as "cold sores". HSV-1 can also cause clinical disease in a wide variety of other anatomic locations including the genitalia, liver, lung, eye, and central nervous system. These infections can be severe, particularly in the setting of immunosuppression, such as inflammatory arthropathy patients on Methotrexate ± biological therapies. Here, we highlight the importance of physician awareness of HSV due to its potential impact for rheumatology patients.

  12. Seroprevalence of HSV-1 and HSV-2 antibodies in Canadian women screened for enrolment in a herpes simplex virus vaccine trial.

    Science.gov (United States)

    Gorfinkel, I S; Aoki, F; McNeil, S; Dionne, M; Shafran, S D; Zickler, P; Halperin, S; Langley, J; Bellamy, A; Schulte, J; Heineman, T; Belshe, R

    2013-05-01

    Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) infections continue to be among the most common and unrecognized sexually transmitted infections in the world. Although treatable, HSV-1 and HSV-2 infections remain incurable. Hence, there is interest in the development of a vaccine to prevent genital herpes. As part of a multicentre, randomized, placebo-controlled trial to test such a vaccine, healthy women 18-30 years were enrolled as volunteers in several Canadian centres between 2005 and 2007. This study reports the seroprevalence of HSV-1 and HSV-2 antibodies in this group. A total of 2694 adult female volunteers in Canada with no known history of herpes simplex were screened for HSV antibodies using Western blot assay (the gold standard for diagnosis of HSV) for potential participation in a randomized, double-blind efficacy field trial of a herpes simplex vaccine. This trial provides a unique opportunity to examine the prevalence of antibodies to HSV-1 and of antibodies to HSV-2 in women with no known history of herpes simplex infection. The prevalence of antibodies to HSV-1 and to HSV-2 is compared with that found in previous Canadian studies that focused on a more general population. The overall seroprevalence of antibody to HSV-1 was 43%; that of HSV-2 was 2.5% and seropositivity to both was 2%. The prevalence of antibody to both HSV-1 and to HSV-2 increased with age. Seronegativity to both HSV-1 and HSV-2 was 56% in participating centres with populations under 250,000 and 46% in participating centres with populations over 250,000. Significant racial differences in seropositivity to HSV-1 and to HSV-2 were noted. The likelihood of participants being seropositive to HSV-1 and to HSV-2 was found to increase with age and to positively correlate with the population of the city in which they resided. Hypotheses are proposed to account for differences in racial seropositivity to HSV-1 and to HSV-2.

  13. Barrier to auto integration factor becomes dephosphorylated during HSV-1 Infection and Can Act as a host defense by impairing viral DNA replication and gene expression.

    Science.gov (United States)

    Jamin, Augusta; Thunuguntla, Prasanth; Wicklund, April; Jones, Clinton; Wiebe, Matthew S

    2014-01-01

    BAF (Barrier to Autointegration Factor) is a highly conserved DNA binding protein that senses poxviral DNA in the cytoplasm and tightly binds to the viral genome to interfere with DNA replication and transcription. To counteract BAF, a poxviral-encoded protein kinase phosphorylates BAF, which renders BAF unable to bind DNA and allows efficient viral replication to occur. Herein, we examined how BAF phosphorylation is affected by herpes simplex virus type 1 (HSV-1) infection and tested the ability of BAF to interfere with HSV-1 productive infection. Interestingly, we found that BAF phosphorylation decreases markedly following HSV-1 infection. To determine whether dephosphorylated BAF impacts HSV-1 productive infection, we employed cell lines stably expressing a constitutively unphosphorylated form of BAF (BAF-MAAAQ) and cells overexpressing wild type (wt) BAF for comparison. Although HSV-1 production in cells overexpressing wtBAF was similar to that in cells expressing no additional BAF, viral growth was reduced approximately 80% in the presence of BAF-MAAAQ. Experiments were also performed to determine the mechanism of the antiviral activity of BAF with the following results. BAF-MAAAQ was localized to the nucleus, whereas wtBAF was dispersed throughout cells prior to infection. Following infection, wtBAF becomes dephosphorylated and relocalized to the nucleus. Additionally, BAF was associated with the HSV-1 genome during infection, with BAF-MAAAQ associated to a greater extent than wtBAF. Importantly, unphosphorylated BAF inhibited both viral DNA replication and gene expression. For example, expression of two regulatory proteins, ICP0 and VP16, were substantially reduced in cells expressing BAF-MAAAQ. However, other viral genes were not dramatically affected suggesting that expression of certain viral genes can be differentially regulated by unphosphorylated BAF. Collectively, these results suggest that BAF can act in a phosphorylation-regulated manner to impair

  14. Nested PCR for detection of HSV-1 in oral mucosa

    OpenAIRE

    Jalouli, Miranda-Masoumeh; Jalouli, Jamshid; Hasséus, Bengt; Öhman, Jenny; Hirsch, Jan-Michaél; Sand, Lars

    2015-01-01

    Background: It has been estimated that 15%-20% of human tumours are driven by infection and inflammation, and viral infections play an important role in malignant transformation. The evidence that herpes simplex virus type 1 (HSV-1) could be involved in the aetiology of oral cancer varies from weak to persuasive. This study aimed to investigate by nested PCR (NPCR) the prevalence of HSV-1 in samples from normal oral mucosa, oral leukoplakia, and oral squamous ...

  15. In Vitro Synergism of Trifluorothymidine and Ganciclovir against HSV-1

    Science.gov (United States)

    Hobden, Jeffery A.; Kumar, Manish; Kaufman, Herbert E.; Clement, Christian; Varnell, Emily D.; Bhattacharjee, Partha S.

    2011-01-01

    Purpose. To determine whether trifluorothymidine (TFT) and ganciclovir (GCV) are synergistic against herpes simplex virus type 1 (HSV-1). Methods. TFT and GCV activity against 12 strains of HSV-1 (including an acyclovir-resistant strain) was measured by plaque-forming unit (PFU) inhibition. Cellular toxicity was assessed with an MTT dye reduction assay. Synergism was determined by calculating fractional inhibitory concentration (FIC indices) based on PFU reduction. Results. Concentrations of TFT resulting in 50% inhibition of PFUs (IC50) of acyclovir-susceptible HSV-1 strains ranged from 3.07 ± 0.36 to 12.52 ± 0.61 μM. GCV IC50 values ranged from 0.40 ± 0.02 to 1.59 ± 0.14 μM. IC50 values of TFT and GCV against the acyclovir-resistant strain were 15.40 ± 3.17 and 93.00 ± 9.64 μM, respectively. Concentrations of TFT or GCV resulting in 50% cell cytotoxicity (CC50) were 0.99 ± 0.01 and 92.91 ± 8.92 μM, respectively. TFT and GCV combined (10:1) were 10 times more potent against all acyclovir-susceptible HSV-1 strains. For 8 of 12 HSV-1 strains, the IC50 of TFT and GCV combined was lower than the CC50 of either drug. For acyclovir-susceptible HSV-1 strains, TFT and GCV combined generated a FIC index of <0.5, suggesting strong synergism between the two drugs. The FIC value for TFT and GCV combined against the acyclovir-resistant HSV-1 strain was 0.84, indicating nonantagonism. Conclusions. TFT and GCV are synergistic against acyclovir-susceptible HSV-1 at concentrations significantly less toxic than if each antiviral were used as a sole agent. PMID:20861476

  16. Direct activation of RIP3/MLKL-dependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense

    Science.gov (United States)

    Wang, Xing; Li, Yun; Liu, Shan; Yu, Xiaoliang; Li, Lin; Shi, Cuilin; He, Wenhui; Li, Jun; Xu, Lei; Hu, Zhilin; Yu, Lu; Yang, Zhongxu; Chen, Qin; Ge, Lin; Zhang, Zili; Zhou, Biqi; Jiang, Xuejun; Chen, She; He, Sudan

    2014-01-01

    The receptor-interacting kinase-3 (RIP3) and its downstream substrate mixed lineage kinase domain-like protein (MLKL) have emerged as the key cellular components in programmed necrotic cell death. Receptors for the cytokines of tumor necrosis factor (TNF) family and Toll-like receptors (TLR) 3 and 4 are able to activate RIP3 through receptor-interacting kinase-1 and Toll/IL-1 receptor domain-containing adapter inducing IFN-β, respectively. This form of cell death has been implicated in the host-defense system. However, the molecular mechanisms that drive the activation of RIP3 by a variety of pathogens, other than the above-mentioned receptors, are largely unknown. Here, we report that human herpes simplex virus 1 (HSV-1) infection triggers RIP3-dependent necrosis. This process requires MLKL but is independent of TNF receptor, TLR3, cylindromatosis, and host RIP homotypic interaction motif-containing protein DNA-dependent activator of IFN regulatory factor. After HSV-1 infection, the viral ribonucleotide reductase large subunit (ICP6) interacts with RIP3. The formation of the ICP6–RIP3 complex requires the RHIM domains of both proteins. An HSV-1 ICP6 deletion mutant failed to cause effective necrosis of HSV-1–infected cells. Furthermore, ectopic expression of ICP6, but not RHIM mutant ICP6, directly activated RIP3/MLKL-mediated necrosis. Mice lacking RIP3 exhibited severely impaired control of HSV-1 replication and pathogenesis. Therefore, this study reveals a previously uncharacterized host antipathogen mechanism. PMID:25316792

  17. Pneumomediastinum and Pneumothorax Associated with Herpes Simplex Virus (HSV) Pneumonia.

    Science.gov (United States)

    López-Rivera, Fermín; Colón Rivera, Xavier; González Monroig, Hernán A; Garcia Puebla, Juan

    2018-01-30

    BACKGROUND Pneumonia is one of the most common causes of death from infectious disease in the United States (US). Although most cases of community-acquired pneumonia (CAP) are secondary to bacterial infection, up to one-third of cases are secondary to viral infection, most commonly due to rhinovirus and influenza virus. Pneumonia due to herpes simplex virus (HSV) is rare, and there is limited knowledge of the pathogenesis and clinical complications. This report is of a fatal case of HSV pneumonia associated with bilateral pneumothorax and pneumomediastinum. CASE REPORT A 36-year-old homeless male Hispanic patient, who was a chronic smoker, with a history of intravenous drug abuse and a medical history of chronic hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infection, not on highly active antiretroviral therapy (HAART), was admitted to hospital as an emergency with a seven-day history of productive purulent cough. The patient was admitted to the medical intensive care unit (MICU) with a diagnosis of CAP, with intubation and mechanical ventilation. Broncho-alveolar lavage (BAL) was performed and was positive for HSV. The patient developed bilateral pneumothorax with pneumomediastinum, which was fatal, despite aggressive clinical management. CONCLUSIONS Pneumonia due to HSV infection is uncommon but has a high mortality. Although HSV pneumonia has been described in immunocompromised patients, further studies are required to determine the pathogenesis, early detection, identification of patients who are at risk and to determine the most effective approaches to prophylaxis and treatment for HSV pneumonia.

  18. Widespread disruption of host transcription termination in HSV-1 infection

    Science.gov (United States)

    Rutkowski, Andrzej J.; Erhard, Florian; L'Hernault, Anne; Bonfert, Thomas; Schilhabel, Markus; Crump, Colin; Rosenstiel, Philip; Efstathiou, Stacey; Zimmer, Ralf; Friedel, Caroline C.; Dölken, Lars

    2015-01-01

    Herpes simplex virus 1 (HSV-1) is an important human pathogen and a paradigm for virus-induced host shut-off. Here we show that global changes in transcription and RNA processing and their impact on translation can be analysed in a single experimental setting by applying 4sU-tagging of newly transcribed RNA and ribosome profiling to lytic HSV-1 infection. Unexpectedly, we find that HSV-1 triggers the disruption of transcription termination of cellular, but not viral, genes. This results in extensive transcription for tens of thousands of nucleotides beyond poly(A) sites and into downstream genes, leading to novel intergenic splicing between exons of neighbouring cellular genes. As a consequence, hundreds of cellular genes seem to be transcriptionally induced but are not translated. In contrast to previous reports, we show that HSV-1 does not inhibit co-transcriptional splicing. Our approach thus substantially advances our understanding of HSV-1 biology and establishes HSV-1 as a model system for studying transcription termination. PMID:25989971

  19. IFI16 restricts HSV-1 replication by accumulating on the hsv-1 genome, repressing HSV-1 gene expression, and directly or indirectly modulating histone modifications.

    Directory of Open Access Journals (Sweden)

    Karen E Johnson

    2014-11-01

    Full Text Available Interferon-γ inducible factor 16 (IFI16 is a multifunctional nuclear protein involved in transcriptional regulation, induction of interferon-β (IFN-β, and activation of the inflammasome response. It interacts with the sugar-phosphate backbone of dsDNA and modulates viral and cellular transcription through largely undetermined mechanisms. IFI16 is a restriction factor for human cytomegalovirus (HCMV and herpes simplex virus (HSV-1, though the mechanisms of HSV-1 restriction are not yet understood. Here, we show that IFI16 has a profound effect on HSV-1 replication in human foreskin fibroblasts, osteosarcoma cells, and breast epithelial cancer cells. IFI16 knockdown increased HSV-1 yield 6-fold and IFI16 overexpression reduced viral yield by over 5-fold. Importantly, HSV-1 gene expression, including the immediate early proteins, ICP0 and ICP4, the early proteins, ICP8 and TK, and the late proteins gB and Us11, was reduced in the presence of IFI16. Depletion of the inflammasome adaptor protein, ASC, or the IFN-inducing transcription factor, IRF-3, did not affect viral yield. ChIP studies demonstrated the presence of IFI16 bound to HSV-1 promoters in osteosarcoma (U2OS cells and fibroblasts. Using CRISPR gene editing technology, we generated U2OS cells with permanent deletion of IFI16 protein expression. ChIP analysis of these cells and wild-type (wt U2OS demonstrated increased association of RNA polymerase II, TATA binding protein (TBP and Oct1 transcription factors with viral promoters in the absence of IFI16 at different times post infection. Although IFI16 did not alter the total histone occupancy at viral or cellular promoters, its absence promoted markers of active chromatin and decreased those of repressive chromatin with viral and cellular gene promoters. Collectively, these studies for the first time demonstrate that IFI16 prevents association of important transcriptional activators with wt HSV-1 promoters and suggest potential

  20. Neurotrophic Factors NGF, GDNF and NTN Selectively Modulate HSV1 and HSV2 Lytic Infection and Reactivation in Primary Adult Sensory and Autonomic Neurons.

    Science.gov (United States)

    Yanez, Andy A; Harrell, Telvin; Sriranganathan, Heather J; Ives, Angela M; Bertke, Andrea S

    2017-02-07

    Herpes simplex viruses (HSV1 and HSV2) establish latency in peripheral ganglia after ocular or genital infection, and can reactivate to produce different patterns and frequencies of recurrent disease. Previous studies showed that nerve growth factor (NGF) maintains HSV1 latency in embryonic sympathetic and sensory neurons. However, adult sensory neurons are no longer dependent on NGF for survival, some populations cease expression of NGF receptors postnatally, and the viruses preferentially establish latency in different populations of sensory neurons responsive to other neurotrophic factors (NTFs). Thus, NGF may not maintain latency in adult sensory neurons. To identify NTFs important for maintaining HSV1 and HSV2 latency in adult neurons, we investigated acute and latently-infected primary adult sensory trigeminal (TG) and sympathetic superior cervical ganglia (SCG) after NTF removal. NGF and glial cell line-derived neurotrophic factor (GDNF) deprivation induced HSV1 reactivation in adult sympathetic neurons. In adult sensory neurons, however, neurturin (NTN) and GDNF deprivation induced HSV1 and HSV2 reactivation, respectively, while NGF deprivation had no effects. Furthermore, HSV1 and HSV2 preferentially reactivated from neurons expressing GFRα2 and GFRα1, the high affinity receptors for NTN and GDNF, respectively. Thus, NTN and GDNF play a critical role in selective maintenance of HSV1 and HSV2 latency in primary adult sensory neurons.

  1. Neurotrophic Factors NGF, GDNF and NTN Selectively Modulate HSV1 and HSV2 Lytic Infection and Reactivation in Primary Adult Sensory and Autonomic Neurons

    Directory of Open Access Journals (Sweden)

    Andy A. Yanez

    2017-02-01

    Full Text Available Herpes simplex viruses (HSV1 and HSV2 establish latency in peripheral ganglia after ocular or genital infection, and can reactivate to produce different patterns and frequencies of recurrent disease. Previous studies showed that nerve growth factor (NGF maintains HSV1 latency in embryonic sympathetic and sensory neurons. However, adult sensory neurons are no longer dependent on NGF for survival, some populations cease expression of NGF receptors postnatally, and the viruses preferentially establish latency in different populations of sensory neurons responsive to other neurotrophic factors (NTFs. Thus, NGF may not maintain latency in adult sensory neurons. To identify NTFs important for maintaining HSV1 and HSV2 latency in adult neurons, we investigated acute and latently-infected primary adult sensory trigeminal (TG and sympathetic superior cervical ganglia (SCG after NTF removal. NGF and glial cell line-derived neurotrophic factor (GDNF deprivation induced HSV1 reactivation in adult sympathetic neurons. In adult sensory neurons, however, neurturin (NTN and GDNF deprivation induced HSV1 and HSV2 reactivation, respectively, while NGF deprivation had no effects. Furthermore, HSV1 and HSV2 preferentially reactivated from neurons expressing GFRα2 and GFRα1, the high affinity receptors for NTN and GDNF, respectively. Thus, NTN and GDNF play a critical role in selective maintenance of HSV1 and HSV2 latency in primary adult sensory neurons.

  2. Photoaddition of p-aminobenzoil acid to thymine and thymidine

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, A.A.; Wainschel, L.A.; Shetlar, M.D. (California Univ., San Francisco, CA (United States). School of Pharmacy)

    1992-05-01

    Several studies in the literature have shown that DNA is damaged after UV irradiation in the presence of the sunscreen agent p-aminobenzoic acid (PABA), both in vivo and in vitro. One type of damage has been shown to be the result of increased yields of pyrimidime cyclobutane dimer formation. However, it has been suggested that other types of lesions are produced as well. We have studied the photochemistry of the thymine-PABA and thymidine-PABA systems and report here the isolation and characterization of thymine-PABA and thymidine-PABA photoadducts. These products have been identified, respectively as 5-(2-amino-5-carboxyphenyl)-5,6-dihydrothymine and isomeric forms of 5-(2-amino-5-carboxyphenyl)-5,6-dihydrothymidine. The quantum yields for the formation of these ducts in deaerated aqueous solutions at pH 7.0 have been determined to be 9.5 x 10{sup -4} and 4.3 x 10{sup -3} for the thymine and thymidine based adducts respectively. A pH profile for the thymine-PABA system indicated a maximum quantum yield for adduct formation at pH 6.5, although it could be detected over the whole pH range studied (pH 3.5-11.0). (Author).

  3. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction.

    Science.gov (United States)

    Sandrini, Michael P B; Piskur, Jure

    2005-05-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions.

  4. Short hairpin-loop-structured oligodeoxynucleotides reduce HSV-1 replication

    Directory of Open Access Journals (Sweden)

    Heinrich Jochen

    2009-04-01

    Full Text Available Abstract The Herpes simplex virus (HSV is known as an infectious agent and widespread in the human population. The symptoms of HSV infections can range from mild to life threatening, especially in immune-compromised individuals. HSV infections are commonly treated with the guanosine analogue Aciclovir, but reports of resistance are increasing. Efforts are made to establish single-stranded antisense oligodeoxynucleotides (as and small interfering ribonucleic acids (siRNAs for antiviral treatment. Recently, another class of short interfering nucleic acids, partially double-stranded hairpin loop-structured 54 mer oligodeoxynucleotides (ODNs, was shown to allow hydrolysis of HIV RNA by binding to the viral RNA. This leads to a substrate for the viral RNase H. To assess the potential of such ODNs for inhibition of HSV-1 replication, five partially double-stranded ODNs were designed based on the sequences of known siRNAs against HSV-1 with antiviral activity. Three of them are directed against early and two against leaky late genes. Primary human lung fibroblasts, MRC-5, and African green monkey kidney cells, Vero, were transfected with ODNs and subsequently infected. The effect on HSV-1 replication was determined by analyzing the virus titer in cell culture supernatants by quantitative PCR and plaque assays. An inhibitory effect was observed with all five selected ODNs, with two cases showing statistical significance in both cell types. The observed effect was sequence-specific and dose dependent. In one case the ODN was more efficient than a previously described siRNA directed against the same target site in the mRNA of UL5, a component of the helicase/primase complex. HSV-1 virions and ODNs can be applied simultaneously without transfection reagent, but at a 50-fold higher concentration to Vero cells with similar efficiencies. The results underline the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.

  5. Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

    Science.gov (United States)

    Bernard, Marie-Clotilde; Barban, Véronique; Pradezynski, Fabrine; de Montfort, Aymeric; Ryall, Robert; Caillet, Catherine; Londono-Hayes, Patricia

    2015-01-01

    HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.

  6. An attenuated herpes simplex virus type 1 (HSV1 encoding the HIV-1 Tat protein protects mice from a deadly mucosal HSV1 challenge.

    Directory of Open Access Journals (Sweden)

    Mariaconcetta Sicurella

    Full Text Available Herpes simplex virus types 1 and 2 (HSV1 and HSV2 are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat. In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ, induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1

  7. Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

    Directory of Open Access Journals (Sweden)

    Marie-Clotilde Bernard

    Full Text Available HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29 has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529 derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a

  8. H2AX phosphorylation and DNA damage kinase activity are dispensable for herpes simplex virus replication.

    Science.gov (United States)

    Botting, Carolyn; Lu, Xu; Triezenberg, Steven J

    2016-01-27

    Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM(-/-) cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection.

  9. Distribution of HIV-1 and HSV-2 epidemics in Chad revealing HSV-2 hot-spot in regions of high-risk HIV spread.

    Science.gov (United States)

    Charpentier, Charlotte; Koyalta, Donato; Ndinaromtan, Montana; Tchobkréo, Bagamla; Jenabian, Mohammad-Ali; Day, Nesrine; Si-Mohamed, Ali; Weiss, Helen; Bélec, Laurent

    2011-02-01

    Herpes Simplex Virus-2 (HSV-2) is known to be a potent co-factor of Human Immunodeficiency type 1 virus (HIV-1) heterosexual transmission. We were interested in assessing the distribution of HIV-1 and HSV-2 epidemics at the national level in Chad. In 2007, a population-based anonymous serosurvey for HIV-1 and HSV-2 infections, using dried blood spots, was conducted. The study included 548 adults living in 15 regions of Chad. After specimen elution, serological testing for HIV and HSV-2 infections was performed. Countrywide, the HIV-1 and HSV-2 seroprevalences were 11.1% and 15.7%, respectively. A positive correlation was observed with the highest HIV-1 prevalence seen in regions of the highest HSV-2 prevalence, especially in two conflict-affected eastern provinces of Darfur. Urgent public health interventions are needed in regions of Chad where high HSV-2 prevalence may be increasing the risk of HIV propagation.

  10. Addition of thymidine to culture media for accurate examination of thymidine-dependent small-colony variants of methicillin-resistant Staphylococcus aureus: a pilot study.

    Science.gov (United States)

    Horiuchi, Kazuki; Matsumoto, Takehisa; Ota, Yusuke; Kasuga, Eriko; Negishi, Tatsuya; Yaguchi, Tomomi; Sugano, Mitsutoshi; Honda, Takayuki

    2015-03-01

    Small-colony variants (SCVs) are slow-growing subpopulations of various auxotrophic bacterial strains. Thymidine-dependent SCVs (TD-SCVs) are unable to synthesize thymidine; hence, these variants fail to grow in a medium without thymidine. In this study, we used 10 TD-SCVs of Staphylococcus aureus, of which four strains possessed mecA. We compared the efficacy of a newly modified medium containing thymidine for the detection of TD-SCVs of methicillin-resistant S. aureus (MRSA) to the efficacy of routinely used laboratory media. We observed that none of the 10 TD-SCVs of S. aureus grew in Mueller-Hinton agar, and four TD-SCVs of MRSA failed to grow on all MRSA screening media, except for the ChromID™ MRSA medium. Laboratory tests conducted using medium with thymidine incorporated showed that thymidine did not affect the minimum inhibitory concentrations of oxacillin and cefoxitin for clinical isolates of S. aureus, and was able to detect MRSA, including TD-SCVs. These findings showed that thymidine-incorporated media are able to detect TD-SCVs of MRSA without altering the properties of other clinically isolated MRSA strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Stress Hormones Epinephrine and Corticosterone Selectively Modulate Herpes Simplex Virus 1 (HSV-1) and HSV-2 Productive Infections in Adult Sympathetic, but Not Sensory, Neurons.

    Science.gov (United States)

    Ives, Angela M; Bertke, Andrea S

    2017-07-01

    Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with the exacerbation of clinical symptoms and the induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive HSV-1 and HSV-2 infections within sensory and autonomic neurons, we analyzed viral DNA and the production of viral progeny after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal ganglion (TG) and sympathetic superior cervical ganglion (SCG) neurons expressed adrenergic receptors (activated by epinephrine) and the glucocorticoid receptor (activated by corticosterone). Productive HSV infection colocalized with these receptors in SCG but not in TG neurons. In productively infected neuronal cultures, epinephrine treatment significantly increased the levels of HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased the levels of HSV-2 DNA replication and production of viral progeny in SCG neurons but not in TG neurons. Thus, the stress-related hormones epinephrine and corticosterone selectively modulate acute HSV-1 and HSV-2 infections in autonomic, but not sensory, neurons. IMPORTANCE Stress exacerbates acute disease symptoms resulting from HSV-1 and HSV-2 infections and is associated with the appearance of recurrent skin lesions in millions of people. Although stress hormones are thought to impact HSV-1 and HSV-2 through immune system suppression, sensory and autonomic neurons that become

  12. False-negative type-specific glycoprotein G antibody responses in STI clinic patients with recurrent HSV-1 or HSV-2 DNA positive genital herpes, The Netherlands.

    Science.gov (United States)

    van Rooijen, Martijn S; Roest, Wim; Hansen, Gino; Kwa, David; de Vries, Henry J C

    2016-06-01

    Herpes simplex virus (HSV) type-discriminating antibody tests (glycoprotein G (gG) directed) are used to identify naïve persons and differentiate acute infections from recurrences. We studied test characteristics of three commercially available antibody tests in patients with recurrent (established by viral PCR tests) herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) genital herpes episodes. Serum samples (at minimum 3 months after t=0) were examined for the presence of gG-1-specific or gG-2-specific antibodies using the HerpeSelect 1 and 2 Immunoblot IgG, the HerpeSelect 1 and 2 enzyme linked immunoassays IgG and the LIAISON HSV-1 and HSV-2 IgG indirect chemiluminescence immunoassays. The immunoblot was HSV-1 positive in 70.6% (95% CI 44.0% to 89.7%), the LIAISON in 88.2% (95% CI 63.5% to 98.5%) and the ELISA in 82.4% (95% CI 56.6% to 96.2%) of the 17 patients with a recurrent HSV-1 episode. From 33 patients with a recurrent HSV-2 episode, the immunoblot was HSV-2 positive in 84.8% (95% CI 68.1% to 94.9%), the LIAISON in 69.7% (95% CI 51.3% to 84.4%) and the ELISA in 84.8% (95% CI 68.1% to 94.9%). Among 15/17 (88.2%; 95% CI 63.5% to 98.5%) patients with HSV-1 and 30/33 (90.1%; 95% CI 75.7% to 98.1%) patients with HSV-2, HSV-1 or HSV-2 antibodies, respectively, were detected in at least one of the three antibody tests. Commercial type-specific gG HSV-1 or HSV-2 antibody assays were false negative in 12-30% of patients with recurrent HSV-1 or HSV-2 DNA positive genital lesions. The clinical and epidemiological use of type-specific HSV serology can be hampered by false-negative results, especially if based on a single test. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  13. Effects of Electroacupuncture on Facial Nerve Function and HSV-1 DNA Quantity in HSV-1 Induced Facial Nerve Palsy Mice

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    Hongzhi Tang

    2014-01-01

    Full Text Available Acupuncture is a common and effective therapeutic method to treat facial nerve palsy (FNP. However, its underlying mechanism remains unclear. This study was aimed to investigate the effects of electroacupuncture on symptoms and content of HSV-1 DNA in FNP mice. Mice were randomized into four groups, an electroacupuncture treatment group, saline group, model animal group, and blank control group. Electroacupuncture was applied at Jiache (ST6 and Hegu (LI4 in electroacupuncture group once daily for 14 days, while electroacupuncture was not applied in model animal group. In electroacupuncture group, mice recovered more rapidly and HSV-1 DNA content also decreased more rapidly, compared with model animal group. We conclude that electroacupuncture is effective to alleviate symptoms and promote the reduction of HSV-1 in FNP.

  14. Effects of Electroacupuncture on Facial Nerve Function and HSV-1 DNA Quantity in HSV-1 Induced Facial Nerve Palsy Mice

    Science.gov (United States)

    Tang, Hongzhi; Feng, Shuwei; Chen, Jiao; Yang, Mingxiao; Zhong, Zhendong; Li, Ying; Liang, Fanrong

    2014-01-01

    Acupuncture is a common and effective therapeutic method to treat facial nerve palsy (FNP). However, its underlying mechanism remains unclear. This study was aimed to investigate the effects of electroacupuncture on symptoms and content of HSV-1 DNA in FNP mice. Mice were randomized into four groups, an electroacupuncture treatment group, saline group, model animal group, and blank control group. Electroacupuncture was applied at Jiache (ST6) and Hegu (LI4) in electroacupuncture group once daily for 14 days, while electroacupuncture was not applied in model animal group. In electroacupuncture group, mice recovered more rapidly and HSV-1 DNA content also decreased more rapidly, compared with model animal group. We conclude that electroacupuncture is effective to alleviate symptoms and promote the reduction of HSV-1 in FNP. PMID:24991226

  15. Performance of Focus ELISA Tests for HSV-1 and HSV-2 Antibodies Among University Students With No History of Genital Herpes

    Science.gov (United States)

    NANDA, JOY P.; ROBERTS, JESSICA; ROMPALO, ANNE; MELENDEZ, JOHAN H.; ZENILMAN, JONATHAN

    2009-01-01

    Objectives To define the performance characteristics of the Focus ELISA HSV-1 and HSV-2 assay among 100 university students. Study Design HSV-1 and HSV-2 Focus ELISA and Western Blot assays were performed on sera from university students who reported no history of genital herpes. Results HSV-2 and HSV-1 seroprevalence by Western Blot were 3.4% and 48%, respectively. In this population, the positive predictive value of the Focus HSV-2 ELISA was 37.5%, the sensitivity was 100%, and specificity was 94.1%. The PPV of the Focus HSV-1 ELISA was 96.7%, the sensitivity was 69.0%, and the specificity was 97.8%. Conclusions In this low-prevalence population, the positive predictive value of the Focus HSV-2 ELISA test was low. This finding, together with those reported elsewhere, indicates that caution is warranted when recommending HSV screening in low-prevalence or heterogeneous populations. Consideration should be given to raising the cutoff index value for defining a positive test result. PMID:17457239

  16. Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy.

    Science.gov (United States)

    Péré, Héléne; Rascanu, Aida; LeGoff, Jérome; Matta, Mathieu; Bois, Frédéric; Lortholary, Olivier; Leroy, Valériane; Launay, Odile; Bélec, Laurent

    2016-03-01

    The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding. © The Author(s) 2015.

  17. Short hairpin RNA-mediated inhibition of HSV-1 gene expression and function during HSV-1 infection in Vero cells.

    Science.gov (United States)

    Liu, Yuan-yuan; Deng, Hai-ying; Yang, Guang; Jiang, Wen-lin; Grossin, Laurent; Yang, Zhan-qiu

    2008-08-01

    To evaluate the efficiency of 3 short hairpin RNA (shRNA) interfering with the herpes simplex virus type 1 (HSV-1) gene coding glycoprotein D (gD) for inhibiting the gD expression and virus replication in vitro. Vero cells were selected for an in vitro model of infection. Three shRNA sequences (shRNAgD1, -gD2, and -gD3) targeting specifically the gD gene of HSV-1 were selected for evaluating the antiviral effects. The antiviral effects of shRNA in the cells infected with HSV-1 were evaluated by cytopathic effect (CPE) observations and plaque assays. The transcription level of viral RNA and the gD expression were studied by RT-PCR, Western blotting, and flow cytometry. With the 3 shRNA at a final concentration of 120 nmol/L, a significant inhibition of CPE in the HSV-1-infected cells was observed. The ED50 of shRNA-gD1, gD2, and gD3 were 48.74+/-2.57, 57.13+/-3.24, and 114.64+/-5.12 nmol/L, respectively. The gD gene decreased significantly after viral infection in the Vero cells pretreated with shRNA compared to the virus group. The expressions of the gD protein, determined by Western blotting and flow cytometry, were also drastically decreased in shRNA-transfected cells. Exogenous shRNA molecules can suppress the HSV-1 gD expression. They are inhibitors of HSV replication during infection in Vero cells.

  18. Use of "biokit HSV-2 Rapid Assay" to improve the positive predictive value of Focus HerpeSelect HSV-2 ELISA.

    Science.gov (United States)

    Morrow, Rhoda Ashley; Friedrich, David; Meier, Amalia; Corey, Lawrence

    2005-10-14

    Commercially available assays to detect antibodies to the herpes simplex virus type 2 (HSV-2)-specific glycoprotein gG-2 have markedly improved serologic diagnosis of HSV-2 infection. However, even tests with high specificity can have low positive predictive values in low prevalence populations. HSV-2 is a chronic, life-long viral infection that requires both medical attention and potential alterations in health care strategy. As such, the concern for false positive diagnoses is high confirmatory testing is routine for other viral serologies such as HIV and hepatitis C. We evaluated such a strategy for HSV-2 serology by using an easily performed commercial test, biokitHSV-2 rapid test ("Biokit"; Biokit USA, Lexington MA) as a confirmatory test for the widely used gG-2 specific serology ("Focus;" HerpeSelect HSV-2 ELISA; Focus Diagnostics, Cypress CA). We tested 782 sera by Focus HSV-2 ELISA, Biokit, and the current gold standard test, Western blot (WB). The positive predictive value of the Focus HSV-2 ELISA increased from 80.5% to 95.6% when Biokit testing was performed on sera that were initially positive by Focus HSV-2 ELISA. Confirmatory testing increased the specificity markedly among sera with Focus EIA values between 1.1 and 3.5: only 35% of low positive (index values 1.1-3.5) Focus HSV-2 ELISA results confirmed as positive by Biokit and WB compared with 92% of those with index values >3.5. Mathematical modeling of the data resulted in expected positive predictive values over 98% for populations with antibody prevalences typical of clinical practices in the US and Europe. Confirmatory Biokit testing of positive Focus HSV-2 ELISA results is fast, easy, and effective in reducing falsely positive HSV-2 antibody results. Patients, clinicians, and laboratories could benefit from the enhanced specificity of this simple HSV-2 serologic test combination.

  19. Use of "biokit HSV-2 Rapid Assay" to improve the positive predictive value of Focus HerpeSelect HSV-2 ELISA

    Directory of Open Access Journals (Sweden)

    Friedrich David

    2005-10-01

    Full Text Available Abstract Background Commercially available assays to detect antibodies to the herpes simplex virus type 2 (HSV-2-specific glycoprotein gG-2 have markedly improved serologic diagnosis of HSV-2 infection. However, even tests with high specificity can have low positive predictive values in low prevalence populations. HSV-2 is a chronic, life-long viral infection that requires both medical attention and potential alterations in health care strategy. As such, the concern for false positive diagnoses is high confirmatory testing is routine for other viral serologies such as HIV and hepatitis C. We evaluated such a strategy for HSV-2 serology by using an easily performed commercial test, biokitHSV-2 rapid test ("Biokit"; Biokit USA, Lexington MA as a confirmatory test for the widely used gG-2 specific serology ("Focus;" HerpeSelect HSV-2 ELISA; Focus Diagnostics, Cypress CA. Methods We tested 782 sera by Focus HSV-2 ELISA, Biokit, and the current gold standard test, Western blot (WB. Results The positive predictive value of the Focus HSV-2 ELISA increased from 80.5% to 95.6% when Biokit testing was performed on sera that were initially positive by Focus HSV-2 ELISA. Confirmatory testing increased the specificity markedly among sera with Focus EIA values between 1.1 and 3.5: only 35% of low positive (index values 1.1–3.5 Focus HSV-2 ELISA results confirmed as positive by Biokit and WB compared with 92% of those with index values >3.5. Mathematical modeling of the data resulted in expected positive predictive values over 98% for populations with antibody prevalences typical of clinical practices in the US and Europe. Conclusion Confirmatory Biokit testing of positive Focus HSV-2 ELISA results is fast, easy, and effective in reducing falsely positive HSV-2 antibody results. Patients, clinicians, and laboratories could benefit from the enhanced specificity of this simple HSV-2 serologic test combination.

  20. Linear Multiepitope (Glyco)peptides for Type-Specific Serology of Herpes Simplex Virus (HSV) Infections.

    Science.gov (United States)

    Risinger, Christian; Sørensen, Kasper K; Jensen, Knud J; Olofsson, Sigvard; Bergström, Tomas; Blixt, Ola

    2017-05-12

    Detection of type-specific antibodies is an important and essential part of accurate diagnosis, even in silent carriers of herpes simplex virus (HSV)-1 (oral) and HSV-2 (genital) infections. Serologic assays that identify HSV-1 and HSV-2 type-specific antibodies have been commercially available for more than a decade but often face problems related to cross-reactivity and similar issues. Attempts to identify type-specific peptide epitopes for use in serology for both HSV-1 and HSV-2 have been limited. We recently demonstrated epitope mapping of envelope glycoprotein G2 and identified a type-specific glycopeptide epitope that broadly recognized HSV-2 infected individuals. In the present work we have performed a comprehensive glycopeptide synthesis and microarray epitope mapping of 14 envelope proteins from HSV-1 and HSV-2, namely, gB, gC, gD, gE, gG, gH, and gI, using sera from HSV-1- and HSV-2-infected individuals and control sera. Several unique type-specific peptide epitopes with high sensitivity were identified and synthesized as one large linear multiepitope sequence using microwave-assisted solid-phase (glyco)peptide synthesis. Microarray validation with clinically defined HSV and Varicella Zoster (VZV) sera confirmed excellent cumulative specificities and sensitivities.

  1. Analysis of HSV oncolytic virotherapy in organotypic cultures.

    Science.gov (United States)

    Fulci, Giulia; Passer, Brent

    2009-01-01

    Tumor-selective replication-competent viral vectors, such as oncolytic herpes simplex virus (HSV) type I (HSV-1), represent an attractive strategy for tumor-based therapies because these viruses can replicate and spread in situ exhibiting cytopathic effects through direct oncolytic activity. These lytic viruses offer a distinct advantage over other forms of cancer therapies in that they are self-perpetuating and can spread not only in the tumor itself, but also to distant micrometastases. Translational studies aimed at identifying novel virotherapies for human cancers are incumbent upon the appropriate experimental models. While animal models are the preferred choice for efficacy studies of HSV virotherapy, we have developed a novel complementary approach toward assessing the effectiveness of oncolytic HSV therapy in both brain and prostate cancers. This experimental model takes advantage of previously published work in which human prostate cancer biopsies and rodent brain slices can be easily maintained ex vivo. The advantage of these systems is that the three-dimensional structure remains intact. Thus, all of the factors that may affect viral entry and replication, such as cell-cell and cell-matrix interactions, and interstitial fluid within this three-dimensional milieu remain preserved. Moreover, with respect to the brain, this system offers the advantage of direct access to brain cells, such as microglia and astrocytes, and circumvents the problems associated with the presence of the blood-brain barrier.

  2. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

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    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-11-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating (/sup 3/H)thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase.

  3. Performance evaluation of the Aptima HSV-1 and 2 assay for the detection of HSV in cutaneous and mucocutaneous lesion specimens.

    Science.gov (United States)

    Sam, Soya S; Caliendo, Angela M; Ingersoll, Jessica; Abdul-Ali, Deborah; Kraft, Colleen S

    2017-12-13

    Timely and precise laboratory diagnosis of Herpes simplex viruses (HSV) is required to guide clinical management. The study evaluated limit of detection (LOD) and performance characteristics of the Aptima HSV 1 & 2 assay in comparison to four assays. The multi-center study compared qualitative detection of HSV-1 and 2 by the Aptima HSV-1 and 2 assay (Hologic) to ELVIS culture, Lyra Direct (Quidel), AmpliVue (Quidel) and a laboratory developed test (LDT). LOD was performed using VTM and STM diluted viral concentrations and clinical performance was evaluated using 505 swab specimens. The Aptima LOD studies performed showed a lower detection limit for STM specimens as 1450 copies/mL and 430 copies/mL for HSV1 and HSV-2 respectively; the LOD for VTM specimens was 9370 copies/mL and 8045 copies/mL for HSV-1 and HSV-2 respectively. When the assays were analyzed based on the positive consensus result established the Aptima had 95% of percent positive agreement (PPA) and 100% negative percent agreement (NPA) for the HSV-1. For the HSV-2, the PPA and NPA for Aptima were 96% and 100% respectively. AmpliVue had 1.8% invalid rate, while Lyra had no invalid results but an inhibition rate of 0.8%. Aptima and LDT did not have any invalid or inhibited results. The results indicate that the Aptima HSV-1 & 2 assay is sensitive and the performance characteristics of the Aptima assay is comparable to the assays analyzed for the detection and differentiation of HSV-1 and 2 from cutaneous and mucocutaneous lesions. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. [H-3]thymidine incorporation into whole liver as an alternative to [H-3]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

    NARCIS (Netherlands)

    de Jong, KP; Brinker, M; van Veen, M; Daemen, T; Scherphof, GL; Slooff, MJH

    1999-01-01

    OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [H-3]thymidine incorporation into liver cell DNA. We hypothesized that [H-3]thymidine incorporation into whole liver tissue parallels [H-3]thymidine incorporation into

  5. HSV-2 Specifically Down Regulates HLA-C Expression to Render HSV-2-Infected DCs Susceptible to NK Cell Killing

    Science.gov (United States)

    Elboim, Moran; Grodzovski, Inna; Djian, Esther; Wolf, Dana G.; Mandelboim, Ofer

    2013-01-01

    Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity. PMID:23555244

  6. Association between Herpes Simplex virus type 2 (HSV 2 and bad obstetric outcomes

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    Hala Mohamed Majeed Hassan

    2014-01-01

    Full Text Available Introduction: HSV is a common human pathogen that lead to lifelong latent infection. Maternal infections may be associate with transmission to the fetus. The risk factors associated with HSV 2 seropositivity in pregnant women in Iraq are not well studied. Aim: The present study conducted to verify the prevalence of HSV 2 infections in women with bad obstetric history (BOH in Kirkuk Governorate. Material and Methods: HSV 2 seropositivity among women aged 14 to 48 years was investigated by determination of HSV 2 IgG and IgM in a prospective, case control descriptive study. Results: The overall HSV 2 seroprevalence was 29.9%, with a non significant difference between women with BOH and women with normal pregnancy. HSV 2 IgM, as an indicator of current infection was demonstrated in 2% of the studied population, and was significantly (P=0.002 higher in women with BOH compared to women with normal pregnancy. Both HSV 2 IgG and IgM were significantly varied with age groups, with trends of increasing with older ages. HSV 2 IgG was statistically significantly higher in working women (P=0.03 as compared to housewife. Conclusions: Significant association was found between HSV 2 seroprevalence and education levels, residence, smoking and animal exposure. Presence of pregnancy in women with HSV-2 latent infection was a risk factor for development of BOH.

  7. HSV Serologic Testing for Pregnant Women: Willingness to Be Tested and Factors Affecting Testing

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    David A. Baker

    2011-01-01

    Full Text Available Objective. This prospective study was undertaken to evaluate pregnant women's willingness to undergo HSV type-specific serologic testing and factors affecting willingness in an obstetrics/gynecology ambulatory unit. Methods. At prenatal Visit 1, pregnant women (n=303 with no history of HSV-2 were tested for HSV-1/HSV-2 before and after they received counseling on genital and neonatal herpes. Results. In both the Unwilling Subgroup and the group that changed from being willing to being unwilling, the most common reasons for choosing not to be tested were not being at risk for genital herpes, being tested is too personal, and concern about what will be done with the results. Of the 134 participants in the Willing/Tested Subgroup, 27 (20% were HSV-2 seropositive and 81 (60% were HSV-1 seropositive. Conclusions. These results support the feasibility of HSV serologic testing and counseling in pregnant women.

  8. HSV serologic testing for pregnant women: willingness to be tested and factors affecting testing.

    Science.gov (United States)

    Baker, David A; Pressley, Andrea; Meek, Lillian; Figueroa, Reinaldo; Yates, Barbara; Dix, Lynn

    2011-01-01

    This prospective study was undertaken to evaluate pregnant women's willingness to undergo HSV type-specific serologic testing and factors affecting willingness in an obstetrics/gynecology ambulatory unit. At prenatal Visit 1, pregnant women (n = 303) with no history of HSV-2 were tested for HSV-1/HSV-2 before and after they received counseling on genital and neonatal herpes. In both the Unwilling Subgroup and the group that changed from being willing to being unwilling, the most common reasons for choosing not to be tested were not being at risk for genital herpes, being tested is too personal, and concern about what will be done with the results. Of the 134 participants in the Willing/Tested Subgroup, 27 (20%) were HSV-2 seropositive and 81 (60%) were HSV-1 seropositive. Conclusions. These results support the feasibility of HSV serologic testing and counseling in pregnant women.

  9. Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy

    DEFF Research Database (Denmark)

    Khan, Z.; Knecht, Wolfgang; Willer, Mette

    2010-01-01

    The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen....... In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT...... suicide gene therapy system in combination with stem cell mediated gene delivery promises new treatment of malignant gliomas....

  10. Herpes Simplex Virus Type 1 Neuronal Infection Perturbs Golgi Apparatus Integrity through Activation of Src Tyrosine Kinase and Dyn-2 GTPase

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    Carolina Martin

    2017-08-01

    Full Text Available Herpes simplex virus type 1 (HSV-1 is a ubiquitous pathogen that establishes a latent persistent neuronal infection in humans. The pathogenic effects of repeated viral reactivation in infected neurons are still unknown. Several studies have reported that during HSV-1 epithelial infection, the virus could modulate diverse cell signaling pathways remodeling the Golgi apparatus (GA membranes, but the molecular mechanisms implicated, and the functional consequences to neurons is currently unknown. Here we report that infection of primary neuronal cultures with HSV-1 triggers Src tyrosine kinase activation and subsequent phosphorylation of Dynamin 2 GTPase, two players with a role in GA integrity maintenance. Immunofluorescence analyses showed that HSV-1 productive neuronal infection caused a scattered and fragmented distribution of the GA through the cytoplasm, contrasting with the uniform perinuclear distribution pattern observed in control cells. In addition, transmission electron microscopy revealed swollen cisternae and disorganized stacks in HSV-1 infected neurons compared to control cells. Interestingly, PP2, a selective inhibitor for Src-family kinases markedly reduced these morphological alterations of the GA induced by HSV-1 infection strongly supporting the possible involvement of Src tyrosine kinase. Finally, we showed that HSV-1 tegument protein VP11/12 is necessary but not sufficient to induce Dyn2 phosphorylation. Altogether, these results show that HSV-1 neuronal infection triggers activation of Src tyrosine kinase, phosphorylation of Dynamin 2 GTPase, and perturbation of GA integrity. These findings suggest a possible neuropathogenic mechanism triggered by HSV-1 infection, which could involve dysfunction of the secretory system in neurons and central nervous system.

  11. UV-C irradiation of HSV-1 infected fibroblasts (HSV-FS) enhances human natural killer (NK) cell activity against these targets

    Energy Technology Data Exchange (ETDEWEB)

    Pettera, L.; Fitzgerald-Bocarsly, P. (New Jersey Medical School, Newark (United States))

    1991-03-11

    Expression of Herpes Simplex Virus Type 1 (HSV-1) immediate early gene products has been bound to be sufficient for NK cell mediated lysis of HSV-1 infected FS. To block the targets at various stages in the infectious cycle, HSV-FS were irradiated with UV light for 1 min at 2, 6, and 20 hr post-infection. NK mediated lysis of 2 hr and 5 hr UV treated HSV-FS was 2-fold higher than non-UV treated HSV-FS despite a {gt}99% inhibition in virus yield. In contrast, 20 hr infected targets were lysed less well than 2 and 6 hr targets despite strong glycoprotein expression and induction of high levels of interferon-alpha (IFN-{alpha}) production by effector PBMC's; this lysis was not enhanced by UV treatment. Since NK lysis of HSV-FS has been found to be dependent on an HLA-DR{sup +} accessory cell (AC), lysis of irradiated HSV-FS by PBMC's depleted of AC was measured. Such depletion eradicated NK lysis of the UV treated HSV-FS indicating that irradiation does not overcome the AC requirement for NK lysis. UV irradiation of another HLA-DR{sup +} dependent target, Vesicular Stomatitis Virus (VSV) infected FS led to a dramatic reduction in both NK lysis and IFN-{alpha} induction. HSV-1 is a DNA virus whose genes are expressed in a cascade fashion whereas VSV is an RNA virus. The authors hypothesize that the enhancement in AC dependent NK activity observed for UV irradiated HSV-FS, but not VSV-FS, targets is due to overproduction of either a cellular or viral gene product which specifically occurs early in the HSV-1 infectious cycle and is downregulated by 20 hr post-infection.

  12. Herpes simplex virus 2 (HSV-2 infected cell proteins are among the most dominant antigens of a live-attenuated HSV-2 vaccine.

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    Joshua J Geltz

    Full Text Available Virion glycoproteins such as glycoprotein D (gD are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2. We have observed that mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748. In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2's thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine's capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.

  13. Characterization of adducts formed in reactions of acrolein with thymidine and calf thymus DNA.

    Science.gov (United States)

    Pawłowicz, Agnieszka J; Kronberg, Leif

    2008-01-01

    Acrolein, an important industrial chemical and environmental contaminant, has been shown to interact with nucleic acids in vitro and in vivo. In this study, we examined the reactivity of acrolein towards thymidine and calf-thymus double- and single-stranded DNA in aqueous buffered solutions. LC-MS Analyses of the reaction mixture of acrolein with thymidine showed the formation of five structurally different adducts. The structures of the products were determined on the basis of mass spectrometry, UV absorbance, and (1)H- and (13)C-NMR spectroscopy. The adducts were identified as 3-(3-oxopropyl)thymidine (dT1), 3-[(tetrahydro-2,4-dihydroxypyran-3-yl)methyl]thymidine (dT2), 2-(hydroxymethyl)-5-(thymidin-3-yl)pent-2-enal (dT3), 3-hydroxy-2-methylidene-5-(thymidin-3-yl)pentanal (dT4), and 2-[(thymidin-3-yl)methyl]penta-2,4-dienal (dT5). The adducts dT2-dT5 were formed in reaction of dT1 with acrolein. In the reaction of acrolein with calf-thymus DNA, dT1 was the only adduct detected in the DNA hydrolysate.

  14. Implementation of Nearest Neighbor using HSV to Identify Skin Disease

    Science.gov (United States)

    Gerhana, Y. A.; Zulfikar, W. B.; Ramdani, A. H.; Ramdhani, M. A.

    2018-01-01

    Today, Android is one of the most widely used operating system in the world. Most of android device has a camera that could capture an image, this feature could be optimized to identify skin disease. The disease is one of health problem caused by bacterium, fungi, and virus. The symptoms of skin disease usually visible. In this work, the symptoms that captured as image contains HSV in every pixel of the image. HSV can extracted and then calculate to earn euclidean value. The value compared using nearest neighbor algorithm to discover closer value between image testing and image training to get highest value that decide class label or type of skin disease. The testing result show that 166 of 200 or about 80% is accurate. There are some reasons that influence the result of classification model like number of image training and quality of android device’s camera.

  15. Transcriptional profiling of thymidine-producing strain recombineered from Escherichia coli BL21

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    Jin-Sook Kim

    2015-12-01

    Full Text Available DNA microarrays were used to compare the expression profiles of a thymidine overproducing strain (BLT013 and its isogenic parent, Escherichia coli BL21(DE3, when each was grown under well-defined thymidine production conditions with glycerol as carbon source. Here we describe the experimental procedures and methods in detail to reproduce the results and provide resource to be applied to similar engineering approach (available at Gene Expression Omnibus database under GSE69963. Taken together, the microarray data provide a basis for new testable hypotheses regarding enhancement of thymidine productivity and attaining a more complete understanding of nucleotide metabolism in bacteria.

  16. Inhibitory effects of lupene-derived pentacyclic triterpenoids from Bursera simaruba on HSV-1 and HSV-2 in vitro replication.

    Science.gov (United States)

    Álvarez, Ángel L; Habtemariam, Solomon; Parra, Francisco

    2015-01-01

    The cytotoxicity and antiviral properties of Bursera simaruba against herpes simplex viruses (HSV-1 and HSV-2) were investigated through a bioactivity-guided isolation protocol. The plant material was fractionated using solvent-solvent partitioning, size-exclusion and thin-layer chromatography. The antiviral compounds present in the most active fractions were identified by means of LC-MS and NMR. Three different methods were compared during the evaluation of antiviral activity of samples. Four lupene-related pentacyclic triterpenes were found to be responsible for the anti-herpesvirus effects of B. simaruba and were isolated from this species for the first time. The selective indexes (SI) of B. simaruba-derived samples ranged from 7.7 to 201.9.

  17. Detection of herpes simplex virus type 2 (HSV-2) -specific cell-mediated immune responses in guinea pigs during latent HSV-2 genital infection.

    Science.gov (United States)

    Perry, Clarice L; Banasik, Brianne N; Gorder, Summer R; Xia, Jingya; Auclair, Sarah; Bourne, Nigel; Milligan, Gregg N

    2016-12-01

    Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to infection with other sexually transmitted pathogens. Understanding immune protection against HSV-2 primary infection and immune modulation of virus shedding events following reactivation of the virus from latency is important for the development of effective prophylactic and therapeutic vaccines. Although the murine model of HSV-2 infection is useful for understanding immunity following immunization, it is limited by the lack of spontaneous reactivation of HSV-2 from latency. Genital infection of guinea pigs with HSV-2 accurately models the disease of humans including the spontaneous reactivation of HSV-2 from latency and provides a unique opportunity to examine virus-host interactions during latency. Although the guinea pig represents an accurate model of many human infections, relatively few reagents are available to study the immunological response to infection. To analyze the cell-mediated immune response of guinea pigs at extended periods of time after establishment of HSV-2 latency, we have modified flow-cytometry based proliferation assays and IFN-γ ELISPOT assays to detect and quantify HSV-specific cell-mediated responses during latent infection of guinea pigs. Here we demonstrate that a combination of proliferation and ELISPOT assays can be used to quantify and characterize effecter function of virus-specific immune memory responses during HSV-latency. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Anti-herpes simplex virus and anti-human cell growth activity of E-5-propenyl-2'-deoxyuridine and the concept of selective protection in antivirus chemotherapy.

    Science.gov (United States)

    Cheng, Y C; Grill, S; Ruth, J; Bergstrom, D E

    1980-12-01

    E-5-Propenyl-2'-deoxyuridine (E-5-propenyl-dUrd) inhibited the growth of herpes simplex virus (HSV) types 1 (HSV-1) and 2 in culture. The concentration of drug required to give a 2-log reduction in virus titer was 5 microM for HSV-1 and 23 microM for HSV-2. The anti-HSV-1 activity of this agent was more potent than 5-propyl-dUrd, equivalent to E-5(3,3,3-trifluoropropenyl)-dUrd, and less potent than E-5-bromovinyl-dUrd. The HSV-1 mutant (B2006) lacking the ability to induce virus-specific thymidine kinase could not be inhibited by E-5-propenyl-dUrd. The binding constants of E-5-propenyl-dUrd to HSV-1, HSV-2, varicella-zoster virus, and human mitochondrial thymidine kinases were established to be 0.2, 6.2, 0.3, and 0.8 microM, respectively. Thymidine phosphorylation catalyzed by human cytosol thymidine kinase could not be inhibited by E-5-propenyl-dUrd at a concentration 10-fold higher than the thymidine in the assay. When thymidine and E-5-propenyl-dUrd were added concomitantly at equal concentrations to virus-infected cells, the antiviral activity was not reversed in HSV-1 and only partially reversed in HSV-2. E-5-Propenyl-dUrd also inhibited the growth of human cells in culture with 50% inhibitory dose of 50 microM. Since this inhibition could be readily reversed by a lower concentration of thymidine, the idea of selective protection is proposed. This approach could avoid the cytotoxic effect of an antiviral agent with properties similar to E-5-propenyl-dUrd without sacrificing antiviral activity.

  19. Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line.

    Science.gov (United States)

    Azizi, Ali; Aidoo, Francisca; Gisonni-Lex, Lucy; McNeil, Bryan

    2013-12-01

    The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Combined anti-tumor therapeutic effect of targeted gene, hyperthermia, radionuclide brachytherapy in breast carcinoma%磁感应加热和HSV-tk自杀基因及核素内照射联合治疗乳腺癌的实验研究

    Institute of Scientific and Technical Information of China (English)

    陈道桢; 唐秋莎; 项静英; 许飞; 张立; 王峻峰

    2011-01-01

    素治疗能有效抑制MCF-7乳腺癌的生长,对乳腺癌治疗有潜在的应用前景.%Objective To investigate the antitumor therapeutic effect of combined therapy of magnetic induction heating by nano-magnetic particles, herpes simplex virus thymidine kinase gene(HSV-tk suicide gene) and internal radiation in mice bearing MCF-7 breast carcinoma. Methods The transfection reagents, plasmids heat shock protein-HSV-tk (pHSP-HSV-tk), ferroso-ferric oxide nano-magnetic fluid flow and 188Re-ganciclovir-bovine serum albumin-nanopaticles (GCV-BSA-NP) were prepared. The heating experiments in vivo were carried out using ferroso-ferric oxide nano-magnetic fluid flow. Sixty mice tumor models bearing MCF-7 breast carcinoma were established and randomly divided into six groups. Group A was the control group, B was gene transfection therapy group, C was hyperthermia group, D was gene transfection therapy combined with radionuclide brachytherapy group, E was gene therapy combined with hyperthermia group, and F was gene therapy, hyperthermia combined with radionuclide brachytherapy group. The tumor growth, tumor mass and histopathological changes were evaluated. The expression of HSV-tk in the groups of B, D, E and F was detected by RT-PCR. Poisson distribution and one-way analysis of variance (ANOVA) were used for statistical analysis by SPSS 10.0 software. Results In the animal heating experiments, the temperature of tumor increased up to 39.6 ℃, 43.2 ℃, and 48.1 ℃ quickly with different injected doses (2, 4 and 6 mg respectively) of nano-magnetic particles and maintained for 40 min. The temperature of tumor tissue reduced to 36.8 ℃, 37.5 ℃ and 37.8 ℃ in 10 min when alternating magnetic field (AMF) stopped. The tumor mass in Groups C ((452.50 ±30.29) mg), D ((240.98 ±35.32)mg), E((231.87 ±27.41) mg) and F ((141.55 ±23.78) mg) were much lower than that in Group A ((719.12±22.65) mg) (F=800.07, P<0. 01), with the most significant treatment effect in Group F.The tumor mass in Group B

  1. Role of thymidine phosphorylase in Fas-induced apoptosis.

    Science.gov (United States)

    Mori, S; Takao, S; Ikeda, R; Noma, H; Mataki, Y; Wang, X; Akiyama, S; Aiko, T

    2001-12-01

    Thymidine phosphorylase (TP) has chemotactic and angiogenic activity in vitro, and it promotes tumor growth and inhibits apoptosis in vivo. It plays a key role in the invasiveness and metastasis of TP-expressing solid tumors. KB/TP cells transfected with a TP cDNA have been shown to be resistant to hypoxia-induced apoptosis, suggesting that TP has effects on tumor growth and cell death independent of its effects on angiogenesis. However, the mechanisms of cell death inhibition by TP are unknown. In the present study, we demonstrate that caspase-8 is cleaved in control transfectant KB cells early on during Fas-induced apoptosis. Caspase-8 activation leads to the loss of mitochondrial membrane potential, followed by the release of cytochrome c, the activation of caspase-3, and apoptosis. In contrast, Fas-induced caspase-8 cleavage is inhibited in KB/TP cells, which lead to inhibition of the downstream apoptotic cascade and inhibition of apoptosis. These findings indicate that TP plays an important role in intracellular apoptotic signal transduction in the Fas-induced apoptotic pathway. Therefore, inhibition of TP may suppress the progression of TP-overexpressing solid tumors by inducing apoptosis.

  2. Ocular HSV-1: Is the Cornea a Reservoir for Viral Latency or a Fast Pit Stop?

    Science.gov (United States)

    Kennedy, David P.; Clement, Christian; Arceneaux, Richard L.; Bhattacharjee, Partha S.; Huq, Tashfin S.; Hill, James M.

    2010-01-01

    Purpose To present a review supporting and refuting evidence from mouse, rabbit, non-human primate, and human studies of herpes simplex virus type 1 (HSV-1) concerning corneal latency. Methods More than 50 research papers on HSV-1 published in peer-reviewed journals were examined. Results Infectious HSV-1 has been found in mouse denervated tissues and in tissues with negative cultures from the corresponding ganglion. However, the different mouse strains have shown varied responses to different strains of HSV, making it difficult to relate such findings to humans. Rabbit studies provide excellent evidence for HSV-1 corneal latency including data on HSV-1 migration from the cornea into the corneoscleral rim and on the distribution of HSV-1 DNA in the cornea. However, the available methods for the detection of infectious HSV-1 may not be sensitive enough to detect low-level infection. Infectious HSV-1 has been successfully isolated from the tears of non-human primates in the absence of detectable corneal lesions. The recurrence of corneal ulcers in non-human primates before the appearance of infectious HSV-1 in tears suggests that the origin of the HSV-1 is the cornea, rather than the TG. Human studies presented evidence of both ganglion and corneal latency. Conclusion Understanding HSV-1 disease progression and the possibility of corneal latency could lead to more effective treatments for herpetic keratitis. However, it is unlikely that operational latency in the cornea will be definitively proven unless a new method with higher sensitivity for the detection of infectious virus is developed. PMID:21304287

  3. Predicting the emergence of drug-resistant HSV-2: new predictions

    Directory of Open Access Journals (Sweden)

    Darby Graham

    2003-03-01

    Full Text Available Abstract Background Mathematical models can be used to predict the emergence and transmission of antiviral resistance. Previously it has been predicted that high usage of antivirals (in immunocompetent populations to treat Herpes Simplex Virus type 2 (HSV-2 would only lead to fairly low levels of antiviral resistance. The HSV-2 predictions were based upon the assumption that drug-resistant strains of HSV-2 would be less infectious than drug-sensitive strains but that the drug-resistant strains would not be impaired in their ability to reactivate. Recent data suggest that some drug-resistant strains of HSV-2 are likely to be impaired in their ability to reactivate. Objectives: (1 To predict the effect of a high usage of antivirals on the prevalence of drug-resistant HSV-2 under the assumption that drug-resistant strains will be less infectious than drug-sensitive strains of HSV-2 and also have an impaired ability to reactivate. (2 To compare predictions with previous published predictions. Methods We generated theoretical drug-resistant HSV-2 strains that were attenuated (in comparison with drug-sensitive strains in both infectivity and ability to reactivate. We then used a transmission model to predict the emergence and transmission of drug-resistant HSV-2 in the immunocompetent population assuming a high usage of antivirals. Results Our predictions are an order of magnitude lower than previous predictions; we predict that even after 25 years of high antiviral usage only 5 out of 10,000 immunocompetent individuals will be shedding drug-resistant virus. Furthermore, after 25 years, 52 cases of HSV-2 would have been prevented for each prevalent case of drug-resistant HSV-2. Conclusions The predicted levels of drug-resistant HSV-2 for the immunocompetent population are so low that it seems unlikely that cases of drug-resistant HSV-2 will be detected.

  4. [Incidence of herpes virus (HSV-2) alterations in squamous cells of vagina epithelium (author's transl)].

    Science.gov (United States)

    Djelmis, J; Zrnic, S

    1981-01-01

    The authors have analysed 17,519 cytological cervix smears. Alterations characteristic of HSV infection were recorded from squamous cells of vaginal and cervical epithelia in 21 cases (0.12 per cent). Incidences of cytologically detected HSV infection of the vagina and cervix were 0.17 per cent among female factory workers and 0.08 per cent among peasant women. No statistically unambiguous difference was found to exist between the incidences of genital HSV infections in the two groups examined (p greater than 0.05). The majority of women with genital HSV infections was between 21 and 25 years old.

  5. Immune response of T cells during herpes simplex virus type 1 (HSV-1) infection.

    Science.gov (United States)

    Zhang, Jie; Liu, Huan; Wei, Bin

    Herpes simplex virus type 1 (HSV-1), a neurotropic member of the alphaherpes virus family, is among the most prevalent and successful human pathogens. HSV-1 can cause serious diseases at every stage of life including fatal disseminated disease in newborns, cold sores, eye disease, and fatal encephalitis in adults. HSV-1 infection can trigger rapid immune responses, and efficient inhibition and clearance of HSV-1 infection rely on both the innate and adaptive immune responses of the host. Multiple strategies have been used to restrict host innate immune responses by HSV-1 to facilitate its infection in host cells. The adaptive immunity of the host plays an important role in inhibiting HSV-1 infections. The activation and regulation of T cells are the important aspects of the adaptive immunity. They play a crucial role in host-mediated immunity and are important for clearing HSV-1. In this review, we examine the findings on T cell immune responses during HSV-1 infection, which hold promise in the design of new vaccine candidates for HSV-1.

  6. Maternal Antiviral Immunoglobulin Accumulates in Neural Tissue of Neonates To Prevent HSV Neurological Disease

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    Yike Jiang

    2017-07-01

    Full Text Available While antibody responses to neurovirulent pathogens are critical for clearance, the extent to which antibodies access the nervous system to ameliorate infection is poorly understood. In this study on herpes simplex virus 1 (HSV-1, we demonstrate that HSV-specific antibodies are present during HSV-1 latency in the nervous systems of both mice and humans. We show that antibody-secreting cells entered the trigeminal ganglion (TG, a key site of HSV infection, and persisted long after the establishment of latent infection. We also demonstrate the ability of passively administered IgG to enter the TG independently of infection, showing that the naive TG is accessible to antibodies. The translational implication of this finding is that human fetal neural tissue could contain HSV-specific maternally derived antibodies. Exploring this possibility, we observed HSV-specific IgG in HSV DNA-negative human fetal TG, suggesting passive transfer of maternal immunity into the prenatal nervous system. To further investigate the role of maternal antibodies in the neonatal nervous system, we established a murine model to demonstrate that maternal IgG can access and persist in neonatal TG. This maternal antibody not only prevented disseminated infection but also completely protected the neonate from neurological disease and death following HSV challenge. Maternal antibodies therefore have a potent protective role in the neonatal nervous system against HSV infection. These findings strongly support the concept that prevention of prenatal and neonatal neurotropic infections can be achieved through maternal immunization.

  7. Immunization with a dominant-negative recombinant Herpes Simplex Virus (HSV type 1 protects against HSV-2 genital disease in guinea pigs

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    Brans Richard

    2010-06-01

    Full Text Available Abstract Background CJ9-gD is a novel dominant-negative recombinant herpes simplex virus type 1 (HSV-1 that is completely replication-defective, cannot establish detectable latent infection in vivo, and expresses high levels of the major HSV-1 antigen glycoprotein D immediately following infection. In the present study, CJ9-gD was evaluated as a vaccine against HSV-2 genital infection in guinea pigs. Results Animals immunized with CJ9-gD developed at least 700-fold higher titers of HSV-2-specific neutralization antibodies than mock-immunized controls. After challenge with wild-type HSV-2, all 10 control guinea pigs developed multiple genital lesions with an average of 21 lesions per animal. In contrast, only 2 minor lesions were found in 2 of 8 CJ9-gD-immunized animals, representing a 40-fold reduction on the incidence of primary genital lesions in immunized animals (p Conclusions Collectively, we demonstrate that vaccination with the HSV-1 recombinant CJ9-gD elicits strong and protective immune responses against primary and recurrent HSV-2 genital disease and significantly reduces the extent of latent infection.

  8. Characterization of a nucleotide kinase encoded by bacteriophage T7.

    Science.gov (United States)

    Tran, Ngoc Q; Tabor, Stanley; Amarasiriwardena, Chitra J; Kulczyk, Arkadiusz W; Richardson, Charles C

    2012-08-24

    Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ~12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) M obtained with dTTP for dTMP kinase is ~3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) M), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.

  9. Characterization of a Nucleotide Kinase Encoded by Bacteriophage T7*

    Science.gov (United States)

    Tran, Ngoc Q.; Tabor, Stanley; Amarasiriwardena, Chitra J.; Kulczyk, Arkadiusz W.; Richardson, Charles C.

    2012-01-01

    Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ∼12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg2+ in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The Km of 4.4 × 10−4 m obtained with dTTP for dTMP kinase is ∼3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10−4 m), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction. PMID:22761426

  10. Analysis of the SUMO2 Proteome during HSV-1 Infection.

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    Elizabeth Sloan

    2015-07-01

    Full Text Available Covalent linkage to members of the small ubiquitin-like (SUMO family of proteins is an important mechanism by which the functions of many cellular proteins are regulated. Sumoylation has roles in the control of protein stability, activity and localization, and is involved in the regulation of transcription, gene expression, chromatin structure, nuclear transport and RNA metabolism. Sumoylation is also linked, both positively and negatively, with the replication of many different viruses both in terms of modification of viral proteins and modulation of sumoylated cellular proteins that influence the efficiency of infection. One prominent example of the latter is the widespread reduction in the levels of cellular sumoylated species induced by herpes simplex virus type 1 (HSV-1 ubiquitin ligase ICP0. This activity correlates with relief from intrinsic immunity antiviral defence mechanisms. Previous work has shown that ICP0 is selective in substrate choice, with some sumoylated proteins such the promyelocytic leukemia protein PML being extremely sensitive, while RanGAP is completely resistant. Here we present a comprehensive proteomic analysis of changes in the cellular SUMO2 proteome during HSV-1 infection. Amongst the 877 potentially sumoylated species detected, we identified 124 whose abundance was decreased by a factor of 3 or more by the virus, several of which were validated by western blot and expression analysis. We found many previously undescribed substrates of ICP0 whose degradation occurs by a range of mechanisms, influenced or not by sumoylation and/or the SUMO2 interaction motif within ICP0. Many of these proteins are known or are predicted to be involved in the regulation of transcription, chromatin assembly or modification. These results present novel insights into mechanisms and host cell proteins that might influence the efficiency of HSV-1 infection.

  11. HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part I. HSV-1 Structure, Replication and Pathogenesis

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    Andreas Jacobs

    1999-11-01

    Full Text Available The design of effective gene therapy strategies for brain tumors and other neurological disorders relies on the understanding of genetic and pathophysiological alterations associated with the disease, on the biological characteristics of the target tissue, and on the development of safe vectors and expression systems to achieve efficient, targeted and regulated, therapeutic gene expression. The herpes simplex virus type 1 (HSV-1 virion is one of the most efficient of all current gene transfer vehicles with regard to nuclear gene delivery in central nervous system-derived cells including brain tumors. HSV-1-related research over the past decades has provided excellent insight into the structure and function of this virus, which, in turn, facilitated the design of innovative vector systems. Here, we review aspects of HSV-1 structure, replication and pathogenesis, which are relevant for the engineering of HSV-1-based vectors.

  12. Pediatric glioma stem cells: biologic strategies for oncolytic HSV virotherapy.

    Science.gov (United States)

    Friedman, Gregory K; Raborn, Joel; Kelly, Virginia M; Cassady, Kevin A; Markert, James M; Gillespie, G Yancey

    2013-01-01

    While glioblastoma multiforme (GBM) is the most common adult malignant brain tumor, GBMs in childhood represent less than 10% of pediatric malignant brain tumors and are phenotypically and molecularly distinct from adult GBMs. Similar to adult patients, outcomes for children with high-grade gliomas (HGGs) remain poor. Furthermore, the significant morbidity and mortality yielded by pediatric GBM is compounded by neurotoxicity for the developing brain caused by current therapies. Poor outcomes have been attributed to a subpopulation of chemotherapy and radiotherapy resistant cells, termed "glioma stem cells" (GSCs), "glioma progenitor cells," or "glioma-initiating cells," which have the ability to initiate and maintain the tumor and to repopulate the recurring tumor after conventional therapy. Future innovative therapies for pediatric HGG must be able to eradicate these therapy-resistant GSCs. Oncolytic herpes simplex viruses (oHSV), genetically engineered to be safe for normal cells and to express diverse foreign anti-tumor therapeutic genes, have been demonstrated in preclinical studies to infect and kill GSCs and tumor cells equally while sparing normal brain cells. In this review, we discuss the unique aspects of pediatric GSCs, including markers to identify them, the microenvironment they reside in, signaling pathways that regulate them, mechanisms of cellular resistance, and approaches to target GSCs, with a focus on the promising therapeutic, genetically engineered oHSV.

  13. Seroprevalence of serum IgG of HSV-1 coinfected with HIV infected ...

    African Journals Online (AJOL)

    Purpose: To determine the seroprevalence of IgG of HSV-1 coinfected HIV patients who attended Offa General Hospital, Highly Active Antiretroviral Therapy Clinic (HAART). Methods: A cross sectional study used to study the seroprevalence of IgG of HSV-1 coinfected HIV infected patients that attended Offa Highly Active ...

  14. Genital HSV Detection among HIV-1-Infected Pregnant Women in Labor

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    Janna Patterson

    2011-01-01

    Full Text Available Objective. To compare genital HSV shedding among HIV-positive and HIV-negative women. Methods. Women with and without known HIV infection who delivered at the University of Washington Medical Center between 1989–1996 had HSV serologies done as part of clinical care. Genital swabs from HSV-2-seropositive women were evaluated by real-time quantitative HSV DNA PCR. Results. HSV-2 seroprevalence was 71% and 30% among 75 HIV-positive and 3051 HIV-negative women, respectively, (P<.001. HSV was detected at delivery in the genital tract of 30.8% of HIV-seropositive versus 9.5% of HIV-negative women (RR=3.2, 95% CI 1.6 to 6.5, P=.001. The number of virion copies shed per mL was similar (log 3.54 for HIV positive versus 3.90 for HIV negative, P=.99. Conclusions. Our study demonstrated that HIV-, HSV-2-coinfected women are more likely to shed HSV at delivery.

  15. Herpes Simplex Virus (HSV-1 Encephalitis Mimicking Glioblastoma: Case Report and Review of the Literature

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    Burke A. Cunha

    2014-12-01

    Full Text Available Glioblastoma multiforme (GBM often presents as a brain mass with encephalitis. In a patient with GBM, subsequent presentation with new onset encephalitis may be due to another GBM or Herpes simplex virus 1 (HSV-1 encephalitis. We present a case of HSV-1 encephalitis mimicking GBM in a patient with previous GBM.

  16. Almond Skin Extracts Abrogate HSV-1 Replication by Blocking Virus Binding to the Cell

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    Carlo Bisignano

    2017-07-01

    Full Text Available The aim of the present research was to determine the effect of almond skin extracts on herpes simplex virus 1 (HSV-1 replication. Drug-resistant strains of HSV frequently develop following therapeutic treatment. Therefore, the discovery of novel anti-HSV drugs deserves great effort. Here, we tested both natural (NS and blanched (BS polyphenols-rich almond skin extracts against HSV-1. HPLC analysis showed that the prevalent compounds in NS and BS extracts contributing to their antioxidant activity were quercetin, epicatechin and catechin. Results of cell viability indicated that NS and BS extracts were not toxic to cultured Vero cells. Furthermore, NS extracts were more potent inhibitors of HSV-1 than BS extracts, and this trend was in agreement with different concentrations of flavonoids. The plaque forming assay, Western blot and real-time PCR were used to demonstrate that NS extracts were able to block the production of infectious HSV-1 particles. In addition, the viral binding assay demonstrated that NS extracts inhibited HSV-1 adsorption to Vero cells. Our conclusion is that natural products from almond skin extracts are an extraordinary source of antiviral agents and provide a novel treatment against HSV-1 infections.

  17. [Influence of the thymidine phosphorylase (platelet-derived endothelial cell growth factor) on tumor angiogenesis. Catalytic activity of enzyme inhibitors].

    Science.gov (United States)

    Miszczak-Zaborska, Elzbieta; Smolarek, Monika; Bartkowiak, Jacek

    2010-01-01

    Thymidine phosphorylase, also known as platelet-derived endothelial cell growth factor, is a potent angiogenic factor. Thymidine phosphorylase is overexpressed in various human tumors and plays an important role in angiogenesis. A novel inhibitor of thymidine phosphorylase (TP), 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI) is about 1000-fold more active than 6-amino-5-chlorouracil, one of the most potent TP inhibitors to 1999 year. Thymidine phosphorylase is also inhibited by 5'-O-trityl-inosine (KIN59) and related compounds, 2-deoxy-L-ribose and glycosides isolated from the bark of Symplocos racemosa.

  18. Autologous antibody is protective against HSV-1 infection of the immunocompromised mouse.

    Science.gov (United States)

    Alexander, T S; Rosenthal, K S

    1990-09-01

    The protective capability of autologous anti-herpes simplex virus type 1 (anti-HSV-1) antibody was analyzed in immunosuppressed mice. Immunologically naive, immunosuppressed mice infected with a low-passage clinical HSV-1 isolate developed local site lesions, monoplegia, paraplegia, and died within 8 days. Mice that had recovered from a previous HSV-1 infection and were immunosuppressed with cyclophosphamide and then rechallenged with live virus showed no symptoms and survived. Untreated mice that had recovered from infection (primed mice) had high titers of anti-HSV-1 antibody and a delayed type hypersensitivity (DTH) response to virus challenge. Cyclophosphamide treatment, but not lethal irradiation, could ablate the DTH response, resulting in a lack of antivirus cell-mediated immunity. Antibody was the only demonstrable protective immune function in the cyclophosphamide-treated animals. This indicates that cell-mediated immunity is not required for protection against HSV-1 challenge in individuals with virus-specific antibody.

  19. Vaccines and microbicides preventing HIV-1, HSV-2, and HPV mucosal transmission.

    Science.gov (United States)

    Nikolic, Damjan S; Piguet, Vincent

    2010-02-01

    HIV-1, herpes simplex virus type 2 (HSV-2), and human papillomavirus (HPV), among other sexually transmitted infections, represent a major burden for global health. Initial insights into the mucosal transmission of these viral pathogens have raised optimism with regard to the rapid generation of protective vaccines. Nevertheless, setbacks for HIV-1 and HSV-2 vaccines have seriously challenged the initial enthusiasm. Recently, two new vaccines that efficiently prevented HPV infection have renewed the hope that vaccinal prevention of viral mucosal sexually transmitted infections is possible. HIV-1 and HSV-2 differ from HPV, and each virus needs to be tackled with a distinct approach. However, vaccines are not the only possible answer. Topically applied agents (microbicides) are an attractive alternative in the prevention of HIV-1 and HSV-2 mucosal transmission. Progress in understanding the mechanisms of genital transmission of HIV-1 and HSV-2 is required for successful vaccine or microbicide candidates to emerge from current approaches.

  20. Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

    Science.gov (United States)

    Houston, David M J; Bugert, Joachim J; Denyer, Stephen P; Heard, Charles M

    2017-01-01

    There is a clinical need for new therapeutic products against Herpes simplex virus (HSV). The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE) and co-administered zinc (II) ions. PRE was used in conjunction with zinc (II) salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit. Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 μg mL-1) a value comparable to aciclovir (EC50 = 0.18 μg mL-1); however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 μg mL-1), whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution. The potentiated virucidal activity of PRE by coadministered zinc (II) has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

  1. Potentiated virucidal activity of pomegranate rind extract (PRE and punicalagin against Herpes simplex virus (HSV when co-administered with zinc (II ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

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    David M J Houston

    Full Text Available There is a clinical need for new therapeutic products against Herpes simplex virus (HSV. The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE and co-administered zinc (II ions.PRE was used in conjunction with zinc (II salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit.Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 μg mL-1 a value comparable to aciclovir (EC50 = 0.18 μg mL-1; however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 μg mL-1, whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution.The potentiated virucidal activity of PRE by coadministered zinc (II has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

  2. Genital Shedding of Herpes Simplex Virus Among Symptomatic and Asymptomatic Persons with HSV-2 Infection

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    Tronstein, Elizabeth; Johnston, Christine; Huang, Meei-Li; Selke, Stacy; Magaret, Amalia; Warren, Terri; Corey, Lawrence; Wald, Anna

    2011-01-01

    Context Since HSV-2 antibody tests have become commercially available, an increasing number of persons learn that they have genital herpes through serologic testing. The course of natural history of HSV-2 in asymptomatic, seropositive persons is uncertain. Objective To evaluate the virologic and clinical course of HSV genital shedding among participants with symptomatic and asymptomatic HSV-2 infection. Design, Setting and Participants Cohort of 498 immunocompetent HSV-2 seropositive persons enrolled in prospective studies of genital HSV shedding at the University of Washington Virology Research Clinic, Seattle, Washington, and Westover Heights Clinic in Portland, Oregon, between 1992 and 2008. Each participant obtained daily self-collected swabs of genital secretions for ≥ 30 days. Main Outcome Measurement The rate of viral shedding measured by quantitative real-time fluorescence polymerase chain reaction (PCR) for HSV DNA from genital swabs. Results HSV was detected on 4,753 of 23,683 days (20.1%; 95% CI, 18.3 to 22.0) in persons with symptomatic genital HSV-2 infection compared with 519 of 5,070 days (10.2%; 95% CI, 7.7 to 13.6) in persons with asymptomatic infection, pgenital viral shedding among persons with symptomatic genital HSV-2 infection compared with 85 of 519 days (16.4%; 95% CI, 11.2 to 23.9) among persons with asymptomatic infection, pgenital tract less frequently than persons with symptomatic infection, but much of the difference is attributable to less frequent genital lesions, as lesions are accompanied by frequent viral shedding. PMID:21486977

  3. Herpes simplex virus serotype and entry receptor availability alter CNS disease in a mouse model of neonatal HSV.

    Science.gov (United States)

    Kopp, Sarah J; Ranaivo, Hantamalala R; Wilcox, Douglas R; Karaba, Andrew H; Wainwright, Mark S; Muller, William J

    2014-12-01

    Outcomes of neonates with herpes simplex virus (HSV) encephalitis are worse after infection with HSV-2 when compared with HSV-1. The proteins herpes virus entry mediator (HVEM) and nectin-1 mediate HSV entry into susceptible cells. Prior studies have shown receptor-dependent differences in pathogenesis that depend on route of inoculation and host developmental age. We investigated serotype-related differences in HSV disease and their relationship to entry receptor availability in a mouse model of encephalitis. Mortality was attenuated in 7-d-old, wild-type (WT) mice inoculated with HSV-1(F) when compared with HSV-2(333). No serotype-specific differences were seen after inoculation of adult mice. HSV-1 pathogenesis was also attenuated relative to HSV-2 in newborn but not adult mice lacking HVEM or nectin-1. HSV-2 requires nectin-1 for encephalitis in adult but not newborn mice; in contrast, nectin-1 was important for HSV-1 pathogenesis in both age groups. Early viral replication was independent of age, viral serotype, or mouse genotype, suggesting host responses influence outcomes. In this regard, significantly greater amounts of inflammatory mediators were detected in brain homogenates from WT newborns 2 d after infection compared with adults and receptor-knockout newborns. Dysregulation of inflammatory responses induced by infection may influence the severity of HSV encephalitis.

  4. Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases.

    Science.gov (United States)

    Sandrini, Michael Paolo Bastner; Söderbom, Fredrik; Mikkelsen, Nils Egil; Piskur, Jure

    2007-06-08

    The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.

  5. OBSERVATIONS ON THE UPTAKE OF TRITIATED THYMIDINE IN THE PRONUCLEI OF FERTILIZED SAND DOLLAR EMBRYOS

    Science.gov (United States)

    Simmel, Eva B.; Karnofsky, David A.

    1961-01-01

    Following fertilization of the egg of the sand dollar Echinarachnius parma, tritiated thymidine (H3TDR) was taken up independently by the male and female pronuclei beginning within about 15 to 20 minutes, and the labeled pronuclei fused at about 30 to 40 minutes. At cleavage 90 minutes later the labeled nuclear material was distributed to both daughter cells. Unfertilized eggs and sperm exposed to H3TDR did not show nuclear localization of thymidine. DNA replication, thus, is initiated in the haploid pronuclei shortly after fertilization and prior to fusion. The major portion of DNA synthesis, as evidenced by thymidine uptake, appears to be during a 20 to 30 minute period after fertilization. Fertilization is associated with the activation of a mechanism which initiates early and independent replication of DNA in both the male and female pronuclei. PMID:19866590

  6. Comparison of thymidine phosphorylase expression and prognostic factors in gallbladder and bile duct cancer

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    You Young

    2010-10-01

    Full Text Available Abstract Background Biliary tract cancers have limitations in information about different location-related pathogenesis and clinico-pathological characteristics. The goal of this study was to investigate anatomical site-related similarities and differences in biliary tract cancers and to assess the expression and clinical significance of functional proteins such as p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1. Methods One hundred and sixty-one patients with biliary tract adenocarcinomas, who underwent curative or palliative surgery in a single institution between October 1994 and December 2003 were evaluated, retrospectively. The level of protein expression of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 was assessed by immunohistochemistry. Results With respect to clinico-pathological characteristics, gallbladder cancer was more frequent in women, and bile duct cancer was more common in men. Perineural invasion was more common in bile duct cancer. Recurrence as a distant metastasis was more common in gallbladder cancer. Immunohistochemical analysis revealed that thymidine phosphorylase expression was significantly higher in gallbladder cancer than in bile duct cancer. Positive thymidine phosphorylase and p53 staining were associated with an advanced stage. Differentiation, vascular invasion, perineural invasion, lymphatic invasion, lymph node metastasis, and TNM stage independently predicted poor prognosis in biliary tract cancer. These correlations were seen more clearly in gallbladder cancer. The immunohistochemical staining patterns of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 showed no prognostic significance in biliary tract cancers. Conclusions We concluded that gallbladder and bile duct cancers are considered to be separate diseases with different clinico-pathological characteristics and prognostic factors. In addition, we hypothesize that high expression of thymidine phosphorylase by

  7. ATYPICAL CSF PICTURE IN VIRAL MENINGITIS HSV- TYPE-2

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    Vikram

    2016-05-01

    Full Text Available INTRODUCTION Acute infections of nervous system are among the most important problems in medicine because early recognition, efficient decision making and rapid institution of therapy can be lifesaving. Making a clinical diagnosis of acute meningitis depends on the cornerstone of cerebrospinal fluid (CSF examination. We present a case with the above-mentioned difficulty and the approach involved in establishing the exact diagnosis and institution of appropriate treatment. CONCLUSION About findings in viral meningitis one should be careful while evaluating a CSF report so as to not make a mistaken diagnosis and delay treatment. The most important analysis in patients whose symptoms are consistent with herpes simplex meningitis is the detection of Herpes simplex Virus deoxy-ribo-nucleic acid (HSV-DNA in CSF with Polymerase Chain Reaction (PCR.

  8. The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions.

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    Svetlana Gershburg

    Full Text Available Herpes simplex virus type 1 (HSV-1 encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP, respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD, release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.

  9. Rabbit and Mouse Models of HSV-1 Latency, Reactivation, and Recurrent Eye Diseases

    Science.gov (United States)

    Webre, Jody M.; Hill, James M.; Nolan, Nicole M.; Clement, Christian; McFerrin, Harris E.; Bhattacharjee, Partha S.; Hsia, Victor; Neumann, Donna M.; Foster, Timothy P.; Lukiw, Walter J.; Thompson, Hilary W.

    2012-01-01

    The exact mechanisms of HSV-1 establishment, maintenance, latency, reactivation, and also the courses of recurrent ocular infections remain a mystery. Comprehensive understanding of the HSV-1 disease process could lead to prevention of HSV-1 acute infection, reactivation, and more effective treatments of recurrent ocular disease. Animal models have been used for over sixty years to investigate our concepts and hypotheses of HSV-1 diseases. In this paper we present descriptions and examples of rabbit and mouse eye models of HSV-1 latency, reactivation, and recurrent diseases. We summarize studies in animal models of spontaneous and induced HSV-1 reactivation and recurrent disease. Numerous stimuli that induce reactivation in mice and rabbits are described, as well as factors that inhibit viral reactivation from latency. The key features, advantages, and disadvantages of the mouse and rabbit models in relation to the study of ocular HSV-1 are discussed. This paper is pertinent but not intended to be all inclusive. We will give examples of key papers that have reported novel discoveries related to the review topics. PMID:23091352

  10. Avidity of Antibodies against HSV-2 and Risk to Neonatal Transmission among Mexican Pregnant Women

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    Antonia Herrera-Ortiz

    2013-01-01

    Full Text Available Objective. To determine HSV-2 seroprevalence, risk factors, and antibody avidity among a sample of Mexican pregnant women. Material and Methods. The avidity test was standardized with different urea concentrations and incubation times; the cut-off point was calculated to determine the low avidity (early infection. IgG antibodies against HSV-2 were detected from pregnant and postpartum women from Morelos, Mexico, and the avidity test was performed to positive samples. Multivariate regression logistic analysis was employed to evaluate demographic and sexual behavior characteristics associated with HSV-2 infection. Results. HSV-2 seroprevalence among Mexican women analyzed was 14.5% (333/2300, demographic factors (location of General Hospital, age, education level, and civil status, and risky sexual behaviors (STI self-report and number of sexual partners during last year were associated with HSV-2 infection. Seventeen women were detected with low avidity antibodies (early infection with a cut-off point of 66.1%. Conclusions. HSV-2 infection was common among this group of women from Mexico; the avidity test detected women with recent infections, and these women were more likely to transmit HSV-2 to their neonates. Neonatal herpes has no epidemiological surveillance, the disease could be overlooked, and so more studies are needed to estimate the magnitude of neonatal infection.

  11. The effect of cellular differentiation on HSV-1 infection of oligodendrocytic cells.

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    Raquel Bello-Morales

    Full Text Available Herpes simplex type 1 (HSV-1 is a neurotropic virus that infects many types of cells. Previous studies have demonstrated that oligodendrocytic cells are highly susceptible to HSV-1 infection. Here we analysed HSV-1 infection of a human oligodendrocytic cell line, HOG, and oligodendrocyte precursor cells (OPCs cultured under growth or differentiation conditions. In addition to cell susceptibility, the role of the major cell receptors for viral entry was assessed. Our results revealed that OPCs and HOG cells cultured under differentiation conditions became more susceptible to HSV-1. On the other hand, viral infection induced morphological changes corresponding to differentiated cells, suggesting that HSV-1 might be inducing cell differentiation. We also observed colocalization of HVEM and nectin-1 with viral particles, suggesting that these two major HSV-1 receptors are functional in HOG cells. Finally, electron microscopy assays indicated that HSV-1 may be also entering OLs by macropinocytosis depending on their differentiation stage. In addition, vesicles containing intracellular enveloped virions observed in differentiated cells point to an endocytic mechanism of virus entry. All these data are indicative of diverse entry pathways dependent on the maturation stage of OLs.

  12. HSV presence in brains of individuals without dementia: the TASTY brain series

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    Jan Olsson

    2016-11-01

    Full Text Available Herpes simplex virus (HSV type 1 affects a majority of the population and recent evidence suggests involvement in Alzheimer's disease aetiology. We investigated the prevalence of HSV type 1 and 2 in the Tampere Autopsy Study (TASTY brain samples using PCR and sero-positivity in plasma, and associations with Alzheimer's disease neuropathology. HSV was shown to be present in human brain tissue in 11/584 (1.9% of samples in the TASTY cohort, of which six had Alzheimer's disease neuropathological amyloid beta (Aβ aggregations. Additionally, serological data revealed 86% of serum samples tested were IgG-positive for HSV. In conclusion, we report epidemiological evidence of the presence of HSV in brain tissue free from encephalitis symptoms in a cohort most closely representing the general population (a minimum prevalence of 1.9%. Whereas 6/11 samples with HSV DNA in the brain tissue had Aβ aggregations, most of those with Aβ aggregations did not have HSV present in the brain tissue.

  13. HSV-2 meningoencephalitis in an immunocompetent young man: what is the pathogenesis and what is the treatment?

    Science.gov (United States)

    Fernandez-Gerlinger, Mp; Greffe, S; Meffre, A; Grenet, J; Au, S; Bojanova, M; Rouveix, E; Rozenberg, F

    2015-08-01

    Herpes simplex encephalitis is rarely caused by herpes simplex virus type 2 (HSV-2) after the neonatal period. The pathogenesis of HSV-2 encephalitis is not known and its treatment has not been discussed. We report a case of mild meningoencephalitis secondary to HSV-2 primary infection after sexual risk behaviour in a healthy young man. The diagnosis was established upon clinical, biological and electroencephalographic criteria. Aciclovir treatment led to rapid clinical improvement. This case highlights HSV-2 as a rare cause of meningoencephalitis, and questions the management of this rare manifestation of HSV-2 infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Evaluation of cell cytotoxicity after ganciclovir treatment by radioiodinated IVDU

    Energy Technology Data Exchange (ETDEWEB)

    Lee, M. J.; Choi, T. H.; Woo, K. S. [Korean Institute of Radiological And Medical Sciences, Seoul (Korea, Republic of)] (and others)

    2005-07-01

    The herpes simplex virus type1 thymidine kinase(HSV1-tk) converts nontoxic nucleoside analogs such as ganciclovir into phosphorylated compounds that act as chain terminators and specially kill dividing cells. Unlike mammalian TK, HSV1-TK which is a nonspecific nucleoside kinase, is encoded by a viral gene that is not present in normal mammalian cells. Various radiolabelled nucleoside analogues are used as specific probes for HSV1-tk and can be freely transported across cell membranes. When phosphorylated by the tranduced HSV1-tk gene, the metabolites of probes subsequently accumulate within the transduced cells.

  15. DNA vaccine-encoded glycoprotein B of HSV-1 fails to protect chronic morphine-treated mice against HSV-1 challenge.

    Science.gov (United States)

    Jamali, Abbas; Roostaee, Mohammad H; Soleimanjahi, Hoorieh; Ghaderi Pakdel, Firouz; Bamdad, Taravat

    2007-03-01

    The use of morphine has been demonstrated to increase susceptibility to infections. Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen among immunocompromised individuals. In the present study, due to the importance of HSV vaccination in morphine abusers, the effects of chronic morphine exposure on the host response to a HSV-1 gB DNA-based vaccine have been investigated. The study is addressing an important aspect of vaccine development among the susceptible (immunocompromised) hosts. BALB/c mice were exposed to morphine over 11 days. They were then vaccinated with DNA vaccine or KOS strain as a live vaccine. The findings showed that the morphine-treated animals failed to respond to DNA vaccination evaluated by the anti-HSV gB antibody titer, delayed type hypersensitivity (DTH) and lethal HSV-1 challenge. Under the same conditions, the KOS vaccine showed a reduced Ab titer and DTH response in morphine-treated mice, but could protect mice against the lethal challenge and was safe for vaccination of morphine-treated animals.

  16. In Situ Detection of Regulatory T Cells in Human Genital Herpes Simplex Virus Type 2 (HSV-2) Reactivation and Their Influence on Spontaneous HSV-2 Reactivation.

    Science.gov (United States)

    Milman, Neta; Zhu, Jia; Johnston, Christine; Cheng, Anqi; Magaret, Amalia; Koelle, David M; Huang, Meei-Li; Jin, Lei; Klock, Alexis; Layton, Erik D; Corey, Lawrence

    2016-07-01

    Herpes simplex virus type 2 (HSV-2) reactivation is accompanied by a sustained influx of CD4(+) and CD8(+) T cells that persist in genital tissue for extended periods. While CD4(+) T cells have long been recognized as being present in herpetic ulcerations, their role in subclinical reactivation and persistence is less well known, especially the role of CD4(+) regulatory T cells (Tregs). We characterized the Treg (CD4(+)Foxp3(+)) population during human HSV-2 reactivation in situ in sequential genital skin biopsy specimens obtained from HSV-2-seropositive subjects at the time of lesion onset up to 8 weeks after healing. High numbers of Tregs infiltrated to the site of viral reactivation and persisted in proximity to conventional CD4(+) T cells (Tconvs) and CD8(+) T cells. Treg density peaked during the lesion stage of the reactivation. The number of Tregs from all time points (lesion, healed, 2 weeks after healing, 4 weeks after healing, and 8 weeks after healing) was significantly higher than in control biopsy specimens from unaffected skin. There was a direct correlation between HSV-2 titer and Treg density. The association of a high Treg to Tconv ratio with high viral shedding suggests that the balance between regulatory and effector T cells influences human HSV-2 disease. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  17. Bacterial incorporation of tritiated thymidine and populations of bacteriophagous fauna in the rhizosphere of wheat

    DEFF Research Database (Denmark)

    Christensen, Henrik; Griffiths, Bryan; Christensen, Søren

    1992-01-01

    Bacterial and microfaunal populations, and bacterial productivity measured by tritiated thymidine (3HTdr) incorporation, in the rhizosphere of wheat seedlings were measured. Soil from planted pots was fractionated into rhizosphere and non-rhizosphere (bulk) soil, while unplanted soil was taken fr...

  18. Radio diagnosis using 125I-fibrinogen and 203Hg-thymidine.

    Science.gov (United States)

    Zimmermann, M; Schmutz, H

    1976-12-30

    A comparative study of 125I-fibrinogen and 203Hg-thymidine using 144 Ehrlich carcinoma-bearing mice, indicates that tumor affinity of carrier substances with covalent labeling could be better used for scintigraphic tumor diagnosis. Halogenated or possibly metallo-organic compounds offer an alternative to the metal complexes in general use up to now.

  19. Microwave-assisted synthesis and biological evaluation of novel uracil derivatives inhibiting human thymidine phosphorylase.

    Science.gov (United States)

    Corelli, Federico; Botta, Maurizio; Lossani, Andrea; Pasquini, Serena; Spadari, Silvio; Focher, Federico

    2004-12-01

    New 5-chloro-6-substituted-uracil derivatives have been prepared by microwave assisted-synthesis and tested in vitro as thymidine phosphorylase inhibitors. One of these compounds showed potent inhibitory activity, with an IC50 value in the submicromolar range. The biological activity of the new compounds is discussed in terms of structure-activity relationship.

  20. Mechanism of inhibition of HSV-1 replication by tumor necrosis factor and interferon gamma.

    Science.gov (United States)

    Feduchi, E; Carrasco, L

    1991-02-01

    Tumor necrosis factor (TNF) synergizes with interferon (IFN gamma) in the blockade of HSV-1 replication. Antibodies against IFN beta block this synergism, implying a role of IFN beta in the antiviral activity of TNF plus IFN gamma. IFN beta 1 added exogenously to Hep-2 cells shows antiviral activity against HSV-1 only at high concentrations, whereas IFN beta 2 (also known as IL-6) alone has no effect on the replication of VSV or HSV-1 even when 1,000 U/ml are present. Our results are in accordance with the idea that TNF induces IFN beta 1 and that both cytokines must be present in the culture medium to synergize with IFN gamma in order to inhibit HSV-1 replication.

  1. Inhibition of HIV and HSV infection by vaginal lactobacilli in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Zabihollahi Rezvan

    2012-10-01

    Full Text Available Abstract Background and the purpose of the study The cervico-vaginal mucosa which is populated with microflora (mostly includes lactobacilli is the portal of entry for sexually transmitted pathogens. Methods The in vitro anti-viral effect of vaginal and non-vaginal lactobacillus was evaluated using single cycle HIV-1 replication and HSV-2 plaque reduction assays. The XTT proliferation assay was used to monitor the cellular toxicity. The in vivo anti-HSV-1 activity was evaluated in BALB/c mouse model by monitoring skin lesion and immune response development. Results and major conclusion DMEM culture supernatant of L. Gasseri and L. fermentum (PH 7.3 did not show toxic effect but inhibited 50% of HIV replication at 12 and 31% concentrations, respectively. Co-culture of L. gasseri (1000 CFU/ target cell showed mild cytotoxicity but inhibited 68% of HIV replication. The supernatant of L. crispatus inhibited 50% of HSV replication at 4% and also co-culture of L. gasseri, L. rhamnosus and L. crispatus revokes almost all of the HSV multiplication. Culture supernatants of L. gasseri and L. crispatus had significant virucidal effect against the HIV and HSV and inhibited HSV infection in a stage before viral entry to the target cells. Alive L. gasseri cells showed high potential for inhibiting HSV-1 infection in vivo condition. Current data indicates that lactobacilli supernatant encompasses components with neutralizing activity against HIV and HSV and it would be a determinant factor for viral diseases transmission and promising lead for anti-viral probiotic design.

  2. Inhibition of HIV and HSV infection by vaginal lactobacilli in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Modarressi

    2012-10-01

    Full Text Available Background and the purpose of the study The cervico-vaginal mucosa which is populated with microflora (mostly includes lactobacilli is the portal of entry for sexually transmitted pathogens.MethodsThe in vitro anti-viral effect of vaginal and non-vaginal lactobacillus was evaluated using single cycle HIV-1 replication and HSV-2 plaque reduction assays. The XTT proliferation assay was used to monitor the cellular toxicity. The in vivo anti-HSV-1 activity was evaluated in BALB/c mouse model by monitoring skin lesion and immune response development. Results and major conclusion DMEM culture supernatant of L. Gasseri and L. fermentum (PH 7.3 did not show toxic effect but inhibited 50% of HIV replication at 12 and 31% concentrations, respectively. Coculture of L. gasseri (1000 CFU/ target cell showed mild cytotoxicity but inhibited 68% of HIV replication. The supernatant of L. crispatus inhibited 50% of HSV replication at 4% and also co-culture of L. gasseri, L. rhamnosus and L. crispatus revokes almost all of the HSV multiplication. Culture supernatants of L. gasseri and L. crispatus had significant virucidal effect against the HIV and HSV and inhibited HSV infection in a stage before viral entry to the target cells. Alive L. gasseri cells showed high potential for inhibiting HSV-1 infection in vivo condition. Current data indicates that lactobacilli supernatant encompasses components with neutralizing activity against HIV and HSV and it would be a determinant factor for viral diseases transmission and promising lead for anti-viral probiotic design.

  3. Inhibitory effect of aqueous extract of Echinacea purpurea and Nerium oleander on HSV-1 multiplication

    OpenAIRE

    Maliheh Farahani

    2014-01-01

    Background: Treatment of HSV-1 infections with the available chemical drugs may have some problems such as drug resistance and virus latency. Therefore, there is a requirement for new antiherpes drugs in today's world. The present study was carried out to analyze the inhibitory effect of Echinacea purpurea and Nerium oleander plants with ethnomedical background on HSV-1 replication. Methods: Plants were extracted by decoction method to obtain aqueous extract. These extracts were screened f...

  4. Assessment of Anti HSV-1 Activity of Aloe Vera Gel Extract: an In Vitro Study.

    Science.gov (United States)

    Rezazadeh, Fahimeh; Moshaverinia, Maryam; Motamedifar, Mohammad; Alyaseri, Montazer

    2016-03-01

    Herpes simplex virus (HSV) infection is one of the most common and debilitating oral diseases; yet, there is no standard topical treatment to control it. The extract of Aloe vera leaves has been previously reported to have anti-inflammatory, antibacterial, and also antiviral effects. There is no data on anti-Herpes simplex virus type 1 (HSV-1) activity of Aloe vera gel. This study aimed to evaluate the anti-HSV-1 activity of Aloe vera gel in Vero cell line. In this study, gel extraction and cytotoxicity of various increasing concentrations of Aloe vera gel (0.2, 0.5, 1, 2, and 5%) was evaluated in Dulbecco's Modified Eagle Medium (DMEM) containing 2% fetal bovine serum (FBS). Having been washed with phosphate buffered saline, 50 plaque-forming units (PFU) of HSV-1 was added to each well. After 1 hour of incubation at 37°C, cell monolayers in 24 well plates were exposed to different increasing concentrations of Aloe vera gel. The anti-HSV-1 activity of Aloe vera gel in different concentrations was assessed by plaque reduction assays. Data were analyzed by using One-way ANOVA. The cytotoxicity assay showed that Aloe vera in prearranged concentrations was cell-compatible. The inhibitory effect of various concentrations of Aloe vera was observed one hour after the Vero cell was infected with HSV-1. However, there was no significant difference between two serial concentrations (p> 0.05). One-way ANOVA also revealed no significant difference between the groups. The findings indicated a dose-dependent antiviral effect of Aloe vera. The findings showed significant inhibitory effect of 0.2-5% Aloe vera gel on HSV-1 growth in Vero cell line. Therefore, this gel could be a useful topical treatment for oral HSV-1 infections without any significant toxicity.

  5. Characterization of Wild-type and Temperature Sensitive Mutants of HSV-1 DNA Polymerase

    Science.gov (United States)

    1988-08-15

    two genomic isomeric arrangements [Ben-Porat , 1979; Plummer, 1973] o Virus: Pseudorabies Virus (Suid herpesvirus 1) Equine Herpesvirus (Equid...Huang, 1975], equine herpesvirus [Cohen et al., 1975; Kemp et al., 1975) and HSV [Keir & Gold, 1963]. Since then, several herpesvirus DNA polymerases... hypersensitive to the inhibitor PAA, based on an in vivo plaque reduction assay, when compared to the levels of PAA needed to inhibit HSV-1 KOS plaque

  6. Plasmacytoid dendritic cells contribute to systemic but not local antiviral responses to HSV infections.

    Directory of Open Access Journals (Sweden)

    Melissa Swiecki

    2013-10-01

    Full Text Available Plasmacytoid dendritic cells (pDC produce type I interferons (IFN-I and proinflammatory cytokines in response to viruses; however, their contribution to antiviral immunity in vivo is unclear. In this study, we investigated the impact of pDC depletion on local and systemic antiviral responses to herpes simplex virus (HSV infections using CLEC4C-DTR transgenic mice. We found that pDC do not appear to influence viral burden or survival after vaginal HSV-2 infection, nor do they seem to contribute to virus-specific CD8 T cell responses following subcutaneous HSV-1 infection. In contrast, pDC were important for early IFN-I production, proinflammatory cytokine production, NK cell activation and CD8 T cell responses during systemic HSV-2 and HSV-1 infections. Our data also indicate that unlike pDC, TLR3-expressing cells are important for promoting antiviral responses to HSV-1 regardless of the route of virus administration.

  7. Herpes Simplex Vaccines: Prospects of Live-attenuated HSV Vaccines to Combat Genital and Ocular infections

    Science.gov (United States)

    Stanfield, Brent; Kousoulas, Konstantin Gus

    2015-01-01

    Herpes simplex virus type-1 (HSV-1) and its closely related type-2 (HSV-2) viruses cause important clinical manifestations in humans including acute ocular disease and genital infections. These viruses establish latency in the trigeminal ganglionic and dorsal root neurons, respectively. Both viruses are widespread among humans and can frequently reactivate from latency causing disease. Currently, there are no vaccines available against herpes simplex viral infections. However, a number of promising vaccine approaches are being explored in pre-clinical investigations with few progressing to early phase clinical trials. Consensus research findings suggest that robust humoral and cellular immune responses may partially control the frequency of reactivation episodes and reduce clinical symptoms. Live-attenuated viral vaccines have long been considered as a viable option for generating robust and protective immune responses against viral pathogens. Varicella zoster virus (VZV) belongs to the same alphaherpesvirus subfamily with herpes simplex viruses. A live-attenuated VZV vaccine has been extensively used in a prophylactic and therapeutic approach to combat primary and recurrent VZV infection indicating that a similar vaccine approach may be feasible for HSVs. In this review, we summarize pre-clinical approaches to HSV vaccine development and current efforts to test certain vaccine approaches in human clinical trials. Also, we discuss the potential advantages of using a safe, live-attenuated HSV-1 vaccine strain to protect against both HSV-1 and HSV-2 infections. PMID:27114893

  8. Experimental Oral Herpes Simplex Virus-1 (HSV-1 Co-infection in Simian Immunodeficiency Virus (SIV-Infected Rhesus Macaques

    Directory of Open Access Journals (Sweden)

    Meropi Aravantinou

    2017-12-01

    Full Text Available Herpes simplex virus 1 and 2 (HSV-1/2 similarly initiate infection in mucosal epithelia and establish lifelong neuronal latency. Anogenital HSV-2 infection augments the risk for sexual human immunodeficiency virus (HIV transmission and is associated with higher HIV viral loads. However, whether oral HSV-1 infection contributes to oral HIV susceptibility, viremia, or oral complications of HIV infection is unknown. Appropriate non-human primate (NHP models would facilitate this investigation, yet there are no published studies of HSV-1/SIV co-infection in NHPs. Thus, we performed a pilot study for an oral HSV-1 infection model in SIV-infected rhesus macaques to describe the feasibility of the modeling and resultant immunological changes. Three SIV-infected, clinically healthy macaques became HSV-1-infected by inoculation with 4 × 108 pfu HSV-1 McKrae on buccal, tongue, gingiva, and tonsils after gentle abrasion. HSV-1 DNA was shed in oral swabs for up to 21 days, and shedding recurred in association with intra-oral lesions after periods of no shedding during 56 days of follow up. HSV-1 DNA was detected in explant cultures of trigeminal ganglia collected at euthanasia on day 56. In the macaque with lowest baseline SIV viremia, SIV plasma RNA increased following HSV-1 infection. One macaque exhibited an acute pro-inflammatory response, and all three animals experienced T cell activation and mobilization in blood. However, T cell and antibody responses to HSV-1 were low and atypical. Through rigorous assessesments, this study finds that the virulent HSV-1 strain McKrae resulted in a low level HSV-1 infection that elicited modest immune responses and transiently modulated SIV infection.

  9. FASTSAT-HSV01 Thermal Math Model Correlation

    Science.gov (United States)

    McKelvey, Callie

    2011-01-01

    This paper summarizes the thermal math model correlation effort for the Fast Affordable Science and Technology SATellite (FASTSAT-HSV01), which was designed, built and tested by NASA's Marshall Space Flight Center (MSFC) and multiple partners. The satellite launched in November 2010 on a Minotaur IV rocket from the Kodiak Launch Complex in Kodiak, Alaska. It carried three Earth science experiments and two technology demonstrations into a low Earth circular orbit with an inclination of 72deg and an altitude of 650 kilometers. The mission has been successful to date with science experiment activities still taking place daily. The thermal control system on this spacecraft was a passive design relying on thermo-optical properties and six heaters placed on specific components. Flight temperature data is being recorded every minute from the 48 Resistance Temperature Devices (RTDs) onboard the satellite structure and many of its avionics boxes. An effort has been made to correlate the thermal math model to the flight temperature data using Cullimore and Ring's Thermal Desktop and by obtaining Earth and Sun vector data from the Attitude Control System (ACS) team to create an "as-flown" orbit. Several model parameters were studied during this task to understand the spacecraft's sensitivity to these changes. Many "lessons learned" have been noted from this activity that will be directly applicable to future small satellite programs.

  10. The case for immunomodulatory approaches in treating HSV encephalitis.

    Science.gov (United States)

    Ramakrishna, Chandran; Openshaw, Harry; Cantin, Edouard M

    2013-03-01

    HSV encephalitis (HSE) is the most prevalent sporadic viral encephalitis. Although safe and effective antiviral therapies and greatly improved noninvasive diagnostic procedures have significantly improved outcomes, mortality (~20%) and debilitating neurological sequelae in survivors remain unacceptably high. An encouraging new development is that the focus is now shifting away from the virus exclusively, to include consideration of the host immune response to infection in the pathology underlying development of HSE. In this article, the authors discuss results from recent studies in experimental mouse models, as well as clinical reports that demonstrate a role for exaggerated host inflammatory responses in the brain in the development of HSE that is motivating researchers and clinicians to consider new therapeutic approaches for treating HSE. The authors also discuss results from a few studies that have shown that immunomodulatory drugs can be highly protective against HSE, which supports a role for deleterious host inflammatory responses in HSE. The impressive outcomes of some immunomodulatory approaches in mouse models of HSE emphasize the urgent need for clinical trials to rigorously evaluate combination antiviral and immunomodulatory therapy in comparison with standard antiviral therapy for treatment of HSE, and support for such an initiative is gaining momentum.

  11. Pediatric glioma stem cells: biologic strategies for oncolytic HSV virotherapy

    Directory of Open Access Journals (Sweden)

    Gregory K Friedman

    2013-02-01

    Full Text Available While glioblastoma multiforme (GBM is the most common adult malignant brain tumor, GBMs in childhood represent less than 10% of pediatric malignant brain tumors and are phenotypically and molecularly distinct from adult GBMs. Similar to adult patients, outcomes for children with high-grade gliomas (HGGs remain poor. Furthermore, the significant morbidity and mortality yielded by pediatric GBM is compounded by neurotoxicity for the developing brain caused by current therapies. Poor outcomes have been attributed to a subpopulation of chemotherapy and radiotherapy resistant cells, termed ‘glioma stem cells’ (GSCs, ‘glioma progenitor cells’, or ‘glioma-initiating cells', which have the ability to initiate and maintain the tumor and to repopulate the recurring tumor after conventional therapy. Future innovative therapies for pediatric HGGs must be able to eradicate these therapy-resistant GSCs. Oncolytic herpes simplex viruses, genetically engineered to be safe for normal cells and to express diverse foreign anti-tumor therapeutic genes, have been demonstrated in preclinical studies to infect and kill GSCs and tumor cells equally while sparing normal brain cells. In this review, we discuss the unique aspects of pediatric GSCs, including markers to identify them, the microenvironment they reside in, signaling pathways that regulate them, mechanisms of cellular resistance, and approaches to target GSCs, with a focus on the promising therapeutic, genetically engineered oncolytic herpes simplex virus (HSV.

  12. Prevalence, incidence and correlates of HSV-2 infection in an HIV incidence adolescent and adult cohort study in western Kenya.

    Directory of Open Access Journals (Sweden)

    Brenda Akinyi

    Full Text Available Herpes simplex virus type 2 (HSV-2 infections are associated with increased risk of HIV transmission. We determined HSV-2 prevalence, incidence and associated risk factors, incidence among persons with indeterminate results, and prevalence of HSV-2/HIV co-infection among young adults (18-34 years and adolescents (16-17 years enrolled in an HIV incidence cohort study in western Kenya.Participants (n = 1106; 846 adults were screened and those HIV-1 negative were enrolled and followed-up quarterly for one year. HSV-2 was assessed using the Kalon enzyme immunoassay. HSV-2 incidence was calculated separately among HSV-2 seronegative participants and those indeterminate at baseline. Logistic regression was used to estimate the odds of HSV-2 infection and Poisson regression was used to assess HSV-2 incidence and associated factors.Overall, HSV-2 prevalence was 26.6% [95% confidence interval (CI: 23.9-29.4] and was higher in adults (31.5% [95% CI: 28.3-34.9] than adolescents (10.7% [95% CI: 7.1-15.3]. Factors associated with prevalent HSV-2 included female gender, increasing age, HIV infection, history of sexually transmitted infection, low level of education, multiple sexual partners, and being married, divorced, separated or widowed. Overall HSV-2 incidence was 4.0 per 100 person-years (/100PY 95% CI: 2.7-6.1 and was higher in adults (4.5/100PY and females (5.1/100PY. In multivariable analysis only marital status was associated with HSV-2 incidence. Among 45 participants with indeterminate HSV-2 results at baseline, 22 seroconverted, resulting in an incidence rate of 53.2 /100PY [95% CI: 35.1-80.9]. Inclusion of indeterminate results almost doubled the overall incidence rate to 7.8 /100 PY [95% CI: 5.9-10.5]. Prevalence of HIV/HSV-2 co-infection was higher in female adults than female adolescents (17.1 [95% CI: 13.6-21.0] versus 3.4 [95% CI: 1.1-7.8].The high incidence rate among persons with indeterminate results underscores the public health

  13. False-negative type-specific glycoprotein G antibody responses in STI clinic patients with recurrent HSV-1 or HSV-2 DNA positive genital herpes, The Netherlands

    NARCIS (Netherlands)

    van Rooijen, Martijn S.; Roest, Wim; Hansen, Gino; Kwa, David; de Vries, Henry J. C.

    2016-01-01

    Herpes simplex virus (HSV) type-discriminating antibody tests (glycoprotein G (gG) directed) are used to identify naïve persons and differentiate acute infections from recurrences. We studied test characteristics of three commercially available antibody tests in patients with recurrent (established

  14. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    The present review on casein kinases focuses mainly on the possible metabolic role of CK-2, with special emphasis on its behavior in pathological tissues. From these data at least three ways to regulate CK-2 activity emerge: (i) CK-2 activity changes during embryogenesis, being high at certain...

  15. Detection of herpes simplex virus-related antigens in the nuclei and cytoplasm of biochemically transformed cells with peroxidase/anti-peroxidase immunological staining and indirect immunofluorescence.

    Science.gov (United States)

    Kurchak, M; Dubbs, D R; Kit, S

    1977-09-15

    Herpes simplex virus (HSV)-related antigens have been demonstrated in the nuclei and cytoplasm of human and mouse cells biochemically transformed by ultraviolet light-irradiated HSV. This was accomplished by using peroxidase/anti-peroxidase immunological staining and indirect immunofluorescence with rabbit antisera that had high neutralizing titers against the HSV-specific thymidine kinase activity and virus infectivity. HSV-1 antisera reacted with antigens in cells biochemically transformed by type 1 HSV, but not with those of cells biochemically transformed by type 2 HSV. Similarly, HSV-2 antisera reacted with antigens in cells biochemically transformed by HSV-2, but not with those in cells biochemically transformed by HSV-1. In contrast, herpes virus-related antigens were detected in cells cytolytically infected with HSV-1 and with HSV-2 by either type 1 or type 2 HSV antisera. These observations suggest that the antigens detected in the biochemically transformed cells were a type-specific subset of the HSV-related antigens synthesized in cells undergoing productive infection by HSV-1 and HSV-2.

  16. Lentiviral HSV-Tk.007-mediated suicide gene therapy is not toxic for normal brain cells.

    Science.gov (United States)

    Hossain, Jubayer A; Ystaas, Lars Rømo; Mrdalj, Jelena; Välk, Kristjan; Riecken, Kristoffer; Fehse, Boris; Bjerkvig, Rolf; Grønli, Janne; Miletic, Hrvoje

    2016-09-01

    Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation. In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis. During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed. We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. HSV-1-induced chemokine expression via IFI16-dependent and IFI16-independent pathways in human monocyte-derived macrophages

    DEFF Research Database (Denmark)

    Søby, Stine; Laursen, Rune R; Østergaard, Lars Jørgen

    2012-01-01

    and monocytes were differentiated to macrophages. Macrophages infected with HSV-1 were analyzed using siRNA-mediated knock-down of IFI16 by real-time PCR, ELISA, and Western blotting. RESULTS: We determined that both CXCL10 and CCL3 are induced independent of HSV-1 replication. IFI16 mediates CCL3 m......RNA accumulation during early HSV-1 infection. In contrast, CXCL10 was induced independently of IFI16. CONCLUSIONS: Our data provide the first evidence of HSV-1-induced innate immune responses via IFI16 in human primary macrophages. In addition, the data suggest that at least one additional unidentified receptor...

  18. Effect of genital herpes on cervicovaginal HIV shedding in women co-infected with HIV AND HSV-2 in Tanzania.

    Science.gov (United States)

    Todd, Jim; Riedner, Gabriele; Maboko, Leonard; Hoelscher, Michael; Weiss, Helen A; Lyamuya, Eligius; Mabey, David; Rusizoka, Mary; Belec, Laurent; Hayes, Richard

    2013-01-01

    To compare the presence and quantity of cervicovaginal HIV among HIV seropositive women with clinical herpes, subclinical HSV-2 infection and without HSV-2 infection respectively; to evaluate the association between cervicovaginal HIV and HSV shedding; and identify factors associated with quantity of cervicovaginal HIV. Four groups of HIV seropositive adult female barworkers were identified and examined at three-monthly intervals between October 2000 and March 2003 in Mbeya, Tanzania: (1) 57 women at 70 clinic visits with clinical genital herpes; (2) 39 of the same women at 46 clinic visits when asymptomatic; (3) 55 HSV-2 seropositive women at 60 clinic visits who were never observed with herpetic lesions; (4) 18 HSV-2 seronegative women at 45 clinic visits. Associations of genital HIV shedding with HIV plasma viral load (PVL), herpetic lesions, HSV shedding and other factors were examined. Prevalence of detectable genital HIV RNA varied from 73% in HSV-2 seronegative women to 94% in women with herpetic lesions (geometric means 1634 vs 3339 copies/ml, p = 0.03). In paired specimens from HSV-2 positive women, genital HIV viral shedding was similar during symptomatic and asymptomatic visits. On multivariate regression, genital HIV RNA (log10 copies/mL) was closely associated with HIV PVL (β = 0.51 per log10 copies/ml increase, 95%CI:0.41-0.60, pgenital HIV than the presence of herpetic lesions. These data support a role of HSV-2 infection in enhancing HIV transmissibility.

  19. Lymphocyte transformation (by (/sup 3/H)thymidine) in mice infected with Trichinella spiralis

    Energy Technology Data Exchange (ETDEWEB)

    Borojevic, D.; Lalic, R.; Cuperlovic, K. (Institut za Primeni Nuklearne Energije u Poljoprivedri, Veterinarstvu i Sumarstvu, Zemun (Yugoslavia)); Dujic, A. (Belgrade Univ. (Yugoslavia). Medicinski Fakultet)

    1983-05-01

    The blastogenic response of infected and normal mice (to stimulation by specific antigen and nonspecific antigens) was followed by the uptake of (/sup 3/H)thymidine. It was established that there was a minimum number of cells (1 x 10/sup 6/) per culture for these responses to be observed and that for specific stimulation by T. spiralis soluble antigen 10..mu..g per culture gave optimal results.

  20. Integrating retroviral cassette extends gene delivery of HSV-1 expression vectors to dividing cells.

    Science.gov (United States)

    de Felipe, P; Izquierdo, M; Wandosell, F; Lim, F

    2001-08-01

    Retroviral vectors have long been used in a wide variety of gene transfer applications but have certain drawbacks, such as small cargo size, limited tropism, and low titers. HSV expression vectors overcome these disadvantages, but, because they persist in target cells as nonreplicative episomes, they are not retained in all the progeny of dividing cells. Chimeric HSV/AAV products that can mediate transgene integration in human mitotic cells have been constructed, but, to date, genetic modification of dividing cells in animal models using HSV products has not been possible. Here, we report the construction of hybrid HSV/retroviral vectors that exhibit up to 50-fold higher transgene integration efficiency compared to vectors containing only HSV-1 components. Efficient integration of a retroviral transgene cassette encoding pac in human cells required expression of the Moloney murine leukemia virus gag-pol genes, but in murine cells, could also be mediated by endogenous activities, albeit at a lower level. Gene delivery was equally efficient in BHK21, a cell line resistant to retroviral infection, and transgene retention and expression were observed to be stable for least one month in Hs683 human glioma cells. These vectors have wide applications for the genetic modification of many cell types.

  1. Isolation, identification and function of a novel anti-HSV-1 protein from Grifola frondosa.

    Science.gov (United States)

    Gu, Chang-Qing; Li, Jun-Wen; Chao, Fuhuan; Jin, Min; Wang, Xin-Wei; Shen, Zhi-Qiang

    2007-09-01

    A novel antiviral protein was purified from an extract of Grifola frondosa fruiting bodies using a procedure that included 40% ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography, and designated GFAHP. This protein inhibited herpes simplex virus type 1 (HSV-1) replication in vitro with an IC(50) value of 4.1 microg/ml and a therapeutic index >29.3. Higher concentrations of GFAHP (125 and 500 microg/ml) also significantly reduced the severity of HSV-1 induced blepharitis, neovascularization, and stromal keratitis in a murine model. Topical administration of GFAHP to the mouse cornea resulted in a significant decrease in virus production (mean virus yields: 3.4log10PFU in the treated group and 4.19log10PFU in the control group). We proved that GFAHP directly inactivates HSV-1 while simultaneously inhibiting HSV-1 penetration into Vero cells. Gel electrophoresis showed that GFAHP had a molecular weight of 29.5 kDa. GFAHP was tryptic digested and analyzed from the PMF of matrix assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and nanoelectrospray ionization tandem mass spectrometry. The N-terminal sequence of GFAHP consisted of an 11 amino acid peptide, NH(2)-REQDNAPCGLN-COOH that did not match any known amino acid sequences, indicating that GFAHP is likely to be a novel antivirus protein. To our knowledge, this is the first report that characterizes an anti-HSV protein from G. frondosa.

  2. Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

    Science.gov (United States)

    Wang, Kening; Tomaras, Georgia D; Jegaskanda, Sinthujan; Moody, M Anthony; Liao, Hua-Xin; Goodman, Kyle N; Berman, Phillip W; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Haynes, Barton F; Cohen, Jeffrey I

    2017-10-01

    The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice. IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were

  3. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    Science.gov (United States)

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  4. Effect of genital herpes on cervicovaginal HIV shedding in women co-infected with HIV AND HSV-2 in Tanzania.

    Directory of Open Access Journals (Sweden)

    Jim Todd

    Full Text Available To compare the presence and quantity of cervicovaginal HIV among HIV seropositive women with clinical herpes, subclinical HSV-2 infection and without HSV-2 infection respectively; to evaluate the association between cervicovaginal HIV and HSV shedding; and identify factors associated with quantity of cervicovaginal HIV.Four groups of HIV seropositive adult female barworkers were identified and examined at three-monthly intervals between October 2000 and March 2003 in Mbeya, Tanzania: (1 57 women at 70 clinic visits with clinical genital herpes; (2 39 of the same women at 46 clinic visits when asymptomatic; (3 55 HSV-2 seropositive women at 60 clinic visits who were never observed with herpetic lesions; (4 18 HSV-2 seronegative women at 45 clinic visits. Associations of genital HIV shedding with HIV plasma viral load (PVL, herpetic lesions, HSV shedding and other factors were examined.Prevalence of detectable genital HIV RNA varied from 73% in HSV-2 seronegative women to 94% in women with herpetic lesions (geometric means 1634 vs 3339 copies/ml, p = 0.03. In paired specimens from HSV-2 positive women, genital HIV viral shedding was similar during symptomatic and asymptomatic visits. On multivariate regression, genital HIV RNA (log10 copies/mL was closely associated with HIV PVL (β = 0.51 per log10 copies/ml increase, 95%CI:0.41-0.60, p<0.001 and HSV shedding (β = 0.24 per log10 copies/ml increase, 95% CI:0.16-0.32, p<0.001 but not the presence of herpetic lesions (β = -0.10, 95%CI:-0.28-0.08, p = 0.27.HIV PVL and HSV shedding were more important determinants of genital HIV than the presence of herpetic lesions. These data support a role of HSV-2 infection in enhancing HIV transmissibility.

  5. Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte; Munch-Petersen, Sune; Berenstein, Dvora

    2006-01-01

    and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP for DNA replication. The cell cycle specific activity of TK1 is regulated at the transcriptional level...

  6. Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene

    NARCIS (Netherlands)

    van der Eb, Marjolijn M.; Geutskens, Sacha B.; van Kuilenburg, André B. P.; van Lenthe, Henk; van Dierendonck, Jan-Hein; Kuppen, Peter J. K.; van Ormondt, Hans; van de Velde, Cornelis J. H.; Wanders, Ronald J. A.; van Gennip, Albert H.; Hoeben, Rob C.

    2003-01-01

    BACKGROUND: Ganciclovir exhibits broad-spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella-zoster virus, Epstein-Barr virus and human herpes virus-6. Ganciclovir is widely applied for anti-herpetic treatment, cytomegalovirus prophylaxis after organ

  7. Fusion of herpes simplex virus thymidine kinase to VP22 does not result in intercellular trafficking of the protein

    NARCIS (Netherlands)

    Beerens, A. M. J.; Rots, M. G.; de Vries, E. F. J.; Haisma, H. J.

    Suicide gene therapy is a promising approach for the treatment of cancer. Current protocols, however, suffer from low efficiency. We tried to alleviate this problem by developing a transgene that will spread from the initially transduced cell to the surrounding cells (transmission). We used herpes

  8. Fast, Affordable, Science and Technology Satellite (FASTSAT) Huntsville-01 (HSV-01) Spacecraft Lessons Learned Report

    Science.gov (United States)

    Smith, Timothy A.

    2012-01-01

    The Fast Affordable Science and Technology Satellite (FASTSAT) project is a path finding effort to produce reliable satellite busses for different applications at an unprecedented speed and low cost. The project is designed to be a generational project and the first satellite produced is the Huntsville -01 (HSV-01) spacecraft. The subject of this report is the lessons learned gained during the development, testing, and up to the delivery of the FASTSAT HSV -01 spacecraft. The purpose of this report is to capture the major findings that will greatly benefit the future FASTSAT satellites and perhaps other projects interested in pushing the boundaries for cost and schedule. The FASTSAT HSV -01 primary objectives, success criteria, and team partners are summarized to give a frame of reference to the lessons learned.

  9. Varicella zoster vaccines and their implications for development of HSV vaccines

    Energy Technology Data Exchange (ETDEWEB)

    Gershon, Anne A., E-mail: aag1@columbia.edu [Department of Pediatrics, Columbia University College of Physicians and Surgeons, 620W. 168th Street, NY, NY 10032 (United States)

    2013-01-05

    Live attenuated vaccines to prevent varicella and zoster have been available in the US for the past 17 years, with a resultant dramatic decrease in varicella incidence and a predicted future decrease in the incidence of zoster. The pathogenesis and immune responses to varicella zoster virus (VZV) as well as the safety and effectiveness of VZV vaccines are reviewed. The lack of sterilizing immunity provided by VZV vaccines has not prevented them from being safe and effective. Virological and pathological information concerning parallels and differences between VZV and herpes simplex virus (HSV) are highlighted. Although VZV and HSV are distinct pathogens, they appear to have similarities in target organs and immunity that provide an expectation of a high likelihood for the success of vaccination against HSV, and predicted to be similar to that of VZV.

  10. A spiroketal-enol ether derivative from Tanacetum vulgare selectively inhibits HSV-1 and HSV-2 glycoprotein accumulation in Vero cells.

    Science.gov (United States)

    Álvarez, Ángel L; Habtemariam, Solomon; Abdel Moneim, Ahmed E; Melón, Santiago; Dalton, Kevin P; Parra, Francisco

    2015-07-01

    The inhibitory effects of Tanacetum vulgare rhizome extracts on HSV-1 and HSV-2 in vitro replication were assessed. Unlike extracts obtained from the aerial parts, adsorption inhibition and virucidal activities seemed not to be relevant for the observed antiviral action of tansy rhizome extracts. Instead, the most significant effects were the inhibition of virus penetration and a novel mechanism consisting of the specific arrest of viral gene expression and consequently the decrease of viral protein accumulation within infected cells. Through a bioactivity-guided fractionation protocol we isolated and identified the spiroketal-enol ether derivative (E)-2-(2,4-hexadiynyliden)-1,6-dioxaspiro[4.5]dec-3-ene as the active compound responsible for this inhibitory effect. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Ying [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Yunnan Academy of Tobacco Science, Kunming, Yunnan 650106 (China); Guo, Lei; Yang, Erxia; Liao, Yun; Liu, Longding; Che, Yanchun; Zhang, Ying; Wang, Lichun; Wang, Jingjing [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Li, Qihan, E-mail: imbcams.lq@gmail.com [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China)

    2014-09-15

    During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31–UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31–UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress. - Highlights: • Cellular TMEM140 protein interacts with HSV-1 UL31 protein during viral infection. • Increasing expression of TMEM140 leads to inhibition of HSV-1 proliferation. • Increasing expression of TMEM140 blocks HSV-1 nucleocapsid egress process. • Binding to UL31 of TMEM140 impedes formation of HSV-1 UL31–UL34 complex.

  12. Risk factors for herpes simplex virus type 2 and HIV among women at high risk in northwestern Tanzania: preparing for an HSV-2 intervention trial

    National Research Council Canada - National Science Library

    Watson-Jones, Deborah; Weiss, Helen A; Rusizoka, Mary; Baisley, Kathy; Mugeye, Kokugonza; Changalucha, John; Everett, Dean; Balira, Rebecca; Knight, Louise; Ross, David; Hayes, Richard J

    2007-01-01

    To determine prevalence of and risk factors for herpes simplex virus type 2 (HSV-2) and HIV among women being screened for a randomized, controlled trial of HSV suppressive therapy in northwestern Tanzania...

  13. Prevalence and Determinants of Herpes Simplex Virus Type 2 (HSV-2)/Syphilis Co-Infection and HSV-2 Mono-Infection among Human Immunodeficiency Virus Positive Men Who Have Sex with Men: a Cross-Sectional Study in Northeast China.

    Science.gov (United States)

    Hu, Qing-Hai; Xu, Jun-Jie; Chu, Zhen-Xing; Zhang, Jing; Yu, Yan-Qiu; Yu, Huan; Ding, Hai-Bo; Jiang, Yong-Jun; Geng, Wen-Qing; Wang, Ning; Shang, Hong

    2017-05-24

    This study assessed the prevalence and determinants of herpes simplex virus type 2 (HSV-2)/syphilis co-infection and HSV-2 mono-infection in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) in China. A cross-sectional study was conducted of 545 HIV-positive MSM in Shenyang between February 2009 and October 2014. Participants underwent physical examinations and serological tests for HSV-2 and syphilis. A multinomial logistic regression was used to identify the risk factors associated with HSV-2/syphilis co-infection and HSV-2 mono-infection. The prevalence of HSV-2 mono-infection, syphilis mono-infection, and HSV-2/syphilis co-infection (95% confidence interval) was 48.6% (44.4-52.8%), 34.3% (30.3-38.3%), and 22.9% (19.4-26.5%), respectively. After controlling within HSV-2/syphilis-seropositive cases, regression analysis revealed that the related factors for HSV-2/syphilis co-infection included age (25-50 vs. ≤ 24 years: adjusted odds ratio [aOR], 4.55; > 50 vs. ≤ 24 years: aOR, 43.02), having regular female sexual partner(s) in the past 6 months (aOR, 0.43), and age at first MSM experience (≤ 18 vs. > 18 years: aOR, 2.59) (all P syphilis co-infection in HIV-positive MSM indicates a high secondary HIV transmission risk. A campaign for detection and treatment of HSV-2 and syphilis is urgently required for HIV-positive MSM in China.

  14. [3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein.

    Science.gov (United States)

    Orlov, S N; Pchejetski, D V; Sarkissian, S D; Adarichev, V; Taurin, S; Pshezhetsky, A V; Tremblay, J; Maximov, G V; deBlois, D; Bennett, M R; Hamet, P

    2003-03-01

    [(3)H]-thymidine is commonly used to analyze the accumulation of [(3)H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [(3)H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [(3)H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [(3)H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [(3)H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [(3)H]-thymidine-labeled DNA. They also demonstrate that [(3)H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na(+)](i)/[K(+)](i) ratio.

  15. Docking of anti-HIV-1 oxoquinoline-acylhydrazone derivatives as potential HSV-1 DNA polymerase inhibitors

    Science.gov (United States)

    Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Santos, Fernanda da Costa; Batalha, Pedro Netto; Brozeguini, Leonardo; Seidl, Peter R.; de Alencastro, Ricardo Bicca; Cunha, Anna Cláudia; de Souza, Maria Cecília B. V.; Ferreira, Vitor F.; Giongo, Viveca A.; Cirne-Santos, Cláudio; Paixão, Izabel C. P.

    2014-09-01

    Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1 activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this enzyme. Therefore, in this work, comparative molecular docking simulations of 1 and three new synthesized oxoquinoline-acylhydrazone HIV-1 inhibitors (3-5), which do not contain those hydrophilic groups, were carried out, in order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC50 = 3.4 ± 0.3 μM). Compounds 2 and 3 were used as templates in the design of four new oxoquinoline-acylhydrazones (6-9) as potential anti-HSV-1 agents to increase the antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate for further development of dual anti-HIV/HSV activity.

  16. Thymine utilization in Escherichia coli K12. On the role of deoxyribose 1-phosphate and thymidine phosphorylase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Leer, Johan Christian; Nygaard, Per

    1973-01-01

    to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1-phosphate and/or the level of thymidine phosphorylase. Thymine incorporation in wild...... to the external thymine concentration. The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine...

  17. An Epigenetic Compound Library Screen Identifies BET Inhibitors That Promote HSV-1 and -2 Replication by Bridging P-TEFb to Viral Gene Promoters through BRD4.

    Directory of Open Access Journals (Sweden)

    Ke Ren

    2016-10-01

    Full Text Available The human HSV-1 and -2 are common pathogens of human diseases. Both host and viral factors are involved in HSV lytic infection, although detailed mechanisms remain elusive. By screening a chemical library of epigenetic regulation, we identified bromodomain-containing protein 4 (BRD4 as a critical player in HSV infection. We show that treatment with pan BD domain inhibitor enhanced both HSV infection. Using JQ1 as a probe, we found that JQ1, a defined BD1 inhibitor, acts through BRD4 protein since knockdown of BRD4 expression ablated JQ1 effect on HSV infection. BRD4 regulates HSV replication through complex formation involving CDK9 and RNAP II; whereas, JQ1 promotes HSV-1 infection by allocating the complex to HSV gene promoters. Therefore, suppression of BRD4 expression or inhibition of CDK9 activity impeded HSV infection. Our data support a model that JQ1 enhances HSV infection by switching BRD4 to transcription regulation of viral gene expression from chromatin targeting since transient expression of BRD4 BD1 or BD1/2 domain had similar effect to that by JQ1 treatment. In addition to the identification that BRD4 is a modulator for JQ1 action on HSV infection, this study demonstrates BRD4 has an essential role in HSV infection.

  18. The endozepine ODN stimulates [{sup 3}H]thymidine incorporation in cultured rat astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gandolfo, P.; Patte, C.; Thoumas, J.L.; Leprince, J.; Vaudry, H.; Tonon, M.C. [European Institute for Peptide Research (IFRMP no. 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U 413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan (France)

    1999-05-15

    High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10{sup -14} to 10{sup -11} M), ODN caused a dose-dependent increase of [{sup 3}H]thymidine incorporation. At higher doses (10{sup -10} to 10{sup -5} M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10{sup -6} M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10{sup -6} M) had no effect. The ODN-induced stimulation of [{sup 3}H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-{beta}-carboline-3-carboxylate (DMCM). The GABA{sub A} receptor antagonist bicuculline (10{sup -4} M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABA{sub A} agonist 3APS (10{sup -10} to 10{sup -4} M) also induced a dose-related increase of [{sup 3}H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABA{sub A} receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  19. Autoradiographic investigation of sperm transit through the male mouse genital tract after tritiated thymidine incorporation

    Energy Technology Data Exchange (ETDEWEB)

    Dadoune, J.P.; Alfonsi, M.F. (Faculte de Medecine Broussais-Hotel-Dieu, U.E.R. Biomedicale des Saints-Peres, 75 - Paris (France))

    1984-01-01

    The transit of spermatozoa in the genital tract of the male mouse was investigated by quantitative light microscopic autoradiography after intraperitoneal injection of tritiated thymidine. Transit duration in the caput and the corpus of the epididymis was shown to be 3 days; the total duration of transit in the genital tract was 5 days. These findings indicate that the time required for the transit of spermatozoa in the epididymal caput and corpus was comparable to that calculated in other mammals studied. However, the duration of sperm storage in the epididymal cauda appeared to be shorter than that previously reported for rodents.

  20. NKT cell activation by local α-galactosylceramide administration decreases susceptibility to HSV-2 infection

    DEFF Research Database (Denmark)

    Iversen, Marie Beck; Jensen, Simon Kok; Hansen, Anne Louise

    2015-01-01

    that received local pre-treatment with αGalCer prior to intra-vaginal HSV-2 infection had a lower mean disease score, mortality and viral load in the vagina following infection, compared to mice that did not receive αGalCer pre-treatment. Further, we found increased numbers of CD45 and NK1.1 positive cells...

  1. The antiviral effect of three plant species of Iran on HSV-1

    Directory of Open Access Journals (Sweden)

    malihe Farahani

    2016-05-01

    Full Text Available Background : plants have had special position in human life and their medicinal application have been observed in manuscripts of many world scientists. Nowadays the treatment of HSV-1 infections with the available  chemical drugs often leads to the problems due to viral resistance and virus latency duration, therefore there is a requirement for new anti-herpes drugs.In this research the antiviral effects of  Camellia sinesis, Echium amoenumL and Nerium oleander, with ethnomedical background on HSV-1 were studied. Materials and Methods:The plants were extracted with decoction method to obtain aqueous extracts and after evaluating their cytotoxicity on Hep-2 cell lines by evaluating CPE, antiherpes effect of the plants extracts were determined by cytopathic effect inhibition assay. Results: Nerium oleander extract had the  most toxicity (> 50 μg/ml on cell line and Camellia sinesis showed the most inhibitory property on HSV-1 multiplication. Echium amoenumL had the lowest antiherpes effect. Camellia sinesis was inhibitor of virus multiplication completely at 50-1000 μg/ml and Echium amoenumL at concentrations >300 μg/ml. Conclusion: Camellia sinesis and Echium amoenumL could inhibit well HSV-1 multiplication completely at concentrations nontoxic. Further researches are needed to find the effect mechanism of these drugs which may be used in the manufacture of new antiherpes drugs.

  2. Inhibitory effect of aqueous extract of Echinacea purpurea and Nerium oleander on HSV-1 multiplication

    Directory of Open Access Journals (Sweden)

    Maliheh Farahani

    2014-02-01

    Full Text Available Background: Treatment of HSV-1 infections with the available chemical drugs may have some problems such as drug resistance and virus latency. Therefore, there is a requirement for new antiherpes drugs in today's world. The present study was carried out to analyze the inhibitory effect of Echinacea purpurea and Nerium oleander plants with ethnomedical background on HSV-1 replication. Methods: Plants were extracted by decoction method to obtain aqueous extract. These extracts were screened for their cytotoxicity against Hep-2 cell line by CPE (cytopathic effect assay. Antiviral effect of the plant extracts were determined by the virus cytopathic effect inhibition assay. Results: Nerium oleander extract had the highest toxicity (>0.1 μg/ml on Hep-2 cells and Echinacea purpurea extract exhibited significant antiherpes effect at nontoxic concentrations used on the cell lines. Findings indicated that Echinacea purpurea extract inhibited HSV-1 multiplication at concentrations >400 μg/ml. Conclusion: Echinacea purpurea plant had no any effect on cells at nontoxic concentrations and inhibited HSV-1 multiplication at concentrations >400 μg/ml. Further research is needed to find out the anti herpetic mechanism of this plant.

  3. PD-L1-Expressing Dendritic Cells Contribute to Viral Resistance during Acute HSV-1 Infection

    Directory of Open Access Journals (Sweden)

    Katie M. Bryant-Hudson

    2012-01-01

    Full Text Available The inhibitory receptor, Programmed Death 1 (PD-1, and its ligands (PD-L1/PD-L2 are thought to play a role in immune surveillance during chronic viral infection. The contribution of the receptor/ligand pair during an acute infection is less understood. To determine the role of PD-L1 and PD-L2 during acute ocular herpes simplex virus type 1 (HSV-1 infection, HSV-1-infected mice administered neutralizing antibody to PD-L1 or PD-L2 were assessed for viral burden and host cellular immune responses. Virus titers were elevated in cornea and trigeminal ganglia (TG of anti-PD-L1-treated mice which corresponded with a reduced number of CD80-expressing dendritic cells, PD-L1+ dendritic cells, and HSV-1-specific CD8+ T cells within the draining (mandibular lymph node (MLN. In contrast, anti-PD-L2 treatment had no effect on viral replication or changes in the MLN population. Notably, analysis of CD11c-enriched MLN cells from anti-PD-L1-treated mice revealed impaired functional capabilities. These studies indicate PD-L1-expressing dendritic cells are important for antiviral defense during acute HSV-1 infection.

  4. Newly Characterized Murine Undifferentiated Sarcoma Models Sensitive to Virotherapy with Oncolytic HSV-1 M002

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    Eric K. Ring

    2017-12-01

    Full Text Available Despite advances in conventional chemotherapy, surgical techniques, and radiation, outcomes for patients with relapsed, refractory, or metastatic soft tissue sarcomas are dismal. Survivors often suffer from lasting morbidity from current treatments. New targeted therapies with less toxicity, such as those that harness the immune system, and immunocompetent murine sarcoma models to test these therapies are greatly needed. We characterized two new serendipitous murine models of undifferentiated sarcoma (SARC-28 and SARC-45 and tested their sensitivity to virotherapy with oncolytic herpes simplex virus 1 (HSV-1. Both models expressed high levels of the primary HSV entry molecule nectin-1 (CD111 and were susceptible to killing by interleukin-12 (IL-12 producing HSV-1 M002 in vitro and in vivo. M002 resulted in a significant intratumoral increase in effector CD4+ and CD8+ T cells and activated monocytes, and a decrease in myeloid-derived suppressor cells (MDSCs in immunocompetent mice. Compared to parent virus R3659 (no IL-12 production, M002 resulted in higher CD8:MDSC and CD8:T regulatory cell (Treg ratios, suggesting that M002 creates a more favorable immune tumor microenvironment. These data provide support for clinical trials targeting sarcomas with oncolytic HSV-1. These models provide an exciting opportunity to explore combination therapies for soft tissue sarcomas that rely on an intact immune system to reach full therapeutic potential.

  5. Hsc70 focus formation at the periphery of HSV-1 transcription sites requires ICP27.

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    Ling Li

    Full Text Available BACKGROUND: The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1 infected cells. We recently reported that cellular RNA polymerase II (RNAP II undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments. METHODOLOGY/PRINCIPLE FINDINGS: Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression. CONCLUSIONS/SIGNIFICANCE: We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

  6. Synthesis and evaluation of antiviral activities of novel sonochemical silver nanorods against HIV and HSV viruses

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    Mazyar Etemadzade

    2016-11-01

    Full Text Available Objective: To evaluate the effect of novel sonochemical silver nanorods on HIV and herpes simplex virus type 1 (HSV-1 viruses in human cervical cancer HeLa cells. Methods: The formation of silver nanorods conjugated with sodium 2-mercaptoethane sulfonate (Ag-MES was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and thermal gravimetric analysis. The antiviral activity of this Ag-MES was examined against HIV and HSV-1 virus replication. Results: The characterizations of Ag-MES and physiochemical structure were determined by scanning electron microscopy, Fourier transform infrared spectroscopy and thermal gravimetric analysis. Approximately entire viral replication was inhibited by Ag-MES at 10 µmol/mL concentration. About 90% of HSV virions failed to replicate in the present of this concentration of nanorods. However, HIV showed more sensitivity to Ag-MES than HSV-1. Conclusions: According to the obtained data, the synthesized sonochemical silver nanorod in this study is a promising candidate for further drug discovery investigation.

  7. Evaluation of infectious titer in a candidate HSV type 2 vaccine by a quantitative molecular approach.

    Science.gov (United States)

    Azizi, Ali; Tang, Mei; Gisonni-Lex, Lucy; Mallet, Laurent

    2013-12-06

    One of the critical tasks in analytical testing is to monitor and assign the infectivity or potency of viral based vaccines from process development to production of final clinical lots. In this study, a high throughput RT-qPCR based approach was developed to evaluate the infectious titre in a replication-defective HSV-2 candidate vaccine, called HSV529. This assay is a combination of viral propagation and quantitative RT-PCR which measures the amount of RNA in infected cells after incubation with test samples. The relative infectious titre of HSV529 candidate vaccine was determined by a RT-qPCR method targeting HSV-2 gD2 gene. The data were analyzed using the parallel-line analysis as described in the European Pharmacopoeia 8th edition. The stability of HSV529 test samples were also investigated in a concordance study between RT-qPCR infectivity assay and a classical plaque assays. A suitable correlation was determined between both assays using an identical sample set in both assays. The RT-qPCR infectivity assay was further characterized by evaluating the intermediate precision and accuracy. The coefficient of variation from the six independent assays was less than 10%. The accuracy of each of the assay was also evaluated in the range of 92.91% to 120.57%. Our data demonstrate that the developed RT-qPCR infectivity assay is a rapid high throughput approach to quantify the infectious titer or potency of live attenuated or defective viral-based vaccines, an attribute which is associated with product quality.

  8. A novel intravaginal ring to prevent HIV-1, HSV-2, HPV, and unintended pregnancy.

    Science.gov (United States)

    Ugaonkar, Shweta R; Wesenberg, Asa; Wilk, Jolanta; Seidor, Samantha; Mizenina, Olga; Kizima, Larisa; Rodriguez, Aixa; Zhang, Shimin; Levendosky, Keith; Kenney, Jessica; Aravantinou, Meropi; Derby, Nina; Grasperge, Brooke; Gettie, Agegnehu; Blanchard, James; Kumar, Narender; Roberts, Kevin; Robbiani, Melissa; Fernández-Romero, José A; Zydowsky, Thomas M

    2015-09-10

    Women urgently need a self-initiated, multipurpose prevention technology (MPT) that simultaneously reduces their risk of acquiring HIV-1, HSV-2, and HPV (latter two associated with increased risk of HIV-1 acquisition) and prevents unintended pregnancy. Here, we describe a novel core-matrix intravaginal ring (IVR), the MZCL IVR, which effectively delivered the MZC combination microbicide and a contraceptive. The MZCL IVR contains four active pharmaceutical ingredients (APIs): MIV-150 (targets HIV-1), zinc acetate (ZA; targets HIV-1 and HSV-2), carrageenan (CG; targets HPV and HSV-2), and levonorgestrel (LNG; targets unintended pregnancy). The elastomeric IVR body (matrix) was produced by hot melt extrusion of the non-water swellable elastomer, ethylene vinyl acetate (EVA-28), containing the hydrophobic small molecules, MIV-150 and LNG. The solid hydrophilic core, embedded within the IVR by compression, contained the small molecule ZA and the macromolecule CG. Hydrated ZA/CG from the core was released by diffusion via a pore on the IVR while the MIV-150/LNG diffused from the matrix continuously for 94 days (d) in vitro and up to 28 d (study period) in macaques. The APIs released in vitro and in vivo were active against HIV-1ADA-M, HSV-2, and HPV16 PsV in cell-based assays. Serum LNG was at levels associated with local contraceptive effects. The results demonstrate proof-of-concept of a novel core-matrix IVR for sustained and simultaneous delivery of diverse molecules for the prevention of HIV, HSV-2 and HPV acquisition, as well as unintended pregnancy. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Therapeutic Drug Monitoring in Neonatal HSV Infection on Continuous Renal Replacement Therapy.

    Science.gov (United States)

    Funaki, Takanori; Miyata, Ippei; Shoji, Kensuke; Enomoto, Yuki; Sakamoto, Seisuke; Kasahara, Mureo; Miyairi, Isao

    2015-07-01

    Optimal acyclovir dosing under continuous renal replacement therapy (CRRT) in neonates is unknown. We monitored serum acyclovir levels and herpes simplex virus 1 (HSV-1) DNA levels in a neonate with disseminated HSV-1 infection and renal failure undergoing CRRT. A full-term, 5-day-old female presented with a 2-day history of lethargy and fever. She developed fulminant hepatitis and was diagnosed with HSV-1 infection by real-time polymerase chain reaction. Acyclovir was initiated at 60 mg/kg/day, which was lowered to 20 mg/kg/day because of development of renal failure. She was placed on continuous hemodialysis. Acyclovir dosing was adjusted according to serum acyclovir levels, and HSV-1 viral load was sequentially monitored. Semiquantification of serum HSV-1 levels was performed by real-time polymerase chain reaction. Acyclovir levels were measured by using liquid chromatography-tandem mass spectrometry. Acyclovir was administered at 20 mg/kg intravenously over 1 hour; peak concentration was 18.9 μg/mL. The half-life of acyclovir was estimated to be 2 to 3 h. Viral load remained high during dosing every 24 hours, with a decline of 0.17 log copies/24 hours. Acyclovir dosing was changed to 20 mg/kg/dose every 8 hours, with an average viral load decline of 0.44 log copies/24 hours. Despite the guideline recommendation of 24-hour redosing, acyclovir was dialyzed at a rate that resulted in suboptimal treatment. Individual therapeutic drug monitoring for acyclovir and dosing adjustment may be required to optimize therapy for patients undergoing CRRT. Copyright © 2015 by the American Academy of Pediatrics.

  10. Interleukin-21 receptor signalling is important for innate immune protection against HSV-2 infections.

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    Sine K Kratholm

    Full Text Available Interleukin (IL -21 is produced by Natural Killer T (NKT cells and CD4(+ T cells and is produced in response to virus infections, where IL-21 has been shown to be essential in adaptive immune responses. Cells from the innate immune system such as Natural Killer (NK cells and macrophages are also important in immune protection against virus. These cells express the IL-21 receptor (IL-21R and respond to IL-21 with increased cytotoxicity and cytokine production. Currently, however it is not known whether IL-21 plays a significant role in innate immune responses to virus infections. The purpose of this study was to investigate the role of IL-21 and IL-21R in the innate immune response to a virus infection. We used C57BL/6 wild type (WT and IL-21R knock out (KO mice in a murine vaginal Herpes Simplex Virus type 2 (HSV-2 infection model to show that IL-21 - IL-21R signalling is indeed important in innate immune responses against HSV-2. We found that the IL-21R was expressed in the vaginal epithelium in uninfected (u.i WT mice, and expression increased early after HSV-2 infection. IL-21R KO mice exhibited increased vaginal viral titers on day 2 and 3 post infection (p.i. and subsequently developed significantly higher disease scores and a lower survival rate compared to WT mice. In addition, WT mice infected with HSV-2 receiving intra-vaginal pre-treatment with murine recombinant IL-21 (mIL-21 had decreased vaginal viral titers on day 2 p.i., significantly lower disease scores, and a higher survival rate compared to infected untreated WT controls. Collectively our data demonstrate the novel finding that the IL-21R plays a critical role in regulating innate immune responses against HSV-2 infection.

  11. A dominant positive and negative selectable gene for use in mammalian cells.

    OpenAIRE

    Schwartz, F; Maeda, N; Smithies, O; Hickey, R; Edelmann, W; Skoultchi, A; Kucherlapati, R

    1991-01-01

    We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusi...

  12. Humanized Thymidine Kinase–NOG Mice Can Be Used to Identify Drugs That Cause Animal-Specific Hepatotoxicity: A Case Study with Furosemide

    Science.gov (United States)

    Xu, Dan; Michie, Sara A.; Zheng, Ming; Takeda, Saori; Wu, Manhong

    2015-01-01

    Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)–NOG mice with humanized livers could prevent this unfortunate outcome (i.e., “rescue” a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide. PMID:25962391

  13. Enhancement of Herpes Simplex Virus (HSV Infection by Seminal Plasma and Semen Amyloids Implicates a New Target for the Prevention of HSV Infection

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    Lilith Torres

    2015-04-01

    Full Text Available Human herpesviruses cause different infectious diseases, resulting in world-wide health problems. Sexual transmission is a major route for the spread of both herpes simplex virus-1 (HSV-1 and -2. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa and, thereby, initiates viral replication. Previously, we demonstrated that the amyloid fibrils semenogelin (SEM and semen-derived enhancer of viral infection (SEVI, and seminal plasma (SP augment cytomegalovirus infection (Tang et al., J. Virol 2013. Whether SEM or SEVI amyloids or SP could also enhance other herpesvirus infections has not been examined. In this study, we found that the two amyloids as well as SP strongly enhance both HSV-1 and -2 infections in cell culture. Along with SP, SEM and SEVI amyloids enhanced viral entry and increased infection rates by more than 10-fold, as assessed by flow cytometry assay and fluorescence microscopy. Viral replication was increased by about 50- to 100-fold. Moreover, viral growth curve assays showed that SEM and SEVI amyloids, as well as SP, sped up the kinetics of HSV replication such that the virus reached its replicative peak more quickly. The interactions of SEM, SEVI, and SP with HSVs are direct. Furthermore, we discovered that the enhancing effects of SP, SEM, and SEVI can be significantly reduced by heparin, a sulfated polysaccharide with an anionic charge. It is probable that heparin abrogates said enhancing effects by interfering with the interaction of the viral particle and the amyloids, which interaction results in the binding of the viral particles and both SEM and SEVI.

  14. Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir.

    Science.gov (United States)

    Hensel, Michael T; Marshall, Jason D; Dorwart, Michael R; Heeke, Darren S; Rao, Eileen; Tummala, Padmaja; Yu, Li; Cohen, Gary H; Eisenberg, Roselyn J; Sloan, Derek D

    2017-05-01

    Several prophylactic vaccines targeting herpes simplex virus 2 (HSV-2) have failed in the clinic to demonstrate sustained depression of viral shedding or protection from recurrences. Although these vaccines have generated high titers of neutralizing antibodies (NAbs), their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the NAb targets gD and gB and the novel T cell antigen and tegument protein UL40, and we compared this vaccine to a whole-inactivated-virus vaccine (formaldehyde-inactivated HSV-2 [FI-HSV-2]). We evaluated different formulations in combination with several Th1-inducing Toll-like receptor (TLR) agonists in vivo In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted most robust, functional HSV-2 antigen-specific CD8 T cell responses and high titers of neutralizing antibodies, demonstrating its superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)-alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of NAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses. IMPORTANCE Millions of people worldwide are infected with herpes simplex virus 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors

  15. The Effects of Daily Distress and Personality on Genital HSV Shedding and Lesions in a Randomized, Double-blind, Placebo-Controlled, Crossover Trial of Acyclovir in HSV-2 Seropositive Women

    OpenAIRE

    Strachan, Eric; Saracino, Misty; Selke, Stacy; Magaret, Amalia; Buchwald, Dedra; Wald, Anna

    2011-01-01

    Herpes simplex virus (HSV) infections are ubiquitous in humans, but the determinants of clinical and virologic severity are not completely understood. Prior research has suggested that psychological distress can be a co-factor in reactivation of latent HSV infection. Personality traits such as extraversion and neuroticism influence stress attributions and may inform the relationship between psychological distress and health outcomes. Earlier studies in this area have primarily focused on subj...

  16. Effects of low-energy electrons on DNA constituents: effective cross sections for condensed thymidine

    Science.gov (United States)

    Panajotovic, Radmila

    2009-05-01

    Since the first experiments of low-energy electron scattering from condensed DNA [1] have been performed, the interest in studying low-energy electron-biomolecule interactions has been increasing. Knowledge of effective cross sections for single- and double-strand breaks of DNA and for vibrational and electronic excitation of nucleic bases and nucleosides are opening the door to better understanding of effects of radiation on live tissue and possibly indicating interaction pathways leading to gene mutations and cancer. The strong variation of effective cross sections for DNA single-strand breaks with incident electron energy and the resonant enhancement at 1 eV suggested that considerable damage is inflicted by very low-energy electrons to DNA, and indicates the important role of π* shape resonances in the bond-breaking process. However, the complexity of DNA, even if studied as a short single-strand chain, imposes a need to perform measurements on its isolated constituents, such as nucleic bases and nucleosides. Thymidine is one of the most important nucleosides of DNA and an important component of antiviral compounds. In the condensed phase, thymidine's 2'-deoxyribose ring is in the pentose sugar ring form, which is a true conformation of this nucleoside in DNA. Results from High-Resolution Electron Energy Loss [2] study of monomolecular films of thymidine will be discussed and the presence of resonances in the effective cross sections at incident energy below 5 eV will be commented as a possible indication of the dissociative electron attachment. In addition, results on the resonance structures in the effective cross sections for electronic excitations for the incident electron energy from 1.5 to 12 eV will be discussed as a possible pathway for strand brakes in DNA. [4pt] [1] Boudaiffa B, Cloutier P, Hunting D, Huels M A and Sanche L 2002 Rad. Res. 157 227-234[0pt] [2] Panajotovic R, Martin F, Cloutier P, Hunting, D, and Sanche L, 2006 Rad.Res. 165 452

  17. Helper virus-free HSV-1 vectors packaged both in the presence of VSV G protein and in the absence of HSV-1 glycoprotein B support gene transfer into neurons in the rat striatum.

    Science.gov (United States)

    Tang, J; Yang, T; Ghosh, H P; Geller, A I

    2001-12-01

    Herpes simplex virus (HSV-1) vectors have potential for gene transfer into quiescent cells, but the gene transfer process could be more efficient. In other vector systems, both the titers and the efficiency of gene transfer have been enhanced by pseudotyping the vector particles with vesicular stomatitis virus (VSV) G protein. In this report, we pseudotyped helper virus-free HSV-1 plasmid vectors with VSV G protein. Packaging was performed in the presence of both VSV G protein and a deletion in an essential HSV-1 glycoprotein, gB. The resulting vector stocks supported gene transfer into both fibroblast and neuronal cell lines. VSV G protein was required for gene transfer because preincubation of these vector stocks with antibodies directed against either VSV G protein or VSV reduced the titer to undetectable levels. Although the titers were lower than those obtained using the unmodified vector system, the titers were not increased by use of chimeric proteins that contain the extracellular domain of VSV G protein and the transmembrane and/or cytoplasmic domains of specific HSV-1 glycoproteins. Also, the titers were not increased by performing the packaging in the presence of deletions in multiple HSV-1 glycoproteins. Nonetheless, pHSVlac pseudotyped with VSV G protein supported gene transfer into striatal neurons in the rat brain. Thus, HSV-1 vectors pseudotyped with VSV G protein may be useful for specific gene transfer studies.

  18. HSV-1/HSV-2 Infection-Related Cancers in Bantu Populations Driving HIV-1 Prevalence in Africa: Tracking the Origin of AIDS at the Onset of the 20th Century

    Directory of Open Access Journals (Sweden)

    Jacqueline Le Goaster

    2016-11-01

    Full Text Available Introduction: At the onset of the 20th century, ancient clinical observations of cancer epidemics in Bantu populations of Sub-Saharan Africa were discovered. They were reported from 1914 to 1960, but remained unexplained. In 1983, in San Francisco, Calif., USA, cancer epidemics were related to infections by the human immunodeficiency virus type 1 (HIV-1 known as AIDS disease. Yet since 1996, it is known that HIV-1 strains are not the only ones involved. In Sub-Saharan Africa, recurrent orobuccal herpes simplex virus type 1 (HSV-1 and genital recurrent herpes simplex virus type 2 (HSV-2 appeared many times prior to infection by HIV-1. Case Reports: Data on these ancient medical observations regarding African cancer epidemics can today be referred to as the relationship between the unfortunate immune deficiency of herpes in Bantu populations and HIV-1 viral strains. For centuries, the Bantu populations dispersed in forests were living in close proximity to chimpanzees infected by simian immunodeficiency virus (SIV and were exposed to SIV contamination which became HIV-1 in human beings. Presently, these unexplained Bantu cancer epidemics can be linked to the viral partnership of HSV-1/HSV-2 to HIV-1 strains. Conclusion: The key issue is now to prevent HSV-1/HSV-2 diseases related to HIV-1. An anti-herpes treatment administered early during childhood to Bantu populations will offer a mean of preventing herpes diseases related to HIV-1 infection and hence avoid cancer epidemics.

  19. Sensing of HSV-1 by the cGAS-STING pathway in microglia orchestrates antiviral defence in the CNS

    DEFF Research Database (Denmark)

    Reinert, Line S; Lopušná, Katarína; Winther, Henriette

    2016-01-01

    Herpes simplex encephalitis (HSE) is the most common form of acute viral encephalitis in industrialized countries. Type I interferon (IFN) is important for control of herpes simplex virus (HSV-1) in the central nervous system (CNS). Here we show that microglia are the main source of HSV-induced t......Herpes simplex encephalitis (HSE) is the most common form of acute viral encephalitis in industrialized countries. Type I interferon (IFN) is important for control of herpes simplex virus (HSV-1) in the central nervous system (CNS). Here we show that microglia are the main source of HSV......-induced type I IFN expression in CNS cells and these cytokines are induced in a cGAS-STING-dependent manner. Consistently, mice defective in cGAS or STING are highly susceptible to acute HSE. Although STING is redundant for cell-autonomous antiviral resistance in astrocytes and neurons, viral replication...

  20. Seroepidemiology of herpes simplex virus type 2 (HSV2) in HIV infected patients in Kermanshah-Iran

    OpenAIRE

    Janbakhash, Alireza; Mansouri, Feizollah; Vaziri, Siavash; Sayad, Babak; Afsharian, Mandana; Abedanpor, Ahmadreza

    2012-01-01

    Background: HSV2 has an important role in acquiring and transmitting HIV through genital ulcers. This study was conducted to determine the prevalence of this virus in HIV infected subject in Kermanshah, Iran.

  1. Use of injectable hormonal contraception and HSV-2 acquisition in a cohort of female sex workers in Vancouver, Canada.

    Science.gov (United States)

    Socías, M Eugenia; Duff, Putu; Shoveller, Jean; Montaner, Julio S G; Nguyen, Paul; Ogilvie, Gina; Shannon, Kate

    2017-06-01

    Increased risk of herpes simplex virus 2 (HSV-2) has been proposed as a possible indirect pathway through which hormonal contraceptives (specifically depot medroxyprogesterone acetate (DMPA)) may increase the risk of HIV acquisition among women. We investigated the effects of DMPA on HSV-2 acquisition among female sex workers. Longitudinal data were drawn from a prospective cohort of sex workers in Vancouver, Canada. The primary outcome was HSV-2 seroconversion. Extended Cox regression analyses were used to model the independent effect of DMPA use on HSV-2 acquisition. Between January 2010 and February 2014, 149 HSV-2 seronegative women were enrolled, contributing to 228 person-years (py) of follow-up. Of these, 19 (13.3%) reported DMPA use. There were 39 HSV-2 seroconversions (12 among DMPA users and 27 among non-users) over the study period (median follow-up of 18.6 months (IQR 8.4-29.9)), resulting in an overall incidence rate of 17.1 cases per 100 py (95% CI 12.4 to 23.6). Incidence rates were higher among DMPA users (57.4 cases per 100 py, 95% CI 31.4 to 105.0) compared with non-users (13.1 cases per 100 py, 95% CI 8.9 to 19.1). After adjusting for key confounders, use of DMPA remained an independent predictor of HSV-2 acquisition (adjusted HR 4.43, 95% CI 1.90 to 10.35). The high observed incidence rates of HSV-2, together with a strong association between DMPA exposure and HSV-2 acquisition, raise serious concerns about the provision of optimal reproductive and sexual healthcare to sex workers in this setting. Given the known links between HSV-2 and HIV, our findings underscore the need for further research to better understand the potential association between DMPA and increased risk of HSV-2 and other STIs to help inform the development of safer reproductive choices for women worldwide. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  2. Imaging expression of adenoviral HSV1-tk suicide gene transfer using the nucleoside analogue FIRU

    Energy Technology Data Exchange (ETDEWEB)

    Nanda, Dharmin [Department of Neurology, Daniel den Hoed Cancer Centre, University Hospital Rotterdam (Netherlands); Department of Neurosurgery, University Hospital Rotterdam (Netherlands); Jong, Marion de; Bakker, Willem; Bijster, Magda; Cox, Peter [Department of Nuclear Medicine, University Hospital Rotterdam (Netherlands); Vogels, Ronald; Havenga, Menzo [Crucell Holland BV, Leiden (Netherlands); Driesse, Maarten; Avezaat, Cees [Department of Neurosurgery, University Hospital Rotterdam (Netherlands); Morin, Kevin; Naimi, Ebrahim; Knaus, Edward; Wiebe, Leonard [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton (Canada); Smitt, Peter Sillevis [Department of Neurology, Daniel den Hoed Cancer Centre, University Hospital Rotterdam (Netherlands)

    2002-07-01

    Substrates for monitoring HSV1-tk gene expression include uracil and acycloguanosine derivatives.The most commonly used uracil derivative to monitor HSV1-tk gene transfer is 1-(2-fluoro-2-deoxy-{beta}-D-arabinofuranosyl)-5-[*I]iodouracil (fialuridine; I*-FIAU), where the asterisk denotes any of the radioactive iodine isotopes that can be used. We have previously studied other nucleosides with imaging properties as good as or better than FIAU, including 1-(2-fluoro-2-deoxy-{beta}-D-ribofuranosyl)-5-[*I]iodouracil (FIRU). The first aim of this study was to extend the biodistribution data of {sup 123}I-labelled FIRU. Secondly, we assessed the feasibility of detecting differences in HSV1-tk gene expression levels following adenoviral gene transfer in vivo with {sup 123}I-FIRU. 9L rat gliosarcoma cells were stably transfected with the HSV1-tk gene (9L-tk+). {sup 123}I-FIRU was prepared by radioiodination of 1-(2-fluoro-2-deoxy-{beta}-D-ribofuranosyl)-5-tributylstannyl uracil (FTMRSU; precursor compound) and purified using an activated Sep-Pak column. Incubation of 9L-tk+ cells and the parental 9L cells with {sup 123}I-FIRU resulted in a 100-fold higher accumulation of radioactivity in the 9L-tk+ cells after an optimum incubation time of 4 h. NIH-bg-nu-xid mice were then inoculated subcutaneously with HSV1-tk (-) 9L cells or HSV1-tk (+) 9L-tk+ cells into both flanks. Biodistribution studies and gamma camera imaging were performed at 15 min and 1, 2, 4 and 24 h p.i. At 15 min, the tumour/muscle, tumour/blood and tumour/brain ratios were 5.2, 1.0 and 30.3 respectively. Rapid renal clearance of the tracer from the body resulted in increasing tumour/muscle, tumour/blood and tumour/brain ratios, reaching values of 32.2, 12.5 and 171.6 at 4 h p.i. A maximum specific activity of 22%ID/g tissue was reached in the 9L-tk+ tumours 4 h after {sup 123}I-FIRU injection. Two Ad5-based adenoviral vectors containing the HSV1-tk gene were constructed: a replication-incompetent vector with

  3. Modelling the effects of sexting on the transmission dynamics of HSV-2 amongst adolescents

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    A. Mhlanga

    2015-12-01

    Full Text Available Prior studies have indicated that adolescents who are into sexting are likely to engage in risky sexual behaviours. In this paper, a mathematical model to assess the impact of sexting and peer influence on the spread of HSV-2 amongst adolescents is developed. The threshold parameters of the model are determined and stabilities are analysed. The impact of filtering and awareness campaigns is explored. Results from the study suggest that HSV-2 prevalence is high amongst adolescents who are into sexting as compared to those who do not. Further, we applied optimal control theory to the proposed model. The controls represent filtering and awareness campaigns. The objective is based on minimising the susceptible sexting adolescents, infected non-sexting adolescents and the infected sexting adolescents. The optimal control is characterised and numerically solved. Overall, the application of optimal control theory suggests that more effort should be devoted to both controls, filtering and awareness campaigns.

  4. Prognostic significance of numeric aberrations of genes for thymidylate synthase, thymidine phosphorylase and dihydrofolate reductase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Søren Astrup; Vainer, B.; Witton, C.J.

    2008-01-01

    BACKGROUND: Most human cancer cells have structural aberrations of chromosomal regions leading to loss or gain of gene specific alleles. This study aimed to assess the range of gene copies per nucleus of thymidylate synthase (TYMS), thymidine phosphorylase (TP) and dihydrofolate reductase (DHFR...

  5. Influence of dehydroepiandrosterone on G-6-PD activity and /sup 3/H-thymidine uptake of human lymphocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ennas, M.G.; Laconi, S.; Dessi, S.; Milia, G.; Murru, M.R.; Manconi, P.E.

    1987-01-01

    Dehydroepiandrosterone (DHEA) was found to inhibit experimental cancer development in mouse and rat lung, colon and mammary gland. Since DHEA is a potent inhibitor of mammalian G-6-PD, the hypothesis that the compound could inhibit cell proliferation through an inhibition of the pentose phosphate pathway has been formulated. We studied the effects of DHEA on the proliferation in vitro of human lymphocytes induced by several mitogens (PHA, ConA and PWM), measuring /sup 3/H-thymidine uptake. DHEA inhibited /sup 3/H-thymidine uptake of mitogen-stimulated cells from both G-6-PD+ and G-6-PD- (mediterranean type deficiency) individuals in a dose-dependent and reversible fashion. The inhibitory effect was found even if DHEA was added to cells in the last hours of culture, simultaneously with the addition of /sup 3/H-thymidine. These data suggest that the inhibition of thymidine uptake induced by DHEA on human lymphocytes probably does not depend on the inhibition of G-6-PD.

  6. Cytoenzymology and 3H-thymidine uptake of retro-ocular connective tissue cultures in experimental endocrino-exophthalmos.

    Science.gov (United States)

    Vaida, E; Petrescu, R; Ghinea, E; Stefaneanu, L

    1976-01-01

    The in vitro retro-ocular connective tissue cultures from guinea pigs with endocrine exophthalmos were studied before and after retro-ocular treatment with cortisol and hyaluronidase. Both cortisol and hyaluronidase inhibited the cell proliferation, the cytoenzymic activities of oxydoreductases, the 3H-thymidine uptake, the number of mitoses and the protein content of cultivated cells.

  7. The antiviral effect of three plant species of Iran on HSV-1

    OpenAIRE

    malihe Farahani

    2016-01-01

    Background : plants have had special position in human life and their medicinal application have been observed in manuscripts of many world scientists. Nowadays the treatment of HSV-1 infections with the available  chemical drugs often leads to the problems due to viral resistance and virus latency duration, therefore there is a requirement for new anti-herpes drugs.In this research the antiviral effects of  Camellia sinesis, Echium amoenumL and Nerium oleander, with ethnomedica...

  8. Platelet Activating Factor (PAF) Receptor Deletion or Antagonism Attenuates Severe HSV-1 Meningoencephalitis.

    Science.gov (United States)

    Vilela, Márcia Carvalho; Lima, Graciela Kunrath; Rodrigues, David Henrique; Lacerda-Queiroz, Norinne; Pedroso, Vinicius Sousa Pietra; de Miranda, Aline Silva; Rachid, Milene Alvarenga; Kroon, Erna Geessien; Campos, Marco Antônio; Teixeira, Mauro Martins; Teixeira, Antonio Lucio

    2016-12-01

    Herpes simplex virus type 1 (HSV-1) is a human pathogen that may cause severe encephalitis. The exacerbated immune response against the virus contributes to the disease severity and death. Platelet activating factor (PAF) is a mediator capable of inducing increase in vascular permeability, production of cytokines on endothelial cells and leukocytes. We aimed to investigate the activation of PAF receptor (PAFR) and its contribution to the severity of the inflammatory response in the brain following HSV-1 infection. C57BL/6 wild-type (WT) and PAFR deficient (PAFR-/-) mice were inoculated intracranially with 104 plaque-forming units (PFU) of HSV-1. Visualization of leukocyte recruitment was performed using intravital microscopy. Cells infiltration in the brain tissue were analyzed by flow cytometry. Brain was removed for chemokine assessment by ELISA and for histopathological analysis. The pharmacological inhibition by the PAFR antagonist UK-74,505 was also analyzed. In PAFR-/- mice, there was delayed lethality but no difference in viral load. Histopathological analysis of infected PAFR-/- mice showed that brain lesions were less severe when compared to their WT counterparts. Moreover, PAFR-/- mice showed less TCD4+, TCD8+ and macrophages in brain tissue. This reduction of the presence of leukocytes in parenchyma may be mechanistically explained by a decrease in leukocytes rolling and adhesion. PAFR-/- mice also presented a reduction of the chemokine CXCL9 in the brain. In addition, by antagonizing PAFR, survival of C57BL/6 infected mice increased. Altogether, our data suggest that PAFR plays a role in the pathogenesis of experimental HSV-1 meningoencephalitis, and its blockade prevents severe disease manifestation.

  9. Methimazole-Induced Hypothyroidism Enhances HSV-1 Infectivity without Altering Circulating Leukocytes in the Rat

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    Hadi Feizi

    2015-04-01

    Full Text Available Backgrounds & objectives: In addition to their genomic actions, thyroid hormones (THs can modulate the immune responses through cell surface receptors. One of these is the antiviral effect of THs. Methimazole, as an anti-thyroid compound, is widely used to treat hyperthyroid patients. It also reduces blood leukocytes, granulocytes in particular, and thereby may affect the immune response. Recently, we reported that methimazole-induced hypothyroidism intensifies herpes simplex virus-1(HSV-1 infectivity. To determine whether the effect is mediated through alterations in circulating leukocytes, we assessed the HSV-1 infectivity and circulating leukocytes in methimazole-induced hypothyroid rat.   Methods: Male Sprague Dawley rats received methimazole (200 μg/ml in their drinking water for 2 weeks. Rats were then inoculated with a non-lethal single dose of HSV-1, and sacrificed 3 days later to harvest their spleen. Spleen extract was prepared, and virus yield was determined by evaluation of cytopathic effects (CPE induced by the extract in a Vero cell culture system. For quantitative analysis, standard method of Reed-Muench was employed. The routine Wright’s staining protocol was used for blood leukocytes differential count.   Results: The CPE development was significantly increased in the cell cultures exposed to the spleen extract of methimazole-treated animals (P < 0.05, indicating a higher virus yield and intensified virus infectivity. However, the effect of methimazole on blood leukocytes was minimal.   Conclusion: Our data suggest that methimazole increases the susceptibility to HSV-1 infection, at least in part, by blocking THs synthesis but not alterations in circulating leukocytes.

  10. Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

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    Todo Tomoki

    2006-09-01

    Full Text Available Abstract Background Oncolytic herpes simplex virus (HSV vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. Results We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39

  11. A phase II trial of thymidine and carboplatin for recurrent malignant glioma: a North American Brain Tumor Consortium Study.

    Science.gov (United States)

    Robins, H. Ian; Chang, Susan M.; Prados, Michael D.; Yung, W. K. Alfred; Hess, Kenneth; Schiff, David; Greenberg, Harry; Fink, Karen; Nicolas, Kelly; Kuhn, John G.; Cloughesy, Timothy; Junck, Larry; Mehta, Minesh

    2002-01-01

    This phase II study in recurrent high-grade glioma evaluated the response rate, toxicities, and time to treatment failure of high-dose carboplatin modulated by a 24-h infusion of thymidine (75 g/m(2)). The trial was based on preclinical data and a prior phase I study ( J. Clin. Oncol. 17, 2922-2931, 1999); a phase II recurrent high-grade glioma study was initiated in July of 1998. Thymidine was given over 24 h; carboplatin was given over 20 min at hour 20 of the thymidine infusion. The starting dose of carboplatin had a value of 7 for the area under the curve (AUC), with allowance for dose escalation of 1 AUC unit per cycle if grade 2 toxicity was observed. Treatment cycles were repeated every 4 weeks. Accrual as of September 1999 was 45 patients [4 were unevaluable]: 76% with glioblastoma multiforme (GBM), 20% with anaplastic oligodendroglioma, 2% with mixed type, and 2% with anaplastic astrocytoma. Most patients had prior chemotherapy (78%). As observed in the earlier phase I study (in which carboplatin pharmacokinetics were unaltered by thymidine or antiseizure medications), thymidine was myeloprotective, resulting in a minimal need for dose reduction for patients having a >2 grade toxicity (in only 4% of the courses of treatment). Of 101 total courses, the number of courses (at the AUCs) was 3 (5), 4 (6), 58 (7), 20 (8), 11 (9), and 5 (10). Grade 3 nonhematologic toxicities included headache (4%), altered consciousness (3%), fatigue (1%), and nausea (3%). Responses included 2 partial (1 oligodendroglioma, 1 GBM; 5%); 3 minor (1 anaplastic astrocytoma, 2 GBM; 7.3%); 6 stable disease (14.6%); and 30 progressive disease (73.2%). For GBM patients, median survival was 23 weeks (with a 95% confidence interval of 20 to 50 weeks), and progression-free survival was 8 weeks (with a 95% confidence interval of 7-16 weeks). These results in GBM were comparable to other phase II GBM trials and thus do not represent a therapeutic advance in the treatment of GBM. Taken

  12. Validity of genito-urinary discharges, genital ulcers and genital rashes as indicators of seroincident HSV-2 infection

    Directory of Open Access Journals (Sweden)

    Eziyi Iche Kalu

    2015-06-01

    Full Text Available Objective: To evaluate the validity of vaginal discharges, urethral discharges, genital rashes, and painful genital ulcers as indicators of early detection of incident herpes simplex virus type 2 (HSV-2 infection among pregnant women in Benin metropolis. Methods: Participants were antenatal clinic attendees of University of Benin Teaching Hospital and Central Hospital, Benin. Baseline sociodemographic, obstetric and HSV-2 serological data were collected. The HSV-2-seronegative returned for a repeat HSV-2 antibody assay before delivery date. Data on incidence of genital rashes, abnormal vaginal discharges, painful genital ulcers and urethral discharges were collected. Results: The sensitivities of abnormal vaginal discharges, genital rashes, urethral discharges and painful genital ulcers were 82.3%, 70.6%, 41.2% and 28.6% respectively; while their positive-predictive values were 53.8%, 60.0%, 58.3% and 66.7% respective. All the symptoms had >95% specificities and 95% negative-predictive values for seroincident HSV-2 infection. Conclusions: Abnormal vaginal discharge, genital rashes, urethral discharges and genital ulcers are valid indicators of seroincident HSV-2 infection and could be useful in formulation of screening tools in resource-limited settings.

  13. Seroepidemiology of herpes simplex virus type 2 (HSV2) in HIV infected patients in Kermanshah-Iran.

    Science.gov (United States)

    Janbakhash, Alireza; Mansouri, Feizollah; Vaziri, Siavash; Sayad, Babak; Afsharian, Mandana; Abedanpor, Ahmadreza

    2012-01-01

    HSV2 has an important role in acquiring and transmitting HIV through genital ulcers. This study was conducted to determine the prevalence of this virus in HIV infected subject in Kermanshah, Iran. This descriptive study was performed among 170 HIV positive patients (case group) and 165 non-HIV cases (control group)) referred to Behavioral Counseling Center of Kermanshah, western of Iran. For the evaluation of HSV2 infection, blood sample was obtained and assessed for IgG antibody of HSV2 using ELISA method. The data were collected and analyzed. Out of 170 cases, 11 were seropositive for HSV2 (6.5%) in case group and 2 of 165 (1.21%) in control group (p=0.015). Seropositivity was 17.6% in female and 5.2% in male, 59% under and 8% age over 40. In HIV infected subjects, seroprevalence in female was 17.6% and in male was 5.2% (p=0.083). It can be derived that the seroprevalence of HSV2 in HIV positive patients in our region is relatively low. Hence, we do not recommend that HSV2 needs to be considered in HIV pretreatment evaluation program.

  14. Effects of Acyclovir and IVIG on Behavioral Outcomes after HSV1 CNS Infection

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    Chandran Ramakrishna

    2017-01-01

    Full Text Available Herpes simplex virus 1 (HSV encephalitis (HSE has serious neurological complications, involving behavioral and cognitive impairments that cause significant morbidity and a reduced quality of life. We showed that HSE results from dysregulated central nervous system (CNS inflammatory responses. We hypothesized that CNS inflammation is casually involved in behavioral abnormalities after HSE and that treatment with ACV and pooled human immunoglobulin (IVIG, an immunomodulatory drug, would improve outcomes compared to mice treated with phosphate buffered saline (PBS or ACV alone. Anxiety levels were high in HSV-infected PBS and ACV-treated mice compared to mice treated with ACV + IVIG, consistent with reports implicating inflammation in anxiety induced by lipopolysaccharide (LPS or stress. Female, but not male, PBS-treated mice were cognitively impaired, and unexpectedly, ACV was protective, while the inclusion of IVIG surprisingly antagonized ACV’s beneficial effects. Distinct serum proteomic profiles were observed for male and female mice, and the antagonistic effects of ACV and IVIG on behavior were paralleled by similar changes in the serum proteome of ACV- and ACV + IVIG-treated mice. We conclude that inflammation and other factors mediate HSV-induced behavioral impairments and that the effects of ACV and IVIG on behavior involve novel mechanisms.

  15. PEDF plus DHA modulate inflammation and stimulate nerve regeneration after HSV-1 infection.

    Science.gov (United States)

    He, Jiucheng; Neumann, Donna; Kakazu, Azucena; Pham, Thang Luong; Musarrat, Farhana; Cortina, M Soledad; Bazan, Haydee E P

    2017-08-01

    Herpes simplex virus type-1 (HSV-1) infection leads to impaired corneal sensation and, in severe cases, to corneal ulceration, melting and perforation. Here, we explore the potential therapeutic action of pigment epithelial-derived factor (PEDF) plus docosahexaenoic acid (DHA) on corneal inflammation and nerve regeneration following HSV-1 infection. Rabbits inoculated with 100,000 PFU/eye of HSV-1 strain 17Syn+ were treated with PEDF + DHA or vehicle. PEDF + DHA treatment resulted in a biphasic immune response with stronger infiltration of CD4+T cells, neutrophils and macrophages at 7-days post-treatment (p.t.) that was significantly decreased by 14 days, compared to the vehicle-treated group. Screening of 14 immune-related genes by q-PCR showed that treatment induced higher expression of IFN-γ and CCL20 and inhibition of IL-18 by 7 days in the cornea. PEDF + DHA-treated animals developed less dendritic corneal lesions, opacity and neovascularization. Corneal nerve density increased at 12-weeks p.t. with functional recovery of corneal sensation. Treatment with PEDF + DHA that was postponed by 3 weeks also showed increased nerve density when compared to vehicle. Our data demonstrate that PEDF + DHA promotes resolution of the inflammatory response to the virus and, most importantly, induces regeneration of damaged corneal nerves vital for maintaining ocular surface homeostasis. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Synthesis and anti-HSV-1 activity of quinolonic acyclovir analogues.

    Science.gov (United States)

    Lucero, Bianca d'A; Gomes, Claudia Regina B; Frugulhetti, Izabel Christina de P P; Faro, Letícia V; Alvarenga, Lise; de Souza, Maria Cecília B V; de Souza, Thiago M L; Ferreira, Vitor F

    2006-02-15

    Several 1-[(2-hydroxy-ethoxy)methyl]-3-carbethoxy-4(1H)quinolones (2a-l) and l-[(2-hydroxy-ethoxy)methyl]-4(1H)quinolone-3-carboxylic acids (3a-j and 3l) were synthesized and 2a-j, 2l and 3a-j, 3l were evaluated against herpes simplex virus type 1 (HSV-1), employing a one-pot reaction: silylation of the desired quinolone (BSTFA 1% TMCS) followed by equimolar amount addition of 1,3-dioxolane, chlorotrimethylsilane and KI, at room temperature. The acyclonucleosides 2a-l were obtained in 40-77% yields. The esters 2a-j and 2l were subsequently converted into the corresponding hydroxyacids 3 in 40-70% yields. Attempts of hydrolysis of 2k produced only a mixture of degradation products. Antiviral activity of 2 and 3 on HSV-1 virus infection was assessed by the virus yield assay. Except for compounds 2i and 3e, the acyclonucleosides were found to reduce the virus yield by 70-99% at the concentration of 50 microM, being the acids, in general, more effective inhibitors than their corresponding esters. Compounds 3j and 2d exhibited antiviral activity against HSV-1 virus with EC50 of 0.7+/-0.04 and 0.8+/-0.09 microM, respectively. Both compounds were not toxic towards the Vero cell line.

  17. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    Science.gov (United States)

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. HSV usurps eukaryotic initiation factor 3 subunit M for viral protein translation: novel prevention target.

    Directory of Open Access Journals (Sweden)

    Natalia Cheshenko

    2010-07-01

    Full Text Available Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by approximately 90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m. Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease.

  19. The Synthesis of the Stable IVDU Derivative for Imaging HSV-1 TK Expression

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun Jung; Choi, Tae Hyun; Ahn, Soon Hyuk; Kim, Byoung Soo; Park, Hyun; Cheon, Gi Jeong; An, Gwang Il [Korea Institute of Radiological and Medical Science, Seoul (Korea, Republic of); Rhee, Hak June [Hanyang University, Ansan (Korea, Republic of)

    2009-10-15

    5-iododeoxyuridine analogues have been exclusively developed for the potential antiviral and antitumor therapeutic agents. In this study, we synthesized carbocyclic radioiododeoxyuridineanalogue (ddIVDU) and carbocyclic intermediate as efficient carbocyclic radiopharmaceuticals. The synthesis is LAH reduction, hetero Diels-Alder reaction as key reactions including Pd(0)-catalyzed coupling reaction together with organotin. MCA-RH7777 (MCA) and MCA-tk (HSV1-tk positive) cells were treated with various concentration of carbocyclic ddIVDU, and GCV. Cytotoxicity was measured by the MTS methods. For in vitro uptake study, MCA and MCA-tk cells were incubated with 1uCi of [{sup 125}I]carbocyclic ddIVDU. Accumulated radioactivity was measured after various incubation times. The synthesis of ddIVDU and precursor for radioiodination were achieved from cyclopentadiene in good overall yield, respectively. The radioiododemetallation for radiolabeling gave more than 80% yield with > 95% radiochemical purity. GCV was more toxic than carbocyclic ddIVDU in MCA-tk cells. Accumulation of [{sup 125}I]carbocyclic ddIVDU was higher in MCA-tk cells than MCA cells. Biological data reveal that ddIVDU is stable in vitro, less toxic than ganciclovir (GCV), and selective in HSV1-tk expressed cells. Thus, this new carbocyclic nucleoside, referred to in this paper as carbocyclic 2',3'-didehydro-2',3'-dideoxy-5- iodovinyluridine (carbocyclic ddIVDU), is a potential imaging probe for HSV1-tk.

  20. Serum HSV-1 and 2 IgM in sexually transmitted diseases - more for screening less for diagnosis: An evaluation of clinical manifestation

    Directory of Open Access Journals (Sweden)

    Dharmishtha G Tada

    2012-01-01

    Full Text Available Background: Herpes simplex virus type 2 (HSV-2 is the cause of most genital herpes. Now, HSV-1 has become an important cause and represents even about 30% of genital herpes in some countries. So, study related to genital herpes should consider both HSV-1 and HSV-2. Aim: To examine trends in HSV-1 and 2 seroprevalence by Serum HSV-1 and 2 IgM in all type of sexually transmitted disease (STD patients and also to evaluate correlation of serum HSV-1 and 2 IgM in STD. Materials and Methods: 150 patients attending the STD clinic attached to a tertiary care hospital of Ahmedabad were included in the study. Serum HSV-1 and 2 IgM correlations with clinical manifestations of recurrent and non-recurrent type of genital herpes patients and other non-herpetic STD patients were studied. Results: The overall serum HSV-1 and 2 IgM in STD seroprevalence were 15.66%. Female has significant higher prevalence (P < 0.05. STD cases and HSV seroprevalence were specially concentrated in persons aged 21 to 30 years. Among those positive with HSV, the distribution of STD are wide spread and found in non-herpetic group at high frequency. Out of total 23 serum HSV-1 and 2 IgM positive, 12 and 11 are distributed in herpetic and non-herpetic STDs, respectively. Discussion and Conclusion: Though serum HSV-1 and 2 IgM in STDs are less diagnostic, they help to see the iceberg part of the infection among the population concerned in recent scenario or in another words, it provides recent infective burden.

  1. [Cytoplasmic kinase inhibitors].

    Science.gov (United States)

    Mano, Hiroyuki

    2010-10-01

    Protein kinases play essential roles in the regulation of cell proliferation. Point mutations or/and fusions of protein kinases are frequently identified in human cancers, and targeting such activated kinases provides us with a chance to eradicate tumor cells. This was first proved by imatinib mesylate that inhibits ABL tyrosine kinase and, thereby, efficiently kills malignant cells in chronic myeloid leukemia. In addition, other clinical trials are ongoing for kinase inhibitors against EML4--ALK in lung cancer, JAK2 in myeloproliferative disorders and BRAF in malignant melanoma. Early reports indeed reveal that such targeting compounds are promising drugs for human cancers with activated kinases.

  2. Inhibition of HSV-1 replication by laser diode-irradiation: possible mechanism of action.

    Science.gov (United States)

    Donnarumma, G; De Gregorio, V; Fusco, A; Farina, E; Baroni, A; Esposito, V; Contaldo, M; Petruzzi, M; Pannone, G; Serpico, R

    2010-01-01

    Herpes labialis are the most frequent clinical manifestations of HSV-1 infection. Epithelial cells are able to respond to HSV-1 presence inducing the expression of IL-6, IL-1, TNF-α and IL-8. These proinflammatory cytokines have a function in the acute-phase response mediation, chemotaxis, inflammatory cell activation and antigen-presenting cells. In the human epithelial cell models, it has been demonstrated that, after an early induction of proinflammatory host response, HSV-1 down-modulates the proinflammatory cytokine production through the accumulation of two viral proteins, ICP4 and ICP27, whose transcription is induced by tegument protein VP16. These viral proteins, through the decreasing of stabilizing the mRNAs of proinflammatory genes, delay cytokine production to an extent that allows the virus to replicate. Moreover, viral transactivating proteins, ICP-0 and VP-16 induce IL-10 expression. The conventional treatment of herpes labialis involves the topical and systemic use of antiviral drugs but it is necessary to find new therapies that can act in a selective and non-cytotoxic manner in viral infection. Laser diode therapy has been considered as a non-invasive alternative treatment to the conventional treatment of herpes labialis in pain therapy, in modulation of inflammation and in wound healing. This study aims to report a possible mechanism of action of laser diode irradiation in prevention and reduction of severity of labial manifestations of herpes labialis virus. We investigated, in an in vitro model of epithelial cells HaCat, the laser-effect on HSV-1 replication and we evaluated the modulation of expression of certain proinflammatory cytokines (TNF-α, IL-1β and IL-6), antimicrobial peptide HBD2, chemokine IL-8 and the immunosuppressive cytokine, IL-10. Our results lead us to hypothesize that LD-irradiation acts in the final stage of HSV-1 replication by limiting viral spread from cell to cell and that laser therapy acts also on the host immune

  3. Association between HSV1 seropositivity and obesity: data from the National Health and Nutritional Examination Survey, 2007-2008.

    Directory of Open Access Journals (Sweden)

    Zuzana Karjala

    Full Text Available BACKGROUND: Herpes simplex virus (HSV is among the most common sexually transmitted pathogens in the United States and worldwide. HSV has a high incidence of undetected cases. In addition, there is no treatment, and there is a lack of knowledge why disparities among populations exist. Research studies suggest that fat tissue may participate in body's immune responses, and the impact of obesity on susceptibility to HSV1 infection is not clear. The purpose of this study was to examine whether obesity is a risk factor for HSV1 infection using a large sample from the general population. METHODS/PRINCIPAL FINDINGS: This cross-sectional study used data from the National Health and Examination and Nutritional Examination Survey (NHANES from 2007-2008. Variables, gender, age, race/ethnicity, marital status, education, poverty level, and diabetes represented potential confounders and were included in analyses. The two-tailed Pearson's chi square, student's t test, and a multiple logistic regression analysis were applied to evaluate associations using a significance value of p≤0.05. Adjusted odds ratios with 95% confidence interval represented the degree of these associations. The prevalence of HSV1 infection in US population between 20 and 49 years old was 60.3% (n = 1,536. In this study, having a BMI classified as the obese group (BMI 30-39.9 was significantly associated with HSV1 infection before [unadjusted OR = 1.74 (95% CI 1.20-2.51, p = 0.006] and after controlling for socio-demographic factors [adjusted OR = 1.50 (95%CI 1.06-2.13], p = 0.026]. This association was stronger than three already established risk factors of age, female gender, and poverty level. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that obesity may play a role in the susceptibility to HSV1 infection. Findings from this study suggest that obesity should be considered when designing preventive measures for HSV1 infection. These results may also

  4. Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

    Science.gov (United States)

    Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

    2018-01-15

    Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways.IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4+ T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

  5. A truncation mutation of the neurovirulence ICP22 protein produced by a recombinant HSV-1 generated by bacterial artificial chromosome technology targets infected cell nuclei.

    Science.gov (United States)

    Bowles, Robert N; Blaho, John A

    2011-12-01

    The major regulatory protein ICP22 is unique among the immediate early proteins of herpes simplex virus. Viruses deleted for ICP22 replicate well in actively dividing cells, but not in quiescent cells or certain rodent lines. Accordingly, ICP22 represents an understudied herpes simplex virus (HSV) neurovirulence marker which is absolutely essential for viral neurogrowth. We utilized the bacterial artificial chromosome methodology to create a novel ICP22 truncation mutant, termed HSV-1(BACX). The integrity of HSV-1(BACX) was confirmed by detailed polymerase chain reaction analyses and immunoblotting using anti-ICP22 antibody. HSV-1(BACX) showed a reduced replication capacity in rabbit skin cells, consistent with previous studies using ICP22-null viruses. Importantly, HSV-1(BACX) localized to nuclei of infected primate Vero cells in a manner similar to wild-type ICP22. Thus, HSV-1(BACX) will serve as a useful tool to decipher the unusual biological properties and functions of the ICP22 protein.

  6. A live-attenuated HSV-2 ICP0 virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine.

    Directory of Open Access Journals (Sweden)

    William P Halford

    2011-03-01

    Full Text Available Glycoprotein D (gD-2 is the entry receptor of herpes simplex virus 2 (HSV-2, and is the immunogen in the pharmaceutical industry's lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0⁻ virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain. In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0⁻ virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein.

  7. Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

    Science.gov (United States)

    Çuburu, Nicolas; Wang, Kening; Goodman, Kyle N; Pang, Yuk Ying; Thompson, Cynthia D; Lowy, Douglas R; Cohen, Jeffrey I; Schiller, John T

    2015-01-01

    No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. Genital herpes is a highly prevalent chronic disease caused by

  8. Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

    Science.gov (United States)

    Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

    2017-01-01

    ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time

  9. Seroprevalence of Herpes Simplex Virus type-2 (HSV-2) among pregnant women who participated in a national HIV surveillance activity in Haiti.

    Science.gov (United States)

    Domercant, Jean Wysler; Jean Louis, Frantz; Hulland, Erin; Griswold, Mark; Andre-Alboth, Jocelyne; Ye, Tun; Marston, Barbara J

    2017-08-18

    Herpes simplex virus type 2 (HSV-2), one the most common causes of genital ulcers, appears to increase both the risk of HIV acquisition and HIV transmission. HSV-2/HIV co-infection among pregnant women may increase the risk of perinatal transmission of HIV. This study describes rates of HSV-2 among pregnant women in Haiti and HSV-2 test performance in this population. Unlinked residual serum specimens from the 2012 National HIV and Syphilis Sentinel Surveillance Survey among pregnant women in Haiti were tested using two commercial kits (Focus HerpeSelect, Kalon) for HSV-2 antibodies. We evaluated rates of HSV-2 seropositivity and HSV-2/HIV co-infection, associations between HSV-2 and demographic characteristics using multivariable Cox proportional hazards modeling, and HSV-2 test performance in this population. Serum samples from 1000 pregnant women (all 164 HIV positive and 836 random HIV negative) were selected. The overall weighted prevalence of HSV-2 was 31.4% (95% CI: 27.7-35.4) and the prevalence of HIV-positivity among HSV-2 positive pregnant women was five times higher than the prevalence among HSV-2 negative women (4.8% [95% CI: 3.9-6.0] vs. 0.9% [95% CI: 0.6-1.3], respectively). Factors significantly associated with HSV-2 positivity were HIV-positivity (PR: 2.27 [95% CI: 1.94-2.65]) and older age (PRs: 1.41 [95% CI: 1.05-1.91] for 20-24 years, 1.71 [95% CI:1.13-2.60] for 30-34 years, and 1.55 [95% CI: 1.10-2.19] for 35 years or greater]), while rural residence was negatively associated with HSV-2 positivity (PR 0.83 [95% CI: 0.69-1.00]), after controlling for other covariables. For this study a conservative Focus index cutoff of 3.5 was used, but among samples with a Focus index value ≥2.5, 98.4% had positive Kalon tests. The prevalence of HSV-2 is relatively high among pregnant women in Haiti. Public health interventions to increase access to HSV-2 screening in antenatal services are warranted.

  10. Local CD4 and CD8 T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence in latently infected human trigeminal ganglia.

    Directory of Open Access Journals (Sweden)

    Monique van Velzen

    2013-08-01

    Full Text Available Herpes simplex virus type 1 (HSV-1 infection results in lifelong chronic infection of trigeminal ganglion (TG neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

  11. HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part II. Vector Systems and Applications

    Directory of Open Access Journals (Sweden)

    Andreas Jacobs

    1999-11-01

    Full Text Available Many properties of HSV-1 are especially suitable for using this virus as a vector to treat diseases affecting the central nervous system (CNS, such as Parkinson's disease or malignant gliomas. These advantageous properties include natural neurotropism, high transduction efficiency, large transgene capacity, and the ability of entering a latent state in neurons. Selective oncolysis in combination with modulation of the immune response mediated by replication-conditional HSV-1 vectors appears to be a highly promising approach in the battle against malignant glioma. Helper virus-free HSV/AAV hybrid amplicon vectors have great promise in mediating long-term gene expression in the PNS and CNS for the treatment of various neurodegenerative disorders or chronic pain. Current research focuses on the design of HSV-1-derived vectors which are targeted to certain cell types and support transcriptionally regulatable transgene expression. Here, we review the recent developments on HSV-1-based vector systems and their applications in experimental and clinical gene therapy protocols.

  12. Identification of replication-competent HSV-1 Cgal+ strain targets in a mouse model of human hepatocarcinoma xenograft.

    Science.gov (United States)

    Santamaría, Enrique; Mora, María I; Carro-Roldán, Elvira; Molina, Manuela; Fernández-Irigoyen, Joaquín; Marconi, Peggy; Manservigi, Roberto; Greco, Anna; Epstein, Alberto L; Prieto, Jesús; Hernández-Alcoceba, Rubén; Corrales, Fernando J

    2009-11-02

    Recent studies based on animal models have shown the advantages and potential of oncolytic viral therapy using HSV-1 -based replication-competent vectors in the treatment of liver tumors, but little is known about the cellular targets that are modulated during viral infection. In the present work, we have studied the effects of intratumoral injections of HSV-1 Cgal(+) strain in a murine model of human hepatoma xenografts. Viral replication was assessed for more than 1month, leading to a significant reduction of tumor growth rate mediated, in part, by a cyclin B dependent cell proliferation arrest. Early events resulting in this effect were analyzed using a proteomic approach. Protein extracts from xenografted human hepatomas treated with saline or HSV-1 Cgal(+) strain during 24h were compared by 2-D DIGE and differential spots were identified by nanoLC-ESI-MS/MS. Alterations on glutathione S transferase 1 Omega, and ERp29 suggest novel HSV-1 Cgal(+) targets in solid liver tumors. Additionally, ERp29 showed a complex differential isoform pattern upon HSV-1 Cgal(+) infection, suggesting regulatory mechanisms based on post-translational modification events.

  13. Comparison of herpes simplex (HSV) proteins synthesized in Vero, HEP-2 and human megakaryocyte-like cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Soslau, G.; Pastorino, M.B.; Morgan, D.A.; Brodsky, I.; Howett, M.K.

    1986-05-01

    The natural human host blood cell capable of supporting herpes virus replication has yet to be defined. They found that a recently cultured human megakaryocyte-like (Meg) cell line can support HSV 1 and 2 replication as demonstrated by growth inhibition, CPE, virus production and HSV DNA synthesis. The HSV proteins synthesized and post-translationally modified in Vero and HEp-2 infected cells were compared to the protein species produced in the infected Meg cell since differences may influence antigenic properties and host range. Host cell protein synthesis was greatly reduced in all three cell lines within hours post infection (pi). However, maximum viral protein synthesis occurs between 4 and 24 hrs pi with the Meg cells as compared to 24-48 hrs pi with the other cell lines. The immunoprecipitated /sup 35/S-methionine labeled HSV protein gel patterns for each infected cell line are qualitatively and quantitatively very different from each other. Dramatic differences were also observed when infected cells were labeled with /sup 32/P-ATP (in vitro method) or /sup 32/Pi (in vivo method). Finally, analysis of /sup 3/H-mannose labeled HSV glycoproteins demonstrates that the post-translational modifications of these proteins are significantly altered in the Meg cell as compared to the Vero and HEp-2 cells.

  14. [Eucaryotic expression and bioactivity analysis of the recombinant HSV-gD1].

    Science.gov (United States)

    Wang, Zhengmao; Li, Lin; Guan, Wenyan; Li, Yuexi

    2010-05-01

    Envelope proteins of herpes simplex virus (HSV) plays a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among the envelope proteins, glycoprotein D (gD), which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. In order to analyze the antigenicity and immunogenicity of HSV-gD1, we chemically synthesized the extracellular domain fragment gene of gD1, cloned it into eucaryotic expression vector pCEP4, and transfected the HEK293 cells with the recombinant vector. Then we identified the recombinant protein by Western blotting, and detected antigenicity of the protein by ELISA. Finally, we used the purified gD1 protein to immunize Kunming mice in 1, 3, 5 weeks, and collected antiserum in 3, 5 and 7 weeks. We titrated the sera for the detection of anti gD1 using an ELISA assay. Gene sequencing analysis demonstrated that the recombinant plasmid pCEP4-gD1 was constructed successfully. Western blotting analysis indicated one major protein band, which molecular weights is approximate 46 kDa corresponding to the truncated forms of gD1 protein, was observed. ELISA assay showed that the expressed recombinant protein gD1 had good antigenicity. After the third immunization, antibody titer of the mouse anti-gD1 was at least 5 x10(3). The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.

  15. Sperm viral infection and male infertility: focus on HBV, HCV, HIV, HPV, HSV, HCMV, and AAV.

    Science.gov (United States)

    Garolla, Andrea; Pizzol, Damiano; Bertoldo, Alessandro; Menegazzo, Massimo; Barzon, Luisa; Foresta, Carlo

    2013-11-01

    Chronic viral infections can infect sperm and are considered a risk factor in male infertility. Recent studies have shown that the presence of HIV, HBV or HCV in semen impairs sperm parameters, DNA integrity, and in particular reduces forward motility. In contrast, very little is known about semen infection with human papillomaviruses (HPV), herpesviruses (HSV), cytomegalovirus (HCMV), and adeno-associated virus (AAV). At present, EU directives for the viral screening of couples undergoing assisted reproduction techniques require only the evaluation of HIV, HBV, and HCV. However, growing evidence suggests that HPV, HSV, and HCMV might play a major role in male infertility and it has been demonstrated that HPV semen infection has a negative influence on sperm parameters, fertilization, and the abortion rate. Besides the risk of horizontal or vertical transmission, the negative impact of any viral sperm infection on male reproductive function seems to be dramatic. In addition, treatment with antiviral and antiretroviral therapies may further affect sperm parameters. In this review we attempted to focus on the interactions between defined sperm viral infections and their association with male fertility disorders. All viruses considered in this article have a potentially negative effect on male reproductive function and dangerous infections can be transmitted to partners and newborns. In light of this evidence, we suggest performing targeted sperm washing procedures for each sperm infection and to strongly consider screening male patients seeking fertility for HPV, HSV, and HCMV, both to avoid viral transmission and to improve assisted or even spontaneous fertility outcome. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. TNF as marker of oral candidiasis, HSV infection, and mucositis onset during chemotherapy in leukemia patients.

    Science.gov (United States)

    Ramírez-Amador, V; Zambrano, J G; Anaya-Saavedra, G; Zentella-Dehesa, A; Irigoyen-Camacho, E; Meráz-Cruz, N; Ponce de León-Rosales, S

    2017-10-01

    To assess changes in the salivary expression of IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-17, and TNF in acute leukemia (AL) patients before and during chemotherapy, and its association with HSV infection, oral candidiasis (OC), and oral mucositis (OM) onset. Cohort study in AL patients >15 years starting induction chemotherapy at a Mexican oncological center (2013-2014). Onset of oral lesions (OLs) was assessed during follow-up, and saliva was obtained at baseline, at visit 2 (days 4-12), and at visit 3 (days 13-21) after chemotherapy, treated with a protease inhibitor and stored at -70°C. An enzyme-linked immunosorbent assay was performed. Cox proportional hazards regression models were constructed to estimate hazard ratios and its 95% CI (HR, 95% CI) for OL development. Forty-one patients were followed up, and 17 (41.5%) developed OLs. OL patients had higher baseline salivary IL-1α than those without lesions (p = 0.040). During visit 2, OL patients had higher levels of IL-1α (p = 0.033), IL-1β (p = 0.016), IL-6 (p = 0.035), and TNF (p = 0.019) than those who did not develop OLs. Patients with HSV infection, OC, and OM showed higher salivary TNF levels during follow-up (HR: 3.52, 95% CI: 1.35-9.14, p = 0.010). AL patients undergoing chemotherapy with high salivary TNF levels were more likely to develop HSV infection, OC, and OM. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

  17. Influence of concomitant infusion of thymidine and inosine on methotrexate activity in normal and P388-bearing mice

    NARCIS (Netherlands)

    Uitendaal, Martin P.; Schornagel, J.H.; Leyva, A.; Pinedo, H.M.

    1984-01-01

    Combinations of thymidine and inosine (ranging from 0 to 7.5 mg/hr) were co-administered during a 72-hr continuous i.v. infusion of 3 μg/hr methotrexate in normal and P388 solid tumor-bearing DBA/2 mice. Methotrexate alone was lethal to all normal mice. Inosine at 1.0–7.5 mg/hr could reverse

  18. Syntheses of pyrimidine acyclic nucleoside phosphonates as potent inhibitors of thymidine phosphorylase (PD-ECGF) from SD-lymphoma

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Votruba, Ivan; Holý, Antonín; Pohl, Radek

    2007-01-01

    Roč. 26, 8/9 (2007), s. 1025-1028 ISSN 1525-7770. [International Roundtable /17./. Bern, 03.09.2006-07.09.2006] R&D Projects: GA MŠk 1M0508 Grant - others: Descartes Prize(XE) HPAW-CT-2002-9001 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * pyrimidines * FPMP derivatives * fluorination Subject RIV: CC - Organic Chemistry Impact factor: 0.723, year: 2007

  19. The Relevance of miRNA-21 in HSV-Induced Inflammation in a Mouse Model

    OpenAIRE

    Bunsoon Choi; Hyoun-Ah Kim; Chang-Hee Suh; Hae Ok Byun; Ju-Yang Jung; Seonghyang Sohn

    2015-01-01

    The purpose of this study was to clarify the correlation between microRNA-21 (miR-21) expression and inflammation in a herpes simplex virus (HSV)-induced Behçet’s Disease (BD) mouse model. miR-21 was compared between BD patients and healthy controls in peripheral blood mononuclear cells (PBMC). For miR-21 inhibition, miR-21 antagomir was applied to BD mice. The change of symptoms was monitored. The levels of cytokines and related molecules were determined by ELISA and real time qPCR. Treatme...

  20. In Vitro Bypass of Thymidine Glycol by DNA Polymerase θ Forms Sequence-Dependent Frameshift Mutations.

    Science.gov (United States)

    Laverty, Daniel J; Greenberg, Marc M

    2017-12-26

    Unrepaired DNA lesions block replication and threaten genomic stability. Several specialized translesion polymerases, including polymerase θ (Pol θ), contribute to replicative bypass of these lesions. The role of Pol θ in double-strand break repair is well-understood, but its contribution to translesion synthesis is much less so. We describe the action of Pol θ on templates containing thymidine glycol (Tg), a major cytotoxic, oxidative DNA lesion that blocks DNA replication. Unrepaired Tg lesions are bypassed in human cells by specialized translesion polymerases by one of two distinct pathways: high-fidelity bypass by the combined action of Pol κ and Pol ζ or weakly mutagenic bypass by Pol θ. Here we report that in vitro bypass of Tg by Pol θ results in frameshift mutations (deletions) in a sequence-dependent fashion. Steady-state kinetic analysis indicated that one- and two-nucleotide deletions are formed 9- and 6-fold more efficiently, respectively, than correct, full-length bypass products. Sequencing of in vitro bypass products revealed that bypass preference decreased in the following order on a template where all three outcomes were possible: two-nucleotide deletion > correct bypass > one-nucleotide deletion. These results suggest that bypass of Tg by Pol θ results in mutations opposite the lesion, as well as frameshift mutations.

  1. The effect of chlorination of nucleotide bases on the conformational properties of thymidine monophosphate

    Directory of Open Access Journals (Sweden)

    T. M. Mukhina

    2015-04-01

    Full Text Available Recent studies on Escherichia coli bacteria cultivation, in which DNA thymine was replaced with 5-chlorouracil have refreshed the problem of understanding the changes to physical properties of DNA monomers resultant from chemical modifications. These studies have shown that the replacement did not affect the normal activities and division of the bacteria, but has significantly reduced its life span. In this paper a comparative analysis was carried out by the methods of computational experiment of a set of 687 possible conformers of natural monomeric DNA unit (2′-deoxyribonucleotide thymidine monophosphate and 660 conformers of 5-chloro-2′-deoxyuridine monophosphate – a similar molecules in which the natural nitrogenous base thymine is substituted with 5-chlorouracil. Structures of stable conformers of the modified deoxyribonucleotide have been obtained and physical factors, which determine their variation from the conformers of the unmodified molecule have been analyzed. A comparative analysis of the elastic properties of conformers­ of investigated molecules and non-covalent interactions present in them was conducted. The results can be used for planning experiments on synthesis of artificial DNA suitable for incorporation into living organisms.

  2. Elevation of radiolabelled thymidine uptake in RIF-1 fibrosarcoma and HT29 colon adenocarcinoma cells after treatment with thymidylate synthase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Yau, Kawai; Price, Patricia; Pillai, Radhakrishma G.; Aboagye, Eric [Imperial College, Imaging Sciences, London (United Kingdom)

    2006-09-15

    We recently showed an increase in tumour uptake of 2-[{sup 11}C]thymidine in patients with gastrointestinal malignancies after thymidylate synthase (TS) inhibition. To understand the phenomenon in more detail, we investigated whether TS inhibition by different TS inhibitors leads to a dose- and time-dependent change in the uptake of radiolabelled thymidine, and whether radiotracer uptake is related to changes in cell viability resulting from treatment. RIF-1 and HT29 cells were treated with the TS inhibitors 5-fluorouracil (5-FU) and AG337 (nolatrexed dihydrochloride), as well as cisplatin as control. The cell viability and net accumulation of [{sup 3}H]thymidine after a 1-h pulse was determined at different times after drug treatment. In both cell lines, [{sup 3}H]thymidine uptake increased after a 2-h treatment with 5-FU, in a dose- and time-dependent manner. [{sup 3}H]thymidine uptake decreased at 24 and 48 h post treatment. AG337 also produced a similar effect. In contrast to the TS inhibitors, cisplatin decreased [{sup 3}H]thymidine uptake in RIF-1 and HT29 cells at all time points. Cell viability was compromised only after 24 h. Using two types of TS inhibitor, we have shown an increase in [{sup 3}H]thymidine uptake, in a dose-dependent manner, a few hours after TS inhibition when the cell viability was not compromised. This effect was not seen with a non-TS inhibitor. These findings suggest that 2-[{sup 11}C]thymidine positron emission tomography can be used to study TS inhibition in vivo at early time points when cell viability is not compromised and may therefore be helpful in the development of new TS inhibitors and in differentiating between patients with tumours sensitive to TS inhibitors and those unlikely to respond. (orig.)

  3. Bacterial production determined by [3H]thymidine incorporation in field rhizospheres as evaluated by comparison to rhizodeposition

    DEFF Research Database (Denmark)

    Christensen, Henrik; Rønn, Regin; Ekelund, Flemming

    1995-01-01

    .2–15 × 104 cells g-1 soil) as well as elevated thymidine incorporation (9.7–12 pmol g-1 soil) in rhizosphere soil compared to bulk soil. Rhizodeposition, as determined by several pulse labellings with 14CO2, was estimated to be 412 µg C g-1 dry wt soil in the 0–15 cm layer. Bacterial production......, as determined by incorporation of 3H-labelled thymidine converted to bacterial C, revealed a plant-induced formation of 1348 µg bacterial C g-1 soil in the 0–15 cm layer. This is probably the first estimate for bacterial production based on thymidine incorporation which has been compared to an estimate of C......In a sandy loam soil cropped to barley bacterial production in the rhizosphere was compared to the results of a parallel investigation on rhizodeposition. Bacterial production was stimulated in the rhizosphere as revealed by an increased biomass of bacteria (643–883 µg C g-1 soil) and protozoa (7...

  4. Joint capsule treatment with enkephalin-encoding HSV-1 recombinant vector reduces inflammatory damage and behavioural sequelae in rat CFA monoarthritis.

    Science.gov (United States)

    Lu, Ying; McNearney, Terry A; Wilson, Steven P; Yeomans, David C; Westlund, Karin N

    2008-03-01

    This study assessed enkephalin expression induced by intra-articular application of recombinant, enkephalin-encoding herpes virus (HSV-1) and the impact of expression on nociceptive behaviours and synovial lining inflammation in arthritic rats. Replication-conditional HSV-1 recombinant vectors with cDNA encoding preproenkephalin (HSV-ENK), or control transgene beta-galactosidase cDNA (HSV-beta-gal; control) were injected into knee joints with complete Freund's adjuvant (CFA). Joint temperatures, circumferences and nociceptive behaviours were monitored on days 0, 7, 14 and 21 post CFA and vector treatments. Lumbar (L4-6) dorsal root ganglia (DRG) and spinal cords were immunostained for met-enkephalin (met-ENK), beta-gal, HSV-1 proteins and Fos. Joint tissues were immunostained for met-ENK, HSV-1 proteins, and inflammatory mediators Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) and cyclo-oxygenase-2, or stained with haematoxylin and eosin for histopathology. Compared to exuberant synovial hypertrophy and inflammatory cell infiltration seen in arthritic rats treated with CFA only or CFA and HSV-beta-gal, the CFA- and HSV-ENK-treated arthritic rats had: (i) striking preservation of synovial membrane cytoarchitecture with minimal inflammatory cell infiltrates; (ii) significantly improved nociceptive behavioural responses to mechanical and thermal stimuli; (iii) normalized Fos staining in lumbar dorsal horn; and (iv) significantly increased met-ENK staining in ipsilateral synovial tissue, lumbar DRG and spinal cord. The HSV-1 and transgene product expression were confined to ipsilateral lumbar DRG (HSV-1, met-ENK, beta-gal). Only transgene product (met-ENK and beta-gal) was seen in lumbar spinal cord sections. Targeted delivery of enkephalin-encoding HSV-1 vector generated safe, sustained opioid-induced analgesia with protective anti-inflammatory blunting in rat inflammatory arthritis.

  5. Daily Acyclovir Delays HIV-1 Disease Progression Among HIV-1/HSV-2 Dually-Infected Persons: A Randomized Trial

    Science.gov (United States)

    Lingappa, Jairam R.; Baeten, Jared M.; Wald, Anna; Hughes, James P.; Thomas, Katherine K.; Mujugira, Andrew; Mugo, Nelly; Bukusi, Elizabeth A.; Cohen, Craig R.; Katabira, Elly; Ronald, Allan; Kiarie, James; Farquhar, Carey; Stewart, Grace John; Makhema, Joseph; Essex, Myron; Were, Edwin; Fife, Kenneth H.; de Bruyn, Guy; Gray, Glenda E.; McIntyre, James; Manongi, Rachel; Kapiga, Saidi; Coetzee, David; Allen, Susan; Inambao, Mubiana; Kayitenkore, Kayitesi; Karita, Etienne; Kanweka, William; Delany, Sinead; Rees, Helen; Vwalika, Bellington; Magaret, Amalia; Wang, Richard S.; Kidoguchi, Lara; Barnes, Linda; Ridzon, Renee; Corey, Lawrence; Celum, Connie

    2010-01-01

    Background Well-tolerated medications that slow HIV-1 disease progression and delay initiation of antiretroviral therapy (ART) are needed. Most HIV-1-infected persons are dually-infected with herpes simplex virus type 2 (HSV-2). Daily HSV-2 suppression reduces plasma HIV-1 levels, but whether HSV-2 suppression delays HIV-1 disease progression is unknown. Methods Within a randomized, placebo-controlled trial of HSV-2 suppressive therapy (acyclovir 400 mg orally bid) to decrease HIV-1 transmission, 3381 HSV-2/HIV-1 dually-infected heterosexual Africans who at enrollment had CD4 counts ≥250 cells/mm3 and were not taking ART were followed for up to 24 months. We evaluated the effect of acyclovir on HIV-1 disease progression, defined by a primary composite endpoint of first occurrence of CD4 count death. As an exploratory analysis, we evaluated the endpoint of CD4 decline to HIV-1 plasma RNA was 4.1 log10 copies/mL. Acyclovir reduced risk of HIV-1 disease progression: 284 participants on acyclovir versus 324 on placebo reached the primary endpoint (hazard ratio [HR] 0.84, 95% confidence interval [CI] 0.71–0.98, p=0.03). Among participants with CD4 counts ≥350 cells/mm3, acyclovir delayed risk of CD4 decline to HIV-1 disease progression by 16% (95% CI 2–29%). The role of HSV-2 suppression in reducing HIV-1 disease progression prior to ART initiation warrants consideration (ClinicalTrials.gov #NCT00194519). PMID:20153888

  6. Prospective cohort study showing persistent HSV-2 shedding in women with genital herpes 2 years after acquisition.

    Science.gov (United States)

    Ramchandani, Meena; Selke, Stacy; Magaret, Amalia; Barnum, Gail; Huang, Meei-Li Wu; Corey, Lawrence; Wald, Anna

    2017-11-25

    Herpes simplex virus type 2 (HSV-2) is a prevalent infection with great variability in clinical and virological manifestations among individuals. This prospective cohort study aims to evaluate the natural history of HSV-2 reactivation in the genital area in the same group of women over time. Eighteen immunocompetent HSV-2 seropositive women were evaluated for viral shedding for 70 consecutive days within a median of 8 months (range 1-24 months) of HSV-2 acquisition and again approximately 2.5 years later from the original study. Participants obtained daily swabs of genital secretions for HSV PCR and recorded genital symptoms. The viral shedding rate was 29% during the initial study and 19% in the follow-up study (32% reduction, P=0.019). Subclinical shedding rate also decreased from 24% to 13% (37% reduction, P=0.032), as did the rate of days with genital lesions from 22% to 15% (33% reduction, P=0.24). The mean copy number during viral shedding remained unchanged over time at 4.8 log 10 c/mL (SD=2.0 and 1.6 during each study, respectively, P=0.33). Women with high viral shedding rates in the past were likely to continue to have high shedding rates (r=0.63, P=0.005). Despite some reduction, high viral shedding rates persist in women with genital HSV-2 greater than 2 years after acquisition. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  7. The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

    Science.gov (United States)

    Drevets, Peter; Chucair-Elliott, Ana; Shrestha, Priyadarsini; Jinkins, Jeremy; Karamichos, Dimitrios; Carr, Daniel J J

    2015-10-01

    To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-β1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-β1. Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.

  8. [Synthesis of optically active (R)- and (S)-tai-ding-an(TDA) and their anti-HSV activity evaluation].

    Science.gov (United States)

    Wang, G X; Wang, L; Zhao, Z Z; Tao, P Z; Wang, S Q

    1996-01-01

    Tai-Ding-An (3-phthalimido-2-oxo-n-butyraldehyde bisthiosemicarbazone, TDA) is an antiviral drug first synthesized in this institute. In order to clarify the difference between the two enantiomeric isomers of TDA, (R)- and (S)-TDA were synthesized from (R)- and (S)-alanine, respectively, via the following steps: fusing with phthalic anhydride gave 2-phthalimido alanine(2a or 2b). The resulting acid was reacted with thionyl chloride to offer the corresponding acid chloride(3a or 3b), which was treated with diazomethane to give the diazoketone(4a or 4b). Bromination of the ketone with hydrobromic acid gave the key intermediate 3-phthalimido-2-oxo-1-bromobutanone (5a or 5b). Compound 5a or 5b was oxidized with DMSO to give 6a or 6b, which was directly condensed with thiosemicarbazide to afford the desired (R)- or (S)-TDA. (R)-TDA, (S)-TDA and (RS)-TDA have been tested in cell culture for anti-Herpes simplex virus I (HSV-1) and HSV-2 activities by plaque reducing method. All of them showed inhibitory effects on HSV-1 and HSV-2 replication with IC50 of 0.0296 mmol.L-1, 0.0359 mmol.L-1 and 0.0418 mmol.L-1 for HSV-1 and 0.88 mmol.L-1, 1.04 mmol.L-1 and 1.06 mmol.L-1 for HSV-2. Not much difference was found among these compounds either on IC50 or on therapeutic indexes.

  9. A hybrid method of natural scene text detection using MSERs masks in HSV space color

    Science.gov (United States)

    Turki, Houssem; Ben Halima, Mohamed; Alimi, Adel M.

    2017-03-01

    Text detection in natural scenes holds great importance in the field of research and still remains a challenge and an important task because of size, various fonts, line orientation, different illumination conditions, weak characters and complex backgrounds in image. The contribution of our proposed method is to filtering out complex backgrounds by combining three strategies. These are enhancing the edge candidate detection in HSV space color, then using MSER candidate detection to get different masks applied in HSV space color as well as gray color. After that, we opt for the Stroke Width Transform (SWT) and heuristic filtering. Such strategies are followed so as to maximize the capacity of zones text pixels candidates and distinguish between text boxes and the rest of the image. The non-text components are filtered by classifying the characters candidates based on Support Vector Machines (SVM) using Histogram of Oriented Gradients (HOG) features. Finally we apply boundary box localization after a stage of word grouping where false positives are eliminated by geometrical properties of text blocks. The proposed method has been evaluated on ICDAR 2013 scene text detection competition dataset and the encouraging experiments results demonstrate the robustness of our method.

  10. Nonsulfated, cinnamic acid-based lignins are potent antagonists of HSV-1 entry into cells.

    Science.gov (United States)

    Thakkar, Jay N; Tiwari, Vaibhav; Desai, Umesh R

    2010-05-10

    In an effort to discover macromolecular mimetics of heparan sulfate (HS), we previously designed sulfated lignins (Raghuraman et al. Biomacromolecules 2007, 8, 1759-1763). To probe the relevance of sulfate groups of HS in viral entry, lignins completely devoid of sulfate moieties, and yet possessing an electrostatic surface equivalent to that of HS, were designed. Two carboxylated lignins based on a 4-hydroxy cinnamic acid scaffold were synthesized using enzymatic oxidative coupling in high yields, fractionated according to their sizes, and tested in cellular assays of herpes simplex virus-1 (HSV-1) infection. The two carboxylated lignins were found to not only inhibit HSV-1 entry into mammalian cells (IC(50) = 8-56 nM), but were more potent than sulfated lignins. In addition, shorter carboxylated lignins were found to be as active as the longer chains, suggesting that structural features, in addition to carboxylate groups, may be important. It can be expected that carboxylated lignins also antagonize the entry of other enveloped viruses, for example, HIV-1, Kaposi's sarcoma-associated herpes virus, and hepatitis C virus, that utilize HS to gain entry into cells. The results present major opportunities for developing lignin-based antiviral formulations for topical use.

  11. HSV amplicon delivery of glial cell line-derived neurotrophic factor is neuroprotective against ischemic injury.

    Science.gov (United States)

    Harvey, B K; Chang, C F; Chiang, Y H; Bowers, W J; Morales, M; Hoffer, B J; Wang, Y; Federoff, H J

    2003-09-01

    Direct intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) is neuroprotective against ischemia-induced cerebral injury. Utilizing viral vectors to deliver and express therapeutic genes presents an opportunity to produce GDNF within localized regions of an evolving infarct. We investigated whether a herpes simplex virus (HSV) amplicon-based vector encoding GDNF (HSVgdnf) would protect neurons against ischemic injury. In primary cortical cultures HSVgdnf reduced oxidant-induced injury compared to the control vector HSVlac. To test protective effects in vivo, HSVgdnf or HSVlac was injected into the cerebral cortex 4 days prior to, or 3 days, after a 60-min unilateral occlusion of the middle cerebral artery. Control stroke animals developed bradykinesia and motor asymmetry; pretreatment with HSVgdnf significantly reduced such motor deficits. Animals receiving HSVlac or HSVgdnf after the ischemic insult did not exhibit any behavioral improvement. Histological analyses performed 1 month after stroke revealed a reduction in ischemic tissue loss in rats pretreated with HSVgdnf. Similarly, these animals exhibited less immunostaining for glial fibrillary acidic protein and the apoptotic marker caspase-3. Taken together, our data indicate that HSVgdnf pretreatment provides protection against cerebral ischemia and supports the utilization of the HSV amplicon for therapeutic delivery of trophic factors to the CNS.

  12. The Relevance of miRNA-21 in HSV-Induced Inflammation in a Mouse Model

    Science.gov (United States)

    Choi, Bunsoon; Kim, Hyoun-Ah; Suh, Chang-Hee; Byun, Hae Ok; Jung, Ju-Yang; Sohn, Seonghyang

    2015-01-01

    The purpose of this study was to clarify the correlation between microRNA-21 (miR-21) expression and inflammation in a herpes simplex virus (HSV)-induced Behçet’s Disease (BD) mouse model. miR-21 was compared between BD patients and healthy controls in peripheral blood mononuclear cells (PBMC). For miR-21 inhibition, miR-21 antagomir was applied to BD mice. The change of symptoms was monitored. The levels of cytokines and related molecules were determined by ELISA and real time qPCR. Treatment with colchicine or pentoxifylline down-regulated the level of miR-21 with improved symptoms in mice. miR-21 inhibition was accompanied by down-regulated serum levels of IL-17 and IL-6. The expression levels of PDCD4, RhoB, PD-1, IL-12p35, and toll-like receptor-4 were also regulated by miR-21 inhibition. miR-21 was correlated with HSV-induced BD-like inflammation in mice and BD patients. The expression of miR-21 was regulated by antagomir in mice. PMID:25849652

  13. The Relevance of miRNA-21 in HSV-Induced Inflammation in a Mouse Model

    Directory of Open Access Journals (Sweden)

    Bunsoon Choi

    2015-04-01

    Full Text Available The purpose of this study was to clarify the correlation between microRNA-21 (miR-21 expression and inflammation in a herpes simplex virus (HSV-induced Behçet’s Disease (BD mouse model. miR-21 was compared between BD patients and healthy controls in peripheral blood mononuclear cells (PBMC. For miR-21 inhibition, miR-21 antagomir was applied to BD mice. The change of symptoms was monitored. The levels of cytokines and related molecules were determined by ELISA and real time qPCR. Treatment with colchicine or pentoxifylline down-regulated the level of miR-21 with improved symptoms in mice. miR-21 inhibition was accompanied by down-regulated serum levels of IL-17 and IL-6. The expression levels of PDCD4, RhoB, PD-1, IL-12p35, and toll-like receptor-4 were also regulated by miR-21 inhibition. miR-21 was correlated with HSV-induced BD-like inflammation in mice and BD patients. The expression of miR-21 was regulated by antagomir in mice.

  14. Evaluation of antibodies against glycoprotein D (gD) and glycoprotein G (gG) in HSV-1 infected individuals' serum samples.

    Science.gov (United States)

    Meshkat, Z; Roostaee, M H; Soleimanjahi, M; Zandi, K

    2012-04-01

    Glycoproteins D (gD) and G (gG) of herpes simplex virus type 1 (HSV-1) are virus envelope glycoproteins that are able to induce HSV-1 antibody production in infected persons. Therefore, those proteins could be in interest to develop the serodiagnostic test(s) for HSV antibody detection. The aim of present study was the comparison of anti-gD and anti-gG antibodies in HSV-1 infected individuals' serum samples. In this study, recombinant gD and gG were prepared and used for western blot test to detect the antibodies against HSV-1. Our data showed the total gD antibody titer was higher than gG antibody titer in the HSV-1 infected patient's sera but the gG antibody titer was high significantly. According to our results, gD and gG can be used for designing the diagnostic laboratory tests to evaluate total antibody against HSV-1 and HSV-2.

  15. Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: Inherent potential for biosynthetic versus live recombinant microbicides

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2008-08-01

    Full Text Available Abstract Background Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of ~120–200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2. Methods and Results Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6% had potential to cleave the HSV-2 genome in > 100 but 400 but Conclusion Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.

  16. Multicenter Evaluation of Meridian Bioscience HSV 1&2 Molecular Assay for Detection of Herpes Simplex Virus 1 and 2 from Clinical Cutaneous and Mucocutaneous Specimens.

    Science.gov (United States)

    Faron, Matthew L; Ledeboer, Nathan A; Patel, Anami; Beqa, Safedin H; Yen-Lieberman, Belinda; Kohn, Debra; Leber, Amy L; Mayne, Donna; Northern, William I; Buchan, Blake W

    2016-08-01

    Herpes simplex virus (HSV) causes acute and relapsing symptoms characterized by ulcerative lesions. Laboratory diagnosis of HSV in cutaneous or mucocutaneous lesions has historically been performed with the use of viral cell culture systems; however, these tests are laborious and suffer decreased sensitivity for advanced-stage lesions. The recent availability of FDA-cleared moderately complex assays has resulted in the increased use of molecular diagnostics for the routine detection of HSV in superficial swab specimens. We performed a clinical evaluation of the recently FDA-cleared illumigene HSV 1&2 loop-mediated isothermal amplification (LAMP) assay (Meridian Bioscience, Cincinnati OH) for the detection and differentiation of HSV-1 and HSV-2 in cutaneous and mucocutaneous swab specimens. A total of 1,153 clinical swab specimens were collected and tested at 7 different clinical centers. Each specimen was tested for the presence of HSV-1 and HSV-2 using the illumigene assay, and results were compared to those of the enzyme-linked virus-inducible system (ELVIS) as the reference method. Overall, the illumigene assay demonstrated a sensitivity and specificity of 94.8% and 95.5%, respectively, for the detection of HSV-1. Detection of HSV-2 was similar, with a sensitivity of 98.9% and a specificity of 95.5%. Discrepant analysis was performed using an alternative molecular test (AmpliVue HSV1+2 assay; Quidel Molecular, San Diego, CA) on 91/99 specimens that were recorded as false positive (FP) or false negative (FN) compared to the reference method. In total, 57/78 (73%) FP and 9/13 (69%) FN illumigene results were supported by the AmpliVue result. The illumigene HSV 1&2 assay demonstrated high sensitivity and specificity to detect and differentiate HSV in clinical specimens and identified 57 additional specimens that were positive for HSV compared to culture. The use of LAMP eliminates the need for the cycling of temperatures and provides results in less than 60 min

  17. Effects of ozone inhalation on polyamine metabolism and tritiated thymidine incorporation into DNA of rat lungs

    Energy Technology Data Exchange (ETDEWEB)

    Elsayed, N.M.; Ellingson, A.S.; Tierney, D.F.; Mustafa, M.G. (Univ. of California, Los Angeles (USA))

    1990-01-01

    We examined the effects of low-level ozone (O3) inhalation on polyamine metabolism and tritiated thymidine (3H-TdR) incorporation into DNA in rat lungs. We have also compared the activities of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, and glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the pentose phosphate cycle and a typical marker of oxidant injury, to assess whether ODC can serve as a sensitive marker of O3 effects on the lung. We exposed 90-day-old male specific-pathogen-free Sprague-Dawley rats to either 0.45 +/- 0.05 ppm (882 +/- 98 micrograms/m3) O3 or filtered room air continuously for 3 days. After exposure, the rats were terminated and the lungs examined for enzyme activities, polyamine contents, DNA content, and 3H-TdR incorporation. We found that in exposed rats, the enzyme activities were significantly increased (p less than 0.05) relative to air controls. G6PD, 25%, ODC, 147%, and S-adenosylmethionine decarboxylase (AdoMet DC), 86%. Polyamine contents were also affected by O3; putrescine increased 80%, p less than 0.05, spermidine did not change, and spermine decreased 23%, p less than 0.05. 3H-TdR incorporation into DNA was significantly elevated, 155%, p less than 0.001, after O3 exposure while total lung DNA content remained unchanged. The concomitant and large increase in ODC activity (reflecting polyamine metabolism) and DNA labeling (reflecting DNA synthesis and/or repair), indicates a strong correlation between the two and suggests that polyamine metabolism may play an important role in the accelerated cell proliferation associated with O3 injury. Moreover, the greater increase in lung ODC activity compared to other enzymes offers a sensitive marker of the lung response to inhaled O3.

  18. Early assessment of therapy response in malignant lymphoma with the thymidine analogue [18F]FLT.

    Science.gov (United States)

    Buck, Andreas K; Kratochwil, Clemens; Glatting, Gerhard; Juweid, Malik; Bommer, Martin; Tepsic, Djurdja; Vogg, Andreas T J; Mattfeldt, Torsten; Neumaier, Bernd; Möller, Peter; Reske, Sven N

    2007-11-01

    The aim of this study was to determine whether the thymidine analogue 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) is adequate for early evaluation of the response of malignant lymphoma to antiproliferative treatment in a mouse xenotransplant model. Immunodeficient mice bearing a follicular lymphoma xenotransplant were treated with high-dose chemotherapy (cyclophosphamide, n = 10), immunotherapy (CD20 mAb, ibritumomab-tiuxetan, n = 10) or radioimmunotherapy ([(90)Y]CD20 mAb, Zevalin, n = 10). Forty-eight hours after treatment, antiproliferative effects were assessed with [(18)F]FLT. Ninety minutes after i.v. injection of 5-10 MBq [(18)F]FLT, mice were sacrificed and radioactivity within the tumour and normal organs was measured using a gamma counter and calculated as % ID/g. The proliferation fraction in tissue samples derived from treated and untreated tumours was evaluated by Ki-67 immunohistochemistry, which served as the reference for proliferative activity. In untreated lymphoma, the mean proliferation fraction was 83.6%. After chemotherapy, the mean proliferation fraction decreased to 39.3% (p = 0.0001), after immunotherapy to 77.6% (p = 0.0078) and after radioimmunotherapy to 78.8% (p = 0.014). In none of the animals was a significant change in tumour size observed. In untreated lymphoma, tumoural [(18)F]FLT uptake was 5.4% ID/g, after chemotherapy it was 1.5% (p = 0.0005), after immunotherapy, 3.9% (non-significant), and after radioimmunotherapy, 5.8% (non-significant). In a lymphoma xenotransplant model, [(18)F]FLT detects early antiproliferative drug activity before changes in tumour size are visible. These findings further support the use of [(18)F]FLT-PET for imaging early response to treatment in malignant lymphoma.

  19. [The thymidine phosphorylase as the platelet-derived endothelial cell growth factor of endometrial cancer].

    Science.gov (United States)

    Kubiak, Robert; Miszczak-Zaborska, Elzbieta; Smolarek, Monika; Wójcik-Krowiranda, Katarzyna; Bartkowiak, Jacek

    2009-08-01

    The aim of the study was to assess the correlation between the activity of thymidine phosphorylase (TP) and the platelet derived-endothelial cell growth factor (PD-ECGF) expression in endometrial carcinoma. The study group consisted of 40 tissue samples taken from patients with endometrial carcinoma, who underwent surgery in First Clinic of Gynecology and Oncologic Gynecology of Medical University in Lodz. The control tissue samples were taken from patients who were operated on for non-oncologic reason. The activity of TP was measured by the spectrophotometric method in the cytosol of tumor cells, and the immunohistochemical staining of PD-ECGF was performed in the same tumors. The results of TP activity were compared with the microvessel density (MD) assessed by immunohistochemical analysis and with clinico-pathological features like tumor grade and FIGO stage. A positive correlation between the enzyme activity and expression of TP/PD-ECGF protein was found. Moreover a significantly higher TP activity was confirmed in malignant tumors from endometrial cancer patients when compared to the controls. A positive correlation between the enzyme activity and MD was also stated, but there was no connection to the grade of tumors and FIGO stage. Since the TP activity proved to be related to PD-ECGF expression and angiogenesis, we can state that TP seems to be an active form of PD-ECGF growth factor in endometrial carcinoma. This is in agreement with the results of many publications on other malignancies. The proper modulation of this activity may be useful in adjuvant therapies.

  20. Genital herpes evaluation by quantitative TaqMan PCR: correlating single detection and quantity of HSV-2 DNA in cervicovaginal lavage fluids with cross-sectional and longitudinal clinical data.

    Science.gov (United States)

    Aumakhan, Bulbulgul; Hardick, Andrew; Quinn, Thomas C; Laeyendecker, Oliver; Gange, Stephen J; Beyrer, Chris; Cox, Christopher; Anastos, Kathryn; Cohen, Mardge; Greenblatt, Ruth M; Merenstein, Daniel J; Minkoff, Howard; Nowicki, Marek; Gaydos, Charlotte A

    2010-11-18

    To evaluate the utility of a single quantitative PCR (qPCR) measurement of HSV (HSV-1&2) DNA in cervicovaginal lavage (CVL) specimens collected from women with predominantly chronic HSV-2 infection in assessing genital HSV shedding and the clinical course of genital herpes (GH) within a cohort with semiannual schedule of follow up and collection of specimens. Two previously described methods used for detection of HSV DNA in mucocutaneous swab samples were adapted for quantification of HSV DNA in CVLs. Single CVL specimens from 509 women were tested. Presence and quantity of CVL HSV DNA were explored in relation to observed cross-sectional and longitudinal clinical data. The PCR assay was sensitive and reproducible with a limit of quantification of ~50 copies per milliliter of CVL. Overall, 7% of the samples were positive for HSV-2 DNA with median log10 HSV-2 DNA copy number of 3.9 (IQR: 2.6-5.7). No HSV-1 was detected. Presence and quantity of HSV-2 DNA in CVL directly correlated with the clinical signs and symptoms of presence of active symptomatic disease with frequent recurrences. Single qPCR measurement of HSV DNA in CVL fluids of women with chronic HSV-2 infection provided useful information for assessing GH in the setting of infrequent sampling of specimens. Observed positive correlation of the presence and quantity of HSV-2 DNA with the presence of active and more severe course of HSV-2 infection may have clinical significance in the evaluation and management of HSV-2 infected patients.

  1. The effects of daily distress and personality on genital HSV shedding and lesions in a randomized, double-blind, placebo-controlled, crossover trial of acyclovir in HSV-2 seropositive women.

    Science.gov (United States)

    Strachan, Eric; Saracino, Misty; Selke, Stacy; Magaret, Amalia; Buchwald, Dedra; Wald, Anna

    2011-10-01

    Herpes simplex virus (HSV) infections are ubiquitous in humans, but the determinants of clinical and virologic severity are not completely understood. Prior research has suggested that psychological distress can be a co-factor in reactivation of latent HSV infection. Personality traits such as extraversion and neuroticism influence stress attributions and may inform the relationship between psychological distress and health outcomes. Earlier studies in this area have primarily focused on subjective reports of HSV lesion recurrence, but such reports may be influenced by both personality traits and distress. We report results from a randomized, double-blind, placebo-controlled, crossover trial of acyclovir in 19 women for whom personality was assessed at baseline and daily assessments of genital lesions, stress, anxiety, and depression levels were collected for 22 weeks. In addition, daily swabs of the genital mucosa were collected to assess HSV-2 viral reactivation. We found that daily stress predicted genital lesion frequency, and that daily stress, anxiety, and depression predicted genital lesion onset approximately 5 days before onset. Anxiety was also associated with genital lesions 3 days after onset. Distress and viral reactivation were not associated; and no personality traits were associated with any of the outcomes. These results support the hypothesis that psychological distress is both a cause and a consequence of genital lesion episodes. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  3. Contribution of N-linked glycans on HSV-2 gB to cell–cell fusion and viral entry

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Sukun [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Hu, Kai [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); He, Siyi; Wang, Ping; Zhang, Mudan; Huang, Xin [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Du, Tao [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Zheng, Chunfu [Soochow University, Institutes of Biology and Medical Sciences, Suzhou 215123 (China); Liu, Yalan [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Hu, Qinxue, E-mail: qhu@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Institute for Infection and Immunity, St George' s University of London, London SW17 0RE (United Kingdom)

    2015-09-15

    HSV-2 is the major cause of genital herpes and its infection increases the risk of HIV-1 acquisition and transmission. HSV-2 glycoprotein B together with glycoproteins D, H and L are indispensable for viral entry, of which gB, as a class III fusogen, plays an essential role. HSV-2 gB has seven potential N-linked glycosylation (N-CHO) sites, but their significance has yet to be determined. For the first time, we systematically analyzed the contributions of N-linked glycans on gB to cell–cell fusion and viral entry. Our results demonstrated that, of the seven potential N-CHO sites on gB, mutation at N390, N483 or N668 decreased cell–cell fusion and viral entry, while mutation at N133 mainly affected protein expression and the production of infectious virus particles by blocking the transport of gB from the endoplasmic reticulum to Golgi. Our findings highlight the significance of N-linked glycans on HSV-2 gB expression and function. - Highlights: • N-linked glycan at N133 is important for gB intracellular trafficking and maturation. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal cell–cell fusion. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal viral entry.

  4. Binding to chromosomes of herpes simplex-related antigens in biochemically transformed cells.

    Science.gov (United States)

    Kit, S; Kurchak, M; Wray, W; Dubbs, D R

    1978-07-01

    Association of herpes simplex virus (HSV)-related antigens with chromosomes was demonstrated in human and mouse cells biochemically transformed by HSV that had been irradiated with ultraviolet light. This was accomplished by using peroxidase-anti-peroxidase immunological staining with rabbit antisera that had high neutralizing titers against both HSV-specific thymidine kinase activity and virus infectivity. Antisera-against HSV did not react with chromosomes of uninfected cells nor did normal sera react with any of the constitutents of biochemically transformed cells. Methanol/acetic acid treatment of biochemically transformed cells eliminated their nuclear staining for HSV-related antigens. In vitro binding of HSV-related antigens to chromosomes was demonstrated by incubating soluble antigens from high salt extracts of HSV-infected cells with methanol/acetic acid-fixed chromosomes of biochemically transformed or uninfected cells, followed by exposure to antiserum against HSV and peroxidase-anti-peroxidase staining. There was no staining when soluble extracts from uninfected cells were substituted for those from HSV-infected cells. The results show that cells biochemically transformed and lytically infected by HSV, respectively, contain antigens, which like the Epstein-Barr virus-associated nuclear antigen (EBNA), bind to chromosomes in vivo and in vitro.

  5. The epidemiology of HIV and HSV-2 infections among women participating in microbicide and vaccine feasibility studies in Northern Tanzania.

    Directory of Open Access Journals (Sweden)

    Saidi H Kapiga

    Full Text Available To prepare for future HIV prevention trials, we conducted prospective cohort studies among women working in food and recreational facilities in northern Tanzania. We examined the prevalence and incidence of HIV and HSV-2, and associated risk factors.Women aged 18-44 years working in food and recreational facilities were screened to determine their eligibility for the studies. Between 2008-2010, HIV-negative women were enrolled and followed for 12 months. At enrolment and 3-monthly, we collected socio-demographic and behavioural data, and performed clinical examinations for collection of biological specimens that were tested for reproductive tract infections. Risk factors for HIV and HSV-2 incidence were investigated using Poisson regression models.We screened 2,229 and enrolled 1,378 women. The median age was 27 years (interquartile range, IQR 22, 33, and median duration working at current facility was 2 years. The prevalences of HIV at screening and HSV-2 at enrolment were 16% and 67%, respectively. Attendance at the 12-month visit was 86%. HIV and HSV-2 incidence rates were 3.7 (95% confidence interval, CI: 2.8,5.1 and 28.6 (95% CI: 23.5,35.0/100 person-years, respectively. Women who were separated, divorced, or widowed were at increased risk of HIV (adjusted incidence rate ratio, aRR = 6.63; 95% CI: 1.97,22.2 and HSV-2 (aRR = 2.00; 95% CI: 1.15,3.47 compared with married women. Women reporting ≥3 partners in the past 3 months were at higher HIV risk compared with women with 0-1 partner (aRR = 4.75; 95% CI: 2.10,10.8, while those who had reached secondary education or above were at lower risk of HSV-2 compared with women with incomplete primary education (aRR = 0.42; 95% CI: 0.22,0.82.HIV and HSV-2 rates remain substantially higher in this cohort than in the general population, indicating urgent need for effective interventions. These studies demonstrate the feasibility of conducting trials to test new interventions in this

  6. Evaluation of 5'-deoxy-5'-[F-18]fluorothymidine as a tracer of intracellular thymidine phosphorylase activity

    Energy Technology Data Exchange (ETDEWEB)

    Grierson, John R. [Department of Radiology, University of Washington, Seattle, WA 98195 (United States)]. E-mail: grierson@u.washington.edu; Brockenbrough, J. Scott [Department of Radiology, University of Washington, Seattle, WA 98195 (United States); Rasey, Janet S. [Department of Radiation Oncology, University of Washington, Seattle, WA 98195 (United States); Wiens, Linda W. [Department of Radiology, University of Washington, Seattle, WA 98195 (United States); Schwartz, Jeffery L. [Department of Radiation Oncology, University of Washington, Seattle, WA 98195 (United States); Jordan, Robert [Department of Radiation Oncology, University of Washington, Seattle, WA 98195 (United States); Vesselle, Hubert [Department of Radiology, University of Washington, Seattle, WA 98195 (United States)

    2007-07-15

    Two human cell lines (A549 and U937) with cytosolic thymidine phosphorylase (TP) activity were used to evaluate the potential of 5'-deoxy-5'-[F-18]fluorothymidine ([F-18]DFT) as a tracer of intracellular TP expression. Cellular metabolism of DFT led to the production of 5-[F-18]fluoro-2,5-dideoxy-D-ribose-1{alpha}-phosphate ([F-18]FddR-1P), in analogy to the metabolism of thymidine, which produces 2-deoxy-D-ribose-1{alpha}-phosphate (dR-1P). A549 cells showed the highest production rate of FddR-1P. After A549 cells were exposed to [F-18]DFT for 40 min, the relative intracellular concentration of [F-18]FddR-1P was more than sevenfold higher in cells than its precursor in the incubating medium. For the same amount of time, a twofold concentration was seen in U937 cells. However, uptake ratios did not rank with the corresponding TP activities found in cell extracts [TP activity ratio (U937:A549)=1.6] that were independently determined with a labeled thymidine/thymine cleavage assay. The discrepancy of TP activity ratios was traced to the instability of FddR-1P in cells. This was evident from the fact that cells accumulated radioactivity by producing FddR-1P, but activity also effluxed from cells over 1 h when the medium was subsequently made tracer free. The dominant labeled molecule released by cells was characterized as a neutral and lipophilic molecule, which was presumed to be a deoxynucleoside. Our results indicate that [F-18]DFT would not be effective for imaging TP expression because its initial metabolite undergoes further conversion to a diffusible secondary metabolite, allowing activity loss from cells.

  7. The effect of adriamycin on dentinogenesis and 3H-thymidine incorporation into the enamel organ of the rat incisor.

    Science.gov (United States)

    Karim, A C

    1990-01-01

    The effect of adriamycin (5 mg/kg) on 3H-thymidine incorporation and on dentin formation was studied in rat incisors. Male Sprague Dawley rats received an intravenous injection of adriamycin. Some of these also received a subcutaneous injection of 3H-thymidine at a dose of 2 mCi/kg one day later. One group of control animals received an intravenous injection of a volume of physiological saline equal to that of the adriamycin dose. Another group received physiological saline, and one hour later was given an additional injection of 3H-thymidine at a similar dose as above. All the animals were killed by perfusion with 2.5% phosphate buffered glutaraldehyde 1 h, 1 d, 4 d, 8 d, 16 d, 28 d, and 32 d after 3H-thymidine treatment. Light microscopy revealed irregular dentin deposits between the mantle and circumpulpal layer of the labial dentin at 16 d. Within these deposits were trapped cells. The latter, through radioautographic labelling, appeared to be cells from the odontoblast layer. Also, the labelling pattern of the enamel organ in both the control and experimental groups indicated that the eruption rate of the tooth was not affected. Serial sectioning and examination of the lingual portion of the incisors at 28 d revealed a lack of dentin formation and a failure in the closure of the apical foramen. Electron microscopic observations showed an irregular and random arrangement of collagen fibers within the deposits of irregular dentin, and the presence of twisted odontoblastic processes. Examination of the lingual surface showed the presence of fibroblasts and collagen fibers bridging the gap that resulted from the failure in dentin formation. These cells, which were similar to periodontal ligament cells, appeared to have arisen from that area. These results indicate that adriamycin has no effect on tooth eruption, but has a reversible effect on the function of secretory odontoblasts, which manifested itself as a periodic deposition of irregular dentin on the labial

  8. Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine.

    Science.gov (United States)

    Li, Yang; Zhu, Xujun; Zhang, Xueyu; Fu, Jing; Wang, Zhiwen; Chen, Tao; Zhao, Xueming

    2016-06-03

    Genome streamlining has emerged as an effective strategy to boost the production efficiency of bio-based products. Many efforts have been made to construct desirable chassis cells by reducing the genome size of microbes. It has been reported that the genome-reduced Bacillus subtilis strain MBG874 showed clear advantages for the production of several heterologous enzymes including alkaline cellulase and protease. In addition to enzymes, B. subtilis is also used for the production of chemicals. To our best knowledge, it is still unknown whether genome reduction could be used to optimize the production of chemicals such as nucleoside products. In this study, we constructed a series of genome-reduced strains by deleting non-essential regions in the chromosome of B. subtilis 168. These strains with genome reductions ranging in size from 581.9 to 814.4 kb displayed markedly decreased growth rates, sporulation ratios, transformation efficiencies and maintenance coefficients, as well as increased cell yields. We re-engineered the genome-reduced strains to produce guanosine and thymidine, respectively. The strain BSK814G2, in which purA was knocked out, and prs, purF and guaB were co-overexpressed, produced 115.2 mg/L of guanosine, which was 4.4-fold higher compared to the control strain constructed by introducing the same gene modifications into the parental strain. We also constructed a thymidine producer by deleting the tdk gene and overexpressing the prs, ushA, thyA, dut, and ndk genes from Escherichia coli in strain BSK756, and the resulting strain BSK756T3 accumulated 151.2 mg/L thymidine, showing a 5.2-fold increase compared to the corresponding control strain. Genome-scale genetic manipulation has a variety of effects on the physiological characteristics and cell metabolism of B. subtilis. By introducing specific gene modifications related to guanosine and thymidine accumulation, respectively, we demonstrated that genome-reduced strains had greatly improved

  9. Detection of circulating tumor cells using oHSV1-hTERT-GFP in lung cancer.

    Science.gov (United States)

    Gao, Hongjun; Liu, Wenjing; Yang, Shaoxing; Zhang, Wen; Li, Xiaoyan; Qin, Haifeng; Wang, Weixia; Zhao, Changyun

    2017-10-25

    This study was conducted to evaluate the clinical utility of the oHSV1-hTERT-GFP circulating tumor cell (CTC) detection method in the peripheral blood of patients with lung cancer by comparing its sensitivity to the CellSearch CTC detection method. The oHSV1-hTERT-GFP and CellSearch CTC detection methods were compared using peripheral blood samples of patients pathologically diagnosed with lung cancer. A total of 240 patients with lung cancer were recruited, including 89 patients who were newly diagnosed and 151 patients who had previously received treatment. Sixty-six newly diagnosed patients were evaluated using both methods. The CTC detection rates were 71.2% and 33.3% using the oHSV1-hTERT-GFP and CellSearch methods, respectively; this difference was statistically significant (P = 0.000). Among the entire cohort (n = 240), the CTC detection rate using the oHSV1-hTERT-GFP method was 76.3%, with a CTC count of 0-81. The CTC detection rates were 76.7%, 68.9%, and 76.3% in patients with squamous cell carcinoma, adenocarcinoma, and small cell lung cancer, respectively. There was no statistically significant difference in the CTC detection rates between these different pathological subtypes (P = 0.738). The CTC detection rates of 79.8% and 74.4% in patients with stage I-III and IV lung cancer, respectively, were not significantly different (P = 0.427). The oHSV1-hTERT-GFP method is highly effective for detecting CTCs in patients with lung cancer, independent of pathological type and disease stage, and is ideal for large-scale clinical applications. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  10. Association between exposure to HSV1 and cognitive functioning in a general population of adolescents. The TRAILS study.

    Directory of Open Access Journals (Sweden)

    Iris Jonker

    Full Text Available Infections with different herpes viruses have been associated with cognitive functioning in psychiatric patients and healthy adults. The aim of this study was to find out whether antibodies to different herpes viruses are prospectively associated with cognitive functioning in a general adolescent population.This study was performed in TRAILS, a large prospective general population cohort (N = 1084, 54% female, mean age 16.2 years (SD 0.6. At age 16, immunoglobulin G antibodies against HSV1, HSV2, CMV and EBV were measured next to high sensitive C-Reactive Protein (hsCRP. Two years later, immediate memory and executive functioning were assessed using the 15 words task and the self ordered pointing task. Multiple linear regression analysis with bootstrapping was performed to study the association between viral infections and cognitive function, adjusting for gender, socioeconomic status, ethnicity, and cannabis use.Presence of HSV1 antibodies was associated with memory function ((B = -0.272, 95% CI = -0.556 to -0.016, p = 0.047, while the association with executive functioning did not reach statistical significance (B = 0.560, 95% CI is -0.053 to 1.184, p = 0.075. The level of HSV1 antibodies was associated with both memory function (B = -0.160, 95% CI = -0.280 to -0.039, p = 0.014 and executive functioning (B = 0.296, 95% CI = 0.011 to 0.578, p = 0.046. Other herpes viruses and hsCRP were not associated with cognitive functioning.Both presence and level of HSV1 antibodies are prospectively associated with reduced cognitive performance in a large cohort of adolescents.

  11. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

    Science.gov (United States)

    Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

    2017-02-28

    Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

  12. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    Science.gov (United States)

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  13. Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

    Science.gov (United States)

    Perevozchikova, Svetlana A; Trikin, Roman M; Heinze, Roger J; Romanova, Elena A; Oretskaya, Tatiana S; Friedhoff, Peter; Kubareva, Elena A

    2014-01-01

    The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

  14. Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-06-01

    Full Text Available Abstract Background Herpes simplex type II (HSV-2 is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M system, we previously identified the Escherichia coli restriction enzyme (REase EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome. Methods and results Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,, and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class, 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00 at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the

  15. Thymidine selectively enhances growth suppressive effects of camptothecin/irinotecan in MSI+ cells and tumors containing a mutation of MRE11

    DEFF Research Database (Denmark)

    Rodriguez, Rene; Hansen, Lasse Tengbjerg; Phear, Geraldine

    2008-01-01

    -thymidine combinations by measuring colony formation, cell cycle distribution, and senescence. Cell strains defective in p53, p21, or Mre11 were used in these assays to investigate the role of these cell cycle regulators. The in vivo antitumor response of xenografts to irinotecan and thymidine combinations was assessed...... in nude mice. RESULTS: Camptothecin-thymidine combinations suppress colony formation of MMR-deficient tumor cells 10- to 3,000-fold relative to that obtained with camptothecin alone and significantly reduce the concentrations of the agents required to induce late S/G(2) arrest and senescence. Sensitivity......PURPOSE: DNA synthesis inhibitors and damaging agents are widely used in cancer therapy; however, sensitivity of tumors to such agents is highly variable. The response of tumor cells in culture to these agents is strongly influenced by the status of DNA damage response pathways. Here, we attempt...

  16. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    Directory of Open Access Journals (Sweden)

    MacLeod Beth

    2007-09-01

    Full Text Available Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt, and cytomegalovirus (CMV antigens. These antigens were determined to be low (HER-2/neu, moderate (tt, and robustly (CMV immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. Results Linear regression analysis indicates that both stimulation index (SI (p = 0.011 and IFN-gamma secreting precursor frequency (p Conclusion These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

  17. Studying Kinetochore Kinases

    NARCIS (Netherlands)

    Saurin, Adrian T; Kops, Geert J P L

    2016-01-01

    Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics

  18. Pyruvate kinase blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003357.htm Pyruvate kinase blood test To use the sharing features on this page, ... energy when oxygen levels are low. How the Test is Performed A blood sample is needed. In the laboratory, white blood ...

  19. Lytic HSV-1 infection induces the multifunctional transcription factor Early Growth Response-1 (EGR-1 in rabbit corneal cells

    Directory of Open Access Journals (Sweden)

    McFerrin Harris E

    2011-05-01

    Full Text Available Abstract Background Herpes simplex virus type-1 (HSV-1 infections can cause a number of diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells, critical for viral replication, is being extensively investigated by many laboratories. In this study, we tested the hypothesis that HSV-1 lytic infection triggers the expression of important multi-functional transcription factor Egr1. The mechanisms of induction are mediated, at least in part, by signaling pathways such as NFκB and CREB. Methods SIRC, VERO, and 293HEK cell lines were infected with HSV-1, and the Egr-1 transcript and protein were detected by RT-PCR and Western blot, respectively. The localization and expression profile of Egr-1 were investigated further by immunofluorescence microscopy analyses. The recruitment of transcription factors to the Egr-1 promoter during infection was studied by chromatin immunoprecipitation (ChIP. Various inhibitors and dominant-negative mutant were used to assess the mechanisms of Egr-1 induction and their effects were addressed by immunofluorescence microscopy. Results Western blot analyses showed that Egr-1 was absent in uninfected cells; however, the protein was detected 24-72 hours post treatment, and the response was directly proportional to the titer of the virus used for infection. Using recombinant HSV-1 expressing EGFP, Egr-1 was detected only in the infected cells. ChIP assays demonstrated that NFкB and cAMP response element binding protein (CREB were recruited to the Egr-1 promoter upon infection. Additional studies showed that inhibitors of NFкB and dominant-negative CREB repressed the Egr-1 induction by HSV-1 infection. Conclusion Collectively, these results demonstrate that Egr-1 is expressed rapidly upon HSV-1 infection and that this novel induction could be due to the NFкB/CREB-mediated transactivation. Egr-1 induction might play a key role in the viral gene

  20. Noninvasive theranostic imaging of HSV-TK/GCV suicide gene therapy in liver cancer by folate-targeted quantum dot-based liposomes

    National Research Council Canada - National Science Library

    Shao, D; Li, J; Pan, Y; Zhang, X; Zheng, X; Wang, Z; Zhang, M; Zhang, H; Chen, L

    2015-01-01

    .... In this work, we have combined an HSV-TK/GCV suicide gene system and near-infrared quantum dots, as the former is quite effective in liver cancer treatment and the latter facilitates tumor imaging...

  1. Prevalence and Incidence Estimation of HSV-2 by Two IgG ELISA Methods among South African Women at High Risk of HIV: e0120207

    National Research Council Canada - National Science Library

    Irith De Baetselier; Joris Menten; Vicky Cuylaerts; Khatija Ahmed; Jennifer Deese; Lut Van Damme; Tania Crucitti

    2015-01-01

    .... Using longitudinal samples from an HIV prevention study, we compared both assays and determined the HSV-2 prevalence and incidence in a South African young female population at elevated risk of acquiring HIV...

  2. Prevalence and incidence estimation of HSV-2 by two IgG ELISA methods among South African women at high risk of HIV

    National Research Council Canada - National Science Library

    De Baetselier, Irith; Menten, Joris; Cuylaerts, Vicky; Ahmed, Khatija; Deese, Jennifer; Van Damme, Lut; Crucitti, Tania

    2015-01-01

    .... Using longitudinal samples from an HIV prevention study, we compared both assays and determined the HSV-2 prevalence and incidence in a South African young female population at elevated risk of acquiring HIV...

  3. Quantification of thymidine kinase (TK1) mRNA in normal and leukemic cells and investigation of structure-function relatiosnhip of recombinant TK1enzyme

    DEFF Research Database (Denmark)

    Kristensen, Tina

    patients with chronic lymphatic leukemia (CLL). 2: Structure-function relationship of recombinant TKI. In the first part a sensitive method (competitive PCR) for quantification of TKI mRNA was established. The TKI mRNA level was quantified in quiescent lymphocytes from control donors (n = 6...... protein was used. TKI protein is cleaved from the GST-part with thrombin. Two TKI mutants, TKI-l 93 and TKI-l 76, with deletions from the C-terminal were constructed by the recombinant PCR method. Deletion of 57 amino acids from the C-terminal (TKI- 176) results in an inactive enzyme. Deletion of 40 amino...... for construction of selective nucleoside analogs, only used by cancer TK isoenzymes. In this Ph.D thesis the cell cycle regulated TKI has been subject for two different approaches. 1: Investigation of the relationship between TK1 mRNA level and TK activity in lymphocytes from healthy donors and in lymphocytes from...

  4. Consequences of chemoresistance for the herpes simplex virus thymidine kinase/ganciclovir-induced bystander effect in a human small cell lung cancer cell line model

    NARCIS (Netherlands)

    Van Dillen, IJ; Mulder, NH; Sluiter, WJ; Meijer, C; De Jong, S; Loncarek, J; Mesnil, M; De Vries, EFJ; Vaalburg, W; Hospers, GAP

    2005-01-01

    This paper focuses on the influence of chemoresistance on the herpes simplex virus (HSVtk)/ganciclovir (GCV)-induced bystander effect (BE), as studied in a human small cell lung cancer (SCLC) cell line (GLC(4)) and its sublines with in vitro acquired resistance to adriamycin (GLC(4)/ADR),

  5. Estimating the cost-effectiveness of pre-exposure prophylaxis to reduce HIV-1 and HSV-2 incidence in HIV-serodiscordant couples in South Africa.

    Directory of Open Access Journals (Sweden)

    Britta L Jewell

    Full Text Available To estimate the cost-effectiveness of daily oral tenofovir-based PrEP, with a protective effect against HSV-2 as well as HIV-1, among HIV-1 serodiscordant couples in South Africa.We incorporated HSV-2 acquisition, transmission, and interaction with HIV-1 into a microsimulation model of heterosexual HIV-1 serodiscordant couples in South Africa, with use of PrEP for the HIV-1 uninfected partner prior to ART initiation for the HIV-1 1infected partner, and for one year thereafter.We estimate the cost per disability-adjusted life-year (DALY averted for two scenarios, one in which PrEP has no effect on reducing HSV-2 acquisition, and one in which there is a 33% reduction. After a twenty-year intervention, the cost per DALY averted is estimated to be $10,383 and $9,757, respectively--a 6% reduction, given the additional benefit of reduced HSV-2 acquisition. If all couples are discordant for both HIV-1 and HSV-2, the cost per DALY averted falls to $1,445, which shows that the impact is limited by HSV-2 concordance in couples.After a 20-year PrEP intervention, the cost per DALY averted with a reduction in HSV-2 is estimated to be modestly lower than without any effect, providing an increase of health benefits in addition to HIV-1 prevention at no extra cost. The small degree of the effect is in part due to a high prevalence of HSV-2 infection in HIV-1 serodiscordant couples in South Africa.

  6. Colony Stimulating Factor-1 Receptor Expressing Cells Infiltrating the Cornea Control Corneal Nerve Degeneration in Response to HSV-1 Infection.

    Science.gov (United States)

    Chucair-Elliott, Ana J; Gurung, Hem R; Carr, Meghan M; Carr, Daniel J J

    2017-09-01

    Herpes simplex virus type-1 (HSV-1) is a leading cause of neurotrophic keratitis, characterized by decreased or absent corneal sensation due to damage to the sensory corneal innervation. We previously reported the elicited immune response to infection contributes to the mechanism of corneal nerve regression/damage during acute HSV-1 infection. Our aim is to further establish the involvement of infiltrated macrophages in the mechanism of nerve loss upon infection. Macrophage Fas-Induced Apoptosis (MAFIA) transgenic C57BL/6 mice were systemically treated with AP20187 dimerizer or vehicle (VEH), and their corneas, lymph nodes, and blood were assessed for CD45+CD11b+GFP+ cell depletion by flow cytometry (FC). Mice were ocularly infected with HSV-1 or left uninfected. At 2, 4, and/or 6 days post infection (PI), corneas were assessed for sensitivity and harvested for FC, nerve structure by immunohistochemistry, viral content by plaque assay, soluble factor content by suspension array, and activation of signaling pathways by Western blot analysis. C57BL6 mice were used to compare to the MAFIA mouse model. MAFIA mice treated with AP20187 had efficient depletion of CD45+CD11b+GFP+ cells in the tissues analyzed. The reduction of CD45+CD11b+GFP+ cells recruited to the infected corneas of AP20187-treated mice correlated with preservation of corneal nerve structure and function, decreased protein concentration of inflammatory cytokines, and decreased STAT3 activation despite no changes in viral content in the cornea compared to VEH-treated animals. Our results suggest infiltrated macrophages are early effectors in the nerve regression following HSV-1 infection. We propose the neurodegeneration mechanism involves macrophages, local up-regulation of IL-6, and activation of STAT3.

  7. Virus-Induced Chaperone-Enriched (VICE domains function as nuclear protein quality control centers during HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Christine M Livingston

    2009-10-01

    Full Text Available Virus-Induced Chaperone-Enriched (VICE domains form adjacent to nuclear viral replication compartments (RC during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70, the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  8. HSV-1 targets lymphatic vessels in the eye and draining lymph node of mice leading to edema in the absence of a functional type I interferon response.

    Science.gov (United States)

    Bryant-Hudson, Katie M; Chucair-Elliott, Ana J; Conrady, Christopher D; Cohen, Alex; Zheng, Min; Carr, Daniel J J

    2013-10-01

    Herpes simplex virus type-1 (HSV-1) induces new lymphatic vessel growth (lymphangiogenesis) in the cornea via expression of vascular endothelial growth factor by virally infected epithelial cells. Here, we extend this observation to demonstrate the selective targeting of corneal lymphatics by HSV-1 in the absence of functional type I interferon (IFN) pathway. Specifically, we examined the impact of HSV-1 replication on angiogenesis using type I IFN receptor deficient (CD118(-/-)) mice. HSV-1-induced lymphatic and blood vessel growth into the cornea proper was time-dependent in immunocompetent animals. In contrast, there was an initial robust growth of lymphatic vessels into the cornea of HSV-1-infected CD118(-/-)mice, but such vessels disappeared by day 5 postinfection. The loss was selective as blood vessel integrity remained intact. Magnetic resonance imaging and confocal microscopy analysis of the draining lymph nodes of CD118(-/-) mice revealed extensive edema and loss of lymphatics compared with wild-type mice. In addition to a loss of lymphatic vessels in CD118(-/-) mice, HSV-1 infection resulted in epithelial thinning associated with geographic lesions and edema within the cornea, which is consistent with a loss of lymphatic vasculature. These results underscore the key role functional type I IFN pathway plays in the maintenance of structural integrity within the cornea in addition to the anti-viral characteristics often ascribed to the type I IFN cytokine family. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. The impact of brief messages on HSV-2 screening uptake among female defendants in a court setting: a randomized controlled trial utilizing prospect theory.

    Science.gov (United States)

    Roth, Alexis M; Van Der Pol, Barbara; Fortenberry, J Dennis; Dodge, Brian; Reece, Michael; Certo, David; Zimet, Gregory D

    2015-01-01

    Epidemiologic data demonstrate that women involved with the criminal justice system in the United States are at high risk for sexually transmitted infections, including herpes simplex virus type 2 (HSV-2). Female defendants were recruited from a misdemeanor court to assess whether brief framed messages utilizing prospect theory could encourage testing for HSV-2. Participants were randomly assigned to a message condition (gain, loss, or control), completed an interviewer-administered survey assessing factors associated with antibody test uptake/refusal and were offered free point-of-care HSV-2 serologic testing. Although individuals in the loss-frame group accepted testing at the highest rate, an overall statistical difference in HSV-2 testing behavior by group (p ≤ .43) was not detected. The majority of the sample (74.6%) characterized receiving a serological test for HSV-2 as health affirming. However, this did not moderate the effect of the intervention nor was it significantly associated with test acceptance (p ≤ .82). Although the effects of message framing are subtle, the findings have important theoretical implications given the participants' characterization of HSV-2 screening as health affirming despite being a detection behavior. Implications of study results for health care providers interested in brief, low cost interventions are also explored.

  10. Efficacy of the ND:YAG laser therapy on EBV and HSV1 contamination in periodontal pockets.

    Science.gov (United States)

    Martelli, Francesco Saverio; Bacci, Giovanni; Martelli, Maria Laura; Nobili, Piero; Boddi, Anna; Rosati, Claudio; Fanti, Elena

    2015-01-01

    The aim of this retrospective multicenter study was to verify the efficacy of Nd:YAG laser in the treatment of periodontal pockets infected by Epstein-Barr Virus (EBV) and Herpes Simplex Virus 1 (HSV1). Subgingival plaque samples of 291 Italian periodontal patients were analyzed by Real Time PCR to evaluate the frequency of both viruses before and after Nd:YAG laser-assisted periodontal treatment. Before treatment, EBV and HSV1 were observed in 29.9% and in 3.8% of periodontal patients respectively, while co-infection with both viruses was detected in 1.7% of cases. Periodontal Nd:YAG laser treatment ("Periodontal Biological Laser-Assisted Therapy", PERIOBLAST) produced statistical significant benefits, especially in EBV periodontal infection: 78.2% of EBV positive patients became EBV-negative following treatment. Results of this preliminary study highlight that EBV is found in periodontal pockets more frequently than HSV1, supporting the theory of the potential role of EBV in the onset and progression of periodontal disease. Moreover, our data showed that Nd:YAG laser-assisted periodontal treatment (Perioblast) is also effective in case of viral infection, validating evidences that it represents a successful alternative approach to traditional periodontal protocols.

  11. Visualization of mouse neuronal ganglia infected by Herpes Simplex Virus 1 (HSV-1) using multimodal non-linear optical microscopy.

    Science.gov (United States)

    Rochette, Pierre-Alexandre; Laliberté, Mathieu; Bertrand-Grenier, Antony; Houle, Marie-Andrée; Blache, Marie-Claire; Légaré, François; Pearson, Angela

    2014-01-01

    Herpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives.

  12. Antiviral Action of Hydromethanolic Extract of Geopropolis from Scaptotrigona postica against Antiherpes Simplex Virus (HSV-1

    Directory of Open Access Journals (Sweden)

    Guilherme Rabelo Coelho

    2015-01-01

    Full Text Available The studies on chemical composition and biological activity of propolis had focused mainly on species Apis mellifera L. (Hymenoptera: Apidae. There are few studies about the uncommon propolis collected by stingless bees of the Meliponini tribe known as geopropolis. The geopropolis from Scaptotrigona postica was collected in the region of Barra do Corda, Maranhão state, Brazil. The chemical analysis of hydromethanolic extract of this geopropolis (HMG was carried out through HPLC-DAD-ESI-MS/MS and the main constituents found were pyrrolizidine alkaloids and C-glycosyl flavones. The presence of alkaloids in extracts of propolis is detected for the first time in this sample. The antiviral activity of HMG was evaluated through viral DNA quantification experiments and electron microscopy experiments. Quantification of viral DNA from herpes virus showed reduction of about 98% in all conditions and concentration tested of the HMG extract. The results obtained were corroborated by transmission electron microscopy, in which the images did not show particle or viral replication complex. The antiviral activity of C-glycosyl flavones was reported for a variety of viruses, being observed at different points in the viral replication. This work is the first report about the antiviral activity of geopropolis from Scaptotrigona postica, in vitro, against antiherpes simplex virus (HSV.

  13. Antiviral Action of Hydromethanolic Extract of Geopropolis from Scaptotrigona postica against Antiherpes Simplex Virus (HSV-1).

    Science.gov (United States)

    Coelho, Guilherme Rabelo; Mendonça, Ronaldo Zucatelli; Vilar, Karina de Senna; Figueiredo, Cristina Adelaide; Badari, Juliana Cuoco; Taniwaki, Noemi; Namiyama, Gisleine; de Oliveira, Maria Isabel; Curti, Suely Pires; Evelyn Silva, Patricia; Negri, Giuseppina

    2015-01-01

    The studies on chemical composition and biological activity of propolis had focused mainly on species Apis mellifera L. (Hymenoptera: Apidae). There are few studies about the uncommon propolis collected by stingless bees of the Meliponini tribe known as geopropolis. The geopropolis from Scaptotrigona postica was collected in the region of Barra do Corda, Maranhão state, Brazil. The chemical analysis of hydromethanolic extract of this geopropolis (HMG) was carried out through HPLC-DAD-ESI-MS/MS and the main constituents found were pyrrolizidine alkaloids and C-glycosyl flavones. The presence of alkaloids in extracts of propolis is detected for the first time in this sample. The antiviral activity of HMG was evaluated through viral DNA quantification experiments and electron microscopy experiments. Quantification of viral DNA from herpes virus showed reduction of about 98% in all conditions and concentration tested of the HMG extract. The results obtained were corroborated by transmission electron microscopy, in which the images did not show particle or viral replication complex. The antiviral activity of C-glycosyl flavones was reported for a variety of viruses, being observed at different points in the viral replication. This work is the first report about the antiviral activity of geopropolis from Scaptotrigona postica, in vitro, against antiherpes simplex virus (HSV).

  14. Ethosomes for the delivery of anti-HSV-1 molecules: preparation, characterization and in vitro activity.

    Science.gov (United States)

    Cortesi, R; Ravani, L; Zaid, A N; Menegatti, E; Romagnoli, R; Drechsler, M; Esposito, E

    2010-10-01

    This paper describes the production, characterization and in vitro activity of ethosomes containing two molecules with antiviral activity, such as acyclovir (ACY) and N1-beta-D-ribofuranosyl-pyrazole [3,4d]pyridazin-7(6p-chlorine-phenyl)-one nucleoside (N1CP). Ethosomes were prepared and morphologically characterized by Cryo-TEM. The encapsulation efficiency was 92.3 +/- 2.5% for ACY and 94.2 +/- 2.8% for N1CP. The release of the drug from vesicles, determined by a Franz cell method, indicated that both drugs were released in a controlled manner. In order to possibly guarantee the stability during long-term storage ethosome suspensions was freeze-dried. It was found that the freeze-dried ethosomes' cakes were compact, glassy characterized by low density and quick re-hydration. However, the storage time slightly influences the percentage of drug encapsulation within ethosomes showing a drug leakage after re-hydration around 10%. The antiviral activity against HSV-1 of both drugs was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that ethosomes allowed a reduction of the ED50 of N1CP evidencing an increase of its antiviral activity. However, ACY remains more active than N1CP. No differences are appreciable between drug-containing ethosomes before and after freeze-drying. Taken together these results, ethosomal formulation could be possibly proposed as mean for topical administration of anti-herpetic molecules.

  15. Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection

    Energy Technology Data Exchange (ETDEWEB)

    Sae-Ueng, Udom; Li, Dong; Zuo, Xiaobing; Huffman, Jamie B.; Homa, Fred L.; Rau, Donald; Evilevitch, Alex

    2014-10-01

    DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that, despite its liquid crystalline state inside the capsid, the DNA is fluid-like, which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37 °C) induces a disordering transition of the encapsidated herpes genome, which reduces interstrand interactions and provides genome mobility required for infection.

  16. (18)F-fluoro-L-thymidine-PET for the evaluation of primary brain tumours in children: a report of three cases.

    NARCIS (Netherlands)

    Gilles, R.; Vogel, W.V.; Gidding, C.E.M.; Janssens, G.O.; Vliet, A.M. van der; Oyen, W.J.G.

    2010-01-01

    BACKGROUND: F-fluoro-L-thymidine (FLT) has been shown to be a useful PET tracer in the evaluation of brain tumours in adults. No studies of this modality in children with brain tumours, however, have been published. OBJECTIVE: In this report three children with brain tumours are presented in which

  17. F-18-fluoro-L-thymidine-PET for the evaluation of primary brain tumours in children: a report of three cases

    NARCIS (Netherlands)

    Gilles, R.; Vogel, W.V.; Gidding, C.E.M.; Janssens, G.O.R.J.; van der Vliet, T.M.; Oyen, W.J.G.

    2010-01-01

    Background F-18-fluoro-L-thymidine (FLT) has been shown to be a useful PET tracer in the evaluation of brain tumours in adults. No studies of this modality in children with brain tumours, however, have been published. Objective In this report three children with brain tumours are presented in which

  18. Long-term foscarnet therapy remodels thymidine analogue mutations and alters resistance to zidovudine and lamivudine in HIV-1

    DEFF Research Database (Denmark)

    Mathiesen, Sofie; Dam, Elisabeth; Roge, Birgit

    2007-01-01

    OBJECTIVE: To study the evolution of multi-drug-resistant HIV-1 in treatment-experienced patients receiving foscarnet (PFA) as part of salvage therapy and to investigate the virological consequences of emerging mutations. METHODS: Genotypic and phenotypic resistance tests were performed on plasma...... viruses from seven patients at baseline and during treatment with PFA. The phenotypic effects of mutations suspected to be associated with PFA resistance were evaluated by site-directed mutagenesis of wild-type or thymidine analogue mutations (TAM)-carrying pNL4-3. Reversion of single mutations...... was performed in a patient-derived recombinant clone. RESULTS: Baseline multi-drug-resistant isolates exhibited hypersusceptibility to PFA. In two patients who received > 12 months of PFA treatment, a novel mutation pattern including K70G, V75T, K219R and L228R emerged. These viruses had 3-6-fold resistance...

  19. Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases.

    Science.gov (United States)

    Fliess, A; Wolfes, H; Rosenthal, A; Schwellnus, K; Blöcker, H; Frank, R; Pingoud, A

    1986-04-25

    We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV.

  20. From Phosphosites to Kinases

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations...... sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available...

  1. [Influence of the selected pyrimidine compounds on the activity of thymidine phosphorylase from normal and tumor endometrial cells].

    Science.gov (United States)

    Miszczak-Zaborska, Elzbieta; Smolarek, Monika; Dramiński, Marcin; Kubiak, Robert; Józwiak, Barbara; Bartkowiak, Jacek

    2009-08-01

    The aim of this study was to evaluate the influence of the selected pyrimidine compounds on the activity of thymidine phosphorylase (TP) of normal and tumor endometrial cells. Influence of 28 chemical compounds on the TP activity in the cytosol of the endometrial cells was studied by the spectrophotometric method. The studied group comprised postmenopausal women with endometrial cancer: adenocarcinoma endometrialis (Adeno Ca E). The second group included women with normal endometrium after surgery due to non-oncologic reasons. The most potent inhibitor of TP activity from cancer and endometrium was synthesized 5-bromo-6-acetyloaminouracil, which at the 0.2 mM concentration, by 0.2 mM concentration thymidine reduced the cytosol TP activity by about 80%. 5-bromo-6-aminouracil, 5-nitrouracil and 5-bromouracil reduced this TP activity in statistically significant manner. From among synthesized 1-N-allyloxymethylpyrimidine derivatives 1-N-allyloxymethylthymine was the strongest inhibitor of the TP activity in endometrium, and 1-N-allyloxymethyl-4-hydrokxy-5-nitro-6-oxopyrimidine in endometrial cancer respectively. The most potent activators of TP in endometrial cancer was 5-bromodeoxyuridine and 1-N-allyloxymethyl-5-nitrouracil, which increased the TP activity about 100%. 5-fluorodeoxyuridine, 5-jododeoxyuridine and 2'-deoxyuridine activated the TP in statistically significant manner too, but stronger in case of endometrial cancer than in normal endometrium. The synthesized 5-bromo-6-acetyloaminouracil strongly inhibited the TP activity of endometrial cells and might be useful in reducing endometrial cancer angiogenesis. On the other hand 5-bromodeoxyuridine and the synthesized 1-N-allyloxymethyl-5-nitrouracil might increase the effect of antitumor therapy with the cytostatics. These conclusions ought to be confirmed by analyzing more tumor cases.

  2. HIV-1, HSV-2 and syphilis among pregnant women in a rural area of Tanzania: Prevalence and risk factors

    Directory of Open Access Journals (Sweden)

    Evjen-Olsen Bjørg

    2008-06-01

    Full Text Available Abstract Background Evidence suggests that a substantial proportion of new HIV infections in African countries are associated with herpes simplex virus type 2 (HSV-2. Thus, the magnitude of HSV-2 infection in an area may suggest the expected course of the HIV epidemic. We determined prevalence of genital herpes, syphilis and associated factors among pregnant women from a remote rural Tanzanian community that has a low but increasing HIV prevalence. Methods We analysed 1296 sera and responses to a standard structured questionnaire collected from pregnant women aged between 15–49 years, attending six different antenatal clinics within rural Manyara and Singida regions in Tanzania. Linked anonymous testing (with informed consent of the serum for specific antibodies against HSV-2 was done using a non-commercial peptide- 55 ELISA. Antibodies against syphilis were screened by using rapid plasma reagin (RPR and reactive samples confirmed by Treponema pallidum haemagglutination assay (TPHA. Results Previous analysis of the collected sera had shown the prevalence of HIV antibodies to be 2%. In the present study the prevalence of genital herpes and syphilis was 20.7% (95% CI: 18.53–23.00 and 1.6% (95% CI: 1.03–2.51, respectively. The presence of HSV-2 antibodies was associated with polygamy (OR 2.2, 95% CI: 1.62 – 3.01 and the use of contraceptives other than condoms (OR 1.7, 95% CI: 1.21 – 2.41. Syphilis was associated with reporting more than one lifetime sexual partner (OR 5.4, 95% CI: 1.88 – 15.76 and previous spontaneous abortion (OR 4.3, 95% CI: 1.52–12.02. Conclusion The low prevalence of HIV infection offers a unique opportunity for strengthening HIV prevention in a cost-effective manner. The identification and control of other prevalent curable STIs other than syphilis and specific intervention of HSV-2 in specific populations like pregnant women would be one among approaches towards preventing incident HIV infections.

  3. An experimental study on cervix cancer with combination of HSV-TK/GCV suicide gene therapy system and 60Co radiotherapy

    Directory of Open Access Journals (Sweden)

    Tang Qiusha

    2010-11-01

    Full Text Available Abstract Background To evaluate the killing effect of HSV-TK/GCV suicide gene therapy system combined with 60Co radiotherapy on human cervical cancer Hela cell line in vitro and in vivo, and to explore the radiosensitization by HSV-TK/GCV system. Methods HSV-TK/GCV suicide gene therapy system and 60Co radiotherapy were used separately or in combination on human cervical cancer Hela cell line in vitro and in vivo to compare their effects. Colony formation test and the rate of radiosensitization effect (E/O were employed to observed the radiosensitization by HSV-TK/GCV system. Results HSV-TK/GCV suicide gene therapy system had strong therapeutic effects on Hela cells in vitro and in vivo (the inhibition rates were 45.8% and 39.5%, respectively, moreover, the combined administration of gene therapy and radiotherapy had stronger therapeutic effects in vitro and in vivo (the inhibition rate was 87.5% in vitro, and the inhibition rate was 87.9% in vivo (P in vitro and 35.8% in vivo. The sensitivity of combined therapy to radiotherapy increased more than that of therapy alone, the ability of colony formation decreased (P 1.4, indicating HSV-TK/GCV system could exert a sensitizing effect on 60Co radiotherapy of the transplanted human cervical cancer cell in nude mice. Conclusion HSV-TK/GCV system had radiosensitization. Gene therapy combined with radiotherapy may be a good supplementary method for cervix cancer synthetic treatment.

  4. Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M

    Science.gov (United States)

    Striebinger, Hannah; Funk, Christina; Raschbichler, Verena; Bailer, Susanne M.

    2016-01-01

    The herpes simplex virus type 1 (HSV-1) glycoprotein N (gN/UL49.5) is a type I transmembrane protein conserved throughout the herpesvirus family. gN is a resident of the endoplasmic reticulum that in the presence of gM is translocated to the trans Golgi network. gM and gN are covalently linked by a single disulphide bond formed between cysteine 46 of gN and cysteine 59 of gM. Exit of gN from the endoplasmic reticulum requires the N-terminal core of gM composed of eight transmembrane domains but is independent of the C-terminal extension of gM. Co-transport of gN and gM to the trans Golgi network also occurs upon replacement of conserved cysteines in gM and gN, suggesting that their physical interaction is mediated by covalent and non-covalent forces. Deletion of gN/UL49.5 using bacterial artificial chromosome (BAC) mutagenesis generated mutant viruses with wild-type growth behaviour, while full deletion of gM/UL10 resulted in an attenuated phenotype. Deletion of gN/UL49.5 in conjunction with various gM/UL10 mutants reduced average plaque sizes to the same extent as either single gM/UL10 mutant, indicating that gN is nonessential for the function performed by gM. We propose that gN functions in gM-dependent as well as gM-independent processes during which it is complemented by other viral factors. PMID:26999189

  5. Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M

    Directory of Open Access Journals (Sweden)

    Hannah Striebinger

    2016-03-01

    Full Text Available The herpes simplex virus type 1 (HSV-1 glycoprotein N (gN/UL49.5 is a type I transmembrane protein conserved throughout the herpesvirus family. gN is a resident of the endoplasmic reticulum that in the presence of gM is translocated to the trans Golgi network. gM and gN are covalently linked by a single disulphide bond formed between cysteine 46 of gN and cysteine 59 of gM. Exit of gN from the endoplasmic reticulum requires the N-terminal core of gM composed of eight transmembrane domains but is independent of the C-terminal extension of gM. Co-transport of gN and gM to the trans Golgi network also occurs upon replacement of conserved cysteines in gM and gN, suggesting that their physical interaction is mediated by covalent and non-covalent forces. Deletion of gN/UL49.5 using bacterial artificial chromosome (BAC mutagenesis generated mutant viruses with wild-type growth behaviour, while full deletion of gM/UL10 resulted in an attenuated phenotype. Deletion of gN/UL49.5 in conjunction with various gM/UL10 mutants reduced average plaque sizes to the same extent as either single gM/UL10 mutant, indicating that gN is nonessential for the function performed by gM. We propose that gN functions in gM-dependent as well as gM-independent processes during which it is complemented by other viral factors.

  6. Adipose tissue mesenchymal stromal cells as therapeutic vehicles against glioblastoma

    OpenAIRE

    Krasheninnikova, Maria Alieva

    2012-01-01

    Lately adipose tissue mesenchymal stem cells (hAMSCs) have emerged as cellular vehicles for therapy of solid tumors, due to their ease of isolation and manipulation, and wound/tumor homing capacity. HAMSCs have been successfully used in suicide gene therapy, employing the prodrug activating system based on Herpes simplex virus type I thymidine kinase (HSV-TK)/ganciclovir (GCV). In the current study we demonstrate an effective model of glioblastoma therapy based on the use of genetically modif...

  7. JAK protein kinase inhibitors.

    Science.gov (United States)

    Thompson, James E

    2005-06-01

    In humans, the Janus protein tyrosine kinase family (JAKs) contains four members: JAK1, JAK2, JAK3 and TYK2. JAKs phosphorylate signal transducers and activators of transcription (STATs) simultaneously with other phosphorylations required for activation, and there are several cellular mechanisms in place to inhibit JAK/STAT signaling. That one might be able to modulate selected JAK/STAT-mediated cellular signals by inhibiting JAK kinase activity to effect a positive therapeutic outcome is a tantalizing prospect, as yet incompletely realized. While current data suggest no therapeutic use for JAK1 and TYK2 inhibition, JAK2 inhibition seems a promising but not definitively tested mechanism for treatment of leukemia. More promising, however, are data indicating a possible therapeutic use of JAK3 inhibition. The restriction of the JAK3-deficient phenotype to the hematopoietic system and the resulting profound immune suppression suggest that JAK3 could be a target for immunosuppressive therapies used to prevent organ transplant rejection.

  8. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  9. Multifunctional Tannic Acid/Silver Nanoparticle-Based Mucoadhesive Hydrogel for Improved Local Treatment of HSV Infection: In Vitro and In Vivo Studies.

    Science.gov (United States)

    Szymańska, Emilia; Orłowski, Piotr; Winnicka, Katarzyna; Tomaszewska, Emilia; Bąska, Piotr; Celichowski, Grzegorz; Grobelny, Jarosław; Basa, Anna; Krzyżowska, Małgorzata

    2018-01-28

    Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection.

  10. The Anti-Tumor Effects of Adipose Tissue Mesenchymal Stem Cell Transduced with HSV-Tk Gene on U-87-Driven Brain Tumor.

    Directory of Open Access Journals (Sweden)

    Suely Maymone de Melo

    Full Text Available Glioblastoma (GBM is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV. U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

  11. A Feasibility Study of Quantifying Longitudinal Brain Changes in Herpes Simplex Virus (HSV Encephalitis Using Magnetic Resonance Imaging (MRI and Stereology.

    Directory of Open Access Journals (Sweden)

    Sylviane Defres

    Full Text Available To assess whether it is feasible to quantify acute change in temporal lobe volume and total oedema volumes in herpes simplex virus (HSV encephalitis as a preliminary to a trial of corticosteroid therapy.The study analysed serially acquired magnetic resonance images (MRI, of patients with acute HSV encephalitis who had neuroimaging repeated within four weeks of the first scan. We performed volumetric measurements of the left and right temporal lobes and of cerebral oedema visible on T2 weighted Fluid Attenuated Inversion Recovery (FLAIR images using stereology in conjunction with point counting.Temporal lobe volumes increased on average by 1.6% (standard deviation (SD 11% in five patients who had not received corticosteroid therapy and decreased in two patients who had received corticosteroids by 8.5%. FLAIR hyperintensity volumes increased by 9% in patients not receiving treatment with corticosteroids and decreased by 29% in the two patients that had received corticosteroids.This study has shown it is feasible to quantify acute change in temporal lobe and total oedema volumes in HSV encephalitis and suggests a potential resolution of swelling in response to corticosteroid therapy. These techniques could be used as part of a randomized control trial to investigate the efficacy of corticosteroids for treating HSV encephalitis in conjunction with assessing clinical outcomes and could be of potential value in helping to predict the clinical outcomes of patients with HSV encephalitis.

  12. A Feasibility Study of Quantifying Longitudinal Brain Changes in Herpes Simplex Virus (HSV) Encephalitis Using Magnetic Resonance Imaging (MRI) and Stereology.

    Science.gov (United States)

    Defres, Sylviane; Keller, Simon S; Das, Kumar; Vidyasagar, Rishma; Parkes, Laura M; Burnside, Girvan; Griffiths, Michael; Kopelman, Michael; Roberts, Neil; Solomon, Tom

    2017-01-01

    To assess whether it is feasible to quantify acute change in temporal lobe volume and total oedema volumes in herpes simplex virus (HSV) encephalitis as a preliminary to a trial of corticosteroid therapy. The study analysed serially acquired magnetic resonance images (MRI), of patients with acute HSV encephalitis who had neuroimaging repeated within four weeks of the first scan. We performed volumetric measurements of the left and right temporal lobes and of cerebral oedema visible on T2 weighted Fluid Attenuated Inversion Recovery (FLAIR) images using stereology in conjunction with point counting. Temporal lobe volumes increased on average by 1.6% (standard deviation (SD 11%) in five patients who had not received corticosteroid therapy and decreased in two patients who had received corticosteroids by 8.5%. FLAIR hyperintensity volumes increased by 9% in patients not receiving treatment with corticosteroids and decreased by 29% in the two patients that had received corticosteroids. This study has shown it is feasible to quantify acute change in temporal lobe and total oedema volumes in HSV encephalitis and suggests a potential resolution of swelling in response to corticosteroid therapy. These techniques could be used as part of a randomized control trial to investigate the efficacy of corticosteroids for treating HSV encephalitis in conjunction with assessing clinical outcomes and could be of potential value in helping to predict the clinical outcomes of patients with HSV encephalitis.

  13. Discordance of HIV and HSV-2 biomarkers and self-reported sexual behaviour among orphan adolescents in Western Kenya.

    Science.gov (United States)

    Cho, Hyunsan; Luseno, Winnie; Halpern, Carolyn; Zhang, Lei; Mbai, Isabella; Milimo, Benson; Hallfors, Denise Dion

    2015-06-01

    This paper examines the discordance between biological data of HIV and herpes simplex virus type 2 (HSV-2) infections and self-reported questionnaire responses among orphan adolescents in Western Kenya. In 2011, 837 orphan adolescents from 26 primary schools were enrolled in an HIV prevention trial. At baseline, blood samples were drawn for HIV and HSV-2 infection biomarker testing, and participants completed an audio computer-assisted self-interviewing survey. Comparing biological data with self-reported responses indicated that 70% of HIV-positive (7 out of 10) and 64% of HSV-2-positive (18 out of 28 positive) participants reported never having had sex. Among ever-married adolescents, 65% (57 out of 88) reported never having had sex. Overall, 10% of study participants appeared to have inconsistently reported their sexual behaviour. Logistic regression analyses indicated that lower educational level and exam scores were significant predictors of inconsistent reporting. Our study demonstrates the discordance between infections measured by biomarkers and self-reports of having had sex among orphan adolescents in Kenya. In order to detect programme effects accurately in prevention research, it is necessary to collect both baseline and endline biological data. Furthermore, it is recommended to triangulate multiple data sources about adolescent participants' self-reported information about marriage and pregnancies from school records and parent/guardians to verify the information. Researchers should recognise potential threats to validity in data and design surveys to consider cognitive factors and/or cultural context to obtain more accurate and reliable information from adolescents regarding HIV/sexually transmitted infection risk behaviours. NCT01501864. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  14. Use of HIV and HSV-2 biomarkers in sub-saharan adolescent prevention research: a comparison of two approaches.

    Science.gov (United States)

    Luseno, Winnie K; Hallfors, Denise Dion; Cho, Hyunsan; Iritani, Bonita J; Adze, Joel; Rusakaniko, Simbarashe; Mbai, Isabella; Milimo, Benson; Hobbs, Marcia

    2014-06-01

    Self-report of sexual behavior among adolescents is notoriously inconsistent, yet such measures are commonly used as outcomes for human immunodeficiency virus (HIV) prevention intervention trials. There has been a growing interest in the use of HIV and other sexually transmitted disease biomarkers as more valid measures of intervention impact in high HIV prevalence areas, particularly in sub-Saharan Africa. We examine the challenges, benefits, and feasibility of including HIV and herpes simplex virus type 2 (HSV-2) biomarker data, with details about different data collection and disclosure methods from two adolescent prevention trials in Kenya and Zimbabwe. In Kenya, whole blood samples were collected using venipuncture; adult guardians were present during biomarker procedures and test results were disclosed to participants and their guardians. In contrast, in Zimbabwe, samples were collected using finger pricks for dried blood spots (DBS); guardians were not present during biomarker procedures, and results were not disclosed to participants and/or their guardians. In both countries, prevalence in the study samples was low. Although the standard of care for testing for HIV and other sexually transmitted infections includes disclosure in the presence of a guardian for adolescents under age 18, we conclude that more research about the risks and benefits of disclosure to adolescents in the context of a clinical trial is needed. Notably, current serological diagnosis for HSV-2 has a low positive predictive value when prevalence is low, resulting in an unacceptable proportion of false positives and serious concerns about disclosing test results to adolescents within a trial. We also conclude that the DBS approach is more convenient and efficient than venipuncture for field research, although both approaches are feasible. Manufacturer validation studies using DBS for HSV-2, however, are needed for widespread use.

  15. Detection of EBV, CMV and HSV-1 in subgingival samples of HIV positive and negative patients with chronic periodontitis.

    Directory of Open Access Journals (Sweden)

    Laura Escalona

    2016-06-01

    Full Text Available Objective: To detect the presence of infection by EBV (Epstein-Barr Virus, CMV (Cytomegalovirus and HSV-1 (Herpes Simplex Virus type 1 in subgingival samples from HIV- positive patients under HAART (High Activity Antiretroviral Therapy, HIV- positive patients without HAART, HIV-negative patients with chronic periodontitis and healthy controls. Methodology: Crevicular fluid samples of 11 HIV+ patients on therapy were evaluated, 6 without antiretroviral therapy, 7 HIV- negative subjects with chronic periodontitis and 7 periodontally-healthy controls. PI (Plaque index, GI (Gingival Index, PD (probing depth and CAL (Clinical Attachment Loss were registered at six sites per each tooth in all teeth and subgingival plaque samples of a tooth were collected per quadrant. Nested PCR was used to detect EBV and endpoint PCR to detect infection by CMV and HSV-1. Results: Clinical parameters showed statistically significant differences between HIV-positive patients and subjects with chronic periodontitis compared with the control group (p<0.05. DNA of EBV was detected mainly in HIV-positive patients under HAART, 91% (10/11. DNA of CMV was detected mainly in patients without HAART, 67% (4/6, while HSV-1 was observed in 27% (3/11 of patients under HAART. In the control group no virus was detected. Coinfection was observed in 50% of HIV patients without HAART, 36% of HIV patients with HAART and 14% of HIV-negative with chronic periodontitis. Conclusion: Viral infection was prevalent in HIV patients under HAART and EBV was the primary viral infection detected in HIV-positive patients with chronic periodontitis.

  16. A novel PET tracer for evaluation of gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Goldman, S.; Monclus, M.; Cool, V. [Cliniques Universitaires de Bruxelles-Hopital Erasme, Brussels (Belgium)] [and others

    1996-05-01

    A promising approach of gene therapy for cancer consist in the transduction of neoplastic cells with the herpes virus thymidine-kinase gene (HSV-tk) which renders transduced cells sensitive to the lethal effect of anti-viral agent such as ganciclovir (GCV). Pet with adapted radiotracers represents an adequate tool to determine in vivo the level of HSV-tk expression and to establish the optimal protocol of gene and GCV administrations in human. We have developed a new potential PET tracer, 9-((1-[F-18]fluoro-3-hydroxy-2-propoxy)methyl)guanine [F-18]FHPG should theoretically accumulate in cells expressing HSV-tk. [F-18]FHPG was obtained by nucleophilic substitution on a ditosylate precursor followed by hydrolysis. To determine the biological behavior of this compound, we synthetized the corresponding non radioactive fluorinated analog (FHPG) and tested its inhibitory activity on HSV-tk transduced 9L gliosarcoma cells maintained in culture. FHPG at 100 {mu}M suppress cell growth by 50% while GCV and acyclovir induced 100% suppression at 10 and 100 {mu}M, respectively. We then tested the in vitro uptake of n.c.a. [F-18]FHPG in cultured cells transduced with HSV-tk or a control gene (neomycin). Ratio of [F-18]FHPG uptake in HSV-tk versus control cells was 240 after 6 hours of incubation. In vivo uptake of [F-18]FHPG was tested in experimental tumors obtained by stereotactical implantion of transduced 9L cells in the brain of male Fischer 344 rats. Ratio of [F-18]FHPG uptake in HSV-tk versus control tumors was 2.5, 3 hours after intravenous tracer injection. Uptake in HSV-tk tumor was 19-fold higher than in the cortex. We concluded that [F-18]FHPG is a promising PET tracer for the evaluation of gene therapy involving viral thymidine kinase genes.

  17. Epidermal growth factor stimulates Rac1 and p21-activated kinase in vascular smooth muscle cells.

    Science.gov (United States)

    Beier, Imke; Düsing, Rainer; Vetter, Hans; Schmitz, Udo

    2008-01-01

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for vascular smooth muscle cells (VSMC) both in vitro and in vivo, thus contributing to the development of atherosclerosis and hypertension. Stimulation of Rho-family GTPases Rac/Cdc42 exerts pleiotropic cellular effects and have been demonstrated to contribute to EGF-induced proliferation in other cell systems. However, the effect of EGF on Rac/Cdc42 activation is unknown for VSMC. In the present report, we evaluated stimulation of Rac/Cdc42 by EGF in VSMC performing PAK-PBD binding assay. EGF treatment of VSMC induced time and concentration dependent binding of GTP-bound Rac1 to PAK-PBD peaking at 1 min and showing sustained activation up to 15 min. However, stimulation of Cdc42 could not be demonstrated. To further evaluate downstream effectors of Rac1 stimulation of p21-activated kinase (PAK) and c-Jun N-terminal kinase (JNK) by EGF was determined. In VSMC, EGF sequentially stimulated PAK, peaking at 5 min, and JNK, peaking at 15 min. Pretreatment of VSMC by EGF receptor specific tyrosine kinase inhibitor AG1478 and non-specific tyrosine kinase inhibitor genistein inhibited EGF-induced activation of Rac1, PAK and JNK, whereas tyrosine kinase inhibitors specific for Src (PP1) and specific for platelet-derived growth factor (AG1296) had no effect. Specific inhibition or Rac1 by NSC23766 attenuated EGF-induced [(3)H] thymidine incorporation in VSMC. Our data provide evidence for EGF-induced Rac1 activation and implicate PAK and JNK as downstream targets of Rac1 in EGF signal transduction in VSMC.

  18. Impact of HIV-1 reverse transcriptase polymorphism F214L on virological response to thymidine analogue-based regimens in antiretroviral therapy (ART)-naive and ART-experienced patients

    DEFF Research Database (Denmark)

    Ceccherini-Silberstein, Francesca; Cozzi-Lepri, Alessandro; Ruiz, Lidia

    2007-01-01

    A negative association between the polymorphism F214L and type 1 thymidine analogue (TA) mutations (TAMs) has been observed. However, the virological response to TAs according to the detection of F214L has not been evaluated....

  19. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  20. A 17-year old patient with DOCK8 deficiency, severe oral HSV-1 and aggressive periodontitis - a case of virally induced periodontitis?

    Science.gov (United States)

    Betts, K; Abusleme, L; Freeman, A F; Sarmadi, M; Fahle, G; Pittaluga, S; Cuellar-Rodriguez, J; Hickstein, D; Holland, S M; Su, H; Moutsopoulos, N M

    2015-02-01

    We present a 17-year old girl with DOCK-8 deficiency, severe untreated oral HSV-1 infection and associated aggressive periodontitis. DOCK-8 deficiency is a primary immunodeficiency, caused by biallelicloss-of-function mutations in the DOCK8 gene, often leading to severe viral and fungal mucocutaneous infections. Nevertheless, to date DOCK8 has not been associated with severe periodontitis and inflammatory bone loss around teeth. Understanding whether DOCK8 deficiency or severe HSV-1 infection underlies susceptibility to periodontitis is central to this case and may provide insights into susceptibility factors for periodontitis in the general population. Our clinical and microbiological data suggest that severe HSV-1 infection is the driver of periodontal inflammation in this case. Published by Elsevier B.V.

  1. X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

    Energy Technology Data Exchange (ETDEWEB)

    Prokofev, I. I.; Lashkov, A. A., E-mail: alashkov83@gmail.com; Gabdulkhakov, A. G.; Balaev, V. V.; Seregina, T. A. [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation); Mironov, A. S. [State Research Institute of Genetics and Selection of Industrial Microorganisms (Russian Federation); Betzel, C. [University of Hamburg (Germany); Mikhailov, A. M. [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation)

    2016-11-15

    In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2′-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation.

  2. Pd-catalyzed Suzuki-Miyaura coupling reaction in the synthesis of 5-aryl-1-[2-(phosphonomethoxy)ethyl]uracils as potential multisubstrate inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Holý, Antonín; Pohl, Radek

    2007-01-01

    Roč. 48, č. 17 (2007), s. 3065-3067 ISSN 0040-4039 R&D Projects: GA MŠk 1M0508 Grant - others: Descartes Prize(XE) HPAW-CT-2002-9001 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * Suzuki coupling * pyrimidine Subject RIV: CC - Organic Chemistry Impact factor: 2.615, year: 2007

  3. Labelling indices after /sup 3/H-thymidine incorporation during organ culture of duodenal mucosa in coeliac disease

    Energy Technology Data Exchange (ETDEWEB)

    Fluge, G.; Aksnes, L. (Bergen Univ. (Norway))

    1980-01-01

    Incorporation of /sup 3/H-thymidine during organ culture of duodenal biopsy specimens from 34 coeliac and 10 non-coeliac patients was studied by autoradiography. High labelling indices were found in flat, coeliac mucosas. Gluten fractions, which provoked histological deterioration during culture, induced labelling of a greater proportion of crypt cells and higher migration rate than parallelly cultured specimens on gluten-free medium. No influence on clypt cell kinetics could be observed after culture with gluten fractions incapable of producing histological damage or with alpha-lactalbumin. In coeliac remission mucosas, labelling indices were at the same level as in non-coeliac biopsis, and no significant effects of gluten were observed. Autoradiography seems to be a fairly sensitive and reliable determinant of gluten toxicity by organ culture in coeliac desease and should supplement the histological appraisal of the biopsies. The increment of labelling indices provoked by gluten exposure seemed not merely to be a concequence of increased desquamation of cells from the biopsy surface but could imply a direct influence of gluten on crypt cell kinetics in coeliac disease.

  4. Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy.

    Science.gov (United States)

    López-Estévez, S; Ferrer, G; Torres-Torronteras, J; Mansilla, M J; Casacuberta-Serra, S; Martorell, L; Hirano, M; Martí, R; Barquinero, J

    2014-07-01

    Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.

  5. Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF can regulate HSV-1 immediate-early transcription via histone modification

    Directory of Open Access Journals (Sweden)

    Hill James M

    2007-06-01

    Full Text Available Abstract Background During primary infection of its human host, Herpes Simplex Virus Type-1 (HSV-1 establishes latency in neurons where the viral genome is maintained in a circular form associated with nucleosomes in a chromatin configration. During latency, most viral genes are silenced, although the molecular mechanisms responsible for this are unclear. We hypothesized that neuronal factors repress HSV-1 gene expression during latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1/Neuronal Restrictive Silencer Element (RE-1/NRSE located between HSV-1 genes ICP22 and ICP4. We predicted that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/NRSF regulates expression of ICP22 and ICP4. Results Transient cotransfection indicated that REST/NRSF inhibited the activity of both promoters. In contrast, cotransfection of a mutant form of REST/NRSF encoding only the DNA-binding domain of the protein resulted in less inhibition. Stably transformed cell lines containing episomal reporter plasmids with a chromatin structure showed that REST/NRSF specifically inhibited the ICP4 promoter, but not the ICP22 promoter. REST/NRSF inhibition of the ICP4 promoter was reversed by histone deacetylase (HDAC inhibitor Trichostatin A (TSA. Additionally, chromatin immuno-precipitation (ChIP assays indicated that the corepressor CoREST was recruited to the proximity of ICP4 promoter and that acetylation of histone H4 was reduced in the presence of REST/NRSF. Conclusion Since the ICP4 protein is a key transactivator of HSV-1 lytic cycle genes, these results suggest that REST/NRSF may have an important role in the establishment and/or maintenance of HSV-1 gene silencing during latency by targeting ICP4 expression.

  6. Prevalence of Conjunctivitis Infection by HSV-1 in Patients Referring to Hospitals Affiliated to Iran University of Medical Sciences (IUMS in 2009

    Directory of Open Access Journals (Sweden)

    L Noor Mohamadian

    2012-05-01

    Full Text Available

    Background and Objectives: The viruses are the most common cause of eye infection. The most common viruses in conjunctivitis infection include Herpes simplex virus, Adeno virus and Entrovirus. It has been estimated that HSV1 causes 60-90% conjunctival infectionsin adults. Recurrent infection by HSV1 results in Herpes keratitis that is the most common cause for blindness in developing countries. Therefore, a prompt laboratory diagnosis is often very useful. Theaim of this study was to evaluate the prevalence of conjunctivitis infection by HSV-1 Hospital affiliated to Iran University of Medical Sciences (IUMS in 2009.

     

    Methods: This descriptive cross-sectional study was doneon 100 tear film and eye swabs specimens from patient with symptoms of viral conjunctivitis. All samples werecollected in viral transport media (VTM and inoculated in Vero cell line. Viral cytopatic effect was compared with control sample. HSV1 DNA wasdetected by polymerase chain reaction (PCR. Chi-square test was used to for data analysis (p<0.05.

     

    Results: Out of 100 samples under study, 2% were positive samples by cell culture, whereas in PCR9% were positive. Patients with 6-29 years old had the highest rate of HSV1 infection (13.5% than other age groups. There was not any significant relationship between age, sex and infection.

     

    Conclusion: Herpes simplex virus infection is the major cause of conjunctivitis. Taking the recurrent infection and similarity in clinical manifestation to other infections into account, it seems necessary to have a quick and specific diagnosis of HSV for bettertreatment.

     

  7. Prevalence of Conjunctivitis Infection by HSV-1 in Patients Referring to Hospitals Affiliated to Iran University of Medical Sciences (IUMS in 2009

    Directory of Open Access Journals (Sweden)

    Ataei Pirkooh A

    2011-08-01

    Full Text Available Background and Objectives: The viruses are the most common cause of eye infection. The most common viruses in conjunctivitis infection include Herpes simplex virus, Adeno virus and Entrovirus. It has been estimated that HSV1 causes 60-90% conjunctival infectionsin adults. Recurrent infection by HSV1 results in Herpes keratitis that is the most common cause for blindness in developing countries. Therefore, a prompt laboratory diagnosis is often very useful. Theaim of this study was to evaluate the prevalence of conjunctivitis infection by HSV-1 Hospital affiliated to Iran University of Medical Sciences (IUMS in 2009.Methods: This descriptive cross-sectional study was doneon 100 tear film and eye swabs specimens from patient with symptoms of viral conjunctivitis. All samples werecollected in viral transport media (VTM and inoculated in Vero cell line. Viral cytopatic effect was compared with control sample. HSV1 DNA wasdetected by polymerase chain reaction (PCR. Chi-square test was used to for data analysis (p<0.05.Results: Out of 100 samples under study, 2% were positive samples by cell culture, whereas in PCR9% were positive. Patients with 6-29 years old had the highest rate of HSV1 infection (13.5% than other age groups. There was not any significant relationship between age, sex and infection.Conclusion: Herpes simplex virus infection is the major cause of conjunctivitis. Taking the recurrent infection and similarity in clinical manifestation to other infections into account, it seems necessary to have a quick and specific diagnosis of HSV for bettertreatment.

  8. Does porphyrin suppres the apoptotic and necrotic effects of bovine herpes virus type-1(BoHV-1) and herpes simplex virus type-1(HSV-1)?

    Science.gov (United States)

    Yavuz, Beyza; Yazici, Zafer

    2016-09-01

    In this study, antiviral effect of porphyrin was investigated. Cooper strain of Bovine Herpes Virus type 1(BoHV-1) and Kos strain of Herpes Simplex Virus type-1 (HSV-1) were used to determine the potential of porphyrins to inhibit infection in vitro (with morphological and cytopathological criteria). Apoptotic and necrotic changes were determined by using DAPI and propidium staining. The non-cytotoxic dose of porphyrin (NCD-p) was initially calculated as 312.50µg/mL on MDBK and Vero cells. The apoptotic cell (APC) count was found 10% with BoHV-1 while it was 5.3% with BoHV-1 treated with porphyrin on MDBK cells between 6th to 24th hours post infection (hpi). Necrotic cell (NEC) count was 51% with BoHV-1 and 37.8% BoHV-1 treated with porphyrin on MDBK cells at 24th hpi. On the other hand, the APC count was found 23% with HSV-1, while 22% with the HSV-1 treated with porphyrin on Vero cells between 6th to 24th hpi. NEC count was 49% with HSV-1 and 34% HSV-1 treated with porphyrin on MDBK cells at 24th hpi. The results show that BoHV-1 was inhibited by porphyrin resulting in decreased apoptotic and necrotic changes in MDBK cells. On the contrary, porphyrine was not effective in the inhibition of HSV-1 in terms of apoptosis but it caused necrotic changes in Vero cells.

  9. [Co-infections of HIV, syphilis and HSV-2 among men who have sex with men at the voluntary HIV counseling and testing clinics in Shanghai].

    Science.gov (United States)

    Liu, Y; Tang, H F; Ning, Z; Zheng, H; He, N; Zhang, Y Y

    2017-10-10

    Objective: To understand the prevalence rates of HIV-syphilis and HIV-herpes simplex virus 2 (HSV-2) co-infections and related factors among men having sex with men (MSM) who had visited the voluntary HIV counseling and testing (VCT) clinics in Shanghai, China. Methods: 756 eligible MSM who attended the VCT clinics of Shanghai Municipality and Putuo district during March to August, 2015 were recruited to participate in a cross-sectional survey with questionnaire interview and blood testing for HIV, syphilis and HSV-2. Results: A total of 732 participants completed a valid questionnaire survey. The prevalence rates were 3.3 % (24/732) for HIV/Syphilis co-infection, 1.9 % (14/732) for HIV/HSV-2 co-infection, and 0.7 % (5/732) for HIV/Syphilis/HSV-2 co-infection, respectively. HIV prevalence appeared significantly higher among syphilis-infected participants (45.3 % , 24/53) than those without Syphilis (7.2 % , 61/679) (χ(2)=63.11, P Syphilis co-infection. Those participants who had high middle school or lower levels of education ( OR =6.87, 95 %CI : 1.86-25.42; OR =9.82, 95 %CI : 2.25-42.85) were under risk on HIV and HSV-2 co-infection. Conclusion: HIV/Syphilis and HIV/HSV-2 co-infection were seen among MSM who attended the VCT clinics in Shanghai that called for special attention, especially on migrants, those with low education or illicit drug users.

  10. Characteristics of HIV-1 discordant couples enrolled in a trial of HSV-2 suppression to reduce HIV-1 transmission: the partners study.

    Directory of Open Access Journals (Sweden)

    Jairam R Lingappa

    Full Text Available The Partners HSV-2/HIV-1 Transmission Study (Partners Study is a phase III, placebo-controlled trial of daily acyclovir for genital herpes (HSV-2 suppression among HIV-1/HSV-2 co-infected persons to reduce HIV-1 transmission to their HIV-1 susceptible partners, which requires recruitment of HIV-1 serodiscordant heterosexual couples. We describe the baseline characteristics of this cohort.HIV-1 serodiscordant heterosexual couples, in which the HIV-1 infected partner was HSV-2 seropositive, had a CD4 count >or=250 cells/mcL and was not on antiretroviral therapy, were enrolled at 14 sites in East and Southern Africa. Demographic, behavioral, clinical and laboratory characteristics were assessed.Of the 3408 HIV-1 serodiscordant couples enrolled, 67% of the HIV-1 infected partners were women. Couples had cohabitated for a median of 5 years (range 2-9 with 28% reporting unprotected sex in the month prior to enrollment. Among HIV-1 susceptible participants, 86% of women and 59% of men were HSV-2 seropositive. Other laboratory-diagnosed sexually transmitted infections were uncommon (500 relative to <350, respectively, p<0.001.The Partners Study successfully enrolled a cohort of 3408 heterosexual HIV-1 serodiscordant couples in Africa at high risk for HIV-1 transmission. Follow-up of this cohort will evaluate the efficacy of acyclovir for HSV-2 suppression in preventing HIV-1 transmission and provide insights into biological and behavioral factors determining heterosexual HIV-1 transmission.ClinicalTrials.gov NCT00194519.

  11. Characteristics of HIV-1 Discordant Couples Enrolled in a Trial of HSV-2 Suppression to Reduce HIV-1 Transmission: The Partners Study

    Science.gov (United States)

    Lingappa, Jairam R.; Kahle, Erin; Mugo, Nelly; Mujugira, Andrew; Magaret, Amalia; Baeten, Jared; Bukusi, Elizabeth A.; Cohen, Craig R.; Katabira, Elly; Ronald, Allan; Kiarie, James; Farquhar, Carey; Stewart, Grace John; Makhema, Joseph; Essex, M.; Were, Edwin; Fife, Kenneth; deBruyn, Guy; Gray, Glenda; McIntyre, James; Manongi, Rachel; Kapiga, Saidi; Coetzee, David; Allen, Susan; Inambao, Mubiana; Kayitenkore, Kayitesi; Karita, Etienne; Kanweka, William; Delany, Sinead; Rees, Helen; Vwalika, Bellington; Coombs, Robert W.; Morrow, Rhoda; Whittington, William; Corey, Lawrence; Wald, Anna; Celum, Connie

    2009-01-01

    Background The Partners HSV-2/HIV-1 Transmission Study (Partners Study) is a phase III, placebo-controlled trial of daily acyclovir for genital herpes (HSV-2) suppression among HIV-1/HSV-2 co-infected persons to reduce HIV-1 transmission to their HIV-1 susceptible partners, which requires recruitment of HIV-1 serodiscordant heterosexual couples. We describe the baseline characteristics of this cohort. Methods HIV-1 serodiscordant heterosexual couples, in which the HIV-1 infected partner was HSV-2 seropositive, had a CD4 count ≥250 cells/mcL and was not on antiretroviral therapy, were enrolled at 14 sites in East and Southern Africa. Demographic, behavioral, clinical and laboratory characteristics were assessed. Results Of the 3408 HIV-1 serodiscordant couples enrolled, 67% of the HIV-1 infected partners were women. Couples had cohabitated for a median of 5 years (range 2–9) with 28% reporting unprotected sex in the month prior to enrollment. Among HIV-1 susceptible participants, 86% of women and 59% of men were HSV-2 seropositive. Other laboratory-diagnosed sexually transmitted infections were uncommon (500 relative to <350, respectively, p<0.001). Conclusions The Partners Study successfully enrolled a cohort of 3408 heterosexual HIV-1 serodiscordant couples in Africa at high risk for HIV-1 transmission. Follow-up of this cohort will evaluate the efficacy of acyclovir for HSV-2 suppression in preventing HIV-1 transmission and provide insights into biological and behavioral factors determining heterosexual HIV-1 transmission. Trial Registration ClinicalTrials.gov NCT00194519 PMID:19404392

  12. The effect of school attendance and school dropout on incident HIV and HSV-2 among young women in rural South Africa enrolled in HPTN 068.

    Science.gov (United States)

    Stoner, Marie C D; Pettifor, Audrey; Edwards, Jessie K; Aiello, Allison E; Halpern, Carolyn T; Julien, Aimée; Selin, Amanda; Twine, Rhian; Hughes, James P; Wang, Jing; Agyei, Yaw; Gomez-Olive, F Xavier; Wagner, Ryan G; MacPhail, Catherine; Kahn, Kathleen

    2017-09-24

    To estimate the association between school attendance, school dropout, and risk of incident HIV and herpes simplex virus type 2 (HSV-2) infection among young women. We used longitudinal data from a randomized controlled trial in rural Mpumalanga province, South Africa, to assess the association between school days attended, school dropout, and incident HIV and HSV-2 in young women aged 13-23 years. We examined inverse probability of exposure weighted survival curves and used them to calculate 1.5, 2.5, and 3.5-year risk differences and risk ratios for the effect of school attendance on incident HIV and HSV-2. A marginal structural Cox model was used to estimate hazard ratios for the effect of school attendance and school dropout on incident infection. Risk of infection increased over time as young women aged, and was higher in young women with low school attendance (school days) compared with high (≥80% school days). Young women with low attendance were more likely to acquire HIV [hazard ratio (HR): 2.97; 95% confidence interval (CI): 1.62, 5.45] and HSV-2 (HR: 2.47; 95% CI: 1.46, 4.17) over the follow-up period than young women with high attendance. Similarly, young women who dropped out of school had a higher weighted hazard of both HIV (HR 3.25 95% CI: 1.67, 6.32) and HSV-2 (HR 2.70; 95% CI 1.59, 4.