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Sample records for imaging hsv-1 tk

  1. Assessment of α-Fetoprotein Targeted HSV1-tk Expression in Hepatocellular Carcinoma with In Vivo Imaging

    PubMed Central

    Park, Ju Hui; Kim, Kwang Il; Lee, Kyo Chul; Lee, Yong Jin; Lee, Tae Sup; Chung, Wee Sup; Lim, Sang Moo

    2015-01-01

    Abstract Tumor-specific enhancer/promoter is applicable for targeting gene expression in tumors and helpful for tumor-targeting imaging and therapy. We aimed to acquire α-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) specific images using adenovirus containing HSV1-tk gene controlled by AFP enhancer/promoter and evaluate in vivo ganciclovir (GCV)-medicated therapeutic effects on AFP-targeted HSV1-tk expression with 18F-FDG positron emission tomography (PET). Recombinant adenovirus expressing HSV1-tk under AFP enhancer/promoter was produced (AdAFP-TK) and the expression levels were evaluated by RT-PCR and 125I-IVDU uptake. GCV-mediated HSV1-tk cytotoxicity was determined by MTT assay. After the mixture of AdAFP-fLuc and AdAFP-TK was administrated, bioluminescent images (BLIs) and 18F-FHBG PET images were obtained in tumor-bearing mice. In vivo therapeutic effects of AdAFP-TK and GCV in the HuH-7 xenograft model were monitored by 18F-FDG PET. When infected with AdAFP-TK, cell viability in HuH-7 was reduced, but those in HT-29 and SK-Hep-1 were not significantly decreased at any GCV concentration less than 100 μM. AFP-targeted fLuc and HSV1-tk expression were clearly visualized by BLI and 18F-FHBG PET images in AFP-producing HCC, respectively. In vivo GCV-mediated tumor growth inhibition by AFP-targeted HSV1-tk expression was monitored by 18F-FDG PET. Recombinant AdAFP-TK could be applied for AFP-targeted HCC gene therapy and imaging in AFP-producing HCC. PMID:25545853

  2. Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis.

    PubMed

    Wang, Zhe; Wang, Fu; Hida, Naoki; Kiesewetter, Dale O; Tian, Jie; Niu, Gang; Chen, Xiaoyuan

    2015-05-01

    Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we describe the design and use of an engineered cyclic herpes simplex virus 1-thymidine kinase (HSV1-TK) PET reporter whose kinase activity is specifically switched on by apoptosis. The expression of cyclic TK (cTK) in healthy cells leads to inactive product, whereas the activation of apoptosis through the caspase-3 pathway cleaves cTK, thus restoring its activity and enabling PET imaging. In addition to detailing the design and construction of the cTK plasmid in this protocol, we include assays for evaluating the function and specificity of the cTK reporter in apoptotic cells, such as assays for measuring the cell uptake of PET tracer in apoptotic cells, correlating doxorubicin (Dox)-induced cell apoptosis to cTK function recovery, and in vivo PET imaging of cancer cell apoptosis, and we also include corresponding data acquisition methods. The time to build the entire cTK reporter is ∼2-3 weeks. The selection of a stable cancer cell line takes ∼4-6 weeks. The time to implement assays regarding cTK function in apoptotic cells and the in vivo imaging varies depending on the experiment. The cyclization strategy described in this protocol can also be adapted to create other reporter systems for broad biomedical applications. PMID:25927390

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

    PubMed Central

    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

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

    PubMed

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

    2010-02-01

    One limitation of HSV1-tk reporter positron emission tomography (PET) 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. 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. 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. Intestinal bacteria in germ-free mice do not contribute to the high intestinal levels of radioactivity following injection of radionucleoside analogues. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration. PMID:20128998

  5. A cyclic HSV1-TK reporter for real-time PET imaging of apoptosis

    PubMed Central

    Wang, Fu; Wang, Zhe; Hida, Naoki; Kiesewetter, Dale O.; Ma, Ying; Yang, Kai; Rong, Pengfei; Liang, Jimin; Tian, Jie; Niu, Gang; Chen, Xiaoyuan

    2014-01-01

    The coordination of cell proliferation and programmed death (apoptosis) is essential for normal physiology, and imbalance in these two opposing processes is implicated in various diseases. Objective and quantitative noninvasive imaging of apoptosis would significantly facilitate rapid screening as well as validation of therapeutic chemicals. Herein, we molecularly engineered an apoptosis switch-on PET-based cyclic herpes simplex virus type 1–thymidine kinase reporter (cTK266) containing a caspase-3 recognition domain as the switch. Translation of the reporter and protein splicing in healthy mammalian cells produce an inactive cyclic chimera. Upon apoptosis, caspase-3–specific cleavage of the circular product occurs, resulting in the restoration of the thymidine kinase activity, which can be detected in living cells and animals by noninvasive PET imaging. Our results showed the high sensitivity of this reporter in dynamic and quantitative imaging of apoptosis in living subjects. This reporter could be applied as a valuable tool for high-throughput functional screening of proapoptotic and antiapoptotic compounds in preclinical models in drug development, and monitoring the destination of therapeutic cells in clinical settings. PMID:24706884

  6. Synthesis and preclinical evaluation of a new C-6 alkylated pyrimidine derivative as a PET imaging agent for HSV1-tk gene expression

    PubMed Central

    Müller, Ursina; Ross, Tobias L; Ranadheera, Charlene; Slavik, Roger; Müller, Adrienne; Born, Mariana; Trauffer, Evelyn; Sephton, Selena Milicevic; Scapozza, Leonardo; Krämer, Stefanie D; Ametamey, Simon M

    2013-01-01

    [18F]FHOMP (6-((1-[18F]-fluoro-3-hydroxypropan-2-yloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione), a C-6 substituted pyrimidine derivative, has been synthesized and evaluated as a potential PET agent for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression. [18F]FHOMP was prepared by the reaction of the tosylated precursor with tetrabutylammonium [18F]-fluoride followed by acidic cleavage of the protecting groups. In vitro cell accumulation of [18F]FHOMP and [18F]FHBG (reference) was studied with HSV1-tk transfected HEK293 (HEK293TK+) cells. Small animal PET and biodistribution studies were performed with HEK293TK+ xenograft-bearing nude mice. The role of equilibrative nucleoside transporter 1 (ENT1) in the transport and uptake of [18F] FHOMP was also examined in nude mice after treatment with ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P). [18F]FHOMP was obtained in a radiochemical yield of ~25% (decay corrected) and the radiochemical purity was greater than 95%. The uptake of [18F]FHOMP in HSV1-TK containing HEK293TK+ cells was 52 times (at 30 min) and 244 times (at 180 min) higher than in control HEK293 cells. The uptake ratios between HEK293TK+ and HEK293 control cells for [18F]FHBG were significantly lower i.e. 5 (at 30 min) and 81 (240 min). In vivo, [18F]FHOMP accumulated to a similar extend in HEK293TK+ xenografts as [18F]FHBG but with a higher general background. Blocking of ENT1 reduced [18F]FHOMP uptake into brain from a standardized uptake value (SUV) of 0.10±0.01 to 0.06±0.02, but did not reduce the general background signal in PET. Although [18F]FHOMP does not outperform [18F]FHBG in its in vivo performance, this novel C-6 pyrimidine derivative may be a useful probe for monitoring HSV1-tk gene expression in vivo. PMID:23342302

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

    PubMed Central

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

    2013-01-01

    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. PMID:24177294

  8. Cold Sores (HSV-1)

    MedlinePlus

    ... Help a Friend Who Cuts? Cold Sores (HSV-1) KidsHealth > For Teens > Cold Sores (HSV-1) Print A A A Text Size What's in ... person's lips, are caused by herpes simplex virus-1 (HSV-1) . But they don't just show ...

  9. Antiviral activity of the marine alga Symphyocladia latiuscula against herpes simplex virus (HSV-1) in vitro and its therapeutic efficacy against HSV-1 infection in mice.

    PubMed

    Park, Hye-Jin; Kurokawa, Masahiko; Shiraki, Kimiyasu; Nakamura, Norio; Choi, Jae-Sue; Hattori, Masao

    2005-12-01

    The antiviral activities of extracts from 5 species of marine algae collected at Haeundae (Pusan, Korea), were examined using plaque reduction assays. Although the activity of a methanol (MeOH) extract of Sargassum ringoldianum (Sargassaceae) was the most potent against several types of viruses, it was also cytotoxic. A MeOH extract of Symphyocladia latiuscula (Rhodomelaceae) and its fractions exhibited antiviral activities against acyclovir (ACV) and phosphonoacetic acid (PAA)-resistant (AP(r)) herpes simplex type 1 (HSV-1), thymidine kinase (TK(-)) deficient HSV-1 and wild type HSV-1 in vitro without cytotoxicity. The major component, 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (TDB) of a CH(2)Cl(2)-soluble fraction was active against wild type HSV-1, as well as AP(r) HSV-1 and TK(-) HSV-1 (IC(50) values of 5.48, 4.81 and 23.3 microg/ml, respectively). The therapeutic effectiveness of the MeOH extract and TDB from S. latiuscula was further examined in BALB/c mice that were cutaneously infected with HSV-1 strain 7401H. Three daily oral administrations of the MeOH extract and TDB significantly delayed the appearance of score 2 skin lesions (local vesicles) and limited the development of further score 6 (mild zosteriform) lesions in infected mice without toxicity compared with controls. In addition, TDB suppressed virus yields in the brain and skin. Therefore TDB should be a promising anti HSV agent. PMID:16327161

  10. IFI16 Restricts HSV-1 Replication by Accumulating on the HSV-1 Genome, Repressing HSV-1 Gene Expression, and Directly or Indirectly Modulating Histone Modifications

    PubMed Central

    Johnson, Karen E.; Bottero, Virginie; Flaherty, Stephanie; Dutta, Sujoy; Singh, Vivek Vikram; Chandran, Bala

    2014-01-01

    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 mechanisms of IFI16

  11. Substrate specificity of three viral thymidine kinases (TK): vaccinia virus TK, feline herpesvirus TK, and canine herpesvirus TK.

    PubMed

    Solaroli, N; Johansson, M; Balzarini, J; Karlsson, A

    2006-01-01

    In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs. PMID:17065088

  12. HSV-1 infection through inhibitory receptor, PILRalpha.

    PubMed

    Satoh, Takeshi; Arase, Hisashi

    2008-06-01

    Paired receptors that consist of highly related activating and inhibitory receptors are widely involved in the regulation of immune response. Several viruses that persistently infect hosts possess genes that encode ligands for inhibitory receptors in order to escape from host immune system. Herpes simplex virus type 1 (HSV-1) is one of the viruses that cause persistent infection. Here, we found that HSV-1-infected cells express a ligand for paired immunoglobulin like-type 2 receptor (PILR)alpha, one of paired inhibitory receptors mainly expressed on myeloid cells such as monocytes, macrophages and dendritic cells. Furthermore, we have identified that glycoprotein B (gB), an envelope protein of HSV-1, is a ligand for PILRalpha by mass spectrometry analysis. Because gB is essential for HSV-1 to infect cells, we analyzed function of PILRalpha in HSV-1 infection. When PILRalpha was transfected into CHO-K1 cells, which is resistant to HSV-1 infection, the PILRalpha-transfected CHO-K1 cells became permissive to HSV-1 infection. We further addressed weather PILRalpha is involved in the HSV-1 infection of primary human cells. CD14-positive monocytes that express both PILRalpha and HVEM, a glycoprotein D receptor, were susceptible to HSV-1 infection. In contrast, HSV-1 did not infect CD14-negative lymphocytes that express HVEM but not PILRalpha. Furthermore, HSV-1 infection of monocyte was blocked by both anti-PILRalpha mAb and anti-HVEM antiserum. These findings indicated that both gB and gD receptors play an important role in HSV-1 infection. We have shown, for the first time, that viruses use an inhibitory immune receptor to enter a cell. Invasion into hematopoietic cells by using inhibitory receptors should be beneficial to the virus because binding to inhibitory receptors may not only provide entry, but also trigger the inhibitory receptor to suppress the immune functions of the infected cell. PMID:19122386

  13. Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.

    PubMed Central

    Focher, F; Maga, G; Bendiscioli, A; Capobianco, M; Colonna, F; Garbesi, A; Spadari, S

    1995-01-01

    L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides. Images PMID:7544886

  14. Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells.

    PubMed

    Bello-Morales, Raquel; Crespillo, Antonio Jesús; Praena, Beatriz; Tabarés, Enrique; Revilla, Yolanda; García, Elena; Fraile-Ramos, Alberto; Baron, Wia; Krummenacher, Claude; López-Guerrero, José Antonio

    2016-01-01

    Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus--unable to replicate in ICP4-defficient cells--showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells. PMID:26807581

  15. Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells

    PubMed Central

    Bello-Morales, Raquel; Crespillo, Antonio Jesús; Praena, Beatriz; Tabarés, Enrique; Revilla, Yolanda; García, Elena; Fraile-Ramos, Alberto; Baron, Wia; Krummenacher, Claude; López-Guerrero, José Antonio

    2016-01-01

    Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus–unable to replicate in ICP4-defficient cells- showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells. PMID:26807581

  16. Genetic incorporation of HSV-1 thymidine kinase into the adenovirus protein IX for functional display on the virion

    SciTech Connect

    Li Jing; Le, Long; Sibley, Don A.; Mathis, J. Michael; Curiel, David T. . E-mail: david.curiel@ccc.uab.edu

    2005-08-01

    Adenoviral vectors have been exploited for a wide range of gene therapy applications. Direct genetic modification of the adenovirus capsid proteins has been employed to achieve alteration of vector tropism. We have defined the carboxy-terminus of the minor capsid protein pIX as a locus capable of presenting incorporated ligands on the virus capsid surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we incorporated the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) within pIX to determine if a larger protein of this type could retain functionality in this context. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. DNA packaging and cytopathic effect were not affected by this genetic modification to the virus, while CAR-dependent binding was only marginally affected. Using an in vitro [{sup 3}H]-thymidine phosphorylation assay, we demonstrated that the kinase activity of the protein IX-TK fusion protein incorporated into adenoviral virions is functional. Analysis of cell killing after adenovirus infection showed that the protein IX-TK fusion protein could also serve as a therapeutic gene by rendering transduced cells sensitive to gancyclovir. Using 9-[4-[{sup 18}F]-fluoro-3-(hydroxymethyl)butyl]guanine ([{sup 18}F]-FHBG; a positron-emitting TK substrate), we demonstrated that we could detect specific cell binding and uptake of adenoviral virions containing the protein IX-TK fusion protein at 1 h post-infection. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. The alternative display of HSV-1 TK on the capsid may offer advantages with respect to direct functional applications of this gene product. In addition, the determination of an expanded upper limit of incorporable

  17. Inhibition of HSV-1 Replication by Gene Editing Strategy

    PubMed Central

    Roehm, Pamela C.; Shekarabi, Masoud; Wollebo, Hassen S.; Bellizzi, Anna; He, Lifan; Salkind, Julian; Khalili, Kamel

    2016-01-01

    HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases. PMID:27064617

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

    PubMed Central

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

    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 cell carcinoma (OSCC). Material and Methods 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. Results 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. Conclusions 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. Key words:HSV-1, nested PCR, PCR. PMID:26449432

  19. Reactivation of HSV-1 following explant of tree shrew brain.

    PubMed

    Li, Lihong; Li, Zhuoran; Li, Xin; Wang, Erlin; Lang, Fengchao; Xia, Yujie; Fraser, Nigel W; Gao, Feng; Zhou, Jumin

    2016-06-01

    Herpes Simplex Virus type I (HSV-1) latently infects peripheral nervous system (PNS) sensory neurons, and its reactivation leads to recurring cold sores. The reactivated HSV-1 can travel retrograde from the PNS into the central nervous system (CNS) and is known to be causative of Herpes Simplex viral encephalitis. HSV-1 infection in the PNS is well documented, but little is known on the fate of HSV-1 once it enters the CNS. In the murine model, HSV-1 genome persists in the CNS once infected through an ocular route. To gain more details of HSV-1 infection in the CNS, we characterized HSV-1 infection of the tree shrew (Tupaia belangeri chinensis) brain following ocular inoculation. Here, we report that HSV-1 enters the tree shrew brain following ocular inoculation and HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 5 days post-infection (p.i.), peaking at 10 days p.i. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the LAT intron region continues to be expressed. Live virus could be recovered from the olfactory bulb and brain stem tissue. Viral proteins could be detected using anti-HSV-1 antibodies and anti-ICP4 antibody, during the acute stage but not beyond. In situ hybridization could detect LAT during acute infection in most brain regions and in olfactory bulb and brain stem tissue well beyond the acute stage. Using a homogenate from these tissues' post-acute infection, we did not recover live HSV-1 virus, supporting a latent infection, but using a modified explant cocultivation technique, we were able to recover reactivated virus from these tissues, suggesting that the HSV-1 virus latently infects the tree shrew CNS. Compared to mouse, the CNS acute infection of the tree shrew is delayed and the olfactory bulb contains most latent virus. During the acute stage, a portion of the infected tree shrews exhibit symptoms similar to human viral encephalitis. These findings, together with the fact that tree shrews are closely

  20. HSV-1 Remodels Host Telomeres To Facilitate Viral Replication

    PubMed Central

    Deng, Zhong; Kim, Eui Tae; Vladimirova, Olga; Dheekollu, Jayaraju; Wang, Zhuo; Newhart, Alyshia; Liu, Dongmei; Myers, Jaclyn L.; Hensley, Scott E.; Moffat, Jennifer; Janicki, Susan M.; Fraser, Nigel W.; Knipe, David M.; Weitzman, Matthew D.; Lieberman, Paul M.

    2015-01-01

    Summary Telomeres protect the ends of cellular chromosomes. We show here that infection with herpes simplex virus 1 (HSV-1) results in chromosomal structural aberrations at telomeres and the accumulation of telomere dysfunction-induced DNA damage foci (TIFs). At the molecular level, HSV-1 induces transcription of telomere repeat-containing RNA (TERRA), followed by the proteolytic degradation of the telomere protein TPP1, and loss of the telomere repeat DNA signal. The HSV-1 encoded E3 ubiquitin ligase ICP0 is required for TERRA transcription and facilitates TPP1 degradation. shRNA depletion of TPP1 increases viral replication, arguing that TPP1inhibits viral replication. Viral replication protein ICP8 forms foci that coincide with telomeric proteins and ICP8 null virus failed to degrade telomere DNA signal. These findings suggest that HSV-1 reorganizes telomeres to form ICP8-associated pre-replication foci and promotes viral genomic replication. PMID:25497088

  1. Retention of the herpes simplex virus type 1 (HSV-1) UL37 protein on single-stranded DNA columns requires the HSV-1 ICP8 protein.

    PubMed Central

    Shelton, L S; Albright, A G; Ruyechan, W T; Jenkins, F J

    1994-01-01

    The UL37 and ICP8 proteins present in herpes simplex virus type 1 (HSV-1)-infected-cell extracts produced at 24 h postinfection coeluted from single-stranded-DNA-cellulose columns. Experiments carried out with the UL37 protein expressed by a vaccinia virus recombinant (V37) revealed that the UL37 protein did not exhibit DNA-binding activity in the absence of other HSV proteins. Analysis of extracts derived from cells coinfected with V37 and an ICP8-expressing vaccinia virus recombinant (V8) and analysis of extracts prepared from cells infected with the HSV-1 ICP8 deletion mutants d21 and n10 revealed that the retention of the UL37 protein on single-stranded DNA columns required a DNA-binding-competent ICP8 protein. Images PMID:8254765

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

    PubMed

    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

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

    PubMed Central

    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

  4. Selective recruitment of host factors by HSV-1 replication centers

    PubMed Central

    LANG, Feng-Chao; LI, Xin; VLADMIROVA, Olga; LI, Zhuo-Ran; CHEN, Gui-Jun; XIAO, Yu; LI, Li-Hong; LU, Dan-Feng; HAN, Hong-Bo; ZHOU, Ju-Min

    2015-01-01

    Herpes simplex virus type 1 (HSV-1) enters productive infection after infecting epithelial cells, where it controls the host nucleus to make viral proteins, starts viral DNA synthesis and assembles infectious virions. In this process, replicating viral genomes are organized into replication centers to facilitate viral growth. HSV-1 is known to use host factors, including host chromatin and host transcription regulators, to transcribe its genes; however, the invading virus also encounters host defense and stress responses to inhibit viral growth. Recently, we found that HSV-1 replication centers recruit host factor CTCF but exclude βH2A.X. Thus, HSV-1 replication centers may selectively recruit cellular factors needed for viral growth, while excluding host factors that are deleterious for viral transcription or replication. Here we report that the viral replication centers selectively excluded modified histone H3, including heterochromatin mark H3K9me3, H3S10P and active chromatin mark H3K4me3, but not unmodified H3. We found a dynamic association between the viral replication centers and host RNA polymerase II. The centers also recruited components of the DNA damage response pathway, including 53BP1, BRCA1 and host antiviral protein SP100. Importantly, we found that ATM kinase was needed for the recruitment of CTCF to the viral centers. These results suggest that the HSV-1 replication centers took advantage of host signaling pathways to actively recruit or exclude host factors to benefit viral growth. PMID:26018857

  5. HSV-1 ICP0: paving the way for viral replication.

    PubMed

    Smith, Miles C; Boutell, Chris; Davido, David J

    2011-04-01

    Herpes simplex virus type 1 (HSV-1) has two distinct phases of its viral life cycle: lytic and latent. One viral immediate-early protein that is responsible for determining the balance between productive lytic replication and reactivation from latency is infected cell protein 0 (ICP0). ICP0 is a 775-amino acid really interesting new gene (RING)-finger-containing protein that possesses E3 ubiquitin ligase activity, which is required for ICP0 to activate HSV-1 gene expression, disrupt nuclear domain (ND) 10 structures, mediate the degradation of cellular proteins, and evade the host cell's intrinsic and innate antiviral defenses. This article examines our current understanding of ICP0's transactivating, E3 ubiquitin ligase, and antihost defense activities and their inter-relationships to one another. Lastly, we will discuss how these properties of ICP0 may be utilized as possible targets for HSV-1 antiviral therapies. PMID:21765858

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

    PubMed

    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. PMID:27018573

  7. Monitoring of tumor growth and metastasis potential in MDA-MB-435s/ tk-luc human breast cancer xenografts

    NASA Astrophysics Data System (ADS)

    Chang, Ya-Fang; Lin, Yi-Yu; Wang, Hsin-Ell; Liu, Ren-Shen; Pang, Fei; Hwang, Jeng-Jong

    2007-02-01

    Molecular imaging of reporter gene expression provides a rapid, sensitive and non-invasive monitoring of tumor behaviors. In this study, we reported the establishment of a novel animal model for longitudinal examination of tumor growth kinetics and metastatic spreading in vivo. The highly metastatic human breast carcinoma MDA-MB-435s cell line was engineered to stably express herpes simplex virus type 1 thymidine kinase (HSV-1- tk) and luciferase ( luc). Both 131I-FIAU and D-luciferin were used as reporter probes. For orthotopic tumor formation, MDA-MB-435s/ tk-luc cells were implanted into the first nipple of 6-week-old female NOD/SCID mice. For metastatic study, cells were injected via the lateral tail vein. Mice-bearing MDA-MB-435s/ tk-luc tumors were scanned for tumor growth and metastatsis using Xenogen IVIS50 system. Gamma scintigraphy and whole-body autoradiography were also applied to confirm the tumor localization. The results of bioluminescence imaging as well as histopathological finding showed that tumors could be detected in femur, spine, ovary, lungs, kidney, adrenal gland, lymph nodes and muscle at 16 weeks post i.v. injection, and correlated photons could be quantified. This MDA-MB-435s/ tk-luc human breast carcinoma-bearing mouse model combined with multimodalities of molecular imaging may facilitate studies on the molecular mechanisms of cancer invasion and metastasis.

  8. Synthesis and In Vitro Evaluation of 5-[18F]Fluoroalkyl Pyrimidine Nucleosides for Molecular Imaging of Herpes Simplex Virus Type-1 Thymidine Kinase Reporter Gene Expression

    PubMed Central

    Chacko, Ann-Marie; Qu, Wenchao; Kung, Hank F.

    2014-01-01

    Two novel series of 5-fluoroalkyl-2′-deoxyuridines (FPrDU, FBuDU, FPeDU) and 2′-fluoro-2′-deoxy-5-fluoroalkylarabinouridines (FFPrAU, FFBuAU, FFPeAU), having three, four or five methylene units (propyl, butyl, or pentyl) at C-5, were prepared and tested as reporter probes for imaging HSV1-tk gene expression. The Negishi coupling methodology was employed to efficiently synthesize the radiolabeling precursors. All six 5-[18F]fluoroalkyl pyrimidines were prepared readily from 3-N-benzoyl-3′,5′-di-O-benzoyl-protected 5-O-mesylate precursors in 17–35% radiochemical yield (decay-corrected). In vitro studies highlighted that all six [18F]labeled nucleosides selectively accumulated in cells expressing the HSV1-TK protein, with negligible uptake in control cells. [18F]FPrDU, [18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU had the best uptake profiles. Despite selective accumulation in HSV1-tk expressing cells, all 5-fluoroalkyl pyrimidine nucleosides had low to negligible cytotoxic activity (CC50>1000–209 μM). Ultimately, results demonstrated that 5-[18F]fluoropropyl, [18F]fluorobutyl, and [18F]fluoropentyl pyrimidine nucleosides have potential as in vivo HSV1-TK PET reporter probes over a dynamic range of reporter gene expression levels. PMID:18800764

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

    PubMed Central

    Groslambert, Marine; Glass, Mandy; Orr, Anne; Hay, Ronald T.; Everett, Roger D.

    2015-01-01

    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. PMID:26200910

  10. A Phase I Clinical Trial of Ad5.SSTR/TK.RGD, a Novel Infectivity-Enhanced Bicistronic Adenovirus, in Patients with Recurrent Gynecologic Cancer

    PubMed Central

    Kim, Kenneth H.; Dmitriev, Igor; O’Malley, Janis P.; Wang, Minghui; Saddekni, Souheil; You, Zhiying; Preuss, Meredith A.; Harris, Raymond D.; Aurigemma, Rosemarie; Siegal, Gene P.; Zinn, Kurt R.; Curiel, David T.; Alvarez, Ronald D.

    2014-01-01

    Purpose Ad5.SSTR/TK.RGD is an infectivity-enhanced adenovirus expressing a therapeutic thymidine kinase suicide gene and a somatostatin receptor that allows for noninvasive gene transfer imaging. The purpose of this study was to identify the MTD, toxicities, clinical efficacy and biologic effects of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer. Experimental Design Eligible patients were treated intraperitoneally (IP) for 3 days with 1×109 to 1×1012 vp/dose of Ad5.SSTR/TK.RGD followed by intravenous ganciclovir for 14 days. Toxicity and clinical efficacy were assessed utilizing CTC Adverse Events grading and RECIST criteria. Imaging utilizing In-111 pentetreotide was obtained before and after treatment. Tissue samples were obtained to evaluate for gene transfer, generation of wild-type virus, viral shedding and antibody response. Results Twelve patients were treated in three cohorts. The most common vector-related clinical toxicities were grade 1–2 constitutional or pain symptoms, experienced most often in patients treated at the highest dose. MTD was not identified. Five patients demonstrated stable disease; all others experienced progressive disease. One patient with stable disease experienced complete resolution of disease and normalization of CA125 on further follow-up. Imaging detected increased In-111 pentetreotide retention in patients treated at the highest dose. Ancillary studies demonstrated presence of Ad5.SSTR/TK.RGD virus and HSV1-tk expression in ascites samples collected at various time points in most patients treated within the higher dose cohorts. Conclusions This study demonstrates the safety, potential efficacy, and possible gene transfer imaging capacity of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer. Further development of this novel gene therapeutic appears to be warranted. PMID:22510347

  11. Recent performance data on the Tektronix TK1024A imager: back-illuminated, MPP, and non-MPP operating modes

    NASA Astrophysics Data System (ADS)

    Woody, Thomas W.; Hayes, Raymond; Gladhill, Kristie W.

    1992-08-01

    The TK1024 four quadrant readout imager has been in production for several years and has evolved into a device with a high level of performance. Current production volumes are several hundred wafer starts per year (4 devices per wafer). In the last year improvements have been made in dark current with the addition of MPP unpiants and the reduction of MOSFET read noise. The paper presented at last year''s symposium focused on general performance data (SPIE vol. 1447 pgs 298 - 309 ). This paper will discuss specific test data recently observed on devices fabricated with the current process. Data on read noise conversion gain and dark current versus temperature in both non-MPP and MPP modes will be the emphasis of this paper. In addition information wifi be presented on Full Well versus operating voltages and optimal timing for MPP operation in the typical slow scan (50 kilopixels/sec) readout mode. 1.

  12. In vivo evaluation of the uptake of [(123)I]FIAU, [(123)I]IVFRU and [(123)I]IVFAU by normal mouse brain: potential for noninvasive assessment of HSV-1 thymidine kinase gene expression in gliomas.

    PubMed

    Li, H-F; Winkeler, A; Moharram, S; Knaus, E E; Dittmar, K; Stöckle, M; Heiss, W D; Wiebe, L I; Jacobs, A H; Jacob, A J

    2008-01-01

    Radioiodinated 5-iodo-1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)uracil (F *IAU) is most commonly used for noninvasive assessment of herpes simplex virus type 1 thymidine kinase (HSV-1-tk) gene expression. However, it does not permeate the intact blood-brain barrier (BBB) because of its moderate lipophilicity. In this work, three iodo-nucleosides, FIAU, IVFRU, and IVFAU, were radiolabeled with iodine-123 and tested for permeation of the BBB in mice and for potential measurement of HSV-1-tk gene expression in gliomas. The results demonstrate that brain uptake and retention of these nucleosides is not directly related to their lipophilicity. The low brain uptake of IVFAU, in conjunction with its higher and constant brain/blood ratio, may reflect greater stability against hydrolysis of the N-glycosidic bond. In vivo PET evaluations of [(124)I]IVFRU and [(124)I]IVFAU in tumor-bearing mice are warranted. PMID:18188770

  13. A case of relapsing-remitting facial palsy and ipsilateral brachial plexopathy caused by HSV-1.

    PubMed

    Alstadhaug, Karl B; Kvarenes, Hanne W; Prytz, Jan; Vedeler, Christian

    2016-05-01

    The etiologies of Bell's palsy and brachial neuritis remain uncertain, and the conditions rarely co-occur or reoccur. Here we present a woman in her twenties who had several relapsing-remitting episodes with left-sided facial palsy and brachial neuropathy. The episodes always started with painful left-sided oral blisters. Repeat PCRs HSV-1 DNA from oral vesicular lesions were positive. Extensive screening did not reveal any other underlying cause. Findings on MRI T2-weighted brachial plexus STIR images, using a 3.0-Tesla scanner during an episode, were compatible with brachial plexus neuritis. Except a mannose-binding lectin deficiency, a congenital complement deficiency that is frequently found in the general Caucasian population, no other immunodeficiency was demonstrated in our patient. In vitro resistance to acyclovir was tested negative, but despite prophylactic treatment with the drug in high doses, relapses recurred. To our knowledge, this is the first ever reported documentation of relapsing-remitting facial and brachial plexus neuritis caused by HSV-1. PMID:26991053

  14. Gamma camera imaging of HSV-tk gene expression with [131I]-FIAU: Clinical applications in gene therapy

    SciTech Connect

    Tjuvajev, J.; Joshi, R.; Kennedy, J.

    1996-05-01

    Develop a method to image gene expression that can be used to monitor successful gene transduction in patients. Currently there are no noninvasive ways to define the extent and spatial location of gene transduction or the level of gene expression in targeted organs or tumors. Wild-type RF2 s.c. tumors were produced by implantation of 10{sup 6} cells into both flanks of Sprague Dawley R-Nu rats. Following a 46 day growth period, the left and right flank tumors reached a 5x4x3 and 3x2x1 cm size. The left tumor was inoculated with 10{sup 6} gp-STK-A2 retroviral vector-producer cells (10{sup 6}-10{sup 7} cfu/ml) in 100 {mu}l of media to induce in vivo transduction with HSV-tk gene. No carrier added 2`-fluoro-1-{beta}-D-arabinofuranosyl-5-[131I]-iodo-uracil [131I]-FIAU was synthesized and 2.8 mCi was injected i.v. 14 days after gp-STK-A2 cell inoculation. Gamma camera imaging was performed in vivo at 4,24 and 36 hours post [131I]-FIAU injection with a dual-headed gamma camera. The 24 and 36 hour images showed specific localization of retained radioactivity only in the transduced tumors. These results were confirmed using quantitative autoradiography (QAR) of the same tumors. QAR also showed significantly higher levels of retained radioactivity (>1% dose/g) in the transduced tumor than in other nontransduced areas (<0.03 %dose/g). The transduced tumor tissue had microscopic features typical of subcutaneously growing RG2 glioma and non vector-producer cells could be identified. Gene therapy trials in patients would benefit greatly from a noninvasive measure and image that could define the location, magnitude and persistence of gene expression overtime. HSV-tk and FIAU can be used as a {open_quotes}marker gene{close_quotes} - {open_quotes}marker substrate{close_quotes} combination for PET ([124-I]) or possibly SPECT ([123-I]) imaging.

  15. ICP4-induced miR-101 attenuates HSV-1 replication

    PubMed Central

    Wang, Xiangling; Diao, Caifeng; Yang, Xi; Yang, Zhen; Liu, Min; Li, Xin; Tang, Hua

    2016-01-01

    Hepes simplex Virus type 1 (HSV-1) is an enveloped DNA virus that can cause lytic and latent infection. miRNAs post-transcriptionally regulate gene expression, and our previous work has indicated that HSV-1 infection induces miR-101 expression in HeLa cells. The present study demonstrates that HSV-1-induced miR-101 is mainly derived from its precursor hsa-mir-101-2, and the HSV-1 immediate early gene ICP4 (infected-cell polypeptide 4) directly binds to the hsa-mir-101-2 promoter to activate its expression. RNA-binding protein G-rich sequence factor 1 (GRSF1) was identified as a new target of miR-101; GRSF1 binds to HSV-1 p40 mRNA and enhances its expression, facilitating viral proliferation. Together, ICP4 induces miR-101 expression, which downregulates GRSF1 expression and attenuates the replication of HSV-1. This allows host cells to maintain a permissive environment for viral replication by preventing lytic cell death. These findings indicate that HSV-1 early gene expression modulates host miRNAs to regulate molecular defense mechanisms. This study provides novel insight into host-virus interactions in HSV-1 infection and may contribute to the development of antiviral therapeutics. PMID:26984403

  16. Visualization of Mouse Neuronal Ganglia Infected by Herpes Simplex Virus 1 (HSV-1) Using Multimodal Non-Linear Optical Microscopy

    PubMed Central

    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. PMID:25133579

  17. Viral Spread to Enteric Neurons Links Genital HSV-1 Infection to Toxic Megacolon and Lethality.

    PubMed

    Khoury-Hanold, William; Yordy, Brian; Kong, Philip; Kong, Yong; Ge, William; Szigeti-Buck, Klara; Ralevski, Alexandra; Horvath, Tamas L; Iwasaki, Akiko

    2016-06-01

    Herpes simplex virus 1 (HSV-1), a leading cause of genital herpes, infects oral or genital mucosal epithelial cells before infecting the peripheral sensory nervous system. The spread of HSV-1 beyond the sensory nervous system and the resulting broader spectrum of disease are not well understood. Using a mouse model of genital herpes, we found that HSV-1-infection-associated lethality correlated with severe fecal and urinary retention. No inflammation or infection of the brain was evident. Instead, HSV-1 spread via the dorsal root ganglia to the autonomic ganglia of the enteric nervous system (ENS) in the colon. ENS infection led to robust viral gene transcription, pathological inflammatory responses, and neutrophil-mediated destruction of enteric neurons, ultimately resulting in permanent loss of peristalsis and the development of toxic megacolon. Laxative treatment rescued mice from lethality following genital HSV-1 infection. These results reveal an unexpected pathogenesis of HSV associated with ENS infection. PMID:27281569

  18. Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.

    PubMed

    Focher, F; Maga, G; Bendiscioli, A; Capobianco, M; Colonna, F; Garbesi, A; Spadari, S

    1995-08-11

    L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides. PMID:7544886

  19. A role for 3-O-sulfotransferase isoform-4 in assisting HSV-1 entry and spread

    SciTech Connect

    Tiwari, Vaibhav; O'Donnell, Christopher D.; Oh, Myung-Jin; Valyi-Nagy, Tibor; Shukla, Deepak . E-mail: dshukla@uic.edu

    2005-12-16

    Many heparan sulfate (HS) 3-O-sulfotransferase (3-OST) isoforms generate cellular receptors for herpes simplex virus type-1 (HSV-1) glycoprotein D (gD). Interestingly, the ability of 3-OST-4 to mediate HSV-1 entry and cell-to-cell fusion has not been determined, although it is predominantly expressed in the brain, a primary target of HSV-1 infections. We report that expression of 3-OST-4 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and a mutant (Rid1) strain of HSV-1. Evidence for generation of gD receptors by 3-OST-4 was suggested by gD-mediated interference assay and the ability of 3-OST-4 expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases-II/III). In addition, 3-OST-4 expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together our results suggest a role of 3-OST-4 in HSV-1 pathogenesis.

  20. Granulocytes in Ocular HSV-1 Infection: Opposing Roles of Mast Cells and Neutrophils

    PubMed Central

    Royer, Derek J.; Zheng, Min; Conrady, Christopher D.; Carr, Daniel J. J.

    2015-01-01

    Purpose. The contributions of mast cells (MCs) to immunologic defense against pathogens in the eye are unknown. We have characterized pericorneal MCs as tissue-resident innate sentinels and determined their impact on the immune response to herpes simplex virus type-1 (HSV-1), a common ocular pathogen. Methods. The impact of mast cells on the immune response to HSV-1 infection was investigated using MC-deficient KitW-sh mice. Virus titers, inflammatory cytokine production, eicosanoid profiles, cellular immune responses, and ocular pathology were evaluated and compared with C57BL/6J mice during an acute corneal HSV-1 infection. Results. Corneas of KitW-sh mice have higher viral titers, increased edema, and greater leukocyte infiltration following HSV-1 infection. Following infection, cytokine profiles were slightly elevated overall in KitW-sh mice. Eicosanoid profiles were remarkably different only when comparing uninfected corneas from both groups. Neutrophils within infected corneas expressed HSV-1 antigen, lytic genes, and served as a disease-causing vector when adoptively transferred into immunocompromised animals. Myeloid-derived suppressor cells did not infiltrate into the cornea or suppress the expansion, recruitment, or cytokine production by CD8+ T cells following acute HSV-1 infection. Conclusions. Collectively, these findings provide new insight into host defense in the cornea and the pathogenesis of HSV-1 infection by identifying previously unacknowledged MCs as protective innate sentinels for infection of the ocular surface and reinforcing that neutrophils are detrimental to corneal infection. PMID:26066745

  1. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    PubMed

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

  2. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

    PubMed Central

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T. C.; Imren, Suzan; Lam, Vivian; Poon, Grace F. T.; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William

    2016-01-01

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

  3. Establishment of HSV1 Latency in Immunodeficient Mice Facilitates Efficient In Vivo Reactivation

    PubMed Central

    Ramakrishna, Chandran; Ferraioli, Adrianna; Calle, Aleth; Nguyen, Thanh K.; Openshaw, Harry; Lundberg, Patric S.; Lomonte, Patrick; Cantin, Edouard M.

    2015-01-01

    The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation. PMID:25760441

  4. 5-[18F]Fluoroalkyl Pyrimidine Nucleosides: Probes for PET Imaging of Herpes Simplex Virus Type-1 Thymidine Kinase Gene Expression

    PubMed Central

    Chacko, Ann-Marie; Blankemeyer, Eric; Lieberman, Brian P.; Qu, Wenchao; Kung, Hank F.

    2009-01-01

    Introduction The preliminary in vivo evaluation of novel 5-[18F]fluoroalkyl-2’-deoxyuridines ([18F]FPrDU, [18F]FBuDU, [18F]FPeDU; [18F]1a–c, respectively) and 2’-fluoro-2’-deoxy-5-[18F]fluoroalkyl-1-β-D-arabinofuranosyl uracils ([18F]FFPrAU, [18F]FFBuAU, [18F]FFPeAU; [18F]1d–f, respectively) as probes for imaging herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene expression are described. Methods [18F]1a–f were successfully synthesized by a rapid and efficient two step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [18F]F-. 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 p.i. revealed that the uptake of [18F]1a–b and [18F]1d–e in RG2TK+ tumors was not significantly different from control tumors. However, [18F]1c and [18F]1f had an average 1.6 and 1.7–fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [18F]1c and [18F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [18F]1c and [18F]1f showed higher initial uptake (3.5–fold and 1.4–fold, respectively) in RG2TK+ tumors than control tumors, with continued washout of activity from both tumors over time. Conclusions Biological evaluations suggest that [18F]1c and [18F]1f may have limited potential for imaging HSV1-tk gene expression due fast washout of activity from the blood thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk expressing tumors. PMID:19181266

  5. The Current State of Vaccine Development for Ocular HSV-1 Infection

    PubMed Central

    Royer, DJ; Cohen, A; Carr, DJJ

    2015-01-01

    Summary HSV-1 continues to be the leading cause of infectious corneal blindness. Clinical trials for vaccines against genital HSV infection have been ongoing for more than three decades. Despite this, no approved vaccine exists, and no formal clinical trials have evaluated the impact of HSV vaccines on eye health. We review here the current state of development for an efficacious HSV-1 vaccine and call for involvement of ophthalmologists and vision researchers. PMID:25983856

  6. Pentagalloylglucose Blocks the Nuclear Transport and the Process of Nucleocapsid Egress to Inhibit HSV-1 Infection.

    PubMed

    Jin, Fujun; Ma, Kaiqi; Chen, Maoyun; Zou, Muping; Wu, Yanting; Li, Feng; Wang, Yifei

    2016-03-23

    Herpes simplex virus type 1 (HSV-1), a widespread virus, causes a variety of human viral diseases worldwide. The serious threat of drug-resistance highlights the extreme urgency to develop novel antiviral drugs with different mechanisms of action. Pentagalloylglucose (PGG) is a natural polyphenolic compound with significant anti-HSV activity; however, the mechanisms underlying its antiviral activity need to be defined by further studies. In this study, we found that PGG treatment delays the nuclear transport process of HSV-1 particles by inhibiting the upregulation of dynein (a cellular major motor protein) induced by HSV-1 infection. Furthermore, PGG treatment affects the nucleocapsid egress of HSV-1 by inhibiting the expression and disrupting the cellular localization of pEGFP-UL31 and pEGFP-UL34, which are indispensable for HSV-1 nucleocapsid egress from the nucleus. However, the over-expression of pEGFP-UL31 and pEGFP-UL34 could decrease the antiviral effect of PGG. In this study, for the first time, the antiviral activity of PGG against acyclovir-resistant virus was demonstrated in vitro, and the possible mechanisms of its anti-HSV activities were identified based on the inhibition of nuclear transport and nucleocapsid egress in HSV-1. It was further confirmed that PGG could be a promising candidate for HSV therapy, especially for drug-resistant strains. PMID:26166506

  7. [18F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility

    PubMed Central

    Eissenberg, Linda G; Rettig, Michael P; Ritchey, Julie K; Prior, Julie L; Schwarz, Sally W; Frye, Jennifer; White, Brian S; Fulton, Robert S; Ghobadi, Armin; Cooper, Matthew L; Couriel, Daniel R; Seegulam, Muhammad Esa; Piwnica-Worms, David; Dehdashti, Farrokh; Cornetta, Kenneth; DiPersio, John F

    2015-01-01

    Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75+-selected donor T cells (1.0–13 × 105)/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-(18F)fluoro-3-hydroxymethyl-butyl]guanine ([18F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [18F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [18F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation. PMID:25807290

  8. [(18)F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility.

    PubMed

    Eissenberg, Linda G; Rettig, Michael P; Ritchey, Julie K; Prior, Julie L; Schwarz, Sally W; Frye, Jennifer; White, Brian S; Fulton, Robert S; Ghobadi, Armin; Cooper, Matthew L; Couriel, Daniel R; Seegulam, Muhammad Esa; Piwnica-Worms, David; Dehdashti, Farrokh; Cornetta, Kenneth; DiPersio, John F

    2015-06-01

    Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75(+)-selected donor T cells (1.0-13 × 10(5))/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-((18)F)fluoro-3-hydroxymethyl-butyl]guanine ([(18)F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [(18)F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [(18)F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation. PMID:25807290

  9. Imaging Transgene Expression with Radionuclide Imaging Technologies1

    PubMed Central

    Gambhir, SS; Herschman, HR; Cherry, SR; Barrio, JR; Satyamurthy, N; Toyokuni, T; Phelps, ME; Larson, SM; Balaton, J; Finn, R; Sadelain, M; Tjuvajev, J

    2000-01-01

    Abstract A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (micro PET) designed specifically for studies of small animals. We review “marker/reporter gene” imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications. PMID:10933072

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

    PubMed Central

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

    2016-01-01

    Statement of the Problem 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. Purpose This study aimed to evaluate the anti-HSV-1 activity of Aloe vera gel in Vero cell line. Materials and Method 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. Results 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. Conclusion 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. PMID:26966709

  11. Cathelicidin LL-37 and HSV-1 Corneal Infection: Peptide Versus Gene Therapy

    PubMed Central

    Lee, Chyan-Jang; Buznyk, Oleksiy; Kuffova, Lucia; Rajendran, Vijayalakshmi; Forrester, John V.; Phopase, Jaywant; Islam, Mohammad M.; Skog, Mårten; Ahlqvist, Jenny; Griffith, May

    2014-01-01

    Purpose To evaluate the potential utility of collagen-based corneal implants with anti–Herpes Simplex Virus (HSV)-1 activity achieved through sustained release of LL-37, from incorporated nanoparticles, as compared with cell-based delivery from model human corneal epithelial cells (HCECs) transfected to produce endogenous LL-37. Methods We tested the ability of collagen-phosphorylcholine implants to tolerate the adverse microenvironment of herpetic murine corneas. Then, we investigated the efficacy of LL-37 peptides delivered through nanoparticles incorporated within the corneal implants to block HSV-1 viral activity. In addition, LL-37 complementary DNA (cDNA) was transferred into HCECs to confer viral resistance, and their response to HSV-1 infection was examined. Results Our implants remained in herpetic murine corneas 7 days longer than allografts. LL-37 released from the implants blocked HSV-1 infection of HCECs by interfering with viral binding. However, in pre-infected HCECs, LL-37 delayed but could not prevent viral spreading nor clear viruses from the infected cells. HCECs transfected with the LL-37 expressed and secreted the peptide. Secreted LL-37 inhibited viral binding in vitro but was insufficient to protect cells completely from HSV-1 infection. Nevertheless, secreted LL-37 reduced both the incidence of plaque formation and plaque size. Conclusion LL-37 released from composite nanoparticle-hydrogel corneal implants and HCEC-produced peptide, both showed anti–HSV-1 activity by blocking binding. However, while both slowed down virus spread, neither was able on its own to completely inhibit the viruses. Translational Relevance LL-37 releasing hydrogels may have potential utility as corneal substitutes for grafting in HSV-1 infected corneas, possibly in combination with LL-37 producing therapeutic cells. PMID:24932432

  12. Circulating herpes simplex type 1 (HSV-1)-specific CD8+ T cells do not access HSV-1 latently infected trigeminal ganglia

    PubMed Central

    2011-01-01

    Background Therapeutic vaccines can be designed to enhance existing T cell memory populations for increased protection against re-infection. In the case of herpes simplex virus type 1, recurrent disease results from reactivation of latent virus in sensory ganglia, which is controlled in part by a ganglia-resident HSV-specific memory CD8+ T cell population. Thus, an important goal of a therapeutic HSV-1 vaccine would be to enhance this population. Methods HSV-1-infected mice were treated with TAK-779 to block CCR5- and CXCR3-mediated CD8+ T cell migration during both acute and latent infections. Additionally, HSV-1-specific CD8+ T cells were transferred into HSV-1 latently infected mice to mimic the effect of a therapeutic vaccine, and their migration into trigeminal ganglia (TG) was traced during steady-state latency, or during recovery of the TG-resident memory CD8+ T cell population following stress-, and corticosterone-induced depletion and HSV-1 reactivation from latency. Bromodeoxy uridine (BrdU) incorporation measured cell proliferation in vivo. Results TAK-779 treatment during acute HSV-1 infection reduced the number of infiltrating CD8+ T cells but did not alter the number of viral genome copies. TAK-779 treatment during HSV latency did not affect the size of the TG-resident memory CD8+ T cell population. Transferred HSV-specific CD8+ T cells failed to access latently infected TG during steady-state latency, or during recovery of the TG resident HSV-specific CD8+ T cell population following exposure of latently infected mice to stress and corticosterone. Recovery of the HSV-specific CD8+ T cell population after stress and corticosterone treatment occurred with homeostatic levels of cell division and did not require CD4+ T cell help. Conclusions Our findings are consistent with the notion that the CD8+ T cells in latently infected TG are a tissue-resident memory (Trm) population that is maintained without replenishment from the periphery, and that when this

  13. Lychee flower extract inhibits proliferation and viral replication of HSV-1-infected corneal epithelial cells

    PubMed Central

    Hsu, Chang-Min; Chiang, Samuel Tung-Hsing; Chang, Yuan-Yen; Chen, Yi-Chen; Yang, Deng-Jye; Chen, Ya-Yu; Lin, Hui-Wen

    2016-01-01

    Purpose Herpes simplex virus type I (HSV-1) is capable of causing a wide array of human ocular diseases. Herpes simplex virus keratitis (HSK)-induced cytopathogenicity together with the chronic immune-inflammatory reaction can trigger stromal scarring, thinning, and neovascularization which may lead to permanent vision impairment. Lychee flower extract (LFE) is known for its antioxidant and anti-inflammatory effects. Therefore, in this study, we investigated the mechanism of the Statens Seruminstitut rabbit corneal (SIRC) epithelial cells infected by HSV-1 and examined the antiviral capabilities of LFE. Methods SIRC cells were pretreated with different concentrations of LFE (0.2, 0.1, and 0.05 μg/ml) and then infected with 1 MOI of HSV-1 for 24 h. The cell viability or morphology was evaluated in this study. In addition, the supernatants and cell extracts were collected for Cell Counting Kit-8 (CCK), plaque assay, and western blotting. Results We found that HSV-1-induced cell proliferation is regulated through inhibition of the mammalian target of rapamycin (mTOR) and p70s6k phosphorylation in response to the LFE. In addition, the LFE enhanced the autophagy protein expression (Beclin-1 and light chain 3, LC3) and decreased the viral titers. Conclusions These results showed the antiviral capabilities and the protective effects of LFE. Taken together, our data indicate that LFE has potential as an anti-HSK (herpes simplex keratitis) for HSV-1 infection. PMID:26937165

  14. Membrane deformation and scission by the HSV-1 nuclear egress complex

    PubMed Central

    Bigalke, Janna M.; Heuser, Thomas; Nicastro, Daniela; Heldwein, Ekaterina E.

    2014-01-01

    The nuclear egress complex (NEC) of herpesviruses such as HSV-1 is essential for the exit of nascent capsids from the cell nucleus. The NEC drives nuclear envelope vesiculation in cells, but the precise budding mechanism and the potential involvement of cellular proteins are unclear. Here we report that HSV-1 NEC alone is sufficient for membrane budding in vitro and thus represents a complete membrane deformation and scission machinery. It forms ordered coats on the inner surface of budded vesicles, suggesting that it mediates scission by scaffolding the membrane bud and constricting the neck to the point of scission. The inward topology of NEC-mediated budding in vitro resembles capsid budding into the inner nuclear membrane during HSV-1 infection and nuclear envelope vesiculation in NEC-transfected cells. We propose that the NEC functions as minimal virus-encoded membrane-budding machinery during nuclear egress and does not require additional cellular factors. PMID:24916797

  15. Characterization of the nuclear import mechanisms of HSV-1 UL31.

    PubMed

    Cai, Mingsheng; Si, Jiang; Li, Xiaowei; Zeng, Zhancheng; Li, Meili

    2016-06-01

    As an important protein, UL31 has been demonstrated to play multiple roles in herpes simplex virus 1 (HSV-1) replication. Previous studies showed that UL31 predominantly locates in the nucleus in chemical fixed cells and live cells, however, the determining mechanisms for its nuclear translocation is not clear. In the present study, by utilizing live cells fluorescent microscopy and co-immunoprecipitation assays, the nuclear import of UL31 was characterized to be dependent on Ran-, importin α1- and transportin-1-mediated pathway. Therefore, these results will promote the understanding of UL31-mediated biological functions in HSV-1 infection cycle. PMID:26854290

  16. An In Vitro HSV-1 Reactivation Model Containing Quiescently Infected PC12 Cells

    PubMed Central

    Hogk, Ina; Kaufmann, Michaela; Finkelmeier, Doris; Rupp, Steffen

    2013-01-01

    Abstract Advances in the understanding of the infection and reactivation process of herpes simplex type 1 (HSV-1) are generally gained by monolayer cultures or extensive and cost-intensive animal models. So far, no reliable in vitro skin model exists either to investigate the molecular mechanisms involved in controlling latency and virus reactivation or to test pharmaceuticals. Here we demonstrate the first in vitro HSV-1 reactivation model generated by using the human keratinocyte cell line HaCaT grown on a collagen substrate containing primary human fibroblasts. We integrated the unique feature of a quiescently infected neuronal cell line, the rat pheochromocytoma line PC12, within the dermal layer of the three-dimensional skin equivalent. Transmission electron microscopy, a cell-based TCID50 assay, and polymerase chain reaction analysis were used to verify cell latency. Thereby viral DNA could be detected, whereas extracellular as well as intracellular virus activity could not be found. Further, the infected PC12 cells show no spontaneous reactivation within the in vitro skin equivalent. In order to simulate a physiologically comparable HSV-1 infection, we achieved a specific and pointed reactivation of quiescently HSV-1 infected PC12 cells by UVB irradiation at 1000 mJ/cm2. PMID:23914331

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

    SciTech Connect

    Guan, Ying; Guo, Lei; Yang, Erxia; Liao, Yun; Liu, Longding; Che, Yanchun; Zhang, Ying; Wang, Lichun; Wang, Jingjing; Li, Qihan

    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.

  18. KSHV-TK is a tyrosine kinase that disrupts focal adhesions and induces Rho-mediated cell contraction

    PubMed Central

    Gill, Michael B; Turner, Rachel; Stevenson, Philip G; Way, Michael

    2015-01-01

    Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV-TK modulates signalling and cell morphology. PMID:25471072

  19. Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene

    SciTech Connect

    Dube, D.K.; Parker, J.D.; French, D.C.; Cahill, D.S.; Dube, S.; Horwitz, M.S.Z.; Munir, K.M.; Loeb, L.A. )

    1991-12-24

    The authors have obtained 42 active artificial mutants of HSV-1 thymidine kinase by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile{sup 166} nor Ala{sup 167} is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine but are unable to grow in the presence of high amounts of thymidine. The high-activity plasmids also have an enhanced ({sup 3}H)dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme. This thermolability could account for the less than expected thymidine kinase activity in the extracts and suggests that amino acid substitutions at Ile{sup 166} and Ala{sup 167} have produced major changes in protein stability.

  20. Inhibitors of signal peptide peptidase (SPP) affect HSV-1 infectivity in vitro and in vivo

    PubMed Central

    Allen, Sariah J.; Mott, Kevin R.; Ghiasi, Homayon

    2014-01-01

    Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology. PMID:24768597

  1. PML plays both inimical and beneficial roles in HSV-1 replication.

    PubMed

    Xu, Pei; Mallon, Stephen; Roizman, Bernard

    2016-05-24

    After entry into the nucleus, herpes simplex virus (HSV) DNA is coated with repressive proteins and becomes the site of assembly of nuclear domain 10 (ND10) bodies. These small (0.1-1 μM) nuclear structures contain both constant [e.g., promyelocytic leukemia protein (PML), Sp100, death-domain associated protein (Daxx), and so forth] and variable proteins, depending on the function of the cells or the stress to which they are exposed. The amounts of PML and the number of ND10 structures increase in cells exposed to IFN-β. On initiation of HSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML and Sp100. The earlier report that IFN-β is significantly more effective in blocking viral replication in murine PML(+/+) cells than in sibling PML(-/-) cells, reproduced here with human cells, suggests that PML acts as an effector of antiviral effects of IFN-β. To define more precisely the function of PML in HSV-1 replication, we constructed a PML(-/-) human cell line. We report that in PML(-/-) cells, Sp100 degradation is delayed, possibly because colocalization and merger of ICP0 with nuclear bodies containing Sp100 and Daxx is ineffective, and that HSV-1 replicates equally well in parental HEp-2 and PML(-/-) cells infected at 5 pfu wild-type virus per cell, but poorly in PML(-/-) cells exposed to 0.1 pfu per cell. Finally, ICP0 accumulation is reduced in PML(-/-) infected at low, but not high, multiplicities of infection. In essence, the very mechanism that serves to degrade an antiviral IFN-β effector is exploited by HSV-1 to establish an efficient replication domain in the nucleus. PMID:27162364

  2. In vitro effect of phototherapy with low-intensity laser on HSV-1 and epithelial cells

    NASA Astrophysics Data System (ADS)

    Eduardo, Fernanda P.; Mehnert, Dolores U.; Monezi, Telma A.; Zezell, Denise M.; Schubert, Mark M.; Eduardo, Carlos P.; Marques, Márcia M.

    2007-02-01

    The effects of phototherapy on herpes lesions have been clinically demonstrated by either preventing the lesion formation or speeding their repair. The aim of this in vitro study was analyze the effect of phototherapy on epithelial cells and HSV-1 in culture. Cultures of HSV-1 and epithelial cells (Vero cell line) were used. The irradiations were done using a GaAlAs laser (660 e 780 nm, 4.0 mm2). One, two and three irradiations with 6 h-intervals were done. The experimental groups were: Control: non-irradiated; 660 nm and 3 J/cm2 (2.8 sec); 660 nm and 5 J/cm2 (3.8 sec); 780 nm and 3 J/cm2 (1.9 sec), and 780 nm and 5 J/cm2 (2.5 sec). The HSV-1 cytopatic effect and the cell viability of irradiated cultures and controls were analyzed in four different conditions: irradiation of non-infected epithelial cells; epithelial cells irradiated prior infection; virus irradiated prior infection; irradiation of HSV infected cells. The mitochondrial activity and cytopathic effects were assessed. The number of irradiations influenced the cell growth positively and proportionally, except for the 660 nm/ 3 J/cm2 group. Any variation in cytopathic effects was observed amongst the experimental groups. The viability of infected cells prior irradiation was significantly higher than that of non-irradiated cultures when 2 irradiations were done. Under the experimental conditions of this study we concluded that phototherapy is capable of enhancing epithelial cell growth and prolonging cell viability of HSV-1 infected cells. Positive benefits of phototherapy could be resultant from prolongation of infected cells viability, corroborating with host defenses.

  3. Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

    PubMed Central

    Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

    2012-01-01

    Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

  4. IFN-γ Primes Keratinocytes for HSV-1-Induced Inflammasome Activation.

    PubMed

    Strittmatter, Gerhard E; Sand, Jennifer; Sauter, Marlies; Seyffert, Michael; Steigerwald, Robin; Fraefel, Cornel; Smola, Sigrun; French, Lars E; Beer, Hans-Dietmar

    2016-03-01

    Inflammasomes are immune complexes that induce an inflammatory response upon sensing of different stress signals. This effect is mainly mediated by activation and secretion of the proinflammatory cytokines proIL-1β and -18. Here we report that infection of human primary keratinocytes with the double-stranded DNA viruses modified vaccinia virus Ankara (MVA) or herpes simplex virus type 1 (HSV-1)-induced secretion of mature IL-1β and -18. This secretion was dependent on several inflammasome complexes; however, the absent in melanoma 2 (AIM2) inflammasome, which is activated by binding of double-stranded DNA, played the most important role. Whereas prestimulation of keratinocytes with IFN-γ moderately increased MVA-induced IL-1β and IL-18 secretion, it was essential for substantial secretion of these cytokines in response to herpes simplex virus type 1 infection. IFN-γ partially restored HSV-1 suppressed proIL-1β expression and was also required for inflammasome activation. Most importantly, IFN-γ strongly suppressed virus replication in keratinocytes in vitro and ex vivo, which was independent of inflammasome activation. Our results suggest that, similar to Herpesviridae infection in mice, HSV-1 replication in human skin is controlled by a positive feedback loop of keratinocyte-derived IL-1/IL-18 and IFN-γ expressed by immune cells. PMID:26739094

  5. Influence of macrophages on HSV-1 induced IL 2 production by human lymphocytes

    SciTech Connect

    Clouse, K.A.; Orosz, C.G.; Sheridan, J.F.

    1986-03-05

    Previous work has demonstrated that human peripheral blood mononuclear cells (PBMC) from HSV-1 seropositive individuals produce interleukin 2 (IL 2) following stimulation in vitro with uv-inactivated herpes simplex virus type 1 (HSV antigen). This study investigated the accessory macrophage (MO) and monokine requirements for IL 2 production by enriched T lymphocytes from HSV-1 seropositive individuals. Following removal of accessory MO populations, enriched T lymphocytes did not secrete IL 2 in response to HSV antigen. However, IL 2 production was restored by the addition of autologous, ..gamma..-irradiated (5000R) MO. HSV antigen-pulsed MO also induced IL 2 production by enriched T lymphocytes. Furthermore, when HSV-pulsed macrophages were treated with paraformaldehyde they no longer caused T lymphocytes to produce IL 2 unless exogenous monokines were provided. Neither exogenous monokines nor purified human IL 1 could support HSV antigen induced IL 2 production in the absence of MO. These studies demonstrated that MO are required for HSV-induced IL 2 production by T lymphocytes from HSV-1 seropositive individuals. Furthermore, these MO appear to provide two functions required for IL 2 production: viral antigen display and monokine production.

  6. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    PubMed

    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. PMID:26459807

  7. The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

    SciTech Connect

    Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.; Courtney, Richard J.

    2008-07-05

    The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.

  8. HSV-1 tegument protein and the development of its genome editing technology.

    PubMed

    Xu, Xingli; Che, Yanchun; Li, Qihan

    2016-01-01

    Herpes simplex virus 1 (HSV-1) is composed of complex structures primarily characterized by four elements: the nucleus, capsid, tegument and envelope. The tegument is an important viral component mainly distributed in the spaces between the capsid and the envelope. The development of viral genome editing technologies, such as the identification of temperature-sensitive mutations, homologous recombination, bacterial artificial chromosome, and the CRISPR/Cas9 system, has been shown to largely contribute to the rapid promotion of studies on the HSV-1 tegument protein. Many researches have demonstrated that tegument proteins play crucial roles in viral gene regulatory transcription, viral replication and virulence, viral assembly and even the interaction of the virus with the host immune system. This article briefly reviews the recent research on the functions of tegument proteins and specifically elucidates the function of tegument proteins in viral infection, and then emphasizes the significance of using genome editing technology in studies of providing new techniques and insights into further studies of HSV-1 infection in the future. PMID:27343062

  9. SOCS1/3 Expression Levels in HSV-1-Infected, Cytokine-Polarized and -Unpolarized Macrophages

    PubMed Central

    Reichard, Adam Craig; Cheemarla, Nagarjuna Reddy

    2015-01-01

    Macrophage subtypes are characterized as proinflammatory (M1) or immunomodulatory and tissue remodeling (M2). Since macrophages play a pivotal role in controlling Herpes simplex virus type-1 (HSV-1) replication, effects of HSV-1 by 24 h of infection were determined in murine J774A.1 macrophages unpolarized (M0) or polarized to either an M1 or M2 phenotype. Morphology, cell viability, and expression of CD14 (co-receptor for lipopolysaccharide), CD86 (B7.2-immune co-stimulatory molecule), and suppressors of cytokine signaling (SOCS1 and SOCS3) were determined. M1 macrophages were flattened and vacuolated, while M2 cells appeared elongated with a few vacuoles. Compared with unpolarized M0 cells, M1 cells showed a 31% decrease in viability, a 2-fold increase in the number of CD14+-CD86+ cells, no change in SOCS1 expression, and an 11-fold decrease in SOCS3 expression. M2 cells exhibited a 9% decrease in viability, a 26.0% decrease in the number of CD14+-CD86+ cells, and no change in SOCS1/SOCS3 expression levels compared with M0 cells. After HSV-1 infection, all phenotypes appeared rounded, cell viabilities decreased as did numbers of M1 cells expressing CD14 and CD86. At 24 h after infection, M0 control and M2 cells showed greater virus yield than did the M1 cells, presumably reflecting the loss of viable M1 cells. SOCS1 expression was predominant in uninfected M1-polarized cells and in virus-infected control (M0) cells. SOCS1/SOCS3 expression ratio was 7:1 in uninfected M1 macrophages and approached 1:1 in M1 cells at 24 h after infection with HSV-1. In contrast, little differences were seen in SOCS1/SOCS3 expression ratios in uninfected M2-polarized cells or virus-infected M2 cells. These observations suggest that SOCS1/SOCS3 expression ratios can be used to characterize HSV-1-infected and uninfected macrophages. PMID:24956148

  10. Type I Interferon and Lymphangiogenesis in the HSV-1 Infected Cornea – Are they Beneficial to the Host?

    PubMed Central

    Bryant-Hudson, Katie; Conrady, Christopher D.; Carr, Daniel J.J.

    2013-01-01

    Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen that can result in significant human morbidity. Within the cornea, it was thought the initial recognition of the pathogen was through Toll-like receptors expressed on/in resident cells that then elicit pro-inflammatory cytokine production, activation of anti-viral pathways, and recruitment of leukocytes. However, our lab has uncovered a novel, TLR-independent innate sensor that supersedes TLR induction of anti-viral pathways following HSV-1 infection. In addition, we have also found HSV-1 induces the genesis of lymphatic vessels into the cornea proper by a mechanism independent of TLRs and unique in the field of neovascularization. This review will focus on these two innate immune events during acute HSV-1 infection of the cornea. PMID:23876483

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

    PubMed

    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. PMID:24586397

  12. HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    PubMed Central

    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. PMID:24586397

  13. A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes.

    PubMed

    Smith, Miles C; Box, Andrew C; Haug, Jeffrey S; Lane, William S; Davido, David J

    2014-01-01

    Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML's activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1. PMID:25513827

  14. Intravesical treatment of advanced urothelial bladder cancers with oncolytic HSV-1 co-regulated by differentially expressed microRNAs.

    PubMed

    Zhang, K-X; Matsui, Y; Lee, C; Osamu, O; Skinner, L; Wang, J; So, A; Rennie, P S; Jia, W W

    2016-05-01

    Urothelial bladder cancer is the most common malignancy of the urinary tract. Although most cases are initially diagnosed as non-muscle-invasive, more than 80% of patients will develop recurrent or metastatic tumors. No effective therapy exists currently for late-stage metastatic tumors. By intravesical application, local administration of oncolytic Herpes Simplex virus (oHSV-1) can provide a promising new therapy for this disease. However, its inherent neurotoxicity has been a perceived limitation for such application. In this study, we present a novel microRNA-regulatory approach to reduce HSV-1-induced neurotoxicity by suppressing viral replication in neurons while maintaining oncolytic selectivity toward urothelial tumors. Specifically, we designed a recombinant virus that utilizes differentially expressed endogenous microR143 (non-cancerous, ubiquitous) and microR124 (neural-specific) to regulate expression of ICP-4, a gene essential for HSV-1 replication. We found that expression of ICP-4 must be controlled by a combination of both miR143 and miR124 to achieve the most effective attenuation in HSV-1-induced toxicity while retaining maximal oncolytic capacity. These results suggest that interaction between miR143 and miR124 may be required to successfully regulate HSV-1 replication. Our resent study is the first proof-in-principle that miRNA combination can be exploited to fine-tune the replication of HSV-1 to treat human cancers. PMID:26905370

  15. A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

    PubMed Central

    Smith, Miles C.; Box, Andrew C.; Haug, Jeffrey S.; Lane, William S.; Davido, David J.

    2014-01-01

    Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML’s activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1. PMID:25513827

  16. HSV-1 as a novel therapy for breast cancer meningeal metastases.

    PubMed

    Kuruppu, D; Tanabe, K K

    2015-10-01

    Meningeal metastasis is a fatal complication of breast cancer that affects 5-8% of patients. When cancer cells seed in the meninges, their subsequent growth results in severe neurological complications involving the cranial nerves, cerebrum and spinal cord, limiting life expectancy to less than 4 months. The incidences of meningeal metastases increase with prolonged lifespan resulting from treatment advances for primary breast cancer and their metastases. Currently, there is no cure. Aggressive multimodal therapies such as radiation and chemotherapy (intra-cerebrospinal fluid (CSF) and systemic) are ineffective. Therapeutic agents are often quickly cleared from the CSF, while higher doses that can achieve a therapeutic response are highly toxic. The secure guarding of the subarachnoid space by the blood-brain barrier on one side and the blood-CSF barrier on the other prevents chemotherapy from reaching cancer cells in the meninges. These challenges with treating meningeal metastases highlight the urgent need for a new therapeutic modality. An ideal treatment would be an agent that avoids rapid clearance, remains within the CSF, reaches the meninges and selectively destroys tumor cells. Replication conditional oncolytic herpes simplex virus type 1 (HSV-1) may be effective in this regard. Viral oncolysis, the destruction of cancer cells by replicating virus, is under clinical investigation for cancers that are unresponsive to current therapies. It is based on the model of multiple cycles of lytic virus replication in cancer cells that amplify the injected dose. The therapeutic potential of oncolytic HSV-1 for breast cancer meningeal metastases is discussed here. HSV-1 could be a potential novel treatment for meningeal metastases that can be translated to the clinic. PMID:26384139

  17. Degeneration and Regeneration of Corneal Nerves in Response to HSV-1 Infection

    PubMed Central

    Chucair-Elliott, Ana J.; Zheng, Min; Carr, Daniel J. J.

    2015-01-01

    Purpose. Herpes simplex virus type 1 (HSV-1) infection is one cause of neurotrophic keratitis, characterized by decreases in corneal sensation, blink reflex, and tear secretion as consequence of damage to the sensory fibers innervating the cornea. Our aim was to characterize changes in the corneal nerve network and its function in response to HSV-1 infection. Methods. C57BL/6J mice were infected with HSV-1 or left uninfected. Corneas were harvested at predetermined times post infection (pi) and assessed for β III tubulin, substance P, calcitonin gene-related peptide, and neurofilament H staining by immunohistochemistry (IHC). Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer. Expression of genes associated with nerve repair was determined in corneas by real time RT-PCR, Western blotting, and IHC. Semaphorin 7A (SEMA 7A) neutralizing antibody or isotype control was subconjunctivally administered to infected mice. Results. The area of cornea occupied by β III tubulin immunoreactivity and sensitivity significantly decreased by day 8 pi. Modified reinnervation was observed by day 30 pi without recovery of corneal sensation. Sensory fibers were lost by day 8 pi and were still absent or abnormal at day 30 pi. Expression of SEMA 7A increased at day 8 pi, localizing to corneal epithelial cells. Neutralization of SEMA 7A resulted in defective reinnervation and lower corneal sensitivity. Conclusions. Corneal sensory nerves were lost, consistent with loss of corneal sensation at day 8 pi. At day 30 pi, the cornea reinnervated but without recovering the normal arrangement of its fibers or function. SEMA 7A expression was increased at day 8pi, likely as part of a nerve regeneration mechanism. PMID:25587055

  18. Tromantadine inhibits HSV-1 induced syncytia formation and viral glycoprotein processing

    SciTech Connect

    Ickes, D.E.

    1989-01-01

    Tromantadine inhibits a late event in Herpes Simplex Virus Type 1 (HSV-1) replication, visualized by the inhibition of both the size and number of syncytia. Tromantadine can be added at any time between 1 and 9 h post infection with complete inhibition of syncytia formation. Glycan synthesis of the viral glycoproteins, important for syncytia formation, is incomplete due to tromantadine treatment. Tromantadine does not inhibit the initiation of glycosylation, since viral glycoproteins, gX{sub t}, synthesized in the presence of tromantadine still incorporate {sup 3}H-glucosamine. Tromantadine does not inhibit the transport of t e viral glycoproteins to the cell surface, since glycoproteins B, C, and D are expressed, as demonstrated by immunofluorescence. Tromantadine inhibition of HSV-1 glycoprotein processing is demonstrated by an increase in mobility of the radioimmunoprecipitated gX{sub t}, on SDS-PAGE. The gX{sub t} of KOS, a non-syncytial strain of HSV-1, had a similar increase in mobility, suggesting that the block in glycoprotein processing is a general effect of tromantadine treatment. Fucose, which is incorporated into oligosaccharides in the medial Golgi, is incorporated into gX{sub t}, indicating that the tromantadine block in glycoprotein processing occurs after this step. Lectin binding studies and SDS-PAGE analysis of gC processed in the presence of tromantadine, gC{sub t}, indicates that it has terminal galactose residues in both N- and O-linked glycans (binds Peanut and Ricin Agglutinins, respectively). The inhibition of sialylation of N-linked glycans by tromantadine was indicated by the extent of the increase in SDS-PAGE mobility of the G protein from Vesicular Stomatitis Virus. O-glycanase digestion and SDS-PAGE analysis of gC{sub t} indicate that the O-linked disaccharide NAcGal-Galactose is present.

  19. CYTOGENETIC ANALYSIS OF THE L5178Y/TK+/- YIELDS TK-/- MOUSE LYMPHOMA MUTAGENESIS ASSAY SYSTEM

    EPA Science Inventory

    The L5178Y/TK t/- TK-/- mouse lymphoma mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK t/- heterozygous cell line, TK t/- 3.7.2C Quantitation of colonies of mutant TK-/- cells in the assay forms the basis for calc...

  20. Neddylation is required for herpes simplex virus type I (HSV-1)-induced early phase interferon-beta production.

    PubMed

    Zhang, Xueying; Ye, Zhenjie; Pei, Yujun; Qiu, Guihua; Wang, Qingyang; Xu, Yunlu; Shen, Beifen; Zhang, Jiyan

    2016-09-01

    Type I interferons such as interferon-beta (IFN-β) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-β production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-β production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-β production through, at least partially, promoting NF-κB activation. PMID:27593482

  1. Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

    PubMed Central

    Lafaille, Fabien G; Pessach, Itai M.; Zhang, Shen-Ying; Ciancanelli, Michael J.; Herman, Melina; Abhyankar, Avinash; Ying, Shui-Wang; Keros, Sotirios; Goldstein, Peter A.; Mostoslavsky, Gustavo; Ordovas-Montanes, Jose; Jouanguy, Emmanuelle; Plancoulaine, Sabine; Tu, Edmund; Elkabetz, Yechiel; Al-Muhsen, Saleh; Tardieu, Marc; Schlaeger, Thorsten M.; Daley, George Q.; Abel, Laurent; Casanova, Jean-Laurent; Studer, Lorenz; Notarangelo, Luigi D.

    2012-01-01

    In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of TLR3 immunity are prone to HSV-1 encephalitis (HSE) 1–3. We tested the hypothesis that the pathogenesis of HSE involves non hematopoietic central nervous system (CNS)-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of IFN-β and/or IFN-γ1 in response to poly(I:C) stimulation was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-γ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele demonstrated that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was further rescued by treatment with exogenous IFN-α/β, but not IFN-γ1.Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3 pathway deficiencies. PMID:23103873

  2. HSV-1 infection of human corneal epithelial cells: Receptor-mediated entry and trends of re-infection

    PubMed Central

    Shah, Arpeet; Farooq, Asim V.; Tiwari, Vaibhav; Kim, Min-Jung

    2010-01-01

    Purpose The human cornea is a primary target for herpes simplex virus-1 (HSV-1) infection. The goals of the study were to determine the cellular modalities of HSV-1 entry into human corneal epithelial (HCE) cells. Specific features of the study included identifying major entry receptors, assessing pH dependency, and determining trends of re-infection. Methods A recombinant HSV-1 virus expressing beta-galactosidase was used to ascertain HSV-1 entry into HCE cells. Viral replication within cells was confirmed using a time point plaque assay. Lysosomotropic agents were used to test for pH dependency of entry. Flow cytometry and immunocytochemistry were used to determine expression of three cellular receptors - nectin-1, herpesvirus entry mediator (HVEM), and paired immunoglobulin-like 2 receptor alpha (PILR-a). The necessity of these receptors for viral entry was tested using antibody-blocking. Finally, trends of re-infection were investigated using viral entry assay and flow cytometry post-primary infection. Results Cultured HCE cells showed high susceptibility to HSV-1 entry and replication. Entry was demonstrated to be pH dependent as blocking vesicular acidification decreased entry. Entry receptors expressed on the cell membrane include nectin-1, HVEM, and PILR-α. Receptor-specific antibodies blocked entry receptors, reduced viral entry and indicated nectin-1 as the primary receptor used for entry. Cells re-infected with HSV-1 showed a decrease in entry, which was correlated to decreased levels of nectin-1 as demonstrated by flow cytometry. Conclusions HSV-1 is capable of developing an infection in HCE cells using a pH dependent entry process that involves primarily nectin-1 but also the HVEM and PILR-α receptors. Re-infected cells show decreased levels of entry, correlated with a decreased level of nectin-1 receptor expression. PMID:21139972

  3. Constitutive and Inducible Innate Responses in Cells Infected by HSV-1-Derived Amplicon Vectors

    PubMed Central

    Tsitoura, Eliza; Epstein, Alberto L

    2010-01-01

    Amplicons are helper-dependent herpes simplex virus type 1 (HSV-1)-based vectors that can deliver very large foreign DNA sequences and, as such, are good candidates both for gene delivery and vaccine development. However, many studies have shown that innate constitutive or induced cellular responses, elicited or activated by the entry of HSV-1 particles, can play a significant role in the control of transgenic expression and in the induction of inflammatory responses. Moreover, transgene expression from helper-free amplicon stocks is often weak and transient, depending on the particular type of infected cells, suggesting that cellular responses could be also responsible for the silencing of amplicon-mediated transgene expression. This review summarizes the current experimental evidence underlying these latter concepts, focusing on the impact on transgene expression of very-early interactions between amplicon particles and the infected cells, and speculates on possible ways to counteract the cellular protective mechanisms, thus allowing stable transgene expression without enhancement of vector toxicity. PMID:20811588

  4. Quantitative comparison of the HSV-1 and HSV-2 transcriptomes using DNA microarray analysis

    SciTech Connect

    Aguilar, J.S. . E-mail: jsaguila@uci.edu; Devi-Rao, G.V.; Rice, M.K.; Sunabe, J.; Ghazal, P.; Wagner, E.K.

    2006-04-25

    The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U{sub L}4, U{sub L}29, U{sub L}30, and U{sub L}31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.

  5. A novel oHSV-1 targeting telomerase reverse transcriptase-positive cancer cells via tumor-specific promoters regulating the expression of ICP4

    PubMed Central

    Zhao, Qian; Zhuang, Xiufen; Deng, Zhenling; Liu, Lingling; Li, Jie; Zhang, Yu; Dong, Ying; Zhang, Youhui; Zhang, Shuren; Liu, Binlei

    2015-01-01

    Virotherapy is a promising strategy for cancer treatment. Using the human telomerase reverse transcriptase promoter, we developed a novel tumor-selective replication oncolytic HSV-1. Here we showed that oHSV1-hTERT virus was cytopathic in telomerase-positive cancer cell lines but not in telomerase-negative cell lines. In intra-venous injection in mice, oHSV1-hTERT was safer than its parental oHSV1-17+. In human blood cell transduction assays, both viruses transduced few blood cells and the transduction rate for oHSV1-hTERT was even less than that for its parental virus. In vivo, oHSV1-hTERT inhibited growth of tumors and prolong survival in telomerase-positive xenograft tumor models. Therefore, we concluded that this virus may be a safe and effective therapeutic agent for cancer treatment, warranting clinical trials in humans. PMID:25972362

  6. Natural killer cell receptor expression in patients with severe and recurrent Herpes simplex virus-1 (HSV-1) infections.

    PubMed

    Carter, C; Savic, S; Cole, J; Wood, P

    2007-04-01

    Herpes simplex virus-1 (HSV-1) is an important human pathogen which in a minority of people causes severe infections. In immunocompetent hosts the infection is self limiting. However, a small minority of people have frequent attacks. As NK cells have been implicated in host protection against HSV-1, the aim of this study was to compare NK cell receptor expression in healthy controls and in patients suffering from recurrent HSV-1 reactivations using monoclonal antibodies against NK cell receptors and 3 colour flow cytometry. Eighteen patients were recruited into the study and the results were compared to a control group. The results obtained showed that overall there was no statistical difference between patient and control groups in the expression of the NK cell receptors. There were however, individuals in the patient group (in particular, two members of one family) with significantly reduced level of activating receptors compared to the control group. PMID:17706187

  7. Anti-Viral Evaluation of Sesquiterpene Coumarins from Ferula assa-foetida against HSV-1

    PubMed Central

    Ghannadi, Alireza; Fattahian, Khadijeh; Shokoohinia, Yalda; Behbahani, Mandana; Shahnoush, Alireza

    2014-01-01

    Several complications attributed with Herpes virus related infections and the emergence of drug resistant viruses prompt scientists to search for new drugs. Several terpenoids and coumarins have shown anti HSV effects while no sesquiterpene coumarins have been previously tested for HSV treatment. Three sesquiterpene coumarins badrakemin acetate (1), kellerin (2) and samarcandin diastereomer (3) were isolated from the gum resin of Ferula assa-foetida, a herbal medicine with antimicrobial, antiprotozoal and antiviral effects. Compounds were identified by 1D and 2D- NMR spectroscopies and comparison with literature data. A comparative evaluation of cytotoxicity and antiviral activity showed that kellerin (2) could significantly inhibit the cytopathic effects and reduce the viral titre of the herpes virus type 1 (HSV-1) DNA viral strain KOS at concentrations of 10, 5 and 2.5 µg/mL. PMID:25237347

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model

    PubMed Central

    Yang, Feng-Yi; Chang, Wen-Yuan; Lin, Wei-Ting; Hwang, Jeng-Jong; Chien, Yi-Chun; Wang, Hsin-Ell; Tsai, Min-Lan

    2015-01-01

    The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with 123I-2′-fluoro-2′-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (123I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with 123I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P < 0.05) of glioma cells on day 16 after implantation. Moreover, combining sonication with GCV significantly inhibited tumor growth compared with GCV alone. This study demonstrated that FUS may be used to deliver a wide variety of theranostic agents to the brain for molecular imaging and gene therapy in brain diseases. PMID:26429860

  10. Anti-HSV1 activity of brown algal polysaccharides and possible relevance to the treatment of Alzheimer's disease.

    PubMed

    Wozniak, Matthew; Bell, Tracey; Dénes, Ádám; Falshaw, Ruth; Itzhaki, Ruth

    2015-03-01

    Herpes simplex virus type 1 (HSV1) induces the formation of the characteristic abnormal molecules of Alzheimer's disease (AD) brains, beta-amyloid, and abnormally phosphorylated, AD-like tau (P-tau). Formation of these molecules is inhibited by treatment with the antiviral agent acyclovir (ACV), which prevents viral DNA replication. A totally different mechanism of antiviral action against herpes simplex viruses is shown by sulfated fucans. The antiviral activity of sulfated fucans from five brown algae (Scytothamnus australis, Marginariella boryana, Papenfussiella lutea, Splachnidium rugosum and Undaria pinnatifida) was investigated in relation to the HSV1-induced formation of beta-amyloid, and AD-like tau. Antiviral activity was also related to specific structural features of these polysaccharides. Four sulfated fucan extracts each prevented the accumulation of HSV1-induced beta-amyloid and AD-like tau in HSV1-infected Vero cells. The structures of these extracts had some similarities but also key differences, indicating that a number of structural features can cause antiviral activity. The most active sulfated fucan combined with acyclovir was particularly effective, so may be particularly suitable for further experimental testing in order to develop treatment protocols for AD patients, with the aim of slowing or stopping disease progression. PMID:25583021

  11. HSV-1-induced activation of NF-κB protects U937 monocytic cells against both virus replication and apoptosis.

    PubMed

    Marino-Merlo, Francesca; Papaianni, Emanuela; Medici, Maria Antonietta; Macchi, Beatrice; Grelli, Sandro; Mosca, Claudia; Borner, Christoph; Mastino, Antonio

    2016-01-01

    The transcription factor nuclear factor-kappa B (NF-κB) is a crucial player of the antiviral innate response. Intriguingly, however, NF-κB activation is assumed to favour herpes simplex virus (HSV) infection rather than restrict it. Apoptosis, a form of innate response to viruses, is completely inhibited by HSV in fully permissive cells, but not in cells incapable to fully sustain HSV replication, such as immunocompetent cells. To resolve the intricate interplay among NF-κB signalling, apoptosis and permissiveness to HSV-1 in monocytic cells, we utilized U937 monocytic cells in which NF-κB activation was inhibited by expressing a dominant-negative IκBα. Surprisingly, viral production was increased in monocytic cells in which NF-κB was inhibited. Moreover, inhibition of NF-κB led to increased apoptosis following HSV-1 infection, associated with lysosomal membrane permeabilization. High expression of late viral proteins and induction of apoptosis occurred in distinct cells. Transcriptional analysis of known innate response genes by real-time quantitative reverse transcription-PCR excluded a contribution of the assayed genes to the observed phenomena. Thus, in monocytic cells NF-κB activation simultaneously serves as an innate process to restrict viral replication as well as a mechanism to limit the damage of an excessive apoptotic response to HSV-1 infection. This finding may clarify mechanisms controlling HSV-1 infection in monocytic cells. PMID:27584793

  12. The antimicrobial agent C31G is effective for therapy for HSV-1 ocular keratitis in the rabbit eye model.

    PubMed

    Hill, James M; Stern, Ethan M; Bhattacharjee, Partha S; Malamud, Daniel; Clement, Christian; Rodriguez, Paulo; Lukiw, Walter J; Ochoa, Augusto C; Foster, Timothy P; Velasco, Cruz; McFerrin, Harris E

    2013-10-01

    The amphoteric C31G solution contains equimolar alkyl dimethlyglycine and alkyl dimethyl amine oxide buffered with citric acid. C31G acts as a broad spectrum antiviral and an antibacterial. No previous in vivo studies have been done to test C31G in an animal model of HSV-1 ocular keratitis. We assessed the anti-herpetic activity of C31G in the rabbit eye model using three treatment groups: (1) 1% trifluorothymidine (TFT); (2) 0.25% C31G plus 0.5% hydroxypropyl methylcellulose (HPMC); and (3) vehicle, 0.5% HPMC. Scarified rabbit corneas were inoculated with the HSV-1 strain McKrae. On post inoculation (PI) day 3, rabbits were placed in three balanced groups based on slit-lamp examination (SLE) scores. Treatment began on PI day 3, five times a day for five consecutive days. In addition to the daily, masked SLE scoring, the eyes were assessed daily for stromal opacity, scleral inflammation, neovascularization, eyelid inflammation, inflammatory discharge, and epiphora. C31G and TFT were very effective in reducing the lesions and pathogenesis associated with HSV-1 ocular keratitis. The vehicle control scores were significantly higher and did not effectively treat HSV-1 keratitis. C31G has the potential to be used to treat herpetic keratitis as well as other herpetic topical lesions in humans. PMID:23860013

  13. NT-38MerTK AS A TARGET IN GLIOBLASTOMA

    PubMed Central

    Wu, Jing; Huey, Lauren; Bash, Ryan E.; Cohen, Stephanie M.; Ewend, Matthew G.; Wang, Xiaodong; Graham, Douglas K.; Frye, Stephen V.; Earp, H. Shelton; Miller, C. Ryan

    2014-01-01

    Glioma-associated macrophages and microglia (GIM) are infiltrating immune cells that modulate the glioblastoma (GBM) micro-environment. Pharmacological targeting of GIM represents a promising therapeutic strategy. MerTK receptor tyrosine kinase triggers macrophage ingestion of apoptotic material and polarizes macrophages to an M2-like, immunosuppressive phenotype that promotes tumor growth. In addition, aberrant MerTK expression in GBM tumor cells can provide pro-survival, pro-invasion and chemo-resistance signals. We examined MerTK expression by double immunofluorescence (IF) in 40 human GBM. Both GFAP+ tumor cells and CD68+ GIM expressed MerTK. Quantification in 12 matched pairs of newly-diagnosed and recurrent GBM showed a 5.5-fold increase in MerTK/CD68+ macrophages (p = 0.002), but no consistent changes in MerTK/GFAP+ tumor cells upon recurrence. Next, we examined the efficacy of a novel UNC-developed small molecule MerTK inhibitor (MerTKI) in a genetically engineered orthotopic allograft model of GBM (TRP). GBM were established for 7 days upon stereotactic injection of luciferase-expressing TRP cells into syngeneic, immune-competent mice. Mice (N = 10-12/group) were then randomized to receive no treatment, daily MerTKI (65mg/kg p.o.), or MerTKI plus fractionated radiation (XRT, 5 Gy q.o.d x3). Median survival was 24, 22, and 41.5 days, respectively (p = 0.003), while historical survival of TRP allograft mice treated with XRT alone was 30 days. Bioluminescence imaging (BLI) showed a significant growth delay in MerTKI + XRT-treated mice (doubling time 14 versus 4-4.5 days, p < 0.0001). Two mice remain alive after 50 days of combination treatment and tumor growth remains stable with 90-99% reduction in pre-treatment BLI. Post-mortem histology and IF are pending. These results suggest that both human GBM tumor cells and GIM express MerTK and that MerTK+ GIM may increase upon disease recurrence. MerTK inhibition in an immune-competent pre-clinical model may

  14. The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons.

    PubMed

    Nicoll, Michael P; Hann, William; Shivkumar, Maitreyi; Harman, Laura E R; Connor, Viv; Coleman, Heather M; Proença, João T; Efstathiou, Stacey

    2016-04-01

    Herpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir. PMID:27055281

  15. The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons

    PubMed Central

    Nicoll, Michael P.; Hann, William; Shivkumar, Maitreyi; Harman, Laura E. R.; Connor, Viv; Coleman, Heather M.; Proença, João T.; Efstathiou, Stacey

    2016-01-01

    Herpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir. PMID:27055281

  16. Ethosomes for the delivery of anti-HSV-1 molecules: preparation, characterization and in vitro activity.

    PubMed

    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. PMID:21105576

  17. Expression of HSV-1 Receptors in EBV-Associated Lymphoproliferative Disease Determines Susceptibility to Oncolytic HSV

    PubMed Central

    Wang, Pin-Yi; Currier, Mark A; Hansford, Loen; Kaplan, David; Chiocca, E. Antonio; Uchida, Hiroaki; Goins, William F.; Cohen, Justus B.; Glorioso, Joseph C.; van Kuppevelt, Toin H.; Mo, Xiaokui; Cripe, Timothy P

    2012-01-01

    Epstein-Barr virus (EBV)-associated B cell lymphoproliferative disease (LPD) after hematopoietic stem cell or solid organ transplantation remains a life-threatening complication. Expression of the virus-encoded gene product, EBER, has been shown to prevent apoptosis via blockade of PKR activation. Because PKR is a major cellular defense against Herpes simplex virus, and oncolytic HSV-1 (oHSV) mutants have shown promising anti-tumor efficacy in preclinical models, we sought to determine whether EBV-LPD cells are susceptible to infection by oHSVs. We tested three primary EBV-infected lymphocyte cell cultures from neuroblastoma (NB) patients as models of naturally acquired EBV-LPD. NB12 was most susceptible, NB122R was intermediate, and NB88R2 was essentially resistant. Despite EBER expression, PKR was activated by oHSV infection. Susceptibility to oHSV correlated with the expression of the HSV receptor, nectin-1. The resistance of NB88R2 was reversed by exogenous nectin-1 expression, whereas down-regulation of nectin-1 on NB12 decreased viral entry. Xenografts derived from the EBV-LPDs exhibited only mild (NB12) or no (NB88R2) response to oHSV injection, compared with a neuroblastoma cell line that showed a significant response. We conclude that EBV-LPDs are relatively resistant to oHSV virotherapy, in some cases due to low virus receptor expression but also due to intact anti-viral PKR signaling. PMID:23254370

  18. Conformational analysis of a quinolonic ribonucleoside with anti-HSV-1 activity

    NASA Astrophysics Data System (ADS)

    Yoneda, Julliane D.; Velloso, Marcia Helena R.; Leal, Kátia Z.; Azeredo, Rodrigo B. de V.; Sugiura, Makiko; Albuquerque, Magaly G.; Santos, Fernanda da C.; Souza, Maria Cecília B. V. de; Cunha, Anna Claudia; Seidl, Peter R.; Alencastro, Ricardo B. de; Ferreira, Vitor F.

    2011-01-01

    The infections caused by the Herpes Simplex Virus are one of the most common sources of diseases in adults and several natural nucleoside analogues are currently used in the treatment of these infections. In vitro tests of a series of quinolonic ribonucleosides derivatives synthesized by part of our group indicated that some of them have antiviral activity against HSV-1. The conformational analysis of bioactive compounds is extremely important in order to better understand their chemical structures and biological activity. In this work, we have carried out a nuclear relaxation NMR study of 6-Me ribonucleoside derivative in order to determine if the syn or anti conformation is preferential. The NMR analysis permits the determination of inter-atomic distances by using techniques which are based on nuclear relaxation and related phenomena. Those techniques are non-selective longitudinal or spin-lattice relaxation rates and NULL pulse sequence, which allow the determination of distances between pairs of hydrogen atoms. The results of NMR studies were compared with those obtained by molecular modeling.

  19. Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection

    SciTech Connect

    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.

  20. Targeted gene transfer to nigrostriatal neurons in the rat brain by helper virus-free HSV-1 vector particles that contain either a chimeric HSV-1 glycoprotein C-GDNF or a gC-BDNF protein

    PubMed Central

    Wang, Xiaodan; Kong, Lingxin; Zhang, Guo-rong; Sun, Mei; Geller, Alfred I.

    2006-01-01

    Direct gene transfer into neurons has potential for both studying neuronal physiology and for developing gene therapy treatments for specific neurological conditions. Due to the heterogeneous cellular composition of the brain, cell-type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. The two prevalent approaches for achieving cell-type-specific expression are to use a cell-type-specific promoter to control recombinant gene expression or to modify a virus vector particle to target gene transfer to a specific cell type. Targeted gene transfer to multiple peripheral cell types has been described, but targeted gene transfer to a specific type of neuron in the brain has yet to be reported. Targeted gene transfer approaches with Herpes Simplex Virus (HSV-1) vectors have focused on modifying glycoprotein C (gC) to remove the heparin binding domain and add a binding activity for a specific protein on the cell surface. This study was designed to develop HSV-1 vectors that target gene transfer to cells that contain receptors for either glial-cell-line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF), such as nigrostriatal neurons. We isolated chimeric gC-GDNF or chimeric gC-BDNF constructs, and the resulting proteins were incorporated into HSV-1 virus particles. We performed helper virus-free HSV-1 vector packaging in the presence of each chimeric protein. The resulting vector stocks supported 2.2- to 5.0-fold targeted gene transfer to nigrostriatal neurons in the rat brain, compared to vector particles that contained wild-type (wt) gC. Gene transfer to nigrostriatal neurons by vector particles that contained chimeric gC-BDNF was reduced by preincubation with an anti-BDNF antibody. Targeted gene transfer to neurons that contain specific neurotrophic factor receptors may benefit specific physiological and gene therapy studies. PMID:15993510

  1. Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M.

    PubMed

    Striebinger, Hannah; Funk, Christina; Raschbichler, Verena; Bailer, Susanne M

    2016-03-01

    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

  2. Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M

    PubMed Central

    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

  3. Differential roles of migratory and resident DCs in T cell priming after mucosal or skin HSV-1 infection

    PubMed Central

    Lee, Heung Kyu; Zamora, Melodie; Linehan, Melissa M.; Iijima, Norifumi; Gonzalez, David; Haberman, Ann

    2009-01-01

    Although mucosal surfaces represent the main portal of entry for pathogens, the mechanism of antigen presentation by dendritic cells (DCs) that patrol various mucosal tissues remains unclear. Instead, much effort has focused on the understanding of initiation of immune responses generated against antigens delivered by injection. We examined the contributions of migratory versus lymph node–resident DC populations in antigen presentation to CD4 and CD8 T cells after needle injection, epicutaneous infection, or vaginal mucosal herpes simplex virus (HSV) 1 infection. We show that upon needle injection, HSV-1 became lymph-borne and was rapidly presented by lymph node–resident DCs to CD4 and CD8 T cells. In contrast, after vaginal HSV-1 infection, antigens were largely presented by tissue-derived migrant DCs with delayed kinetics. In addition, migrant DCs made more frequent contact with HSV-specific T cells after vaginal infection compared with epicutaneous infection. Thus, both migrant and resident DCs play an important role in priming CD8 and CD4 T cell responses, and their relative importance depends on the mode of infection in vivo. PMID:19153243

  4. Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells.

    PubMed

    Siracusano, Gabriel; Venuti, Assunta; Lombardo, Daniele; Mastino, Antonio; Esclatine, Audrey; Sciortino, Maria Teresa

    2016-01-01

    Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes. PMID:27509841

  5. Early collection of saliva specimens from Bell's palsy patients: quantitative analysis of HHV-6, HSV-1, and VZV.

    PubMed

    Turriziani, Ombretta; Falasca, Francesca; Maida, Paola; Gaeta, Aurelia; De Vito, Corrado; Mancini, Patrizia; De Seta, Daniele; Covelli, Edoardo; Attanasio, Giuseppe; Antonelli, Guido

    2014-10-01

    Bell's palsy is the most common cause of facial paralysis. Although it has been associated with diabetes mellitus, hypertension, pregnancy, and preeclampsia, the etiology of Bell's palsy remains unknown. The reactivation of latent herpes simplex virus (HSV) or varicella-zoster virus (VZV) with subsequent inflammation and entrapment of the facial nerve in the narrow labyrinthine segment has been implicated as a cause of facial paralysis, but the active role of these viruses in Bell's palsy is still discussed. This study quantified HSV-1 DNA, VZV DNA, and HHV-6 DNA in 95 saliva samples collected from patients within 48 hr from the onset of paralysis. HSV-1, VZV, and HHV-6 were detected in 13%, 3%, and 61% of patients, respectively. The detection rate did not differ significantly between patients and a control group of healthy donors. Interestingly, however, the value of HHV-6 DNA copies was significantly higher than that detected in healthy donors. In addition, the mean value of HHV-6 DNA recorded in patients who had at least a one grade improvement of palsy at the first visit was significantly lower than that detected in patients who showed no change in facial palsy grade or an increase of at least one grade. These findings call into question the role of HSV-1 and VZV in the etiology of Bell's palsy, and suggest that HHV-6 may be involved in the development of the disease or that the underlying disease mechanism might predispose patients to HHV-6 reactivation. PMID:24619963

  6. Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells

    PubMed Central

    Siracusano, Gabriel; Venuti, Assunta; Lombardo, Daniele; Mastino, Antonio; Esclatine, Audrey; Sciortino, Maria Teresa

    2016-01-01

    Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes. PMID:27509841

  7. Optical imaging of reporter gene expression using a positron-emission-tomography probe

    NASA Astrophysics Data System (ADS)

    Liu, Hongguang; Ren, Gang; Liu, Shuanglong; Zhang, Xiaofen; Chen, Luxi; Han, Peizhen; Cheng, Zhen

    2010-11-01

    Reporter gene/reporter probe technology is one of the most important techniques in molecular imaging. Lately, many reporter gene/reporter probe systems have been coupled to different imaging modalities such as positron emission tomography (PET) and optical imaging (OI). It has been recently found that OI techniques could be used to monitor radioactive tracers in vitro and in living subjects. In this study, we further demonstrate that a reporter gene/nuclear reporter probe system [herpes simplex virus type-1 thymidine kinase (HSV1-tk) and 9-(4-18F-fluoro-3-[hydroxymethyl] butyl) guanine ([18F]FHBG)] could be successfully imaged by OI in vitro and in vivo. OI with radioactive reporter probes will facilitate and broaden the applications of reporter gene/reporter probe techniques in medical research.

  8. HSV-1 infection suppresses TGF-β1 and SMAD3 expression in human corneal epithelial cells

    PubMed Central

    Nie, Yuhong; Cui, Dongmei; Pan, Zhujuan; Deng, Jiangyun; Huang, Qiang

    2008-01-01

    Purpose The present study was undertaken to investigate whether transforming growth factor-β (TGF-β) isoforms (TGF-β1, TGF-β2, and TGF-β3) and SMADs (SMAD2 and SMAD3) are involved in herpes simplex virus type 1 (HSV-1) corneal infection. Methods Human corneal epithelial cells (HCE) were infected with HSV-1 at a multiplicity of infection of 5. Cell morphological changes were observed under phase-contrast microscopy. Levels of mRNA for TGF-β isoforms 1, 2, and 3 as well as for SMAD2 and SMAD3 were measured by reverse transcription polymerase chain reaction (RT–PCR) at 0 h, 4 h, 8 h, 12 h, and 24 h after infection. Protein expression of TGF-β1, TGF-β2, SMAD3, and phospho-SMAD3 were analyzed by indirect immunofluorescence at 0 h, 12 h, and 24 h post-infection (p.i.) in HCE cells. Protein expression of TGF-β1 was also evaluated by ELISA. Results Following HSV-1 infection, a cytopathic effect in HCE cells was observed at 8 h p.i. and became significant at 24 h p.i. Compared with normal cells, the mRNA levels of TGF-β1 in HSV-1 infected HCE cells decreased significantly at 8 h, 12 h, and 24 h p.i. (p<0.01), and the expression of SMAD3 was also dramatically decreased 12 h and 24 h p.i. (p<0.01). No noticeable changes were found as a result of infection with respect to levels of TGF-β2, TGF-β3, and SMAD2 in HCE cells. Protein expression of TGF-β1, SMAD3, and phospho-SMAD3 decreased in infected cells at 12 h and 24 h p.i. compared with normal cells, but TGF-β2 had no change. Conclusions TGF-β1 and SMAD3 may be involved in the pathology of corneal diseases associated with HSV-1 infection. PMID:18776948

  9. HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression.

    PubMed

    Christensen, Maria H; Jensen, Søren B; Miettinen, Juho J; Luecke, Stefanie; Prabakaran, Thaneas; Reinert, Line S; Mettenleiter, Thomas; Chen, Zhijian J; Knipe, David M; Sandri-Goldin, Rozanne M; Enquist, Lynn W; Hartmann, Rune; Mogensen, Trine H; Rice, Stephen A; Nyman, Tuula A; Matikainen, Sampsa; Paludan, Søren R

    2016-07-01

    Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome. PMID:27234299

  10. Down-RANKing the Threat of HSV-1: RANKL Upregulates MHC-Class-I-Restricted Anti-Viral Immunity in Herpes Simplex Virus Infection.

    PubMed

    Finsterbusch, Katja; Piguet, Vincent

    2015-11-01

    Herpes simplex virus (HSV-1) is a major cause of viral skin infection in humans. Klenner and colleagues now show that the epidermal receptor activator of NFκB ligand (RANKL) is critical for the induction of anti-viral CD8(+) effector T cells (CTL) during cutaneous HSV-1 infection. Activation via RANKL prevents Langerhans cell apoptosis, thus leading to enhanced antigen transport to regional lymph nodes, increasing the CTL-priming capacity of lymph node dendritic cells. PMID:26548487

  11. A STING-dependent innate-sensing pathway mediates resistance to corneal HSV-1 infection via upregulation of the antiviral effector tetherin.

    PubMed

    Royer, D J; Carr, D J J

    2016-07-01

    Type 1 interferons (IFNs; IFNα/β) mediate immunological host resistance to numerous viral infections, including herpes simplex virus type 1 (HSV-1). The pathways responsible for IFNα/β signaling during the innate immune response to acute HSV-1 infection in the cornea are incompletely understood. Using a murine ocular infection model, we hypothesized that the stimulator of IFN genes (STING) mediates resistance to HSV-1 infection at the ocular surface and preserves the structural integrity of this mucosal site. Viral pathogenesis, tissue pathology, and host immune responses during ocular HSV-1 infection were characterized by plaque assay, esthesiometry, pachymetry, immunohistochemistry, flow cytometry, and small interfering RNA transfection in wild-type C57BL/6 (WT), STING-deficient (STING(-/-)), and IFNα/β receptor-deficient (CD118(-/-)) mice at days 3-5 postinfection. The presence of STING was critical for sustained control of HSV-1 replication in the corneal epithelium and resistance to viral neuroinvasion, but loss of STING had a negligible impact with respect to gross tissue pathology. Auxiliary STING-independent IFNα/β signaling pathways were responsible for maintenance of corneal integrity. Lymphatic vessels, mast cells, and sensory innervation were compromised in CD118(-/-) mice concurrent with increased tissue edema. STING-dependent signaling led to the upregulation of tetherin, a viral restriction factor we identify is important in containing the spread of HSV-1 in vivo. PMID:26627457

  12. Inactivation of HSV-1 and HSV-2 and prevention of cell-to-cell virus spread by Santolina insularis essential oil.

    PubMed

    De Logu, A; Loy, G; Pellerano, M L; Bonsignore, L; Schivo, M L

    2000-12-01

    The essential oil obtained in toto from Santolina insularis was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The IC(50) values, determined by plaque reduction assays, were 0.88 and 0.7 microg/ml for HSV-1 and HSV-2, respectively, while the CC(50) determined by the MTT test on Vero cells was 112 microg/ml, indicating a CC(50)/IC(50) ratio of 127 for HSV-1 and 160 for HSV-2. Results obtained by plaque reduction assays also indicated that the antiviral activity of S. insularis was principally due to direct virucidal effects. Antiviral activity against HSV-1 and HSV-2 was not observed in a post-attachment assay, and attachment assays indicated that virus adsorption was not inhibited. Up to 80% inhibition of HSV-1 was achieved at the concentration of 40 microg/ml by yield reduction assay. Furthermore, reduction of plaque formation assays also showed that S. insularis essential oil inhibits cell-to-cell transmission of both HSV-1 and HSV-2. PMID:11164504

  13. Molecular modeling studies of 1,4-dihydro-4-oxoquinoline ribonucleosides with anti-HSV-1 activity

    NASA Astrophysics Data System (ADS)

    Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Seidl, Peter Rudolf; de Alencastro, Ricardo Bicca

    2011-12-01

    Eight human herpes viruses ( e.g., herpes simplex, varicella-zoster, Epstein-Barr, cytomegalovirus, Kaposi's sarcoma) are responsible for several diseases from sub-clinic manifestations to fatal infections, mostly in immunocompromised patients. The major limitations of the currently available antiviral drug therapy are drug resistance, host toxicity, and narrow spectrum of activity. However, some non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives ( e.g., PNU-183792) [4] shows broad spectrum antiviral activity. We have developed molecular modeling studies, including molecular docking and molecular dynamics simulations, based on a model proposed by Liu and co-workers [14] in order to understand the mechanism of action of a 6-chloro substituted 1,4-dihydro-4-oxoquinoline ribonucleoside, synthesized by the synthetic group, which showed anti-HSV-1 activity [9]. The molecular docking simulations confirmed the Liu's model showing that the ligand needs to dislocate template residues from the active site in order to interact with the viral DNA polymerase enzyme, reinforcing that the interaction with the Val823 residue is pivotal for the inhibitory activity of non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives, such as PNU-183792, with the HSV-1. The molecular dynamics simulations showed that the 6-chloro-benzyl group of PNU-183792 maintains its interaction with residues of the HSV-1 DNA polymerase hydrophobic pocket, considered important according to the Liu's model, and also showed that the methyl group bounded to the nitrogen atom from PNU-183792 is probably contributing to a push-pull effect with the carbonyl group.

  14. Nonthermal Dielectric Barrier Discharge (DBD) Plasma Suppresses Herpes Simplex Virus Type 1 (HSV-1) Replication in Corneal Epithelium

    PubMed Central

    Alekseev, Oleg; Donovan, Kelly; Limonnik, Vladimir; Azizkhan-Clifford, Jane

    2014-01-01

    Purpose Herpes keratitis (HK) is the leading cause of cornea-derived and infection-associated blindness in the developed world. Despite the availability of effective antivirals, some patients develop refractory disease, drug-resistant infection, and topical toxicity. A nonpharmaceutical treatment modality may offer a unique advantage in the management of such cases. This study investigated the antiviral effect of nonthermal dielectric barrier discharge (DBD) plasma, a partially ionized gas that can be applied to organic substances to produce various biological effects. Methods Human corneal epithelial cells and explanted corneas were infected with herpes simplex virus type 1 (HSV-1) and exposed to culture medium treated with nonthermal DBD plasma. The extent of infection was measured by plaque assay, quantitative PCR, and Western blot. Corneal toxicity assessment was performed with fluorescein staining, histologic examination, and 8-OHdG detection. Results Application of DBD plasma–treated medium to human corneal epithelial cells and explanted corneas produced a dose-dependent reduction of the cytopathic effect, viral genome replication, and the overall production of infectious viral progeny. Toxicity studies showed lack of detrimental effects in explanted human corneas. Conclusions Nonthermal DBD plasma substantially suppresses corneal HSV-1 infection in vitro and ex vivo without causing pronounced toxicity. Translational Relevance Nonthermal plasma is a versatile tool that holds great biomedical potential for ophthalmology, where it is being investigated for wound healing and sterilization and is already in use for ocular microsurgery. The anti-HSV-1 activity of DBD plasma demonstrated here could be directly translated to the clinic for use against drug-resistant herpes keratitis. PMID:24757592

  15. Suppressor of Cytokine Signaling 1 (SOCS1) Mitigates Anterior Uveitis and Confers Protection Against Ocular HSV-1 Infection

    PubMed Central

    Yu, Cheng-Rong; Hayashi, Kozaburo; Lee, Yun Sang; Mahdi, Rashid M.; Shen, De Fen; Chan, Chi-Chao; Egwuagu, Charles E.

    2014-01-01

    Immunological responses to pathogens are stringently regulated in the eye to prevent excessive inflammation that damage ocular tissues and compromise vision. Suppressors of cytokine signaling (SOCS) regulate intensity/duration of inflammatory responses. We have used SOCS1-deficient mice and retina-specific SOCS1 transgenic rats to investigate roles of SOCS1 in ocular herpes simplex virus (HSV-1) infection and non-infectious uveitis. We also genetically engineered cell-penetrating SOCS proteins (membrane-translocating sequence (MTS)-SOCS1, MTS-SOCS3) and examined whether they can be used to inhibit inflammatory cytokines. Overexpression of SOCS1 in transgenic rat eyes attenuated ocular HSV-1 infection while SOCS1-deficient mice developed severe non-infectious anterior uveitis, suggesting that SOCS1 may contribute to mechanism of ocular immune privilege by regulating trafficking of inflammatory cells into ocular tissues. Furthermore, MTS-SOCS1 inhibited IFN-γ-induced signal transducers and activators of transcription 1 (STAT1) activation by macrophages while MTS-SOCS3 suppressed expansion of pathogenic Th17 cells that mediate uveitis, indicating that MTS-SOCS proteins maybe used to treat ocular inflammatory diseases of infectious or autoimmune etiology. PMID:24993154

  16. Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons

    PubMed Central

    Kim, Ju Youn; Mandarino, Angelo; Chao, Moses V.; Mohr, Ian; Wilson, Angus C.

    2012-01-01

    Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons. PMID:22383875

  17. ET: EPICS TCL/TK interface

    SciTech Connect

    Daly, B.

    1995-02-01

    This document describes the tc1 command and command types which are used to communicate with EPICS database servers. The application libraries upon which et is built include tc1, tk, tc1-dp, and blt. The reader of this document is assumed to be familiar with tc1/tk.

  18. Assessment of IgG Antibodies Against HSV1, HSV2, CMV and EBV in Patients with Pemphigus Vulgaris Versus Healthy People

    PubMed Central

    Ghalayani, Parichehr; Rashidi, Fateme; Saberi, Zahra

    2015-01-01

    Objectives: Regarding the implication of viruses particularly herpes in pemphigus vulgaris, we sought to assess and compare the level of immunoglobulin G (IgG) antibodies against herpes simplex virus types 1 and 2 (HSV1 and HSV2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in patients with pemphigus vulgaris and healthy people. Materials and Methods: In this cross-sectional study, 25 patients with pemphigus vulgaris and 27 healthy individuals comprised the experimental and control groups, respectively. Serum samples were taken from both groups; the levels of IgG antibodies against HSV1, HSV2, CMV and EBV were measured using ELISA. Results: Immunoglobulin G titer was higher for all four viruses in the patient group in comparison to the control group. This difference was significant for anti-EBV (P= 0.005), anti-CMV (P=0.0001) and anti-HSV2 (P=0.001) but not significant for anti-HSV1 (P= 0.36). Conclusion: Viruses including EBV, CMV, and HSV2 probably play a role in the pathogenesis of pemphigus in addition to the effects of genetics, toxins and other predisposing factors. In this study, no statistically significant relationship was observed between HSV1 and pemphigus vulgaris, which was probably due to the high titer of anti-HSV1 IgG in healthy individuals in the community. More studies must be done in this regard. PMID:27507994

  19. Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C--Staphylococcus A protein

    PubMed Central

    Cao, Haiyan; Zhang, Guo-rong; Geller, Alfred I.

    2010-01-01

    Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC--glial cell line-derived neurotrophic factor or gC--brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC--ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2 months) expression. PMID:20599821

  20. Cellular responses to HSV-1 infection are linked to specific types of alterations in the host transcriptome.

    PubMed

    Hu, Benxia; Li, Xin; Huo, Yongxia; Yu, Yafen; Zhang, Qiuping; Chen, Guijun; Zhang, Yaping; Fraser, Nigel W; Wu, Dongdong; Zhou, Jumin

    2016-01-01

    Pathogen invasion triggers a number of cellular responses and alters the host transcriptome. Here we report that the type of changes to cellular transcriptome is related to the type of cellular functions affected by lytic infection of Herpes Simplex Virus type I in Human primary fibroblasts. Specifically, genes involved in stress responses and nuclear transport exhibited mostly changes in alternative polyadenylation (APA), cell cycle genes showed mostly alternative splicing (AS) changes, while genes in neurogenesis, rarely underwent these changes. Transcriptome wide, the infection resulted in 1,032 cases of AS, 161 incidences of APA, 1,827 events of isoform changes, and up regulation of 596 genes and down regulations of 61 genes compared to uninfected cells. Thus, these findings provided important and specific links between cellular responses to HSV-1 infection and the type of alterations to the host transcriptome, highlighting important roles of RNA processing in virus-host interactions. PMID:27354008

  1. Cellular responses to HSV-1 infection are linked to specific types of alterations in the host transcriptome

    PubMed Central

    Hu, Benxia; Li, Xin; Huo, Yongxia; Yu, Yafen; Zhang, Qiuping; Chen, Guijun; Zhang, Yaping; Fraser, Nigel W.; Wu, Dongdong; Zhou, Jumin

    2016-01-01

    Pathogen invasion triggers a number of cellular responses and alters the host transcriptome. Here we report that the type of changes to cellular transcriptome is related to the type of cellular functions affected by lytic infection of Herpes Simplex Virus type I in Human primary fibroblasts. Specifically, genes involved in stress responses and nuclear transport exhibited mostly changes in alternative polyadenylation (APA), cell cycle genes showed mostly alternative splicing (AS) changes, while genes in neurogenesis, rarely underwent these changes. Transcriptome wide, the infection resulted in 1,032 cases of AS, 161 incidences of APA, 1,827 events of isoform changes, and up regulation of 596 genes and down regulations of 61 genes compared to uninfected cells. Thus, these findings provided important and specific links between cellular responses to HSV-1 infection and the type of alterations to the host transcriptome, highlighting important roles of RNA processing in virus-host interactions. PMID:27354008

  2. Long-term inducible expression in striatal neurons from helper virus-free HSV-1 vectors that contain the tetracycline-inducible promoter system

    PubMed Central

    Gao, Qingshen; Sun, Mei; Wang, Xiaodan; Zhang, Guo-rong; Geller, Alfred I.

    2006-01-01

    Direct gene transfer into neurons in the brain via a virus vector system has potential for both examining neuronal physiology and for developing gene therapy treatments for neurological diseases. Many of these applications require precise control of the levels of recombinant gene expression. The preferred method for controlling the levels of expression is by use of an inducible promoter system, and the tetracycline (tet)-inducible promoter system is the preferred system. Helper virus-free Herpes Simplex Virus (HSV-1) vectors have a number of the advantages, including their large size and efficient gene transfer. Also, we have reported long-term (14 months) expression from HSV-1 vectors that contain a modified neurofilament heavy gene promoter. A number of studies have reported short-term, inducible expression from helper virus-containing HSV-1 vector systems. However, long-term, inducible expression has not been reported using HSV-1 vectors. The goal of this study was to obtain long-term, inducible expression from helper virus-free HSV-1 vectors. We examined two different vector designs for adapting the tet promoter system to HSV-1 vectors. One design was an autoregulatory design; one transcription unit used a tet-regulated promoter to express the tet-regulated transcription factor tet-off, and another transcription unit used a tet-regulated promoter to express the Lac Z gene. In the other vector design, one transcription unit used the modified neurofilament heavy gene promoter to express tet-off, and another transcription unit used a tet-regulated promoter to express the Lac Z gene. The results showed that both vector designs supported inducible expression in cultured fibroblast or neuronal cell lines and for a short time (4 days) in the rat striatum. Of note, only the vector design that used the modified neurofilament promoter to express tet-off supported long-term (2 months) inducible expression in striatal neurons. PMID:16545782

  3. A tale of two HSV-1 helicases: roles of phage and animal virus helicases in DNA replication and recombination.

    PubMed

    Marintcheva, B; Weller, S K

    2001-01-01

    Helicases play essential roles in many important biological processes such as DNA replication, repair, recombination, transcription, splicing, and translation. Many bacteriophages and plant and animal viruses encode one or more helicases, and these enzymes have been shown to play many roles in their respective viral life cycles. In this review we concentrate primarily on the roles of helicases in DNA replication and recombination with special emphasis on the bacteriophages T4, T7, and A as model systems. We explore comparisons between these model systems and the herpesviruses--primarily herpes simplex virus. Bacteriophage utilize various pathways of recombination-dependent DNA replication during the replication of their genomes. In fact the study of recombination in the phage systems has greatly enhanced our understanding of the importance of recombination in the replication strategies of bacteria, yeast, and higher eukaryotes. The ability to "restart" the replication process after a replication fork has stalled or has become disrupted for other reasons is a critical feature in the replication of all organisms studied. Phage helicases and other recombination proteins play critical roles in the "restart" process. Parallels between DNA replication and recombination in phage and in the herpesviruses is explored. We and others have proposed that recombination plays an important role in the life cycle of the herpesviruses, and in this review, we discuss models for herpes simplex virus type 1 (HSV-1) DNA replication. HSV-1 encodes two helicases. UL9 binds specifically to the origins of replication and is believed to initiate HSV DNA replication by unwinding at the origin; the heterotrimeric helicase-primase complex, encoded by UL5, UL8, and UL52 genes, is believed to unwind duplex viral DNA at replication forks. Structure-function analyses of UL9 and the helicase-primase are discussed with attention to the roles these proteins might play during HSV replication. PMID

  4. B7 Costimulation Molecules Encoded by Replication-Defective, vhs-Deficient HSV-1 Improve Vaccine-Induced Protection against Corneal Disease

    PubMed Central

    Schrimpf, Jane E.; Tu, Eleain M.; Wang, Hong; Wong, Yee M.; Morrison, Lynda A.

    2011-01-01

    Herpes simplex virus 1 (HSV-1) causes herpes stromal keratitis (HSK), a sight-threatening disease of the cornea for which no vaccine exists. A replication-defective, HSV-1 prototype vaccine bearing deletions in the genes encoding ICP8 and the virion host shutoff (vhs) protein reduces HSV-1 replication and disease in a mouse model of HSK. Here we demonstrate that combining deletion of ICP8 and vhs with virus-based expression of B7 costimulation molecules created a vaccine strain that enhanced T cell responses to HSV-1 compared with the ICP8−vhs− parental strain, and reduced the incidence of keratitis and acute infection of the nervous system after corneal challenge. Post-challenge T cell infiltration of the trigeminal ganglia and antigen-specific recall responses in local lymph nodes correlated with protection. Thus, B7 costimulation molecules expressed from the genome of a replication-defective, ICP8−vhs− virus enhance vaccine efficacy by further reducing HSK. PMID:21826207

  5. An unusual case of acute transverse myelitis caused by HSV-1 infection.

    PubMed

    Figueroa, Danisha; Isache, Carmen; Sands, Michael; Guzman, Nilmarie

    2016-01-01

    Transverse myelitis is a neurological disorder of the spinal cord that can have a variety of etiologies. Herpes simplex virus (HSV) infection has been described as one of the causes, most commonly HSV type 2. We report here a case of an 18 year old male who presented with weakness that started in his upper extremities and rapidly evolved to quadriplegia. Magnetic resonance imaging of spine was consistent with transverse myelitis. HSV type 1 PCR testing on cerebrospinal fluid (CSF) was positive. He was started on acyclovir and steroids, but despite therapy, patient did not recover motor function. PMID:27419072

  6. Protection provided by a herpes simplex virus 2 (HSV-2) glycoprotein C and D subunit antigen vaccine against genital HSV-2 infection in HSV-1-seropositive guinea pigs.

    PubMed

    Awasthi, Sita; Balliet, John W; Flynn, Jessica A; Lubinski, John M; Shaw, Carolyn E; DiStefano, Daniel J; Cai, Michael; Brown, Martha; Smith, Judith F; Kowalski, Rose; Swoyer, Ryan; Galli, Jennifer; Copeland, Victoria; Rios, Sandra; Davidson, Robert C; Salnikova, Maya; Kingsley, Susan; Bryan, Janine; Casimiro, Danilo R; Friedman, Harvey M

    2014-02-01

    A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease. PMID:24284325

  7. Quantitative Trait Locus Based Virulence Determinant Mapping of the HSV-1 Genome in Murine Ocular Infection: Genes Involved in Viral Regulatory and Innate Immune Networks Contribute to Virulence

    PubMed Central

    Larsen, Inna; Craven, Mark; Brandt, Curtis R.

    2016-01-01

    Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence. PMID:26962864

  8. Quantitative Trait Locus Based Virulence Determinant Mapping of the HSV-1 Genome in Murine Ocular Infection: Genes Involved in Viral Regulatory and Innate Immune Networks Contribute to Virulence.

    PubMed

    Kolb, Aaron W; Lee, Kyubin; Larsen, Inna; Craven, Mark; Brandt, Curtis R

    2016-03-01

    Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence. PMID:26962864

  9. Virucidal activity of a beta-triketone-rich essential oil of Leptospermum scoparium (manuka oil) against HSV-1 and HSV-2 in cell culture.

    PubMed

    Reichling, Jürgen; Koch, Christine; Stahl-Biskup, Elisabeth; Sojka, Cornelia; Schnitzler, Paul

    2005-12-01

    The inhibitory activity of manuka oil against Herpes simplex virus type 1 (HSV-1) and Herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells (monkey kidney cells) using a plaque reduction assay. In order to determine the mode of antiviral action of the essential oil, manuka oil was added at different times to the cells or viruses during the infection cycle. Both HSV types were significantly inhibited when the viruses were pretreated with manuka oil 1 h prior to cell infection. At non-cytotoxic concentrations of the essential oil, plaque formation was significantly reduced by 99.5 % and 98.9 % for HSV-1 and HSV-2, respectively. The 50 % inhibitory concentration (IC (50)) of manuka oil for virus plaque formation was determined at 0.0001 % v/v ( = 0.96 microg/mL) and 0.00006 % v/v ( = 0.58 microg/mL) for HSV-1 and HSV-2, respectively. On the other hand, pretreatment of host cells with the essential oil before viral infection did not affect plaque formation. After virus penetration into the host cells only replication of HSV-1 particle was significantly inhibited to about 41 % by manuka oil. Flavesone and leptospermone, two characteristic ss-triketones of manuka oil, inhibited the virulence of HSV-1 in the same manner as the essential oil itself. When added at non-cytotoxic concentrations to the virus 1 h prior to cell infection, plaque formation was reduced by 99.1 % and 79.7 % for flavesone and leptospermone, respectively. PMID:16395648

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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. PMID:26559832

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

    PubMed Central

    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

  12. Repeated social stress enhances the innate immune response to a primary HSV-1 infection in the cornea and trigeminal ganglia of Balb/c mice

    PubMed Central

    Dong-Newsom, P.; Powell, N.D.; Bailey, M.T.; Padgett, D.A.; Sheridan, J.F.

    2009-01-01

    Three to 5 days after a primary HSV-1 infection, macrophages infiltrate into the trigeminal ganglia (TG) and produce anti-viral cytokines to reduce viral replication. Previous research demonstrated that social disruption stress (SDR) enhances the trafficking of monocytes/macrophages from the bone marrow to the spleen and increases pro-inflammatory cytokine production in vitro and in vivo. The impact of SDR on the trafficking of these cells to loci of herpes simplex virus type 1 (HSV-1) infection and subsequent function has not been examined. The following studies were designed to determine whether SDR would enhance the innate immune response during a primary HSV-1 infection by increasing the number of macrophages in the cornea and TG, thus increasing anti-viral cytokine production and reducing viral replication. BALB/c mice were exposed to six cycles of SDR prior to ocular infection with HSV-1 McKrae virus. Flow cytometric analysis of cells from the TG revealed an increase in the percentage of CD11b+ macrophages in SDR mice compared to controls. Immune cell infiltration into the cornea, however, could not be determined due to low cell numbers. Although gene expression of IFN-β was decreased, SDR increased gene expression of IFN-α, and TNF-α, in the cornea and TG. Examination of viral proteins showed decreased expression of infected cell protein 0 (ICP0), glycoprotein B (gB), glycoprotein H (gH) and latency-associated transcript (LAT) in the TG, however, expression of ICP0 and gB were elevated in the cornea of SDR mice. These results indicate that the innate immune response to HSV-1 was altered and enhanced by the experience of repeated social defeat. PMID:19822203

  13. Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8+ T Cells

    PubMed Central

    Yu, Cheng-Rong; He, Chang; Mahdi, Rashid M.; Chan, Chi-Chao; Wang, Hongsheng; Morse, Herbert C.; Egwuagu, Charles E.

    2016-01-01

    Interferon Regulatory Factor-8 (IRF8) is constitutively expressed in monocytes and B cell lineages and plays important roles in immunity to pathogens and cancer. Although IRF8 expression is induced in activated T cells, the functional relevance of IRF8 in T cell-mediated immunity is not well understood. In this study, we used mice with targeted deletion of Irf8 in T-cells (IRF8KO) to investigate the role of IRF8 in T cell-mediated responses during herpes simplex virus 1 (HSV-1) infection of the eye. In contrast to wild type mice, HSV-1-infected IRF8KO mice mounted a more robust anti-HSV-1 immune response, which included marked expansion of HSV-1-specific CD8+ T cells, increased infiltration of inflammatory cells into the cornea and trigeminal ganglia (TG) and enhanced elimination of virus within the trigeminal ganglion. However, the consequence of the enhanced immunological response was the development of ocular inflammation, limbitis, and neutrophilic infiltration into the cornea of HSV-1-infected IRF8KO mice. Surprisingly, we observed a marked increase in virus-specific memory precursor effector cells (MPEC) in IRF8KO mice, suggesting that IRF8 might play a role in regulating the differentiation of effector CD8+ T cells to the memory phenotype. Together, our data suggest that IRF8 might play a role in restraining excess lymphocyte proliferation. Thus, modulating IRF8 levels in T cells can be exploited therapeutically to prevent immune-mediated ocular pathology during autoimmune and infectious diseases of the eye. PMID:27171004

  14. Microtubule plus end–associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells

    PubMed Central

    Jovasevic, Vladimir

    2015-01-01

    Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end–tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170–mediated capture to initiate transport in primary human cells. PMID:26504169

  15. Antiviral Action of Hydromethanolic Extract of Geopropolis from Scaptotrigona postica against Antiherpes Simplex Virus (HSV-1).

    PubMed

    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). PMID:25861357

  16. Antiviral Action of Hydromethanolic Extract of Geopropolis from Scaptotrigona postica against Antiherpes Simplex Virus (HSV-1)

    PubMed Central

    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

    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). PMID:25861357

  17. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon.

    PubMed

    Stein, Benjamin; Alonso, María Teresa; Zufall, Frank; Leinders-Zufall, Trese; Chamero, Pablo

    2016-01-01

    In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. PMID:27195771

  18. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon

    PubMed Central

    Stein, Benjamin; Alonso, María Teresa; Zufall, Frank; Leinders-Zufall, Trese; Chamero, Pablo

    2016-01-01

    In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. PMID:27195771

  19. The Nectin-1α Transmembrane Domain, But Not The Cytoplasmic Tail, Influences Cell Fusion Induced by HSV-1 Glycoproteins

    PubMed Central

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

    2006-01-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion. PMID:16005040

  20. The nectin-1{alpha} transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    SciTech Connect

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J. . E-mail: rgeragh@uky.edu

    2005-09-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1{alpha} involved in cell fusion, we measured the ability of nectin-1{alpha}/nectin-2{alpha} chimeras, nectin-1{alpha}/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1{alpha} to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1{alpha} cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1{alpha} and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1{alpha} interaction in fusion.

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

    PubMed

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

    2016-01-01

    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. PMID:27275821

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

    PubMed Central

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

    2016-01-01

    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. PMID:27275821

  3. Activation of NF-κB signaling pathway in HSV-1-induced mouse facial palsy: Possible relation to therapeutic effect of glucocorticoids.

    PubMed

    Liu, W; Fan, Z; Han, Y; Xu, L; Wang, M; Zhang, D; Mao, Y; Li, J; Wang, H

    2015-03-19

    It has been documented that infection of herpes simplex virus type 1 (HSV-1) contributes to the initiation of Bell's palsy. However, the exact mechanisms responsible for this disorder have not been fully elucidated to date. A mouse model of facial palsy induced by HSV-1 provides an opportunity to investigate the alteration in activities of nuclear factor-kappa B (NF-κB) and its consequent effect on two key inflammatory factors, i.e., tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2), as well as the effect of glucocorticoids (GCs) in this work. I-kappa B (IκB)-α phosphorylation and NF-κB nuclear translocation were measured by western blotting, and NF-κB/DNA binding activity was assessed by electrophoretic mobility shift assay (EMSA). Results showed the IκB-α phosphorylation and degradation as well as NF-κB activation in a time-dependent manner. The expression of TNF-α and COX-2 were determined by real-time polymerase chain reaction (PCR), western blotting and/or enzyme-linked immunosorbent assay (ELISA) respectively. Concomitant with the activation, the expression and secretion of TNF-α and COX-2 were rapidly induced in HSV-1-infected paralyzed mice. Conversely, the activation of NF-κB and up-regulation of TNF-α and COX-2 were blocked by pretreatment with NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) before being inoculated with HSV-1 to mice. In addition, GCs inhibited the nuclear translocation and DNA binding activity of NF-κB via inhibiting IκB-α degradation. Meanwhile, TNF-α production and COX-2 expression were significantly reduced by GCs. In conclusion, HSV-1 inoculation induced the activation of NF-κB, expression and secretion of TNF-α and COX-2 in the facial paralyzed mice, while, glucocorticoid effectively down-regulated TNF-α and COX-2 expression in HSV-1-induced paralyzed mice. PMID:25595974

  4. Defense against HSV-1 in a murine model is mediated by iNOS and orchestrated by the activation of TLR2 and TLR9 in trigeminal ganglia

    PubMed Central

    2014-01-01

    Background Herpes simplex 1 (HSV-1) causes various human clinical manifestations, ranging from simple cold sores to encephalitis. Innate immune cells recognize pathogens through Toll-like receptors (TLRs), thus initiating the immune response. Previously, we demonstrated that the immune response against HSV-1 is dependent on TLR2 and TLR9 expression and on IFN gamma production in the trigeminal ganglia (TG) of infected mice. In this work, we further investigated the cells, molecules, and mechanisms of HSV-1 infection control, especially those that are TLR-dependent. Methods C57BL/6 wild-type (WT), TLR2−/−, TLR9−/−, and TLR2/9−/− mice were intranasally infected with HSV-1. On the viral peak day, the TG and brains were collected from mice and TLR expression was measured in the TG and brain and inducible nitric oxide synthase (iNOS) expression was measured in the TG by real-time PCR. Immunofluorescence assays were performed in mice TG to detect iNOS production by F4/80+ cells. Intraperitoneal macrophages nitric oxide (NO) production was evaluated by the Griess assay. WT, CD8−/−, RAG−/−, and iNOS−/− mice were intranasally infected in a survival assay, and their cytokine expression was measured in the TG by real-time PCR. Results Infected WT mice exhibited significantly increased TLR expression, compared with their respective controls, in the TG but not in the brain. TLR-deficient mice had moderately increased TLR expression in the TG and brain in compare with the non-infected animals. iNOS expression in the WT infected mice TG was higher than in the other groups with increased production by macrophages in the WT infected mice, which did not occur in the TLR2/9−/− mice. Additionally, the intraperitoneal macrophages of the WT mice had a higher production of NO compared with those of the TLR-deficient mice. The CD8−/−, RAG−/−, and iNOS−/− mice had 100% mortality after the HSV-1 infection compared with 10% of the WT mice. Cytokines

  5. Genome wide nucleosome mapping for HSV-1 shows nucleosomes are deposited at preferred positions during lytic infection.

    PubMed

    Oh, Jaewook; Sanders, Iryna F; Chen, Eric Z; Li, Hongzhe; Tobias, John W; Isett, R Benjamin; Penubarthi, Sindura; Sun, Hao; Baldwin, Don A; Fraser, Nigel W

    2015-01-01

    HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200 bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6 hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152 kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes. PMID:25710170

  6. Genome Wide Nucleosome Mapping for HSV-1 Shows Nucleosomes Are Deposited at Preferred Positions during Lytic Infection

    PubMed Central

    Oh, Jaewook; Sanders, Iryna F.; Chen, Eric Z.; Li, Hongzhe; Tobias, John W.; Isett, R. Benjamin; Penubarthi, Sindura; Sun, Hao; Baldwin, Don A.; Fraser, Nigel W.

    2015-01-01

    HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes. PMID:25710170

  7. Bortezomib-induced unfolded protein response increases oncolytic HSV-1 replication resulting in synergistic, anti-tumor effects

    PubMed Central

    Yoo, Ji Young; Hurwitz, Brian S; Bolyard, Chelsea; Yu, Jun-Ge; Zhang, Jianying; Selvendiran, Karuppaiyah; Rath, Kellie S; He, Shun; Bailey, Zachary; Eaves, David; Cripe, Timothy P; Parris, Deborah S.; Caligiuri, Michael A.; Yu, Jianhua; Old, Matthew; Kaur, Balveen

    2014-01-01

    Background Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic HSV-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for anti-tumor efficacy. Methods The synergistic interaction between oHSV and bortezomib was calculated using Chou-Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western-blot assays were used to evaluate induction of ER stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Anti-tumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan-Meier curves and two-sided log rank test. Results Combination treatment with bortezomib and oHSV, 34.5ENVE, displayed strong synergistic interaction in ovarian cancer, head & neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK and IRE1α (western blot analysis) and the UPR (induction of hsp40, 70 and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (p value <0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced anti-tumor efficacy in multiple different tumor models in vivo. Conclusions The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib- induced UPR and warrants future clinical testing in patients. PMID:24815720

  8. Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1 (HSV-1) and electroporation: methods and optogenetic applications

    PubMed Central

    Zou, Ming; De Koninck, Paul; Neve, Rachael L.; Friedrich, Rainer W.

    2014-01-01

    The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1) or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications. PMID:24834028

  9. Identification of Replication-competent HSV-1 Cgal+ Strain Signaling Targets in Human Hepatoma Cells by Functional Organelle Proteomics*S⃞

    PubMed Central

    Santamaría, Enrique; Mora, María I.; Potel, Corinne; Fernández-Irigoyen, Joaquín; Carro-Roldán, Elvira; Hernández-Alcoceba, Rubén; Prieto, Jesús; Epstein, Alberto L.; Corrales, Fernando J.

    2009-01-01

    In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal+) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal+ infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca2+-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal+ infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK½, ERK½) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal+ in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest. PMID:19098277

  10. Regulation of viral gene expression by the herpes simplex virus 1UL24 protein (HSV-1UL24 inhibits accumulation of viral transcripts).

    PubMed

    Sanabria-Solano, Carolina; Gonzalez, Carmen Elena; Richerioux, Nicolas; Bertrand, Luc; Dridi, Slimane; Griffiths, Anthony; Langelier, Yves; Pearson, Angela

    2016-08-01

    UL24 is conserved among all Herpesviridae. In herpes simplex virus 1 (HSV-1), UL24 mutations lead to reduced viral titers both in cell culture and in vivo, and reduced pathogenicity. The human cytomegalovirus ortholog of UL24 has a gene regulatory function; however, it is not known whether other UL24 orthologs also affect gene expression. We discovered that in co-transfection experiments, expression of UL24 correlated with a reduction in the expression of several viral proteins and transcripts. Substitution mutations targeting conserved residues in UL24 impaired this function. Reduced transcript levels did not appear attributable to changes in mRNA stability. The UL24 ortholog of Herpes B virus exhibited a similar activity. An HSV-1 mutant that does not express UL24 produced more viral R1 and R2 transcripts than the wild type or rescue virus relative to the amount of viral DNA. These results reveal a new role for HSV-1UL24 in regulating viral mRNA accumulation. PMID:27214229

  11. HSV-1 amplicon vectors that direct the in situ production of foot-and-mouth disease virus antigens in mammalian cells can be used for genetic immunization.

    PubMed

    D'Antuono, Alejandra; Laimbacher, Andrea S; La Torre, Jose; Tribulatti, Virginia; Romanutti, Carina; Zamorano, Patricia; Quattrocchi, Valeria; Schraner, Elisabeth M; Ackermann, Mathias; Fraefel, Cornel; Mattion, Nora

    2010-10-28

    HSV-1 amplicon vectors encoding heterologous antigens were capable to mediate in situ generation of protein synthesis and to generate a specific immune response to the corresponding antigens. In this study, foot-and-mouth disease (FMD) virus antigens were used to generate a genetic vaccine prototype. The amplicons were designed to provide a high safety profile as they do not express any HSV-1 genes when packaged using a helper virus-free system, and they are able to encapsidate several copies of the transgene or allow the simultaneous expression of different genes. Virus-like particles were produced after cell processing of the delivered DNA. Inoculation of mice with 5 × 10(5) transducing units of amplicon vectors resulted in FMDV-specific humoral responses in the absence of adjuvants, which were dependent on the in situ de novo production of the vector-encoded antigens. Challenge of mice vaccinated with these amplicons with a high dose of live virus, resulted in partial protection, with a significant reduction of viremia. This work highlights the potential use of a HSV-1 amplicon vector platform for generation of safe genetic vaccines. PMID:20851082

  12. Lack of effect of treatment with penciclovir or acyclovir on the establishment of latent HSV-1 in primary sensory neurons in culture.

    PubMed

    Smith, R L; Morroni, J; Wilcox, C L

    2001-10-01

    Recent studies suggest reductions in establishment of herpes simplex virus, type 1 (HSV-1) latency using the nucleoside analog penciclovir compared with acyclovir in the murine model. These observations raise the possibility that the new analogs may have novel activities that directly interfere with the establishment of the latent infection, suggesting a mechanism other than simply blocking the productive infection. To determine if penciclovir has a direct action on the establishment of latency, we compared the effects of penciclovir versus acyclovir in an in vitro model of HSV-1 latency in rat dorsal root ganglia neurons in culture. In neurons in culture, both penciclovir and acyclovir were highly effective in blocking the productive infection. However, neither penciclovir nor acyclovir blocked establishment of latency as demonstrated by similar percentages of neurons expressing the latency-associated transcript (LAT). Following removal of the respective nucleoside analog, latency was maintained until reactivation was induced by nerve growth factor deprivation. Similar virus titers were recovered after induction of reactivation of latent infections, which were established in the presence of either penciclovir or acyclovir. These results indicate that neither penciclovir nor acyclovir treatment directly prevents the establishment of latent HSV-1 infections in primary sensory neurons in culture. PMID:11530184

  13. The nucleotide sequence of the gB glycoprotein gene of HSV-2 and comparison with the corresponding gene of HSV-1.

    PubMed

    Bzik, D J; Debroy, C; Fox, B A; Pederson, N E; Person, S

    1986-12-01

    The nucleotide sequence of the gB glycoprotein gene of HSV-2 has been determined and compared with the homologous gene of HSV-1. The two genes are specified by the same total number of codons (904); eight additional codons of the HSV-1 gene are found within the signal sequence, and eight additional codons of the HSV-2 gene are found at three different sites in the gene. The signal cleavage, membrane-spanning, and eight potential N-linked oligosaccharide sites, as well as 5'- and 3'-regulatory signals are largely conserved. The overall amino acid homology is 85%; least conserved are the N- and C-terminal regions of the protein. Secondary structure plots were determined for the two proteins, and the structures were compared with each other and with alterations in structure due to several mutations in the HSV-1 gB gene for which sequence analysis is available. The high homology in primary and secondary structure suggests a conserved, essential function for the gene. PMID:3024391

  14. Experiences in effective use of Tcl/Tk

    SciTech Connect

    Lee, R.W.

    1995-06-01

    Tcl/Tk (Toot Command Language and Tool Kit, pronounced ``tickle tee-kay``) is a scripting language supporting Motifm style X Window interfaces. It is extendible, allowing developers to embed additional functionality as commands in the language. However, the power and flexibility of the system leads to many variations or possibilities in its usage. We describe effective methods for taking advantage of Tcl/Tk to increase productivity and enhance the flexibility and adaptability of applications: writing simple Tcl/Tk scripts, extending the Tcl/Tk widget set, wrapping Tcl commands around existing classes and functions, and building Tcl/Tk and 3GL coprocesses. Examples are presented from working applications.

  15. Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system.

    PubMed

    Zhang, Zhihong; Lin, Juqiang; Chu, Jun; Ma, Yan; Zeng, Shaoqun; Luo, Qingming

    2008-01-01

    Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system. PMID:18601533

  16. Comparison of Simplexa HSV 1 & 2 PCR with culture, immunofluorescence, and laboratory-developed TaqMan PCR for detection of herpes simplex virus in swab specimens.

    PubMed

    Gitman, Melissa R; Ferguson, David; Landry, Marie L

    2013-11-01

    The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type. PMID:24006008

  17. 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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  18. Protein encoded by HSV-1 stimulation-related gene 1 (HSRG1) interacts with and inhibits SV40 large T antigen.

    PubMed

    Guo, H X; Cun, W; Liu, L D; Dong, S Z; Wang, L C; Dong, C H; Li, Q H

    2006-12-01

    Herpes simplex virus (HSV)-1 stimulation-related gene 1 (HSRG1) protein expression is induced in HSV-1 infected cells. We found that HSRG1 interacts with SV40 large T antigen (LT) in yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) assay. This interaction alters LT's regulation of the SV40 promoter and its ability to influence the cell cycle. Choramphenicol acetyl-transferase (CAT) assays revealed that initiation of gene transcription by LT is changed by HSRG1 expression. HSRG1 inhibits the ability of LT to activate SV40 late gene transcription. Further data indicate that the ability of LT protein to stimulate S-phase entry is also inhibited by the expression of HSRG1. The results of a colony-forming assay suggested that expression of HSRG1 in cells transfected by LT gene decreased the rate of colony formation. Yeast two-hybrid beta-galactosidase assay revealed that amino acid residues 132-450 in LT bind HSRG1. PMID:17109635

  19. Structural Characterization and Anti-HSV-1 and HSV-2 Activity of Glycolipids from the Marine Algae Osmundaria obtusiloba Isolated from Southeastern Brazilian Coast

    PubMed Central

    de Souza, Lauro M.; Sassaki, Guilherme L.; Romanos, Maria Teresa Villela; Barreto-Bergter, Eliana

    2012-01-01

    Glycolipids were extracted from the red alga Osmundaria obtusiloba from Southeastern Brazilian coast. The acetone insoluble material was extracted with chloroform/methanol and the lipids, enriched in glycolipids, were fractionated on a silica gel column eluted with chloroform, acetone and then methanol. Three major orcinol-positive bands were found in the acetone and methanol fractions, being detected by thin layer chromatography. The structures of the corresponding glycolipids were elucidated by ESI-MS and 1H/13C NMR analysis, on the basis of their tandem-MS behavior and HSQC, TOCSY fingerprints. For the first time, the structure of sulfoquinovosyldiacylglycerol from the red alga Osmundaria obtusiloba was characterized. This molecule exhibited potent antiviral activity against HSV-1 and HSV-2 with EC50 values of 42 µg/mL to HSV-1 and 12 µg/mL to HSV-2, respectively. Two other glycolipids, mono- and digalactosyldiacylglycerol, were also found in the alga, being characterized by ESI-MS/MS. The structural elucidation of algae glycolipids is a first step for a better understanding of the relation between these structures and their biological activities. PMID:22690151

  20. A conformational epitope mapped in the bovine herpesvirus type 1 envelope glycoprotein B by phage display and the HSV-1 3D structure.

    PubMed

    Almeida, Greyciele R; Goulart, Luiz Ricardo; Cunha-Junior, Jair P; Bataus, Luiz A M; Japolla, Greice; Brito, Wilia M E D; Campos, Ivan T N; Ribeiro, Cristina; Souza, Guilherme R L

    2015-08-01

    The selected dodecapeptide (1)DRALYGPTVIDH(12) from a phage-displayed peptide library and the crystal structure of the envelope glycoprotein B (Env gB) from Herpes Simplex Virus type 1 (HSV-1) led us to the identification of a new discontinuous epitope on the Bovine herpesvirus type 1 (BoHV-1) Env gB. In silico analysis revealed a short BoHV-1 gB motif ((338)YKRD(341)) within a epitope region, with a high similarity to the motifs shared by the dodecapeptide N-terminal region ((5)YxARD(1)) and HSV-1 Env gB ((326)YARD(329)), in which the (328)Arg residue is described to be a neutralizing antibody target. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has the potential to be used as a reagent for virus diagnosis by phage-ELISA assay, which discriminated BoHV-1 infected serum samples from negative ones. PMID:26267086

  1. Theranostic Imaging of Cancer Gene Therapy.

    PubMed

    Sekar, Thillai V; Paulmurugan, Ramasamy

    2016-01-01

    Gene-directed enzyme prodrug therapy (GDEPT) is a promising therapeutic approach for treating cancers of various phenotypes. This strategy is independent of various other chemotherapeutic drugs used for treating cancers where the drugs are mainly designed to target endogenous cellular mechanisms, which are different in various cancer subtypes. In GDEPT an external enzyme, which is different from the cellular proteins, is expressed to convert the injected prodrug in to a toxic metabolite, that normally kill cancer cells express this protein. Theranostic imaging is an approach used to directly monitor the expression of these gene therapy enzymes while evaluating therapeutic effect. We recently developed a dual-GDEPT system where we combined mutant human herpes simplex thymidine kinase (HSV1sr39TK) and E. coli nitroreductase (NTR) enzyme, to improve therapeutic efficiency of cancer gene therapy by simultaneously injecting two prodrugs at a lower dose. In this approach we use two different prodrugs such as ganciclovir (GCV) and CB1954 to target two different cellular mechanisms to kill cancer cells. The developed dual GDEPT system was highly efficacious than that of either of the system used independently. In this chapter, we describe the complete protocol involved for in vitro and in vivo imaging of therapeutic cancer gene therapy evaluation. PMID:27424910

  2. Genetics Home Reference: TK2-related mitochondrial DNA depletion syndrome, myopathic form

    MedlinePlus

    ... DNA depletion syndrome, myopathic form TK2-related mitochondrial DNA depletion syndrome, myopathic form Enable Javascript to view ... Open All Close All Description TK2 -related mitochondrial DNA depletion syndrome, myopathic form ( TK2 -MDS) is an ...

  3. Nucleotide sequences of Herpes Simplex Virus type 1 (HSV-1) affecting virus entry, cell fusion, and production of glycoprotein gB (VP7)

    SciTech Connect

    DeLuca, N.; Bzik, D.J.; Bond, V.C.; Person, S.; Snipes, W.

    1982-10-30

    The tsB5 strain of Herpes Simplex Virus type 1 (HSV-1) contains at least two mutations; one mutation specifies the syncytial phenotype and the other confers temperature sensitivity for virus growth. These functions are known to be located between the prototypic map coordinates 0.30 and 0.42. In this study it was demonstrated that tsB5 enters human embryonic lung (HEL) cells more rapidly than KOS, another strain of HSV-1. The EcoRI restriction fragment F from the KOS strain (map coordinates 0.315 to 0.421) was mapped with eight restriction endonucleases, and 16 recombinant plasmids were constructed which contained varying portions of the KOS genome. Recombinant viruses were generated by marker-rescue and marker-transfer cotransfection procedures, using intact DNA from one strain and a recombinant plasmid containing DNA from the other strain. The region of the crossover between the two nonisogenic strains was inferred by the identification of restriction sites in the recombinants that were characteristic of the parental strains. The recombinants were subjected to phenotypic analysis. Syncytium formation, rate of virus entry, and the production of gB were all separable by the crossovers that produced the recombinants. The KOS sequences which rescue the syncytial phenotype of tsB5 were localized to 1.5 kb (map coordinates 0.345 to 0.355), and the temperature-sensitive mutation was localized to 1.2 kb (0.360 to 0.368), giving an average separation between the mutations of 2.5 kb on the 150-kb genome. DNA sequences that specify a functional domain for virus entry were localized to the nucleotide sequences between the two mutations. All three functions could be encoded by the virus gene specifying the gB glycoprotein.

  4. USE OF DNA PURIFIED IN SITU FROM CELLS EMBEDDED IN AGAROSE PLUGS FOR THE MOLECULAR ANALYSIS OF TK-/-MUTANTS RECOVERED IN THE L5178Y TK+/- 3.7.2C MUTAGEN ASSAY SYSTEM

    EPA Science Inventory

    We have reported that tk-/- mutants recovered in the mouse L5178Y TK+/- 3.7.2C mutagen assay have often lost the tk+ allele. llele loss in tk-/- mutants is mented on Southern blots as the absence of a 6.3-kb Nco I fragment seen in both tk+/+ and tk+/- cell DNAs. or the routine sc...

  5. Using CamiTK for rapid prototyping of interactive Computer Assisted Medical Intervention applications

    PubMed Central

    Promayon, Emmanuel; Fouard, Celine; Bailet, Mathieu; Deram, Aurelien; Fiard, Gaelle; Hungr, Nikolai; Luboz, Vincent; Payan, Yohan; Sarrazin, Johan; Saubat, Nicolas; Selmi, Sonia Yuki; Voros, Sandrine; Cinquin, Philippe; Troccaz, Jocelyne

    2013-01-01

    Computer Assisted Medical Intervention (CAMI hereafter) is a complex multi-disciplinary field. CAMI research requires the collaboration of experts in several fields as diverse as medicine, computer science, mathematics, instrumentation, signal processing, mechanics, modeling, automatics, optics, etc. CamiTK1 is a modular framework that helps researchers and clinicians to collaborate together in order to prototype CAMI applications by regrouping the knowledge and expertise from each discipline. It is an open-source, cross-platform generic and modular tool written in C++ which can handle medical images, surgical navigation, biomedicals simulations and robot control. This paper presents the Computer Assisted Medical Intervention ToolKit (CamiTK) and how it is used in various applications in our research team. PMID:24110841

  6. Enzyme-Assisted Extraction of Bioactive Material from Chondrus crispus and Codium fragile and Its Effect on Herpes simplex Virus (HSV-1)

    PubMed Central

    Kulshreshtha, Garima; Burlot, Anne-Sophie; Marty, Christel; Critchley, Alan; Hafting, Jeff; Bedoux, Gilles; Bourgougnon, Nathalie; Prithiviraj, Balakrishnan

    2015-01-01

    Codium fragile and Chondrus crispus are, respectively, green and red seaweeds which are abundant along the North Atlantic coasts. We investigated the chemical composition and antiviral activity of enzymatic extracts of C. fragile (CF) and C. crispus (CC). On a dry weight basis, CF consisted of 11% protein, 31% neutral sugars, 0.8% sulfate, 0.6% uronic acids, and 49% ash, while CC contained 27% protein, 28% neutral sugars, 17% sulfate, 1.8% uronic acids, and 25% ash. Enzyme-assisted hydrolysis improved the extraction efficiency of bioactive materials. Commercial proteases and carbohydrases significantly improved (p ≤ 0.001) biomass yield (40%–70% dry matter) as compared to aqueous extraction (20%–25% dry matter). Moreover, enzymatic hydrolysis enhanced the recovery of protein, neutral sugars, uronic acids, and sulfates. The enzymatic hydrolysates exhibited significant activity against Herpes simplex virus (HSV-1) with EC50 of 77.6–126.8 μg/mL for CC and 36.5–41.3 μg/mL for CF, at a multiplicity of infection (MOI) of 0.001 ID50/cells without cytotoxity (1–200 μg/mL). The extracts obtained from proteases (P1) and carbohydrases (C3) were also effective at higher virus MOI of 0.01 ID50/cells without cytotoxity. Taken together, these results indicate the potential application of enzymatic hydrolysates of C. fragile and C. crispus in functional food and antiviral drug discovery. PMID:25603348

  7. Ocular neovascularization caused by HSV-1 infection results from breakdown of binding between VEGF-A and its soluble receptor1

    PubMed Central

    Suryawanshi, Amol; Mulik, Sachin; Sharma, Shalini; Reddy, Pradeep B. J.; Sehrawat, Sharvan

    2014-01-01

    The normal cornea is transparent which is essential for normal vision and although the angiogenic factor VEGF-A is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs following ocular infection with HSV, that causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis (SK). We demonstrate that HSV-1 infection causes increased production of VEGF-A, but reduces sVR-1 levels resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, MMP-7 and MMP-9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, or inhibition of sVR-1 breakdown with the MMP inhibitor (MMPi) marimostat, or the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of SK, an important cause of human blindness. PMID:21325621

  8. Lytic Gene Expression Is Frequent in HSV-1 Latent Infection and Correlates with the Engagement of a Cell-Intrinsic Transcriptional Response

    PubMed Central

    Ma, Joel Z.; Russell, Tiffany A.; Spelman, Tim

    2014-01-01

    Herpes simplex viruses (HSV) are significant human pathogens that provide one of the best-described examples of viral latency and reactivation. HSV latency occurs in sensory neurons, being characterized by the absence of virus replication and only fragmentary evidence of protein production. In mouse models, HSV latency is especially stable but the detection of some lytic gene transcription and the ongoing presence of activated immune cells in latent ganglia have been used to suggest that this state is not entirely quiescent. Alternatively, these findings can be interpreted as signs of a low, but constant level of abortive reactivation punctuating otherwise silent latency. Using single cell analysis of transcription in mouse dorsal root ganglia, we reveal that HSV-1 latency is highly dynamic in the majority of neurons. Specifically, transcription from areas of the HSV genome associated with at least one viral lytic gene occurs in nearly two thirds of latently-infected neurons and more than half of these have RNA from more than one lytic gene locus. Further, bioinformatics analyses of host transcription showed that progressive appearance of these lytic transcripts correlated with alterations in expression of cellular genes. These data show for the first time that transcription consistent with lytic gene expression is a frequent event, taking place in the majority of HSV latently-infected neurons. Furthermore, this transcription is of biological significance in that it influences host gene expression. We suggest that the maintenance of HSV latency involves an active host response to frequent viral activity. PMID:25058429

  9. Distinct APC Subtypes Drive Spatially Segregated CD4+ and CD8+ T-Cell Effector Activity during Skin Infection with HSV-1

    PubMed Central

    Macleod, Bethany L.; Bedoui, Sammy; Hor, Jyh Liang; Mueller, Scott N.; Russell, Tiffany A.; Hollett, Natasha A.; Heath, William R.; Tscharke, David C.

    2014-01-01

    Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4+ and CD8+ T-cells during skin infection with HSV-1. IFN-γ-producing CD4+ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8+ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4+ T cells, CD8+ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8+ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4+ and CD8+ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC). PMID:25121482

  10. Phytochemical analysis and in vitro evaluation of the biological activity against herpes simplex virus type 1 (HSV-1) of Cedrus libani A. Rich.

    PubMed

    Loizzo, Monica Rosa; Saab, Antoine; Tundis, Rosa; Statti, Giancarlo A; Lampronti, Ilaria; Menichini, Francesco; Gambari, Roberto; Cinatl, Jindrich; Doerr, Hans Wilhelm

    2008-01-01

    Cedrus libani are widely used as traditional medicine in Lebanon for treatment of different infection diseases. In the present study we reported the phytochemical composition analyzed by GC-MS of wood essential oil and cones and leaves ethanol extracts. The main components of wood essential oil were himachalol (22.50%), beta-himachalene (21.90%), and alpha-himachalene (10.50%). Leaves ethanol extract was characterized by a high content of germacrene d (29.40%). The same extract obtained from cones essentially contained alpha-pinene (51.0%) and beta-myrcene (13.0%). Moreover, we investigated extracts, essential oil, and identified compounds for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1). Cytotoxicity was evaluated by MTT assay in Vero cells. Cones and leaves ethanol extracts exhibited an interesting activity with IC50 of 0.50 and 0.66 mg/ml, respectively, at non-cytotoxic concentration. A comparable activity was found when essential oil was tested (IC50 of 0.44 mg/ml). PMID:17482448

  11. 2-Arachidonoyl-glycerol- and arachidonic acid-stimulated neutrophils release antimicrobial effectors against E. coli, S. aureus, HSV-1, and RSV

    PubMed Central

    Chouinard, François; Turcotte, Caroline; Guan, Xiaochun; Larose, Marie-Chantal; Poirier, Samuel; Bouchard, Line; Provost, Véronique; Flamand, Louis; Grandvaux, Nathalie; Flamand, Nicolas

    2016-01-01

    The endocannabinoid 2-AG is highly susceptible to its hydrolysis into AA, which activates neutrophils through de novo LTB4 biosynthesis, independently of CB activation. In this study, we show that 2-AG and AA stimulate neutrophils to release antimicrobial effectors. Supernatants of neutrophils activated with nanomolar concentrations of 2-AG and AA indeed inhibited the infectivity of HSV-1 and RSV. Additionally, the supernatants of 2-AG- and AA-stimulated neutrophils strongly impaired the growth of Escherichia coli and Staphylococcus aureus. This correlated with the release of a large amount (micrograms) of α-defensins, as well as a limited amount (nanograms) of LL-37. All the effects of AA and 2-AG mentioned above were prevented by inhibiting LTB4 biosynthesis or by blocking BLT1. Importantly, neither CB2 receptor agonists nor antagonists could mimic nor prevent the effects of 2-AG, respectively. In fact, qPCR data show that contaminating eosinophils express ~100-fold more CB2 receptor mRNA than purified neutrophils, suggesting that CB2 receptor expression by human neutrophils is limited and that contaminating eosinophils are likely responsible for the previously documented CB2 expression by freshly isolated human neutrophils. The rapid conversion of 2-AG to AA and their subsequent metabolism into LTB4 promote 2-AG and AA as multifunctional activators of neutrophils, mainly exerting their effects by activating the BLT1. Considering that nanomolar concentrations of AA or 2-AG were sufficient to impair viral infectivity, this suggests potential physiological roles for 2-AG and AA as regulators of host defense in vivo. PMID:23242611

  12. A tyrosine hydroxylase-neurofilament chimeric promoter enhances long-term expression in rat forebrain neurons from helper virus-free HSV-1 vectors.

    PubMed

    Zhang, G R; Wang, X; Yang, T; Sun, M; Zhang, W; Wang, Y; Geller, A I

    2000-12-01

    Helper virus-free herpes simplex virus (HSV-1) plasmid vectors are attractive for neural gene transfer, but a promoter that supports neuronal-specific, long-term expression is required. Although expression from many promoters is unstable, a 6.8-kb, but not a 766-bp, fragment of the tyrosine hydroxylase (TH) promoter supports long-term expression. Thus, 5' upstream sequences in this promoter may enhance expression. In this study, we evaluated expression from vectors that contain 5' upstream sequences from this promoter (-0.5 to -6.8 kb) inserted at the 5' end of either a neurofilament heavy subunit (NF-H) promoter or the cytomegalovirus (CMV) immediate early promoter. The TH-NFH promoter supported expression for 6 months in the striatum, 2 months in the hippocampus, and for 1 month in both perirhinal and postrhinal cortex (the longest time points examined). Expression was targeted to neurons. The enhanced expression may require specific sequences in the TH promoter fragment because replacing this fragment with a similar sized fragment of bacteriophage lambda DNA did not enhance expression. The reverse orientation of the TH promoter fragment also enhanced expression. Insertion of insulators from the chicken beta-globin locus between the TH-NFHlac transcription unit and the vector backbone may support a modest additional enhancement in expression. Other eucaryotic sequences may also enhance expression; a S. cerevisiae (40-kb fragment)-NFH promoter enhanced expression. In contrast, the TH-CMV promoter did not enhance expression. Thus, the TH-NFH promoter may support some physiological studies that require long-term expression in forebrain neurons. PMID:11113528

  13. Effect of Azone upon the in vivo antiviral efficacy of cidofovir or acyclovir topical formulations in treatment/prevention of cutaneous HSV-1 infections and its correlation with skin target site free drug concentration in hairless mice.

    PubMed

    Afouna, Mohsen I; Fincher, Timothy K; Zaghloul, Abdel-Azim A; Reddy, Indra K

    2003-03-01

    The purpose of this study is to examine the influence of Azone upon the skin target site free drug concentration (C(*)) and its correlation with the in vivo antiviral efficacies of cidofovir (HPMPC) and acyclovir (ACV) against HSV-1 infections. Formulations of HPMPC and ACV with or without Azone were used. The in vitro skin flux experiments were performed and the C(*) values were calculated. For the in vivo efficacy studies, hairless mice cutaneously infected with HSV-1 were used and three different treatment protocols were carried out. The protocols were chosen based upon when therapy is initiated and terminated in such a way to assess the efficacy of the test drug to cure and/or prevent HSV-1 infections. A finite dose of the formulation was topically applied twice a day for the predetermined time course for each protocol and the lesions were scored on the fifth day. For ACV formulation with Azone, the C(*) values and hence the in vivo efficacy were much higher than those for that without Azone. In protocol #1, however, early treatment did not increase the in vivo efficacy of ACV when compared with the standard treatment protocol #3. In protocol #2 where the treatment was terminated on the day of virus inoculation, the efficacies for both ACV formulations were completely absent. Although the estimated C(*) values for HPMPC formulations with and without Azone were comparable, formulation with Azone was much more effective than that without Azone in all treatment protocols. HPMPC formulations with Azone at similar flux values were much more effective in "treating and preventing" HSV-1 infections than those without Azone. For ACV formulations, in contrast, addition of Azone has failed to show any effect on the preventive in vivo antiviral efficacy and the enhancement of ACV in vivo antiviral efficacy was merely the skin permeation enhancement effect of Azone. PMID:12593946

  14. Characterization of a Novel Melt Curve by Use of the Roche LightCycler HSV 1/2 Analyte-Specific Reagent Real-Time PCR Assay: Frequencies of This Novel (Low) Melt Curve and Commonly Encountered (Intermediate) Melt Curves

    PubMed Central

    Almradi, Amro; Espy, Mark J.; Prada, Anne E.; Gibson, John P.; Pritt, Bobbi S.

    2014-01-01

    We characterize a novel probe binding-site polymorphism detectable solely by melt curve analysis using the Roche LightCycler HSV 1/2 analyte-specific reagent real-time PCR assay. The frequencies of this novel (47°C) and previously described intermediate (60 to 62°C) melt curves were 0.016% and 4.9%, respectively. PMID:24352998

  15. Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue

    PubMed Central

    Shinn, Helen Ki; Yan, Chunri; Kim, Tae-Hwan; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Moon, Sung-Kwon; Kim, Nam-Hyung; Kim, Jayoung; Cha, Eun-Jong

    2016-01-01

    Purpose: Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. Methods: A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. Results: Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). Conclusions: These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections. PMID:27377944

  16. MicroRNA-H4-5p encoded by HSV-1 latency-associated transcript promotes cell proliferation, invasion and cell cycle progression via p16-mediated PI3K-Akt signaling pathway in SHSY5Y cells

    PubMed Central

    Zhao, Huiliang; Zhang, Chunying; Hou, Guangjun; Song, Jijun

    2015-01-01

    Herpes simplex virus 1 (HSV-1) microRNAs (miRNAs) mostly located in transcription-associated transcript (LAT) region have been identified that play critical roles in the intricate host-pathogen interaction networks. Increasing evidences throw new insight into the role of miRNA-mediated miRNA-mRNA cross-talk in HSV-1 latent or acute infection. In the present study, we found that hsv-1 miR-H4-5p (here termed as miR-H4b) can down-regulate the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A, p16) in neuroblastoma (SHSY5Y) cell lines. Decreased expression of miR-H4b was directly related to attenuated cell proliferation and invasion as well as malfunction of cell cycle in recombinant SHSY5Y cells that stably expressing miR-H4b. Bioinformatics analysis and luciferase assays demonstrated miR-H4b can directly target p16 mRNA. MiR-H4b exerts its pro-proliferation function through inhibition of the p16-related PI3K-Akt pathways. Our findings provide, for the first time, significant clues regarding the role of herpesvirus-encoded miRNAs as a viral modulator to host cells. PMID:26221296

  17. A case of extravascular hemolysis with Tk-activation

    PubMed Central

    Yamada, Chisa; Davenport, Robertson D

    2014-01-01

    Key Clinical Message A 50-year-old female with ovarian cancer for 4 years presented with abdominal pain. She started antibiotics for possible infection, and developed extravascular hemolysis. All antigen detection tests were negative, but lectin panel suggested Tk-activation. Additional laboratory testing in conjunction with blood bank is essential to investigate rare cause of hemolysis. PMID:25356271

  18. ImTK: an open source multi-center information management toolkit

    NASA Astrophysics Data System (ADS)

    Alaoui, Adil; Ingeholm, Mary Lou; Padh, Shilpa; Dorobantu, Mihai; Desai, Mihir; Cleary, Kevin; Mun, Seong K.

    2008-03-01

    The Information Management Toolkit (ImTK) Consortium is an open source initiative to develop robust, freely available tools related to the information management needs of basic, clinical, and translational research. An open source framework and agile programming methodology can enable distributed software development while an open architecture will encourage interoperability across different environments. The ISIS Center has conceptualized a prototype data sharing network that simulates a multi-center environment based on a federated data access model. This model includes the development of software tools to enable efficient exchange, sharing, management, and analysis of multimedia medical information such as clinical information, images, and bioinformatics data from multiple data sources. The envisioned ImTK data environment will include an open architecture and data model implementation that complies with existing standards such as Digital Imaging and Communications (DICOM), Health Level 7 (HL7), and the technical framework and workflow defined by the Integrating the Healthcare Enterprise (IHE) Information Technology Infrastructure initiative, mainly the Cross Enterprise Document Sharing (XDS) specifications.

  19. Urinary MicroRNAs of Prostate Cancer: Virus-Encoded hsv1-miRH18 and hsv2-miR-H9-5p Could Be Valuable Diagnostic Markers

    PubMed Central

    Yun, Seok Joong; Jeong, Pildu; Kang, Ho Won; Kim, Ye-Hwan; Kim, Eun-Ah; Yan, Chunri; Choi, Young-Ki; Kim, Dongho; Kim, Jung Min; Kim, Seon-Kyu; Kim, Seon-Young; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Koh, Gou-Young; Moon, Sung-Kwon; Kim, Isaac Yi; Kim, Jayoung; Choi, Yung-Hyun; Kim, Wun-Jae

    2015-01-01

    Purpose: MicroRNAs (miRNAs) in biological fluids are potential biomarkers for the diagnosis and assessment of urological diseases such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The aim of the study was to identify and validate urinary cell-free miRNAs that can segregate patients with PCa from those with BPH. Methods: In total, 1,052 urine, 150 serum, and 150 prostate tissue samples from patients with PCa or BPH were used in the study. A urine-based miRNA microarray analysis suggested the presence of differentially expressed urinary miRNAs in patients with PCa, and these were further validated in three independent PCa cohorts, using a quantitative reverse transcriptionpolymerase chain reaction analysis. Results: The expression levels of hsa-miR-615-3p, hsv1-miR-H18, hsv2-miR-H9-5p, and hsa-miR-4316 were significantly higher in urine samples of patients with PCa than in those of BPH controls. In particular, herpes simplex virus (hsv)-derived hsv1-miR-H18 and hsv2-miR-H9-5p showed better diagnostic performance than did the serum prostate-specific antigen (PSA) test for patients in the PSA gray zone. Furthermore, a combination of urinary hsv2-miR-H9-5p with serum PSA showed high sensitivity and specificity, providing a potential clinical benefit by reducing unnecessary biopsies. Conclusions: Our findings showed that hsv-encoded hsv1-miR-H18 and hsv2-miR-H9-5p are significantly associated with PCa and can facilitate early diagnosis of PCa for patients within the serum PSA gray zone. PMID:26126436

  20. Enhanced nigrostriatal neuron-specific, long-term expression by using neural-specific promoters in combination with targeted gene transfer by modified helper virus-free HSV-1 vector particles

    PubMed Central

    Cao, Haiyan; Zhang, Guo-rong; Wang, Xiaodan; Kong, Lingxin; Geller, Alfred I

    2008-01-01

    Background Direct gene transfer into neurons has potential for developing gene therapy treatments for specific neurological conditions, and for elucidating neuronal physiology. Due to the complex cellular composition of specific brain areas, neuronal type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. One approach is to target gene transfer to a specific type of neuron. We developed modified Herpes Simplex Virus (HSV-1) particles that contain chimeric glycoprotein C (gC) – glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) proteins. HSV-1 vector particles containing either gC – GDNF or gC – BDNF target gene transfer to nigrostriatal neurons, which contain specific receptors for GDNF or BDNF. A second approach to achieve neuronal type-specific expression is to use a cell type-specific promoter, and we have used the tyrosine hydroxylase (TH) promoter to restrict expression to catecholaminergic neurons or a modified neurofilament heavy gene promoter to restrict expression to neurons, and both of these promoters support long-term expression from HSV-1 vectors. To both improve nigrostriatal-neuron specific expression, and to establish that targeted gene transfer can be followed by long-term expression, we performed targeted gene transfer with vectors that support long-term, neuronal-specific expression. Results Helper virus-free HSV-1 vector packaging was performed using either gC – GDNF or gC – BDNF and vectors that contain either the TH promoter or the modified neurofilament heavy gene promoter. Vector stocks were injected into the midbrain proximal to the substantia nigra, and the rats were sacrificed at either 4 days or 1 month after gene transfer. Immunofluorescent costaining was performed to detect both recombinant gene products and nigrostriatal neurons. The combination of targeted gene transfer with neuronal-specific promoters improved nigrostriatal

  1. 3H-Penciclovir (3H-PCV) Uptake Assay

    PubMed Central

    Sekar, Thillai V; Paulmurugan, Ramasamy

    2016-01-01

    Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as 18F-FHBG and 124I-FIAU. 3H-Penciclovir (PCV) uptake assay is a method of choice used to determine the expression level of HSV1-TK in mammalian cells and tissues. HSV1-TK phosphorylate PCV and result in the formation of penciclovir monophosphate, and its subsequent phopsphorylation by cellular TK lead to the formation of penciclovir triphosphate, which is trapped selectively in cells expressing HSV-TK. 3H-Penciclovir enables the detection of penciclovir uptake of mammalian cells and tissues by radioactive procedures such as scintillation counting. Here we describe the protocol to carry out 3H-Penciclovir uptakes in mammalian cells.

  2. Human gamma interferon and tumor necrosis factor exert a synergistic blockade on the replication of herpes simplex virus.

    PubMed Central

    Feduchi, E; Alonso, M A; Carrasco, L

    1989-01-01

    The replication of herpes simplex virus type 1 (HSV-1) is not inhibited in either HeLa or HEp-2 cells treated with human alpha interferon (HuIFN-alpha), particularly when high multiplicities of infection are used. However, HuIFN-gamma partially inhibits HSV-1 translation in HEp-2 cells infected at low multiplicities. Under these conditions, the transcription of genes alpha 22, TK, and gamma 0 is greatly diminished. The combined addition of human tumor necrosis factor (TNF) and HuIFN-gamma to HEp-2 cells exerts a synergistic inhibition of HSV-1 translation. Cells treated with both cytokines continue synthesizing cellular proteins, even 20 h after HSV-1 infection. As little as 10 U of IFN-gamma per ml blocked HSV-1 DNA replication, provided that TNF was also present in the medium. Analyses of HSV-1 gene transcription suggest that the action of both TNF and IFN-gamma blocked a step that comes at or prior to early HSV-1 gene expression. This early step in HSV-1 replication inhibited by TNF and IFN-gamma occurs after virus attachment and entry into cells, since the internalization of radioactive HSV-1 virion particles was not blocked by the presence of the two cytokines. Therefore, we conclude that the synergistic action of TNF plus IFN-gamma affects a step in HSV-1 replication that comes after virus entry but before or at the transcription of immediate-early genes. Images PMID:2536838

  3. Embryonic Stem Cell Grafting in Normal and Infarcted Myocardium: Serial Assessment with MR Imaging and PET Dual Detection

    PubMed Central

    Qiao, Hui; Zhang, Hualei; Zheng, Yuanjie; Ponde, Datta E.; Shen, Dinggang; Gao, Fabao; Bakken, Ashley B.; Schmitz, Alexander; Kung, Hank F.; Ferrari, Victor A.; Zhou, Rong

    2009-01-01

    Purpose: To use magnetic resonance (MR) imaging and positron emission tomography (PET) dual detection of cardiac-grafted embryonic stem cells (ESCs) to examine (a) survival and proliferation of ESCs in normal and infarcted myocardium, (b) host macrophage versus grafted ESC contribution to serial MR imaging signal over time, and (c) cardiac function associated with the formation of grafts and whether improvement in cardiac function is related to cardiac differentiation of ESCs. Materials and Methods: All animal procedures were approved by the institutional animal care and use committee. Murine ESCs were stably transfected with a mutant version of herpes simplex virus type 1 thymidine kinase, HSV1-sr39tk, and also were labeled with superparamagnetic iron oxide (SPIO) particles. Cells were injected directly in the border zone of the infarcted heart or in corresponding regions of normal hearts in athymic rats. PET and MR imaging were performed longitudinally for 4 weeks in the same animals. Results: ESCs survived and underwent proliferation in the infarcted and normal hearts, as demonstrated by serial increases in 9-(4-[18F]fluoro-3-hydroxymethylbutyl) guanine PET signals. In parallel, the hypointense areas on MR images at the injection sites decreased over time. Double staining for host macrophages and SPIO particles revealed that the majority of SPIO-containing cells were macrophages at week 4 after injection. Left ventricular ejection fraction increased in the ESC-treated rats but decreased in culture media–treated rats, and border-zone function was preserved in ESC-treated animals; however, cardiac differentiation of ESCs was less than 0.5%. Conclusion: Dual-modality imaging permits complementary information in regard to cell survival and proliferation, graft formation, and effects on cardiac function. © RSNA, 2009 PMID:19244049

  4. MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY MUTAGENS IN THE TK GENE OF MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY BROMATE AND N- ETHYL-N-NITROSOUREA IN THE TK GENE OF MOUSE L YMPHOMA CELLS

    The mouse lymphoma assay is widely used to identify chemical mutagens The Tk +1- gene located on an autosome in mouse lymphoma cells may recover a wide ra...

  5. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

    PubMed Central

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P.; Geller, Alfred I.

    2009-01-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an ∼9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported ∼90 % glutamatergic neuron-specific expression. The GAD67 promoter supported ∼90 % GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic

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

    NASA Astrophysics Data System (ADS)

    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.

  7. The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) protects cells against cold-shock-induced apoptosis by maintaining phosphorylation of protein kinase B (AKT).

    PubMed

    Carpenter, Dale; Hsiang, Chinhui; Jiang, Xianzhi; Osorio, Nelson; BenMohamed, Lbachir; Jones, Clinton; Wechsler, Steven L

    2015-10-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) blocks apoptosis and inhibits caspase-3 activation. We previously showed that serum starvation (removal of serum from tissue culture media), which takes several days to induce apoptosis, results in decreased levels of both AKT (protein kinase B) and phosphorylated AKT (pAKT) in cells not expressing LAT. In contrast in mouse neuroblastoma cells expressing LAT, AKT, and pAKT levels remained high. AKT is a serine/threonine protein kinase that promotes cell survival. To examine the effect of LAT on AKT-pAKT using a different and more rapid method of inducing apoptosis, a stable cell line expressing LAT was compared to non-LAT expressing cells as soon as 15 min following recovery from cold-shock-induced apoptosis. Expression of LAT appeared to inhibit dephosphorylation of pAKT. This protection correlated with blocking numerous pro-apoptotic events that are inhibited by pAKT. These results support the hypothesis that inhibiting dephosphorylation of pAKT may be one of the pathways by which LAT protects cells against apoptosis. PMID:26071090

  8. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    SciTech Connect

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  9. An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca2+.

    PubMed

    Sinsereekul, Nitat; Foophow, Tita; Yamanouchi, Mai; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2011-06-01

    Pro-Tk-SP from Thermococcus kodakaraensis consists of the four domains: N-propeptide, subtilisin (EC 3.4.21.62) domain, β-jelly roll domain and C-propeptide. To analyze the maturation process of this protein, the Pro-Tk-SP derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*) were constructed. Pro-Tk-S359C was purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first. The subsequent maturation process was analyzed using ProC-Tk-S359C, instead of the ProN:ProC-Tk-S359C complex. The C-propeptide was autoprocessed and degraded when ProC-Tk-S359C was incubated at 80 °C in the absence of Ca(2+). However, it was not autoprocessed in the presence of Ca(2+). Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca(2+) suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca(2+). We propose that Pro-Tk-SP derivative lacking N-propeptide (Val114-Gly640) represents a mature form of Pro-Tk-SP in a natural environment. The enzymatic activity of ProC-Tk-S359C was higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. However, the T(m) value of ProC-Tk-S359C determined by far-UV CD spectroscopy was higher than that of Tk-S359C by 25.9 °C in the absence of Ca(2+) and 7.5 °C in the presence of Ca(2+), indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C. PMID:21443525

  10. Isolation of an enhancer from the rat tyrosine hydroxylase promoter that supports long-term, neuronal-specific expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

    PubMed Central

    Gao, Qingshen; Sun, Mei; Wang, Xiaodan; Geller, Alfred I.

    2009-01-01

    Direct gene transfer into neurons, using a virus vector, has been used to study neuronal physiology and learning, and has potential for supporting gene therapy treatments for specific neurological diseases. Many of these applications require high-level, long-term recombinant gene expression, in forebrain neurons. We previously showed that addition of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a neurofilament heavy gene (NF-H) promoter supports long-term expression in forebrain neurons, from helper virus-free Herpes Simplex Virus (HSV-1) vectors. This element in the TH promoter satisfied the definition of an enhancer; it displayed activity at a distance from the basal promoter, and in both orientations. This enhancer supported physiological studies that required long-term expression; a modified neurofilament promoter, containing an insulator upstream of the TH-NFH promoter, supported expression in ∼11,400 striatal neurons at 6 months after gene transfer, and expression for 7, 8, or 14 months, the longest times tested. In contrast, the NF-H promoter alone does not support long-term expression, indicating that the critical sequences are in the 6.3 kb fragment of the TH promoter. In this study, we performed a deletion analysis to identify the critical sequences in the TH promoter that support long-term expression. We localized these critical sequences to an ∼320 bp fragment, and two subfragments of ∼100 bp each. Vectors that contained each of these small fragments supported levels of long-term, neuronal-specific expression that were similar to the levels supported by a vector that contained the initial 6.3 kb fragment of the TH promoter. These small fragments of the TH promoter may benefit construction of vectors for physiological studies, and may support studies on the mechanism by which this enhancer supports long-term expression. PMID:17169349

  11. Analysis of in vivo mutation in the Hprt and Tk genes of mouse lymphocytes.

    PubMed

    Dobrovolsky, Vasily N; Shaddock, Joseph G; Heflich, Robert H

    2014-01-01

    Assays measuring mutant frequencies in endogenous reporter genes are used for identifying potentially genotoxic environmental agents and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe methods for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation in both the Hprt and Tk genes. PMID:24623234

  12. Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

    SciTech Connect

    Intine, R.V.; Rainbow, A.J. )

    1990-01-01

    A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.

  13. The Syk Kinase SmTK4 of Schistosoma mansoni Is Involved in the Regulation of Spermatogenesis and Oogenesis

    PubMed Central

    Beckmann, Svenja; Buro, Christin; Dissous, Colette; Hirzmann, Jörg; Grevelding, Christoph G.

    2010-01-01

    The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes. PMID:20169182

  14. B Plant, TK-21-1, analytical results for the final report

    SciTech Connect

    Fritts, L.L., Westinghouse Hanford

    1996-12-09

    This document is the final laboratory report for B Plant Tk-21-1. A Resource Conservation and Recovery Act (RCRA) sample was taken from Tk-21 -1 September 26, 1996. This sample was received at 222-S Analytical Laboratory on September 27, 1996. Analyses were performed in accordance with the accompanying Request for Sample Analysis (RSA) and Letter of Instruction B PLANT RCRA SAMPLES TO 222S LABORATORY, LETTER OF INSTRUCTION (LOI) 2B-96-LOI-012-01 (LOI) (Westra, 1996). LOI was issued subsequent to RSA and replaces Letter of Instruction 2C-96-LOI-004-01 referenced in RSA.

  15. Structural analysis of herpes simplex virus by optical super-resolution imaging

    PubMed Central

    Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

    2015-01-01

    Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument. PMID:25609143

  16. Structural analysis of herpes simplex virus by optical super-resolution imaging

    NASA Astrophysics Data System (ADS)

    Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

    2015-01-01

    Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

  17. MUTAGENICITY AND CLASTOGENICITY OF ADRIAMYCIN IN L5178Y/TK+/- 3.7.2C MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    Adriamycin was a potent mutagen at the tk locus of L5178Y/TK+/- -3.7.2C mouse lymphoma cells. A dose of 5 ng/ml gave total mutant frequencies of 417/ten to the 6th power survivors (background = 110/ten to the 6th power; survival = 62%) and 350/ten to the 6th power survivors (back...

  18. CHROMOSOME 11 ABERRATIONS IN SMALL COLONY L5178Y TK-/-MUTANTS EARLY IN THEIR CLONAL HISTORY

    EPA Science Inventory

    The authors have developed a cytogenetic technique that allows observation of chromosome rearrangements associated with TK-/- mutagenesis of the L5178Y/TK+/-3.7.2C cell line early in mutant clonal history. For a series of mutagenic treatments they show that the major proportion (...

  19. Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells.

    PubMed

    Zhan, Li; Sakamoto, Hiroko; Sakuraba, Mayumi; Wu, De Sheng; Zhang, Li Shi; Suzuki, Takayoshi; Hayashi, Makoto; Honma, Masamitsu

    2004-01-10

    Toxic cyanobacteria (blue-green algae) water blooms have become a serious problem in several industrialized areas of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacteria. In the present study, we used human lymphoblastoid cell line TK6 to investigate the in vitro genotoxicity of MCLR. In a standard 4h treatment, MCLR did not induce a significant cytotoxic response at <80 microg/ml. In a prolonged 24h treatment, in contrast, it induced cytotoxic as well as mutagenic responses concentration-dependently starting at 20 microg/ml. At the maximum concentration (80 microg/ml), the micronucleus frequency and the mutation frequency at the heterozygous thymidine kinase (TK) locus were approximately five-times the control values. Molecular analysis of the TK mutants revealed that MCLR specifically induced loss of heterozygosity at the TK locus, but not point mutations or other small structural changes. These results indicate that MCLR had a clastogenic effect. We discuss the mechanisms of MCLR genotoxicity and the possibility of its being a hepatocarcinogen. PMID:14706513

  20. PURIFICATION AND CHARACTERIZATION OF A HIGHLY THERMOSTABLE ALPHA-L-ARABINOFURANOSIDASE FROM GEOBACILLUS CALDOXYLOLYTICUS TK4

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene encoding an alpha-L-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalyt...

  1. DIFFERENTIAL RECOVERY OF 'TK' AND 'HGPRT' INDUCED MUTANTS IN MAMMALIAN CELLS

    EPA Science Inventory

    Human genetic disease is known to result from both point mutations and chromosomal aberrations. It is therefore critical that short-term in vitro mammalian tests be evaluated as to their capabilities for detecting both types of lesions. Research to date indicates that L5178Y/TK p...

  2. Retrovirus transduction: Segregation of the viral transforming function and the Herpes Simplex virus tk gene in infectious friend spleen focus-forming virus thymidine kinase vectors

    SciTech Connect

    Joyner, A.L.; Bernstein, A.

    1983-12-01

    A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotranfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotranfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK/sup -/ fibroblasts to a TK/sup +/ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.

  3. Investigation of the apoptotic way induced by digallic acid in human lymphoblastoid TK6 cells

    PubMed Central

    2012-01-01

    Background The digallic acid (DGA) purified from Pistacia lentiscus. L fruits was investigated for its antiproliferative and apoptotic activities on human lymphoblastoid TK6 cells. Methods We attempt to characterize the apoptotic pathway activated by DGA. Apoptosis was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activities. Results The inhibition of lymphoblastoid cell proliferation was noted from 8.5 μg/ml of DGA. The induction of apoptosis was confirmed by DNA fragmentation and PARP cleavage. We have demonstrated that DGA induces apoptosis by activating the caspase-8 extrinsic pathway. Caspase-3 was also activated in a dose dependent manner. Conclusion In summary, DGA exhibited an apoptosis inductor effect in TK6 cells revealing thus its potential as a cancer-preventive agent. PMID:22686580

  4. Crystal structure of the TK2203 protein from Thermococcus kodakarensis, a putative extradiol dioxygenase.

    PubMed

    Nishitani, Yuichi; Simons, Jan Robert; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

    2016-06-01

    The TK2203 protein from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (262 residues, 29 kDa) is a putative extradiol dioxygenase catalyzing the cleavage of C-C bonds in catechol derivatives. It contains three metal-binding residues, but has no significant sequence similarity to proteins for which structures have been determined. Here, the first crystal structure of the TK2203 protein was determined at 1.41 Å resolution to investigate its functional role. Structure analysis reveals that this protein shares the same fold and catalytic residues as other extradiol dioxygenases, strongly suggesting the same enzymatic activity. Furthermore, the important region contributing to substrate selectivity is discussed. PMID:27303894

  5. TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells.

    PubMed

    Miles, Mark A; Shekhar, Tanmay M; Hall, Nathan E; Hawkins, Christine J

    2016-05-01

    When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of therapy-related cancers. These data suggest that TRAIL too may provoke oncogenic damage to the genomes of surviving cells. PMID:26943263

  6. MerTK Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells.

    PubMed

    Healy, Luke M; Perron, Gabrielle; Won, So-Yoon; Michell-Robinson, Mackenzie A; Rezk, Ayman; Ludwin, Samuel K; Moore, Craig S; Hall, Jeffery A; Bar-Or, Amit; Antel, Jack P

    2016-04-15

    Multifocal inflammatory lesions featuring destruction of lipid-rich myelin are pathologic hallmarks of multiple sclerosis. Lesion activity is assessed by the extent and composition of myelin uptake by myeloid cells present in such lesions. In the inflamed CNS, myeloid cells are comprised of brain-resident microglia, an endogenous cell population, and monocyte-derived macrophages, which infiltrate from the systemic compartment. Using microglia isolated from the adult human brain, we demonstrate that myelin phagocytosis is dependent on the polarization state of the cells. Myelin ingestion is significantly enhanced in cells exposed to TGF-β compared with resting basal conditions and markedly reduced in classically activated polarized cells. Transcriptional analysis indicated that TGF-β-treated microglia closely resembled M0 cells. The tyrosine kinase phagocytic receptor MerTK was one of the most upregulated among a select number of differentially expressed genes in TGF-β-treated microglia. In contrast, MerTK and its known ligands, growth arrest-specific 6 and Protein S, were downregulated in classically activated cells. MerTK expression and myelin phagocytosis were higher in CNS-derived microglia than observed in monocyte-derived macrophages, both basally and under all tested polarization conditions. Specific MerTK inhibitors reduced myelin phagocytosis and the resultant anti-inflammatory biased cytokine responses for both cell types. Defining and modulating the mechanisms that regulate myelin phagocytosis has the potential to impact lesion and disease evolution in multiple sclerosis. Relevant effects would include enhancing myelin clearance, increasing anti-inflammatory molecule production by myeloid cells, and thereby permitting subsequent tissue repair. PMID:26962228

  7. Low temperature ageing of silicas Gasil-I and TK800

    NASA Astrophysics Data System (ADS)

    Collins, K. E.; Gonçalves, M. C.; Romero, R. B.; Conz, R. F.; de Camargo, V. R.; Collins, C. H.

    2008-04-01

    Gasil-I (a mesoporous silica) and TK800 (a non-porous pyrogenic silica) were investigated in the early 1970s as standard reference materials. Since then the specific surface areas of both silicas have decreased to ˜85% of their initial values, suggesting that the surface character and the ageing mechanism may be the same for both. Comparisons of the shapes of nitrogen-adsorption isotherms, confirmed by comparisons of the shape ratios for Gasil-I and TK800, indicate that Gasil-I has greater microbore character and a higher absorption at p/ p0 > 0.5 than TK800 and that the isotherm shapes have changed little since 1974. The specific volume of Gasil-I has remained nearly constant during the ageing period but the pore size distribution (PSD) has shifted markedly to higher values. Electron micrographs show that low (room) temperature gas-solid ageing results in similar enlargement at the point of contact between attached secondary particles as that which occurs in hydrothermal ageing. In the gas-solid case, this change, which accounts for the decrease in overall surface area, is attributed to the surface transport of silica material in the presence of near monolayer quantities of adsorbed water. Ageing in this manner is geometry-limited so that the rate of ageing is expected to approach zero, resulting in time-stable silicas.

  8. Inhibition of MerTK increases chemosensitivity and decreases oncogenic potential in T-cell acute lymphoblastic leukemia.

    PubMed

    Brandao, L N; Winges, A; Christoph, S; Sather, S; Migdall-Wilson, J; Schlegel, J; McGranahan, A; Gao, D; Liang, X; Deryckere, D; Graham, D K

    2013-01-01

    Pediatric leukemia survival rates have improved dramatically over the past decades. However, current treatment protocols are still largely ineffective in cases of relapsed leukemia and are associated with a significant rate of chronic health conditions. Thus, there is a continued need for new therapeutic options. Here, we show that mer receptor tyrosine kinase (MerTK) was abnormally expressed in approximately one half of pediatric T-cell leukemia patient samples and T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Stimulation of MerTK by the ligand Gas6 led to activation of the prosurvival proteins Erk 1/2 and Stat5, and MerTK-dependent activation of the STAT pathway in leukemia represents a novel finding. Furthermore, inhibition of MerTK expression increased the sensitivity of T-ALL cells to treatment with chemotherapeutic agents and decreased the oncogenic potential of the Jurkat T-ALL cell line in a methylcellulose colony-forming assay. Lastly, inhibition of MerTK expression significantly increased median survival in a xenograft mouse model of leukemia (30.5 days vs 60 days, P<0.0001). These results suggest that inhibition of MerTK is a promising therapeutic strategy for the treatment of leukemia and may allow for dose reduction of currently used chemotherapeutics resulting in decreased rates of therapy-associated toxicities. PMID:23353780

  9. Inhibition of MerTK increases chemosensitivity and decreases oncogenic potential in T-cell acute lymphoblastic leukemia

    PubMed Central

    Brandao, L N; Winges, A; Christoph, S; Sather, S; Migdall-Wilson, J; Schlegel, J; McGranahan, A; Gao, D; Liang, X; DeRyckere, D; Graham, D K

    2013-01-01

    Pediatric leukemia survival rates have improved dramatically over the past decades. However, current treatment protocols are still largely ineffective in cases of relapsed leukemia and are associated with a significant rate of chronic health conditions. Thus, there is a continued need for new therapeutic options. Here, we show that mer receptor tyrosine kinase (MerTK) was abnormally expressed in approximately one half of pediatric T-cell leukemia patient samples and T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Stimulation of MerTK by the ligand Gas6 led to activation of the prosurvival proteins Erk 1/2 and Stat5, and MerTK-dependent activation of the STAT pathway in leukemia represents a novel finding. Furthermore, inhibition of MerTK expression increased the sensitivity of T-ALL cells to treatment with chemotherapeutic agents and decreased the oncogenic potential of the Jurkat T-ALL cell line in a methylcellulose colony-forming assay. Lastly, inhibition of MerTK expression significantly increased median survival in a xenograft mouse model of leukemia (30.5 days vs 60 days, P<0.0001). These results suggest that inhibition of MerTK is a promising therapeutic strategy for the treatment of leukemia and may allow for dose reduction of currently used chemotherapeutics resulting in decreased rates of therapy-associated toxicities. PMID:23353780

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

    PubMed

    Shao, Dan; Zeng, Qinghui; Fan, Zheng; Li, Jing; Zhang, Ming; Zhang, Youlin; Li, Ou; Chen, Li; Kong, Xianggui; Zhang, Hong

    2012-06-01

    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 successfully linked with CdTe/CdS core/shell quantum dots (QDs) via EDC/NHS coupling method. From confocal microscopy it was demonstrated that plasmid TK intracellular trafficking can be effectively and distinctly traced via monitoring the luminescence of the QDs up to 96 h after transfection of QDs-TK conjugates into Hela cells. MTT results show that the QDs-TK conjugates have a high efficient cytotoxicity after adding GCV into Hela cells, whereas the QDs exert no detectable deleterious effects on the cellular processes. The apoptosis induced by QDs-TK conjugates with GCV is distinctly traced partly due to the strong luminescence of the QDs. Our results indicate that photonic nanomaterials, e.g. QDs, provide a tool for monitoring TK gene delivery and anti-cancer activity. PMID:22440046

  11. Differential mutant quantitation at the mouse lymphoma tk and CHO hgprt loci.

    PubMed

    Moore, M M; Harrington-Brock, K; Doerr, C L; Dearfield, K L

    1989-09-01

    Recent reports by several laboratories indicate that not all non-essential target loci are equally capable of detecting chromosomal mutations. The present study was undertaken to determine if both the tk locus in mouse lymphoma cells and the hgprt locus in Chinese hamster ovary (CHO) cells can be used to quantitate chromosomal mutations. Seven known mutagens for the tk locus were selected. These compounds were evaluated in the mouse lymphoma assay and in a suspension adapted CHO assay for their mutagenicity. In addition to the specific locus mutagenesis analysis, mouse lymphoma and CHO cells were evaluated for the frequency of gross chromosome aberrations. From these investigations, it appears that only those compounds [2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl] aminopropylamino)-acridine-dihydrochloride (ICR 170), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)] that induce significant numbers of large-colony thymidine kinase (TK) mutants also induce significant numbers of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) mutants. The four acrylates evaluated (methyl acrylate, ethyl acrylate, trimethylolpropane triacrylate and tetraethyleneglycol diacrylate) induced almost exclusively small-colony TK mutants and very few if any HGPRT mutants. Aberration analysis revealed that both the mouse lymphoma and CHO cells responded to the clastogenicity of the compounds (except for ICR 170 which was not positive in CHO cells) and that neither cell line was clearly more sensitive than the other to the clastogens tested. It is significant that the four acrylates give little or no evidence of genotoxicity when evaluated using selection for HGPRT-deficient mutants, yet are clearly clastogenic to the same cells in the same experiment. These results are consistent with the hypothesis that the hgprt locus may not be useful as a marker to evaluate the clastogenic component of a genotoxic compound. The present study adds to the increasing number of studies

  12. tkLayout: a design tool for innovative silicon tracking detectors

    NASA Astrophysics Data System (ADS)

    Bianchi, G.

    2014-03-01

    A new CMS tracker is scheduled to become operational for the LHC Phase 2 upgrade in the early 2020's. tkLayout is a software package developed to create 3d models for the design of the CMS tracker and to evaluate its fundamental performance figures. The new tracker will have to cope with much higher luminosity conditions, resulting in increased track density, harsher radiation exposure and, especially, much higher data acquisition bandwidth, such that equipping the tracker with triggering capabilities is envisaged. The design of an innovative detector involves deciding on an architecture offering the best trade-off among many figures of merit, such as tracking resolution, power dissipation, bandwidth, cost and so on. Quantitatively evaluating these figures of merit as early as possible in the design phase is of capital importance and it is best done with the aid of software models. tkLayout is a flexible modeling tool: new performance estimates and support for different detector geometries can be quickly added, thanks to its modular structure. Besides, the software executes very quickly (about two minutes), so that many possible architectural variations can be rapidly modeled and compared, to help in the choice of a viable detector layout and then to optimize it. A tracker geometry is generated from simple configuration files, defining the module types, layout and materials. Support structures are automatically added and services routed to provide a realistic tracker description. The tracker geometries thus generated can be exported to the standard CMS simulation framework (CMSSW) for full Monte Carlo studies. tkLayout has proven essential in giving guidance to CMS in studying different detector layouts and exploring the feasibility of innovative solutions for tracking detectors, in terms of design, performance and projected costs. This tool has been one of the keys to making important design decisions for over five years now and has also enabled project engineers

  13. Analysis of cellular response by exposure to acute or chronic radiation in human lymphoblastoid TK-6 cells

    NASA Astrophysics Data System (ADS)

    Ohnishi, T.; Yasumoto, J.; Takahashi, A.; Ohnishi, K.

    To clarify the biological effects of low-dose rate radiation on human health for long-term stay in space, we analyzed the induction of apoptosis and apoptosis-related gene expression after irradiation with different dose-rate in human lymphoblastoid TK-6 cells harboring wild-type p53 gene. We irradiated TK-6 cells by X-ray at 1.5 Gy (1 Gy/min) and then sampled at 25 hr after culturing. We also irradiated by gamma-ray at 1.5 Gy (1 mGy/min) and then sampled immediately or 25 hr after irradiation. For DNA ladder analysis, we extracted DNA from these samples and electrophoresed with 2% agarose gel. In addition, we extracted mRNA from these samples for DNA-array analysis. mRNA from non-irradiated cells was used as a control. After labeling the cDNA against mRNA with [α -33P]-dCTP and hybridizing onto DNA array (Human Apoptosis Expression Array, R&D Systems), we scanned the profiles of the spots by a phosphorimager (BAS5000, FUJI FILM) and calculated using a NIH Image program. The data of each DNA-array were normalized with eight kinds of house keeping genes. We analyzed the expression level of apoptosis-related genes such as p53-related, Bcl-2 family, Caspase family and Fas-related genes. DNA ladders were obviously detected in the cells exposed to a high dose-rate radiation. We detected the induction of the gene expression of apoptosis-promotive genes. In contrast, almost no apoptosis was observed in the cells exposed to the chronic radiation at a low dose-rate. In addition, we detected the induction of the gene expression of apoptosis-suppressive genes as compared with apoptosis promotive-genes immediately after chronic irradiation. These results lead the importance of biological meaning of exposure to radiation at low dose-rate from an aspect of carcinogenesis. Finally, the effects of chronic irradiation become a highly important issue in space radiation biology for human health.

  14. Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product.

    PubMed Central

    Weeks, M O; Hager, G L; Lowe, R; Scolnick, E M

    1985-01-01

    The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells. Images PMID:2985821

  15. Responses of the L5178Y tk/sup +//tk/sup -/ mouse lymphoma cell forward mutation assay. II. 18 coded chemicals

    SciTech Connect

    McGregor, D.B.; Brown, A.; Cattanach, P.; Edwards, I.; McBride, D.; Caspary, W.J.

    1988-01-01

    Eighteen chemicals were tested for their mutagenic potential in the L5178Y tk/sup +///sup -/ mouse lymphoma cell forward mutation assay by the use of procedures based upon those described previously. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 ..mu..g/ml. The chemicals were tested at least twice. Significant responses were obtained with benzofuran, benzyl chloride, bromodichloromethane, butylated hydroxytoluene, chlorendic acid, o-chlorobenzalmalonitrile, 1,2,3,4-diepoxybutane, dimethyl formamide, dimethyl hydrogen phosphite, furfural, glutaraldehyde, hydroquinone, 8-hydroxyquinoline, and resorcinol. Apart from bromodichloromethane, butylated hydroxytoluene and dimethyl hydrogen phosphite, rat liver S9 mix was not a requirement for the activity of any of these compounds. Chemicals not identified as mutagens were water, tert-butyl alcohol, pyridine, and witch hazel.

  16. Identification of the herpes simplex virus DNA sequences present in six herpes simplex virus thymidine kinase-transformed mouse cell lines.

    PubMed Central

    Leiden, J M; Frenkel, N; Rapp, F

    1980-01-01

    We have used a novel filter hybridization approach to detect and map the herpes simplex virus (HSV) DNA sequences which are present in four HSV thymidine kinase (HSVtk+)-transformed cell lines which were derived by exposure of thymidine kinase negative (tk-) mouse cells to UV light-irradiated HSV type 2 (HSV-2). In addition, we have mapped the HSV-1 DNA sequences which are present in two HSV-1tk+-transformed cell lines produced by transfection of tk- mouse cells with sheared HSV-1 DNA. The results of these studies can be summarized as follows. (i) The only HSV DNA sequences which were common to all HSVtk+-transformed cells were those located between map coordinates 0.28 and 0.32. Thus, this region contains all of the viral DNA sequences which are necessary for the expression of HSV-mediated tk transformation. (ii) Many of the cell lines also contained variable amounts of non-tk gene viral DNA sequences located between map coordinates 0.11 to 0.57 and 0.82 to 1.00, suggesting that incorporation of the viral DNA sequences located between these map coordinates is a relatively random event. (iii) The viral DNA sequences located between map coordinates 0 to 0.11 and 0.57 to 0.82 were uniformly absent from all of the HSVtk+ cell lines tested, suggesting that there is a strong negative selective pressure against incorporation of these viral DNA sequences. Images PMID:6245232

  17. Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability.

    PubMed

    Foophow, Tita; Tanaka, Shun-ichi; Angkawidjaja, Clement; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2010-07-23

    Tk-SP is a hyperthermostable subtilisin-like serine protease from Thermococcus kodakaraensis and is autoprocessed from its precursor (Pro-Tk-SP) with N- and C-propeptides. The crystal structure of the active-site mutant of Pro-Tk-SP lacking C-propeptide, ProN-Tk-S359A, was determined at 2.0 A resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and beta-jelly roll domains. Two Ca(2+) ions bind to the beta-jelly roll domain. The overall structure of ProN-Tk-S359A without the beta-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca(2+) ions. To analyze the role of the beta-jelly roll domain of Tk-SP, we constructed a series of the active-site mutants of Tk-SP with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibited protease activities in gel assay, indicating that the beta-jelly roll domain is not required for folding or activity. However, the T(m) value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl(2) was lower than that of Tk-S359A by 29.4 degrees C. The T(m) value of Tk-S359A was decreased by 29.5 degrees C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca(2+)-bound form. Tk-SP highly resembles subtilisin-like serine proteases from Pyrococcus furiosus, Thermococcus gammatolerans, and Thermococcus onnurineus in size and amino acid sequence. We propose that attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment. PMID:20595040

  18. TK gene combined with mIL-2 and mGM-CSF genes in treatment of gastric cancer

    PubMed Central

    Guo, Shan-Yu; Gu, Qin-Long; Zhu, Zheng-Gang; Hong, He-Qun; Lin, Yan-Zhen

    2003-01-01

    AIM: Cancer gene therapy has received more and more attentions in the recent decade. Various systems of gene therapy for cancer have been developed. One of the most promising choices is the suicide gene. The product of thymidine kinase (TK) gene can convert ganciclovir (GCV) to phosphorylated GCV, which inhibits the synthesis of cell DNA, and then induces the cells to death. Cytokines play an important role in anti-tumor immunity. This experiment was designed to combine the TK gene and mIL-2/mGM-CSF genes to treat gastric cancer, and was expected to produce a marked anti-tumor effect. METHODS: TK gene was constructed into the retroviral vector pLxSN, and the mIL-2 and mGM-CSF genes were inserted into the eukaryotic expressing vector pIRES. The gastric cancer cells were transfected by retroviral serum that was harvested from the package cells. In vitro study, the transfected gastric cancer cells were maintained in the GCV- contained medium, to assay the cell killing effect and bystander effect. In vivo experiment, retroviral serum and cytokines plasmid were transfected into tumor-bearing mice, to observe the changes of tumor volumes and survival of the mice. RESULTS: In vitro experiment, 20% TK gene transduced cells could cause 70%-80% of total cells to death. In vivo results showed that there was no treatment effect in control group and TK/GCV could inhibit the tumor growth. The strongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis of the cancer in the treated groups. CONCLUSION: TK/GCV can kill tumor cells and inhibit the tumor growth in vivo. IL-2 and GM-CSF strongly enhance the anti-tumor effect. Through the retrovirus and liposome methods, the suicide gene and cytokine genes are all expressed in the tissues. PMID:12532437

  19. Towards a consistent eddy-covariance processing: an intercomparison of EddyPro and TK3

    NASA Astrophysics Data System (ADS)

    Fratini, G.; Mauder, M.

    2014-07-01

    A comparison of two popular eddy-covariance software packages is presented, namely, EddyPro and TK3. Two approximately 1-month long test data sets were processed, representing typical instrumental setups (i.e., CSAT3/LI-7500 above grassland and Solent R3/LI-6262 above a forest). The resulting fluxes and quality flags were compared. Achieving a satisfying agreement and understanding residual discrepancies required several iterations and interventions of different nature, spanning from simple software reconfiguration to actual code manipulations. In this paper, we document our comparison exercise and show that the two software packages can provide utterly satisfying agreement when properly configured. Our main aim, however, is to stress the complexity of performing a rigorous comparison of eddy-covariance software. We show that discriminating actual discrepancies in the results from inconsistencies in the software configuration requires deep knowledge of both software packages and of the eddy-covariance method. In some instances, it may be even beyond the possibility of the investigator who does not have access to and full knowledge of the source code. Being the developers of EddyPro and TK3, we could discuss the comparison at all levels of details and this proved necessary to achieve a full understanding. As a result, we suggest that researchers are more likely to get comparable results when using EddyPro (v5.1.1) and TK3 (v3.11) - at least with the setting presented in this paper - than they are when using any other pair of EC software which did not undergo a similar cross-validation. As a further consequence, we also suggest that, to the aim of assuring consistency and comparability of centralized flux databases, and for a confident use of eddy fluxes in synthesis studies on the regional, continental and global scale, researchers only rely on software that have been extensively validated in documented intercomparisons.

  20. Improving the safety of cell therapy with the TK-suicide gene.

    PubMed

    Greco, Raffaella; Oliveira, Giacomo; Stanghellini, Maria Teresa Lupo; Vago, Luca; Bondanza, Attilio; Peccatori, Jacopo; Cieri, Nicoletta; Marktel, Sarah; Mastaglio, Sara; Bordignon, Claudio; Bonini, Chiara; Ciceri, Fabio

    2015-01-01

    While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy. PMID:25999859

  1. Improving the safety of cell therapy with the TK-suicide gene

    PubMed Central

    Greco, Raffaella; Oliveira, Giacomo; Stanghellini, Maria Teresa Lupo; Vago, Luca; Bondanza, Attilio; Peccatori, Jacopo; Cieri, Nicoletta; Marktel, Sarah; Mastaglio, Sara; Bordignon, Claudio; Bonini, Chiara; Ciceri, Fabio

    2015-01-01

    While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy. PMID:25999859

  2. A Zinc-Dependent Protease AMZ-tk from a Thermophilic Archaeon is a New Member of the Archaemetzincin Protein Family.

    PubMed

    Jia, Baolei; Li, Zhengqun; Liu, Jinliang; Sun, Ying; Jia, Xiaomeng; Xuan, Yuan Hu; Zhang, Jiayan; Jeon, Che Ok

    2015-01-01

    A putative zinc-dependent protease (TK0512) in Thermococcus kodakarensis KOD1 shares a conserved motif with archaemetzincins, which are metalloproteases found in archaea, bacteria, and eukarya. Phylogenetic and sequence analyses showed that TK0512 and its homologues in Thermococcaceae represent new members in the archaemetzincins family, which we named AMZ-tk. We further confirmed its proteolytic activity biochemically by overexpression of the recombinant AMZ-tk in Escherichia coli and characterization of the purified enzyme. In the presence of zinc, the purified enzyme degraded casein, while adding EDTA strongly inhibited the enzyme activity. AMZ-tk also exhibited self-cleavage activity that required Zn(2+). These results demonstrated that AMZ-tk is a zinc-dependent protease within the archaemetzincin family. The enzyme displayed activity at alkaline pHs ranging from 7.0 to 10.0, with the optimal pH being 8.0. The optimum temperature for the catalytic activity of AMZ-tk was 55°C. Quantitative reverse transcription-PCR revealed that transcription of AMZ-tk was also up-regulated after exposing the cells to 55 and 65°C. Mutant analysis suggested that Zn(2+) binding histidine and catalytic glutamate play key roles in proteolysis. AMZ-tk was thermostable on incubation for 4 h at 70°C in the presence of EDTA. AMZ-tk also retained >50% of its original activity in the presence of both laboratory surfactants and commercial laundry detergents. AMZ-tk further showed antibacterial activity against several bacteria. Therefore, AMZ-tk is of considerable interest for many purposes in view of its activity at alkaline pH, detergents, and thermostability. PMID:26733945

  3. A Zinc-Dependent Protease AMZ-tk from a Thermophilic Archaeon is a New Member of the Archaemetzincin Protein Family

    PubMed Central

    Jia, Baolei; Li, Zhengqun; Liu, Jinliang; Sun, Ying; Jia, Xiaomeng; Xuan, Yuan Hu; Zhang, Jiayan; Jeon, Che Ok

    2015-01-01

    A putative zinc-dependent protease (TK0512) in Thermococcus kodakarensis KOD1 shares a conserved motif with archaemetzincins, which are metalloproteases found in archaea, bacteria, and eukarya. Phylogenetic and sequence analyses showed that TK0512 and its homologues in Thermococcaceae represent new members in the archaemetzincins family, which we named AMZ-tk. We further confirmed its proteolytic activity biochemically by overexpression of the recombinant AMZ-tk in Escherichia coli and characterization of the purified enzyme. In the presence of zinc, the purified enzyme degraded casein, while adding EDTA strongly inhibited the enzyme activity. AMZ-tk also exhibited self-cleavage activity that required Zn2+. These results demonstrated that AMZ-tk is a zinc-dependent protease within the archaemetzincin family. The enzyme displayed activity at alkaline pHs ranging from 7.0 to 10.0, with the optimal pH being 8.0. The optimum temperature for the catalytic activity of AMZ-tk was 55°C. Quantitative reverse transcription-PCR revealed that transcription of AMZ-tk was also up-regulated after exposing the cells to 55 and 65°C. Mutant analysis suggested that Zn2+ binding histidine and catalytic glutamate play key roles in proteolysis. AMZ-tk was thermostable on incubation for 4 h at 70°C in the presence of EDTA. AMZ-tk also retained >50% of its original activity in the presence of both laboratory surfactants and commercial laundry detergents. AMZ-tk further showed antibacterial activity against several bacteria. Therefore, AMZ-tk is of considerable interest for many purposes in view of its activity at alkaline pH, detergents, and thermostability. PMID:26733945

  4. B Plant canyon sample TK-21-1 analytical results for the final report

    SciTech Connect

    Steen, F.H.

    1998-04-10

    This document is the analytical laboratory report for the TK-21-1 sample collected from the B Plant Canyon on February 18, 1998. The sample was analyzed in accordance with the Sampling and Analysis Plan for B Plant Solutions (SAP) (Simmons, 1997) in support of the B Plant decommissioning project. Samples were analyzed to provide data both to describe the material which would remain in the tanks after the B Plant transition is complete and to determine Tank Farm compatibility. The analytical results are included in the data summary table (Table 1).

  5. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

    NASA Astrophysics Data System (ADS)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

    2006-05-01

    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the ACV-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  6. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

    NASA Astrophysics Data System (ADS)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

    2006-09-01

    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  7. Effect of caffeine on radiation-induced apoptosis in TK6 cells

    SciTech Connect

    Zhen, W.; Vaughan, A.T.M.

    1995-02-01

    Apoptosis has been measured in cells of the human TK6 lymphoblastoid cell line by recording the release of endonuclease-digested DNA from affected cells using flow cytometry. In asynchronously dividing cells, DNA degradation characteristic of apoptosis was first seen 12 h after irradiation as a defined DNA fluorescent peak of sub-G{sub 1}-phase content, reaching a maximum of 30-50% of the population by 24-72 h. Treating cells with 2 mM caffeine either before or up to 3 h after irradiation eliminated the degradation of DNA entirely. In addition, the percentage of cells in which apoptosis could be detected microscopically decreased from 62.4 {+-} 0.95% to 16.7 {+-} 1.5% 72 h after caffeine treatment. Delaying caffeine treatment for 12 h after irradiation reduced DNA degradation by approximately 50% compared to cells receiving radiation alone. DNA degradation induced by serum deprivation was unaffected by caffeine treatment. These data support the contention that irradiation of TK6 cells produces a long-lived cellular signal which triggers apoptosis. Apoptosis produced by serum deprivation does not operate through the same pathway. 36 refs., 5 figs.

  8. Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

    PubMed Central

    Ghorbel, Sofiane; Smirnov, Aleksey; Chouayekh, Hichem; Sperandio, Brice; Esnault, Catherine; Kormanec, Jan; Virolle, Marie-Joelle

    2006-01-01

    The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed. PMID:16923894

  9. Oncolytic Adenoviruses Armed with Thymidine Kinase Can Be Traced by PET Imaging and Show Potent Antitumoural Effects by Ganciclovir Dosing

    PubMed Central

    Abate-Daga, Daniel; Andreu, Nuria; Camacho-Sánchez, Juan; Alemany, Ramon; Herance, Raúl; Millán, Olga; Fillat, Cristina

    2011-01-01

    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. PMID:22028820

  10. Responses of the L51781Y tk/sup +//tk/sup -/ mouse lymphoma cell forward mutation assay: III. 72 coded chemicals

    SciTech Connect

    McGregor, D.B.; Brown, A.; Cattanach, P.; Edwards, I.; McBride, D.; Riach, C.; Caspary, W.J.

    1988-01-01

    Seventy-two chemicals were tested for their mutagenic potential in the L51781Y tk/sup +///sup -/ mouse lymphoma cell forward mutation assay, using procedures based upon those described previously. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before planting in soft agar with or without trifluorothymidine (TFT), 3 ..mu..g/ml. The chemicals were tested at least twice. Significant responses were obtained with allyl isothiocyanate, p-benzoquinone dioxime, benzyl acetate, 2-biphenylamine HCl, bis(2-chloro-1-methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2-chloroethanol, chlorothalonil, cytarabine x HCl, p,p'-DDE, diazinon, 2,6-dichloro-p-phenylenediamine, N,N-diethylthiourea, diglycidylresorcinol ether, 2,4-dimethoxy aniline x HCl, disperse yellow 3, endosulfan, 1,2-epoxyhexadecane, ethyl acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4'-methylenedianiline x 2 HCl, methyl viologen, nickel sulfate x 6H/sub 2/O, 4,4'-oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6-tetrachloro-4-nitro-anisole, 1,1,1,2-tetrachloroethane, trichlorfon, 2,4,6-trichlorophenol, 2,4,5-trimethoxybenzaldehyde, 1,1,3-trimethyl-2-thiourea, 1-vinyl-3-cyclopetene dioxide, vinyl toluene, and ziram. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these benzyl alcohol, 1,4-dichlorobenzene, phenol, succinic acid-2,2-dimethyl hydrazide, and toluene.

  11. A predictive toxicogenomics signature to classify genotoxic versus non-genotoxic chemicals in human TK6 cells

    PubMed Central

    Williams, Andrew; Buick, Julie K.; Moffat, Ivy; Swartz, Carol D.; Recio, Leslie; Hyduke, Daniel R.; Li, Heng-Hong; Fornace, Albert J.; Aubrecht, Jiri; Yauk, Carole L.

    2015-01-01

    Genotoxicity testing is a critical component of chemical assessment. The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the DNA damage response pathways involved in response, is becoming more common. In companion papers previously published in Environmental and Molecular Mutagenesis, Li et al. (2015) [6] developed a dose optimization protocol that was based on evaluating expression changes in several well-characterized stress-response genes using quantitative real-time PCR in human lymphoblastoid TK6 cells in culture. This optimization approach was applied to the analysis of TK6 cells exposed to one of 14 genotoxic or 14 non-genotoxic agents, with sampling 4 h post-exposure. Microarray-based transcriptomic analyses were then used to develop a classifier for genotoxicity using the nearest shrunken centroids method. A panel of 65 genes was identified that could accurately classify toxicants as genotoxic or non-genotoxic. In Buick et al. (2015) [1], the utility of the biomarker for chemicals that require metabolic activation was evaluated. In this study, TK6 cells were exposed to increasing doses of four chemicals (two genotoxic that require metabolic activation and two non-genotoxic chemicals) in the presence of rat liver S9 to demonstrate that S9 does not impair the ability to classify genotoxicity using this genomic biomarker in TK6cells. PMID:26425668

  12. Auditing the TK and TPACK Confidence of Pre-Service Teachers: Are They Ready for the Profession?

    ERIC Educational Resources Information Center

    Jamieson-Proctor, Romina; Finger, Glenn; Albion, Peter

    2010-01-01

    Teacher education graduates need appropriate levels of confidence and capabilities in relation to technological knowledge (TK) as a basis for having technological pedagogical content knowledge (TPACK) to meet the challenges of learning and teaching in the 21st century. However, it should not be assumed that tomorrow's teachers enter the profession…

  13. Removal of methanethiol, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide from contaminated air by Thiobacillus thioparus TK-m

    SciTech Connect

    Kanagawa, T.; Mikami, E.

    1989-03-01

    Methanethiol, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide were efficiently removed from contaminated air by Thiobacillus thioparus TK-m and oxidized to sulfate stoichiometrically. More than 99.99% of dimethyl sulfide was removed when the load was less than 4.0 g of dimethyl sulfide per g (dry cell weight) per day.

  14. Development of a novel mouse tk{sup +/-} embryonic stem cell line for use in mutagenicity studies

    SciTech Connect

    Dobrovolsky, V.N.; Casciano, D.A.; Heflich, R.H.

    1996-12-31

    A tk{sup +/-} mouse embryonic stem (ES) cell line, designated 1G2, has been created in which one allele of the thymidine kinase (tk) gene was inactivated by targeted homologous recombination. This line is an analog of the mouse lymphoma tk{sup +/-} L5178Y cell line, which is used widely to assess the mutagenicity of chemical agents. Treatment of 1G2 cells with the alkylating agent N-ethyl-N-nitrosourea (ENU) resulted in a dose-related increase in tribluorothymidine-resistant colonies. Mutant frequencies of 152 and 296 per 10{sup 6} cells were determined for 0.1 and 0.3 mg/ml doses of ENU, compared with a spontaneous mutant frequency of 15 per 10{sup 6} cells. The data indicate that tk{sup +/-} 1G2 ES cells may be useful for the creation of a transgenic mouse model for assessing in vivo mutation using an endogenous autosomal gene. 45 refs., 2 figs., 1 tab.

  15. Sequence, Structure, and Binding Analysis of Cyclodextrinase (TK1770) from T. kodakarensis (KOD1) Using an In Silico Approach

    PubMed Central

    Ali, Ramzan; Shafiq, Muhammad Imtiaz

    2015-01-01

    Thermostable cyclodextrinase (Tk1770 CDase) from hyperthermophilic archaeon Thermococcus kodakarensis (KOD1) hydrolyzes cyclodextrins into linear dextrins. The sequence of Tk1770 CDase retrieved from UniProt was aligned with sequences of sixteen CD hydrolyzing enzymes and a phylogenetic tree was constructed using Bayesian inference. The homology model of Tk1770 CDase was constructed and optimized with Modeller v9.14 program. The model was validated with ProSA server and PROCHECK analysis. Four conserved regions and the catalytic triad consisting of Asp411, Glu437, and Asp502 of GH13 family were identified in catalytic site. Also an additional fifth conserved region downstream to the fourth region was also identified. The structure of Tk1770 CDase consists of an additional N′-domain and a helix-loop-helix motif that is conserved in all archaeal CD hydrolyzing enzymes. The N′-domain contains an extended loop region that forms a part of catalytic domain and plays an important role in stability and substrate binding. The docking of substrate into catalytic site revealed the interactions with different conserved residues involved in substrate binding and formation of enzyme-substrate complex. PMID:26819569

  16. Histone and TK0471/TrmBL2 form a novel heterogeneous genome architecture in the hyperthermophilic archaeon Thermococcus kodakarensis.

    PubMed

    Maruyama, Hugo; Shin, Minsang; Oda, Toshiyuki; Matsumi, Rie; Ohniwa, Ryosuke L; Itoh, Takehiko; Shirahige, Katsuhiko; Imanaka, Tadayuki; Atomi, Haruyuki; Yoshimura, Shige H; Takeyasu, Kunio

    2011-02-01

    Being distinct from bacteria and eukaryotes, Archaea constitute a third domain of living things. The DNA replication, transcription, and translation machineries of Archaea are more similar to those of eukaryotes, whereas the genes involved in metabolic processes show more similarity to their bacterial counterparts. We report here that TK0471/TrmB-like 2 (TrmBL2), in addition to histone, is a novel type of abundant chromosomal protein in the model euryarchaeon Thermococcus kodakarensis . The chromosome of T. kodakarensis can be separated into regions enriched either with histone, in which the genetic material takes on a “beads-on-a-string” appearance, or with TK0471/TrmBL2, in which it assumes a thick fibrous structure. TK0471/TrmBL2 binds to both coding and intergenic regions and represses transcription when bound to the promoter region. These results show that the archaeal chromosome is organized into heterogeneous structures and that TK0471/TrmBL2 acts as a general chromosomal protein as well as a global transcriptional repressor. PMID:21148291

  17. ANALYSIS OF TRIFLUOROTHYMIDINE-RESISTANT (TFT(SUP R)) MUTANTS OF L5178Y/TK(SUP +/-) MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    Three classes of TFTr variants of L5178Y/TK+/- -3.72C mouse lymphoma cells can be identified - large colony (lambda), small colony (sigma),and tiny colony (tau). The sigma and lambda mutants are detectable in the routine mutagenesis assay using soft agar cloning. The tau mutants ...

  18. Towards a consistent eddy-covariance processing: an intercomparison of EddyPro and TK3

    NASA Astrophysics Data System (ADS)

    Fratini, G.; Mauder, M.

    2014-03-01

    A comparison of two popular eddy-covariance (EC) software packages is presented, namely EddyPro and TK3. Two about one-month long test datasets were processed, representing typical instrumental setups, i.e. CSAT3/LI-7500 above grassland and Solent R3/LI-6262 above a forest. The resulting fluxes and quality flags were compared. Achieving a satisfying agreement and understanding residual discrepancies required several iterations and interventions of different nature, spanning from simple software reconfiguration to actual code manipulations. In this paper, we document our comparison exercise and show that the two software packages can provide utterly satisfying agreement when properly configured. Our main aim, however, is to stress the complexity of performing a rigorous comparison of EC software. We show that discriminating actual discrepancies in the results from inconsistencies in the software configuration requires deep knowledge of both software packages and of the eddy-covariance method itself. In some instances, it may be even beyond the possibility of the investigator who does not control the source code. Being the developers of EddyPro and TK3, we could discuss the comparison at all levels of details and this proved necessary to achieve a full understanding. As a further consequence, we also suggest that, to the aim of assuring consistency and comparability of centralized flux databases, and for a confident use of eddy fluxes in synthesis studies on the regional, continental and global scale, researchers rely on established software, notably those that have been extensively validated in documented intercomparisons.

  19. Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

    PubMed

    Cheung, Jennifer R; Dickinson, Donna A; Moss, Jocelyn; Schuler, Maik J; Spellman, Richard A; Heard, Pamela L

    2015-01-01

    The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds. PMID:25726170

  20. Effect of Chemical Mutagens and Carcinogens on Gene Expression Profiles in Human TK6 Cells

    PubMed Central

    Godderis, Lode; Thomas, Reuben; Hubbard, Alan E.; Tabish, Ali M.; Hoet, Peter; Zhang, Luoping; Smith, Martyn T.; Veulemans, Hendrik; McHale, Cliona M.

    2012-01-01

    Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose–response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control. PMID:22723965

  1. Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

    PubMed

    Zhang, Shulin; Li, Fang-Yuan; Bass, Harold N; Pursley, Amber; Schmitt, Eric S; Brown, Blaire L; Brundage, Ellen K; Mardach, Rebecca; Wong, Lee-Jun

    2010-01-01

    Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects. PMID:19815440

  2. Towards a consistent eddy-covariance processing: a comparison between EddyPro and TK3

    NASA Astrophysics Data System (ADS)

    Fratini, Gerardo; Mauder, Matthias; Griessbaum, Frank; Foken, Thomas

    2013-04-01

    The eddy-covariance processing sequence, needed to obtain accurate mass and energy fluxes starting from turbulence data is complex, depending on the instruments of choices and their deployment, the site characteristics, and the atmospheric turbulence peculiarities, at a minimum. Eddy-covariance software available to the community support different implementations, all valid in principle, and often the same procedures are therein implemented in different ways, or different order. In addition, many groups use "in-house" collections of scripts that may include customized implementations. It is often found that such differences do show up to the researcher who attempts a software inter-comparison, as either systematic or random differences in resulting fluxes. In this work we present a comparison of two popular eddy-covariance software, namely EddyPro and TK3. The aim of the comparison is twofold: on the one side, we want to show that the two software can provide perfectly matching fluxes. On the other side, we want to stress on what it takes to achieve this result. In fact, performing a fair and rigorous software comparison is not a trivial task, and discriminating actual discrepancies in the results from inaccuracies in the software configuration may be beyond the possibility of the researcher who does not control the source code. Being the developers of EddyPro and TK3 gave us the opportunity to discuss the comparison at all levels of details, and this proved necessary to get to a full agreement. However, normally this is not possible to the software user. As a conclusion, we want to warn against "quick and dirty" inter-comparisons as a means to validate eddy-covariance software. To the aim of assuring consistency and inter-comparability of centralized flux databases, and for a confident use of eddy fluxes on the regional, continental and global scale synthesis studies, we also warn against the proliferation of in-house software. We rather suggest researchers to

  3. Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

    PubMed Central

    Lengronne, Armelle; Pasero, Philippe; Bensimon, Aaron; Schwob, Etienne

    2001-01-01

    Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants. PMID:11266543

  4. Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6T)

    SciTech Connect

    Zeytun, Ahmet; Sikorski, Johannes; Nolan, Matt; Lapidus, Alla L.; Lucas, Susan; Han, James; Tice, Hope; Cheng, Jan-Fang; Tapia, Roxanne; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Ivanova, N; Mavromatis, K; Mikhailova, Natalia; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Ngatchou, Olivier Duplex; Land, Miriam L; Hauser, Loren John; Chang, Yun-Juan; Jeffries, Cynthia; Han, Cliff; Detter, J. Chris; Ubler, Susanne; Rohde, Manfred; Tindall, Brian; Wirth, Reinhard; Woyke, Tanja; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Klenk, Hans-Peter; Kyrpides, Nikos C

    2011-01-01

    Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6T is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO2 via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  5. Humic acids reduce the genotoxicity of mitomycin C in the human lymphoblastoid cell line TK6.

    PubMed

    Ferrara, G; Loffredo, E; Senesi, N; Marcos, R

    2006-01-31

    The antimutagenic/desmutagenic activity of a leonardite humic acid (LHA) and a soil humic acid (SHA) was studied in the cultured human lymphoblastoid cell line TK6 treated with mitomycin C (MMC) as reference mutagen by evaluating the induction of micronuclei (MN). Two different concentrations of HA were used, 2.5 and 10 microg/ml, in three different treatments: (1) HA alone (genotoxic test); (2) HA after 2-h pre-incubation with 0.3 microM of MMC (desmutagenic test) and (3) combinations of HA and MMC at 0.3 microM without pre-incubation (antimutagenic test). Neither of the HA used alone did produce genotoxic effects, but both HAs reduced significantly the frequencies of MN induced by MMC, especially in the desmutagenic test. A slight cell-protective effect against the cytotoxicity of MMC was also exhibited by the two HAs in the desmutagenic test. The LHA showed a desmutagenic/antimutagenic activity that was more pronounced than that of SHA, which is possibly related to the higher carboxylic group content and lower phenolic group content of LHA. These results confirm the antigenotoxic action exerted by HAs in human cells, similarly to what has been previously observed in various plant species. PMID:16386451

  6. Coupling cytotoxicity biomarkers with DNA damage assessment in TK6 human lymphoblast cells.

    PubMed

    Shi, Jing; Springer, Sandra; Escobar, Patricia

    2010-02-01

    There is considerable discussion within the scientific community as to the appropriate measures of cytotoxicity to use when deciding on the maximum concentration of a substance to test in vitro for its ability to induce DNA damage using the Comet assay. Conventional cytotoxicity assessment methods, such as trypan blue dye exclusion or relative cell number (cell counts) may not be the most biologically relevant measurement for cytotoxicity in this assay. Thus, we evaluated for decreased levels of adenosine triphosphate (ATP) and activation of Caspase-3/7 as well as relative cell number and trypan blue exclusion in order to understand the correlation among test compound concentration, cytotoxicity and genotoxicity outcomes in the Comet assay. We tested two non-genotoxic and non-cytotoxic compounds (d-glucose and ethanol), two non-genotoxic but cytotoxic compounds (2,4-dichlorophenol and tunicamycin) and four genotoxic and cytotoxic compounds (methyl methanesulfonate, ethyl methanesulfonate, etoposide and 4-nitroquinoline-N-oxide) in TK6 human lymphoblast cells. Our data show that measuring ATP and Caspase-3/7 levels provides more rapid and perhaps more biologically relevant measures of cytotoxicity compared with trypan blue dye exclusion and relative cell number. Furthermore, incorporating these two assays into the Comet assay also provided insight on the cytotoxic mode of action of the chemicals tested. By extrapolation, such assays may also be useful in other in vitro genotoxicity assays. PMID:20100597

  7. TK3 eBook software to author, distribute, and use electronic course content for medical education.

    PubMed

    Morton, David A; Foreman, K Bo; Goede, Patricia A; Bezzant, John L; Albertine, Kurt H

    2007-03-01

    The methods for authoring and distributing course content are undergoing substantial changes due to advancement in computer technology. Paper has been the traditional method to author and distribute course content. Paper enables students to personalize content through highlighting and note taking but does not enable the incorporation of multimedia elements. Computers enable multimedia content but lack the capability of the user to personalize the content. Therefore, we investigated TK3 eBooks as a potential solution to incorporate the benefits of both paper and computer technology. The objective of our study was to assess the utility of TK3 eBooks in the context of authoring and distributing dermatology course content for use by second-year medical students at the University of Utah School of Medicine during the spring of 2004. We incorporated all dermatology course content into TK3 eBook format. TK3 eBooks enable students to personalize information through tools such as "notebook," "hiliter," "stickies," mark pages, and keyword search. Students were given the course content in both paper and eBook formats. At the conclusion of the dermatology course, students completed a questionnaire designed to evaluate the effectiveness of the eBooks compared with paper. Students perceived eBooks as an effective way to distribute course content and as a study tool. However, students preferred paper over eBooks to take notes during lecture. In conclusion, the present study demonstrated that eBooks provide a convenient method for authoring, distributing, and using course content but that students preferred paper to take notes during lecture. PMID:17327584

  8. A semi-mechanistic integrated toxicokinetic–toxicodynamic (TK/TD) model for arsenic(III) in hepatocytes

    PubMed Central

    Stamatelos, Spyros K.; Androulakis, Ioannis P.; Kong, Ah-Ng Tony; Georgopoulos, Panos G.

    2014-01-01

    Background A systems engineering approach is presented for describing the kinetics and dynamics that are elicited upon arsenic exposure of human hepatocytes. The mathematical model proposed here tracks the cellular reaction network of inorganic and organic arsenic compounds present in the hepatocyte and analyzes the production of toxicologically potent by-products and the signaling they induce in hepatocytes. Methods and results The present modeling effort integrates for the first time a cellular-level semi-mechanistic toxicokinetic (TK) model of arsenic in human hepatocytes with a cellular-level toxicodynamic (TD) model describing the arsenic-induced reactive oxygen species (ROS) burst, the antioxidant response, and the oxidative DNA damage repair process. The antioxidant response mechanism is described based on the Keap1-independent Nuclear Factor-erythroid 2-related factor 2 (Nrf2) signaling cascade and accounts for the upregulation of detoxifying enzymes. The ROS-induced DNA damage is simulated by coupling the TK/TD formulation with a model describing the multistep pathway of oxidative DNA repair. The predictions of the model are assessed against experimental data of arsenite-induced genotoxic damage to human hepatocytes; thereby capturing in silico the mode of the experimental dose–response curve. Conclusions The integrated cellular-level TK/TD model presented here provides significant insight into the underlying regulatory mechanism of Nrf2-regulated antioxidant response due to arsenic exposure. While computational simulations are in a fair good agreement with relevant experimental data, further analysis of the system unravels the role of a dynamic interplay among the feedback loops of the system in controlling the ROS upregulation and DNA damage response. This TK/TD framework that uses arsenic as an example can be further extended to other toxic or pharmaceutical agents. PMID:23069314

  9. Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

    SciTech Connect

    Yu, Zhendong; Wang, Hao; Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li; Li, Pengfei

    2009-09-04

    CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.

  10. Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

    NASA Technical Reports Server (NTRS)

    Evans, Helen H.; Horng, Min-Fen; Ricanati, Marlene; Diaz-Insua, Mireya; Jordan, Robert; Schwartz, Jeffrey L.

    2002-01-01

    Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.

  11. 8-Hydroxy-2'-deoxyguanosine as a biomarker of oxidative DNA damage induced by perfluorinated compounds in TK6 cells.

    PubMed

    Yahia, Doha; Haruka, Igarashi; Kagashi, Yae; Tsuda, Shuji

    2016-02-01

    8-Hydroxy-2'-deoxyguanosine (8-OHdG) is the most common biomarker of oxidative DNA damage, it is formed by chemical carcinogens and can be measured in any species. Perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) are suspected genotoxic carcinogens through induction of reactive oxygen species that are responsible for oxidative DNA damage. This study was conducted to investigate the in vitro genotoxicity of PFOA and PFNA in human lymphoblastoid (TK6) cell line. TK6 cells were exposed to PFOA at 0, 125, 250, and 500 ppm and PFNA at 125 and 250 ppm for 2 h. Single cell gel electrophoresis (comet assay) was used to measure DNA damage; at least 50 cells per sample were analyzed using comet Assay Software Project (CASP). 8-OHdG was measured in DNA of exposed cells using high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Results showed that both PFOA and PFNA induced DNA damage indicated by increased tail length (DNA migration). The level of 8-OHdG was increased in a dose-dependent manner in both PFOA and PFNA exposure. We concluded that PFOA and PFNA induced DNA damage and the biomarker of oxidative DNA damage (8-OHdG) could be measured by HPLC-MS/MS. In addition, PFNA produced high level of 8-OHdG at concentrations lower than PFOA, this may indicate that PFNA is more potent genotoxicant for TK6 cells than PFOA. PMID:25113910

  12. Exogenous p53 and ASPP2 expression enhances rAdV-TK/GCV-induced death in hepatocellular carcinoma cells lacking functional p53

    PubMed Central

    Guo, Xianghua; Wei, Feili; Yin, Jiming; Zang, Yunjin; Li, Ning; Chen, Dexi

    2016-01-01

    Suicide gene therapy using herpes simplex virus-1 thymidine kinase (HSV-TK) in combination with ganciclovir (GCV) has emerged as a potential new method for treating cancer. We hypothesize that the efficacy of HSV-TK/GCV therapy is at least partially dependent on p53 status in hepatocellular carcinoma (HCC) patients. Using recombinant adenoviral vectors (rAdV), TK, p53, and ASPP2 were overexpressed individually and in combination in Hep3B (p53 null) and HepG2 (p53 wild-type) cell lines and in primary HCC tumor cells. p53 overexpression induced death in Hep3B cells, but not HepG2 cells. ASPP2 overexpression increased rAdV-TK/GCV-induced HepG2 cell death by interacting with endogenous p53. Similarly, ASPP2 reduced survival in rAdV-TK/GCV-treated primary HCC cells expressing p53 wild-type but not a p53 R249S mutant. Mutated p53 was unable to bind to ASPP2, suggesting that the increase in rAdV-TK/GCV-induced cell death resulting from ASPP2 overexpression was dependent on its interaction with p53. Additionally, γ-H2AX foci, ATM phosphorylation, Bax, and p21 expression increased in rAdV-TK/GCV-treated HepG2 cells as compared to Hep3B cells. This suggests that the combined use of HSV-TK, GCV, rAdV-p53 and rAdV-ASPP2 may improve therapeutic efficacy in HCC patients lacking functional p53. PMID:26934443

  13. Concentration of thymidine kinase 1 in serum (S-TK1) is a more sensitive proliferation marker in human solid tumors than its activity.

    PubMed

    He, Qimin; Zhang, Pinggn; Zou, Li; Li, Hongxun; Wang, Xiuqin; Zhou, Shan; Fornander, Tommy; Skog, Sven

    2005-10-01

    Activity of thymidine kinase 1 in serum (STK) is a useful marker for leukaemia and lymphoma, but not for solid tumors. We investigate thymidine kinase 1 concentration in serum (S-TK1) as a potential tumor marker. S-TK1 concentration and STK activity levels were determined in 9 human malignant diseases (breast, gastric, rectal, colorectal, lung, brain cancer, hepatoma, lymphoma, leukaemia) and in benign and non-cancerous diseases, representing 850 preoperative cases. Healthy volunteers (n=43) were used as positive controls. S-TK1 concentration was determined by ECL dot blot assay and STK activity levels by an RIA assay. S-TK1 concentrations and STK activity levels in preoperative malignant patients were significantly higher than in healthy individuals, in patients with benign tumors and in those with non-cancerous diseases. Significant correlations between concentration and activity level were only found in healthy individuals, in patients with benign tumors, and in some patients with malignancies, i.e. leukaemia, and breast and gastric cancers. About 90-95 percent of the malignant patients showed S-TK1 concentrations above those of the healthy controls. The corresponding value for STK activity was about 75 percent. When sera from malignant patients were diluted with sera from healthy individuals, S-TK1 concentrations and STK activity levels decreased more than expected. This indicates the presence of a compound (or compounds) in the serum of healthy individuals that destabilises S-TK1. We conclude that S-TK1 concentration is a more sensitive tumor marker in solid malignancies than is STK activity. PMID:16142366

  14. Purification and biological evaluation of the metabolites produced by Streptomyces sp. TK-VL_333.

    PubMed

    Kavitha, Alapati; Prabhakar, Peddikotla; Vijayalakshmi, Muvva; Venkateswarlu, Yenamandra

    2010-06-01

    An Actinobacterium strain isolated from laterite soils of the Guntur region was identified as Streptomyces sp. TK-VL_333 by 16S rRNA analysis. Cultural, morphological and physiological characteristics of the strain were recorded. The secondary metabolites produced by the strain cultured on galactose-tyrosine broth were extracted and concentrated followed by defatting of the crude extract with cyclohexane to afford polar and non-polar residues. Purification of the two residues by column chromatography led to isolation of five polar and one non-polar fraction. Bioactivity- guided fractions were rechromatographed on a silica gel column to obtain four compounds, namely 1H-indole-3-carboxylic acid, 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one and acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester from three active polar fractions and 8-methyl decanoic acid from one non-polar fraction. The structure of the compounds was elucidated on the basis of FT-IR, mass and NMR spectroscopy. The antimicrobial activity of the bioactive compounds produced by the strain was tested against the bacteria and fungi and expressed in terms of minimum inhibitory concentration. Antifungal activity of indole-3-carboxylic acid was further evaluated under in vitro and in vivo conditions. This is the first report of 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one, acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester and 8-methyl decanoic acid from the genus Streptomyces. PMID:20403429

  15. Induction of centrosome amplification by formaldehyde, but not hydroquinone, in human lymphoblastoid TK6 cells.

    PubMed

    Ji, Zhiying; McHale, Cliona M; Bersonda, Jessica; Tung, Judy; Smith, Martyn T; Zhang, Luoping

    2015-07-01

    Benzene and formaldehyde (FA) are important industrial chemicals and environmental pollutants that cause leukemia by inducing DNA damage and chromosome aberrations in hematopoietic stem cells (HSC), the target cells for leukemia. Our previous studies showed that workers exposed to benzene and FA exhibit increased levels of aneuploidy in their blood cells. As centrosome amplification is a common phenomenon in human cancers, including leukemia, and is associated with aneuploidy in carcinogenesis, we hypothesized that benzene and FA would induce centrosome amplification in vitro. We treated human lymphoblastoid TK6 cells with a range of concentrations of hydroquinone (HQ, a benzene metabolite) or FA for 24 h, allowed the cells to recover in fresh medium for 24 h, and examined centrosome amplification; chromosomal gain, loss, and breakage; and cytotoxicity. We included melphalan and etoposide, chemotherapeutic drugs that cause therapy-related acute myeloid leukemia and that have been shown to induce centrosome amplification as well as chromosomal aneuploidy and breakage, as positive controls. Melphalan and etoposide induced centrosome amplification and chromosome gain and breakage in a dose-dependent manner, at cytotoxic concentrations. HQ, though cytotoxic, did not induce centrosome amplification or any chromosomal aberration. FA-induced centrosome amplification and cytotoxicity, but did not induce chromosomal aberrations. Our data suggest, for the first time, that centrosome amplification is a potential mechanism underlying FA-induced leukemogenesis, but not benzene-induced leukemogenesis, as mediated through HQ. Future studies are needed to delineate the mechanisms of centrosome amplification and its association with DNA damage, chromosomal aneuploidy and carcinogenesis, following exposure to FA. PMID:25821186

  16. Molecular conformations, interactions, and properties associated with drug efficiency and clinical performance among VEGFR TK inhibitors

    SciTech Connect

    McTigue, Michele; Murray, Brion William; Chen, Jeffrey H.; Deng, Ya-Li; Solowiej, James; Kania, Robert S.

    2012-09-17

    We performed analyses of compounds in clinical development which have shown that ligand efficient-molecules with privileged physical properties and low dose are less likely to fail in the various stages of clinical testing, have fewer postapproval withdrawals, and are less likely to receive black box safety warnings. However, detailed side-by-side examination of molecular interactions and properties within single drug classes are lacking. As a class, VEGF receptor tyrosine kinase inhibitors (VEGFR TKIs) have changed the landscape of how cancer is treated, particularly in clear cell renal cell carcinoma, which is molecularly linked to the VEGF signaling axis. Despite the clear role of the molecular target, member molecules of this validated drug class exhibit distinct clinical efficacy and safety profiles in comparable renal cell carcinoma clinical studies. The first head-to-head randomized phase III comparative study between active VEGFR TKIs has confirmed significant differences in clinical performance [Rini BI, et al. (2011) Lancet 378:193–1939]. To elucidate how fundamental drug potency–efficiency is achieved and impacts differentiation within the VEGFR TKI class, we determined potencies, time dependence, selectivities, and X-ray structures of the drug–kinase complexes using a VEGFR2 TK construct inclusive of the important juxtamembrane domain. Collectively, the studies elucidate unique drug–kinase interactions that are dependent on distinct juxtamembrane domain conformations, resulting in significant potency and ligand efficiency differences. Finally, the identified structural trends are consistent with in vitro measurements, which translate well to clinical performance, underscoring a principle that may be broadly applicable to prospective drug design for optimal in vivo performance.

  17. HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized mice

    PubMed Central

    Gschweng, Eric H.; McCracken, Melissa N.; Kaufman, Michael L.; Ho, Michelle; Hollis, Roger P.; Wang, Xiaoyan; Saini, Navdeep; Koya, Richard C.; Chodon, Thinle; Ribas, Antoni; Witte, Owen N.; Kohn, Donald B.

    2014-01-01

    Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell-receptors (TCR) or chimeric antigen receptors (CAR) generates a continual supply of effector T-cells, potentially providing superior anti-cancer efficacy compared with the infusion of terminally differentiated T-cells. Here we demonstrate the in vivo generation of functional effector T-cells from CD34-enriched human peripheral blood stem cells (PBSC) modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, co-expressing the PET/suicide gene sr39TK. Ex vivo analysis of T-cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir (GCV), but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. PMID:25038231

  18. Integration of metabolic activation with a predictive toxicogenomics signature to classify genotoxic versus nongenotoxic chemicals in human TK6 cells.

    PubMed

    Buick, Julie K; Moffat, Ivy; Williams, Andrew; Swartz, Carol D; Recio, Leslie; Hyduke, Daniel R; Li, Heng-Hong; Fornace, Albert J; Aubrecht, Jiri; Yauk, Carole L

    2015-07-01

    The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion article, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or nongenotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and nongenotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4 hr and collected 0 hr, 4 hr, and 20 hr postexposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24 hr. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as nongenotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24 hr, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. PMID:25733247

  19. Fragile-to-fragile liquid transition at Tg and stable-glass phase nucleation rate maximum at the Kauzmann temperature TK

    NASA Astrophysics Data System (ADS)

    Tournier, Robert F.

    2014-12-01

    An undercooled liquid is unstable. The driving force of the glass transition at Tg is a change of the undercooled-liquid Gibbs free energy. The classical Gibbs free energy change for a crystal formation is completed including an enthalpy saving. The crystal growth critical nucleus is used as a probe to observe the Laplace pressure change Δp accompanying the enthalpy change -Vm×Δp at Tg where Vm is the molar volume. A stable glass-liquid transition model predicts the specific heat jump of fragile liquids at T≤Tg, the Kauzmann temperature TK where the liquid entropy excess with regard to crystal goes to zero, the equilibrium enthalpy between TK and Tg, the maximum nucleation rate at TK of superclusters containing magic atom numbers, and the equilibrium latent heats at Tg and TK. Strong-to-fragile and strong-to-strong liquid transitions at Tg are also described and all their thermodynamic parameters are determined from their specific heat jumps. The existence of fragile liquids quenched in the amorphous state, which do not undergo liquid-liquid transition during heating preceding their crystallization, is predicted. Long ageing times leading to the formation at TK of a stable glass composed of superclusters containing up to 147 atom, touching and interpenetrating, are evaluated from nucleation rates. A fragile-to-fragile liquid transition occurs at Tg without stable-glass formation while a strong glass is stable after transition.

  20. The Anti-Tumor Effects of Adipose Tissue Mesenchymal Stem Cell Transduced with HSV-Tk Gene on U-87-Driven Brain Tumor

    PubMed Central

    de Melo, Suely Maymone; Bittencourt, Simone; Ferrazoli, Enéas Galdini; da Silva, Clivandir Severino; da Cunha, Flavia Franco; da Silva, Flavia Helena; Stilhano, Roberta Sessa; Denapoli, Priscila Martins Andrade; Zanetti, Bianca Ferrarini; Martin, Priscila Keiko Matsumoto; Silva, Leonardo Martins; dos Santos, Adara Aurea; Baptista, Leandra Santos; Longo, Beatriz Monteiro; Han, Sang Won

    2015-01-01

    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. PMID:26067671

  1. Radioimmunoassay for herpes simplex virus (HSV) thymidine kinase

    SciTech Connect

    McGuirt, P.V.; Keller, P.M.; Elion, G.B.

    1982-01-30

    A sensitive RIA for HSV-1 thymidine kinase (TK) has been developed. This assay is based on competition for the binding site of a rabbit antibody against purified HSV-1 TK, between a purified /sup 3/H-labeled HSV-1 TK and a sample containing an unknown amount of viral TK. The assay is capable of detecting 8 ng or more of the HSV enzyme. Purified HSV-1 TK denatured to <1% of its original kinase activity is as effective in binding to the antibody as is native HSV-1 TK. Viral TK is detectable at ranges of 150-460 ng/mg protein of cell extract from infected cells or cells transformed by HSV or HSV genetic material. HSV-2 TK appears highly cross-reactive, VZV TK is slightly less so, and the vaccinia TK shows little or no cross-reactivity. This RIA may serve as a tool for monitoring the expression of the HSV TK during an active herpes virus infection, a latent ganglionic infection, or in neoplastic cells which may have arisen by viral transformation.

  2. Design, synthesis and antitumor activity of novel pyrazolo[3,4-d]pyrimidine derivatives as EGFR-TK inhibitors.

    PubMed

    Abdelgawad, Mohamed A; Bakr, Rania B; Alkhoja, Olla A; Mohamed, Wafaa R

    2016-06-01

    A novel series of 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-substitutedbenzylidene)acetohydrazide (12a-g) was prepared and their structures were confirmed by spectral and elemental analyses. The cytotoxic activity of the newly synthesized compounds was evaluated against breast carcinoma (MCF-7), non-small cell lung cancer (A549) and human colorectal adenocarcinoma (HT-29) cell lines using MTT and colony formation assays. The tested compounds showed a marked anticancer activity against all the tested cell lines, especially compound 12g, which was the most potent anticancer agent with half maximal inhibitory concentrations (IC50) between 5.36 and 9.09μM. Docking studies into ATP binding site of EGFR protein tyrosine kinase were performed to predict their scores and mode of binding to amino acids, In addition, the inhibitory activity of the target compounds against epidermal growth factor receptor tyrosine kinase (EGFR-TK) was evaluated. Results indicated the ability of the target compounds to inhibit EGFR-TK with half maximal inhibitory concentrations (IC50) in the range of 4.18-35.88μM. Furthermore, The most active compounds 12g, 12c and 12d were assayed against Fibroblast Growth Factor Receptor (FGFR), Insulin Receptor (IR) and Vascular Endothelial Growth Factor Receptor (VEGFR). The activity of the reported compounds warrants further optimization as novel members in cancer treatment protocols. PMID:27043178

  3. Expression of HSV-TK suicide gene in primary T lymphocytes: the dog as a preclinical model.

    PubMed

    Weissinger, E M; Franz, M; Voss, C; Bonini, C; Kremmer, E; Kolb, H J

    2000-03-01

    Expression of suicide genes (e.g. herpes simplex virus thymidine kinase,HSV-TK) in T cells is an appealing approach to regulate graft-versus-host disease in adoptive immunotherapy. Here we report the optimization of retroviral infection of canine T cells. Canine T cells were stimulated either with phytohemagglutinin (PHA, 2 microg/ml) for 24-72 hours or with 100 U/ml interleukin-2 for seven days. Stimulated cells were co-cultivated with irradiated virus-producing cells. Transduction efficiencies ranged from 4% to 45% using PG13, a gibbon ape leukemia virus envelope (env) pseudotyped packaging cell line. Infection of cells with GPenvAM12, expressing the amphotropic Moloney murine leukemia virus env, did not yield a satisfactory percentage of transduced cells. Enrichment of transduced cells was performed using immunoselection, and gave a purity of up to 98%. Transfusion of 1 x 10(6) transduced cells per kilogram body weight showed that transduced cells could convert mixed chimerism to 100% and transfer immunity to a specific antigen. Transduced cells were repeatedly detected in peripheral blood and bone marrow by polymerase chain reaction with primers specific for the HSV-TK gene. We have demonstrated the feasibility of using the canine model to study gene therapy as a preclinical model. PMID:10976536

  4. A fiber-modified adenovirus co-expressing HSV-TK and Coli.NTR enhances antitumor activities in breast cancer cells

    PubMed Central

    Zhan, Yang; Yu, Bin; Wang, Zhen; Zhang, Yu; Zhang, Hai-Hong; Wu, Hao; Feng, Xiao; Geng, Ran-Shen; Kong, Wei; Yu, Xiang-Hui

    2014-01-01

    Breast cancers especially in late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Suicide gene therapy based on adenoviral technology will be promising strategies for such advanced diseases. We previously showed that co-expression of herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli nitroreductase (Coli.NTR) by an hTERT-driven adenovirus vector resulted in additive anti-tumor effects in breast cancer cells in vitro and in vivo. As many tumor tissue and cancer cells express low level of coxsackie-adenovirus receptor (CAR), which is the functional receptor for the fiber protein of human adenovirus serotype 5 (Ad5), novel Ad5 vectors containing genetically modifi ed fiber are attractive vehicles for achieving targeted gene transfer and improving suicide gene expression in these cancer cells. In the present study, we first built a simplified Ad5 vector platform for fiber modification and quick detection for gene transfer. Then a fiber-modified adenovirus vector containing an RGD motif in the HI loop of the fiber knob was constructed. After recombined with HSV-TK and Coli.NTR gene, this fiber-modified Ad5 vector (Ad-RGD-hT-TK/NTR) was compared with that of our previously constructed Ad5 vector (Ad-hT-TK/NTR) for its therapeutic effects in human breast cancer cell lines. The anti-tumor activity of Ad-RGD-hT-TK/NTR was significantly enhanced compared with Ad-hT-TK/NTR both in vitro and in vivo. This new vector platform provided a robust and simplified approach for capsid modification, and the fiber-modified Ad5 with double suicide genes under the control of hTERT promoter would be a useful gene therapy strategy for breast cancer. PMID:25031704

  5. The in vitro PIG-A gene mutation assay: glycosylphosphatidylinositol (GPI)-related genotype-to-phenotype relationship in TK6 cells.

    PubMed

    Krüger, Christopher T; Fischer, Bettina M; Armant, Olivier; Morath, Volker; Strähle, Uwe; Hartwig, Andrea

    2016-07-01

    In our previous work, we established an in vitro variant of the currently developed in vivo PIG-A assay as promising mutagenicity test system. We applied the human B-lymphoblastoid cell line TK6 for the in vitro assay development, which is based on the cellular glycosylphosphatidylinositol (GPI) status. At least 22 genes are involved in GPI biosynthesis, leading to the complex situation that, in principle, multiple genes could induce a GPI-deficient phenotype by acquiring inactivating mutations. However, only the PIG-A gene is located on the X-chromosome, rendering PIG-A more sensitive compared to autosomal linked, GPI-relevant genes. In this work, we investigated the GPI-related genotype-to-phenotype relationship in TK6 cells. By a next-generation sequencing approach, we identified a heterozygous chromosomal deletion on chromosome 17, where the PIG-L gene is located. In the analyzed TK6 cell clones, the GPI-deficient phenotype was induced either by mutations in PIG-A, by the complete absence of PIG-A mRNA, or by deletions in the remaining functional PIG-L gene, causing loss of heterozygosity. The identified PIG-L heterozygosity could also be responsible for the increased sensitivity toward mutagenic ethyl methanesulfonate or UV-C treatments of p53-proficient TK6 compared to the TK6-related, but p53-deficient WI-L2-NS cell line. Moreover, the WI-L2-NS cell line was found to exhibit a much lower number of GPI-deficient mutant cells in the purchased cell batch, and WI-L2-NS exerted a lower spontaneous rate of GPI deficiency compared to TK6 cells. PMID:27100116

  6. MUTANT FREQUENCIES AND LOSS OF HETEROZYGOSITY INDUCED BY N-ETHYL-N-NITROSOUREA (ENU) IN THE THYMIDINE KINASE (TK) GENE OF L5178YTK+/-3.7.2C MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    MUTANT FREQUENCIES AND LOSS HETEROZYGOSITY INDUCED BY N-ETHYK-N-NITROSOUREA (ENU) IN THE THYMIDINE KINASE (tk) GENE IF l5178Y/TK+/-3.7.2C MOUSE LYMPHOMA CELLS

    N-ethyl-N-nitrosourea (ENU) is a potent monofunctional-ethylating agent that has been found to be mutagenic in a w...

  7. Evaluation of a lipopeptide biosurfactant from Bacillus natto TK-1 as a potential source of anti-adhesive, antimicrobial and antitumor activities

    PubMed Central

    Cao, Xiao-Hong; Liao, Zhen-Yu; Wang, Chun-Ling; Yang, Wen-Yan; Lu, Mei-Fang

    2009-01-01

    A lipopeptide biosurfactant produced by Bacillus natto TK-1 has a strong surface activity. The biosurfactant was found to be an anti-adhesive agent against several bacterial strains, and also showed a broad spectrum of antimicrobial activity. The biosurfactant induced a significant reduction in tumor cells viability in a dose-dependent manner. PMID:24031375

  8. IN SITU ANALYSIS OF TRIFLUOROTHYMIDINE-RESISTANT (TFT(SUP R) MUTANTS OF L5178Y/TK(SUP +/-) MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    TFTr mutants of L5178Y/TK+/- mouse lymphoma cells are analyzed as they appear in situ following cloning and incubation for 9-11 days in soft agar cloning medium. These TFTr mutants can be divided by colony size into sigma, small colony, and lambda, large colony, mutants. The use ...

  9. SEQUENCE ANALYSIS OF MUTATIONS INDUCED BY N-ETHYL-N-NITROSOUREA IN THE TK AND HPRT GENES OF MOUSE LYMPHOMA CELLS.

    EPA Science Inventory

    The mouse lymphoma assay is widely used to identify chemicals that are capable of inducing mutational damages. The Tk+/- gene located on an autosome in mouse lymphoma cells may recover a wider range of mutational events than the X-linked Hprt locus. However, chemical-induced muta...

  10. EFFECTS OF A LIGNIN PEROXIDASE-EXPRESSING RECOMBINANT STREPTOMYCES LIVIDANS TK23.1 ON BIOGEOCHEMICAL CYCLING AND THE NUMBERS AND ACTIVITIES OF MICROORGANISMS IN SOIL

    EPA Science Inventory

    A recombinant actinomycete, Streptomyces lividans TK23.1, expressing a pIJ702-encoded extracellular lignin peroxidase gene cloned from the chromosome of Streptomyces virodosporus T7A, was released into soil in flask- and microcosm-scale studies to determine its effects on humific...

  11. How TK-TD and population models for aquatic macrophytes could support the risk assessment for plant protection products.

    PubMed

    Hommen, Udo; Schmitt, Walter; Heine, Simon; Brock, Theo Cm; Duquesne, Sabine; Manson, Phil; Meregalli, Giovanna; Ochoa-Acuña, Hugo; van Vliet, Peter; Arts, Gertie

    2016-01-01

    This case study of the Society of Environmental Toxicology and Chemistry (SETAC) workshop MODELINK demonstrates the potential use of mechanistic effects models for macrophytes to extrapolate from effects of a plant protection product observed in laboratory tests to effects resulting from dynamic exposure on macrophyte populations in edge-of-field water bodies. A standard European Union (EU) risk assessment for an example herbicide based on macrophyte laboratory tests indicated risks for several exposure scenarios. Three of these scenarios are further analyzed using effect models for 2 aquatic macrophytes, the free-floating standard test species Lemna sp., and the sediment-rooted submerged additional standard test species Myriophyllum spicatum. Both models include a toxicokinetic (TK) part, describing uptake and elimination of the toxicant, a toxicodynamic (TD) part, describing the internal concentration-response function for growth inhibition, and a description of biomass growth as a function of environmental factors to allow simulating seasonal dynamics. The TK-TD models are calibrated and tested using laboratory tests, whereas the growth models were assumed to be fit for purpose based on comparisons of predictions with typical growth patterns observed in the field. For the risk assessment, biomass dynamics are predicted for the control situation and for several exposure levels. Based on specific protection goals for macrophytes, preliminary example decision criteria are suggested for evaluating the model outputs. The models refined the risk indicated by lower tier testing for 2 exposure scenarios, while confirming the risk associated for the third. Uncertainties related to the experimental and the modeling approaches and their application in the risk assessment are discussed. Based on this case study and the assumption that the models prove suitable for risk assessment once fully evaluated, we recommend that 1) ecological scenarios be developed that are also

  12. Whole genome and normalized mRNA sequencing reveal genetic status of TK6, WTK1, and NH32 human B-lymphoblastoid cell lines.

    PubMed

    Revollo, Javier; Petibone, Dayton M; McKinzie, Page; Knox, Bridgett; Morris, Suzanne M; Ning, Baitang; Dobrovolsky, Vasily N

    2016-01-01

    Closely related TK6, WTK1, and NH32 human B-lymphoblastoid cell lines differ in their p53 functional status. These lines are used frequently in genotoxicity studies and in studies aimed at understanding the role of p53 in DNA repair. Despite their routine use, little is known about the genetic status of these cells. To provide insight into their genetic composition, we sequenced and analyzed the entire genome of TK6 cells, as well as the normalized transcriptomes of TK6, WTK1, and NH32 cells. Whole genome sequencing (WGS) identified 21,561 genes and 5.17×10(6) small variants. Within the small variants, 50.54% were naturally occurring single nucleotide polymorphisms (SNPs) and 49.46% were mutations. The mutations were comprised of 92.97% single base-pair substitutions and 7.03% insertions or deletions (indels). The number of predicted genes, SNPs, and small mutations are similar to frequencies observed in the human population in general. Normalized mRNA-seq analysis identified the expression of transcripts bearing SNPs or mutations for TK6, WTK1, and NH32 as 2.88%, 2.04%, and 1.71%, respectively, and several of the variant transcripts identified appear to have important implications in genetic toxicology. These include a single base deletion mutation in the ferritin heavy chain gene (FTH1) resulting in a frame shift and protein truncation in TK6 that impairs iron metabolism. SNPs in the thiopurine S-methyltransferase (TPMT) gene (TPMT*3A SNP), and in the xenobiotic metabolizing enzyme, NADPH quinine oxidoreductase 1 (NQO1) gene (NQO1*2 SNP), are both associated with decreased enzyme activity. The clinically relevant TPMT*3A and NQO1*2 SNPs can make these cell lines useful in pharmacogenetic studies aimed at improving or tailoring drug treatment regimens that minimize toxicity and enhance efficacy. PMID:26774668

  13. Kinetic studies with N2-phenylguanines and with L-thymidine indicate that herpes simplex virus type-1 thymidine kinase and thymidylate kinase share a common active site.

    PubMed

    Maga, G; Focher, F; Wright, G E; Capobianco, M; Garbesi, A; Bendiscioli, A; Spadari, S

    1994-08-15

    It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates. PMID:8068016

  14. Kinetic studies with N2-phenylguanines and with L-thymidine indicate that herpes simplex virus type-1 thymidine kinase and thymidylate kinase share a common active site.

    PubMed Central

    Maga, G; Focher, F; Wright, G E; Capobianco, M; Garbesi, A; Bendiscioli, A; Spadari, S

    1994-01-01

    It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates. PMID:8068016

  15. Attenuation of Vaccinia Tian Tan Strain by Removal of Viral TC7L-TK2L and TA35R Genes

    PubMed Central

    Kan, Shifu; Wang, Yuhang; Sun, Lili; Jia, Peng; Qi, Yanxin; Su, Jiaqiang; Liu, Lei; Yang, Guohua; Liu, Liming; Wang, Zhuoyue; Wang, Jinhui; Liu, Guangchen; Jin, Ningyi; Li, Xiao; Ding, Zhuang

    2012-01-01

    Vaccinia Tian Tan (VTT) was attenuated by deletion of the TC7L-TK2L and TA35R genes to generate MVTT3. The mutant was generated by replacing the open reading frames by a gene encoding enhanced green fluorescent protein (EGFP) flanked by loxP sites. Viruses expressing EGFP were then screened for and purified by serial plaque formation. In a second step the marker EGFP gene was removed by transfecting cells with a plasmid encoding cre recombinase and selecting for viruses that had lost the EGFP phenotype. The MVTT3 mutant was shown to be avirulent and immunogenic. These results support the conclusion that TC7L-TK2L and TA35R deletion mutants can be used as safe viral vectors or as platform for vaccines. PMID:22363781

  16. Novel 5-vinyl pyrimidine nucleosides with potent anti-hepatitis B virus activity.

    PubMed

    Kumar, R; Tyrrell, D L

    2001-11-19

    Synthesis and antiviral activities of novel N-1 alkyl substituted pyrimidines, 1-[(2-hydroxyethoxy)methyl]-5-vinyluracil (5), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-vinyluracil (6), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-vinyluracil (7) are reported. Compounds 6 and 7 were potent inhibitors of DHBV in cell culture, in contrast, all of the compounds described were devoid of activity against TK(+) HSV-1 and TK(-) HSV-1. PMID:11677126

  17. Identification and Characterization of Leucocyclicin Q, a Novel Cyclic Bacteriocin Produced by Leuconostoc mesenteroides TK41401▿

    PubMed Central

    Masuda, Yoshimitsu; Ono, Hiroshi; Kitagawa, Hiroshi; Ito, Haruo; Mu, Fuqin; Sawa, Naruhiko; Zendo, Takeshi; Sonomoto, Kenji

    2011-01-01

    The culture supernatant of Leuconostoc mesenteroides TK41401, isolated from Japanese pickles, possessed antimicrobial activity against broad range of a bacterial genera and particularly strong activity against Bacillus coagulans, the major contaminant of pickles. An antimicrobial peptide was purified in three chromatographic steps, and its molecular mass was determined to be 6,115.59 Da by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). The primary structure of this peptide was determined by amino acid and DNA sequencing, and these analyses revealed that it was translated as a 63-residue precursor. This precursor showed high similarity to the precursor of lactocyclicin Q, a cyclic bacteriocin produced by Lactococcus sp. strain QU 12. The molecular weight calculated after cyclization, which was presumed to involve the same process as in lactocyclicin Q (between L3 and W63), agreed with that estimated by ESI-TOF MS. This peptide was proved to be a novel cyclic bacteriocin, and it was termed leucocyclicin Q. The antimicrobial spectrum of this bacteriocin clearly differed from that of lactocyclicin Q, even though their primary structures were quite similar. This is the first report of a cyclic bacteriocin produced by a strain of the genus Leuconostoc. PMID:21948835

  18. Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

    PubMed Central

    Yoon, K S; Ishii, M; Igarashi, Y; Kodama, T

    1996-01-01

    2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively. PMID:8655524

  19. Biomarkers of Exposure and Effect in Human Lymphoblastoid TK6 Cells Following [13C2]-Acetaldehyde Exposure

    PubMed Central

    Swenberg, James A.

    2013-01-01

    The dose-response relationship for biomarkers of exposure (N2-ethylidene-dG adducts) and effect (cell survival and micronucleus formation) was determined across 4.5 orders of magnitude (50nM–2mM) using [13C2]-acetaldehyde exposures to human lymphoblastoid TK6 cells for 12h. There was a clear increase in exogenous N 2-ethylidene-dG formation at exposure concentrations ≥ 1µM, whereas the endogenous adducts remained nearly constant across all exposure concentrations, with an average of 3.0 adducts/107 dG. Exogenous adducts were lower than endogenous adducts at concentrations ≤ 10µM and were greater than endogenous adducts at concentrations ≥ 250µM. When the endogenous and exogenous adducts were summed together, statistically significant increases in total adduct formation over the endogenous background occurred at 50µM. Cell survival and micronucleus formation were monitored across the exposure range and statistically significant decreases in cell survival and increases in micronucleus formation occurred at ≥ 1000µM. This research supports the hypothesis that endogenously produced reactive species, including acetaldehyde, are always present and constitute the majority of the observed biological effects following very low exposures to exogenous acetaldehyde. These data can replace default assumptions of linear extrapolation to very low doses of exogenous acetaldehyde for risk prediction. PMID:23425604

  20. Noninvolvement of the X chromosome in radiation-induced chromosome translocations in the human lymphoblastoid cell line TK6

    SciTech Connect

    Jordan, R.; Schwartz, J.L. )

    1994-03-01

    Fluorescence in situ hybridization procedures were used to examine the influence of chromosome locus on the frequency and type of chromosome aberrations induced by [sup 60]Co [gamma] rays in the human lymphoblastoid cell line TK6. Aberrations involving the X chromosome were compared to those involving the similarly sized autosome chromosome 7. When corrected for DNA content, acentric fragments were induced with equal frequency in the X and 7 chromosomes. Dose-dependent increases in chromosomal interchanges involving chromosome 7 were noted, and the frequencies of balanced translocations and dicentrics produced were approximately equal. Chromosome interchanges involving the X chromosome were rare and showed no apparent dose dependence. Thus, while chromosomes 7 and X are equally sensitive to the induction of chromosome breaks, the X chromosome is much less likely to interact with autosomes than chromosome 7. The noninvolvement of the X chromosome in translocations with autosomes may reflect a more peripheral and separate location for the X chromosome in the mammalian nucleus. 20 refs., 2 figs., 1 tab.

  1. Safety and immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the E2 protein of classical swine fever virus in pigs.

    PubMed

    Lei, Jian-Lin; Xia, Shui-Li; Wang, Yimin; Du, Mingliang; Xiang, Guang-Tao; Cong, Xin; Luo, Yuzi; Li, Lian-Feng; Zhang, Lingkai; Yu, Jiahui; Hu, Yonghao; Qiu, Hua-Ji; Sun, Yuan

    2016-06-01

    Classical swine fever (CSF) and pseudorabies (PR) are both major infectious diseases of pigs, causing enormous economic losses to the swine industry in many countries. A marker vaccine that enables differentiation of infected from vaccinated animals (DIVA) is highly desirable for control and eradication of these two diseases in endemic areas. Since late 2011, PR outbreaks have been frequently reported in many Bartha-K61-vaccinated pig farms in China. It has been demonstrated that a pseudorabies virus (PRV) variant with altered antigenicity and increased pathogenicity was responsible for the outbreaks. Previously, we showed that rPRVTJ-delgE/gI/TK, a gE/gI/TK-deleted PRV variant, was safe for susceptible animals and provided a complete protection against lethal PRV variant challenge, indicating that rPRVTJ-delgE/gI/TK can be used as an attractive vaccine vector. To develop a safe bivalent vaccine against CSF and PR, we generated a recombinant virus rPRVTJ-delgE/gI/TK-E2 expressing the E2 protein of classical swine fever virus (CSFV) based on rPRVTJ-delgE/gI/TK and evaluated its safety and immunogenicity in pigs. The results indicated that pigs (n=5) immunized with rPRVTJ-delgE/gI/TK-E2 of different doses did not exhibit clinical signs or viral shedding following immunization, the immunized pigs produced anti-PRV or anti-CSFV neutralizing antibodies and the pigs immunized with 10(6) or 10(5) TCID50 rPRVTJ-delgE/gI/TK-E2 were completely protected against the lethal challenge with either CSFV Shimen strain or variant PRV TJ strain. These findings suggest that rPRVTJ-delgE/gI/TK-E2 is a promising bivalent DIVA vaccine candidate against CSFV and PRV coinfections. PMID:27113530

  2. Characterization of a novel highly thermostable esterase from the Gram-positive soil bacterium Streptomyces lividans TK64.

    PubMed

    Wang, Baojuan; Wang, Ao; Cao, Zhengyu; Zhu, Guoping

    2016-05-01

    A novel esterase gene (estW) from soil bacterium Streptomyces lividans TK64 was successfully cloned using a pair of homologous primers. The estW gene encoded a protein (EstW) of 289 amino acid residues with a predicted molecular weight of 31.43 kDa. Sequence alignment revealed that EstW show relatively high levels of homology to other lipolytic enzymes characterized from Streptomyces and phylogenetic analysis suggested EstW belongs to the bacterial lipase/esterase family I. The estW gene was expressed at a high level in Escherichia coli and the recombinant enzyme was purified to homogeneity. The purified EstW was characterized via hydrolysis of various p-nitrophenyl esters and the best substrate was found to be p-nitrophenyl acetate (pNPA). Maximal activity of the recombinant protein was observed at pH 8.0 and 50 °C with pNPA as the substrate. The calculated activation energy (Ea ) of the esterase reaction was 9.12 kcal/mol. Half-life of EstW at 95 °C was approximately 12.5 H, making it the most thermostable esterase among all of the known lipolytic enzymes from Streptomyces, and the thermostability of EstW was similar to those of some enzymes characterized from the thermophilic bacteria. EstW exhibited relatively high tolerance to several detergents and required no cations for its maximal activity. The unique properties of EstW, namely its high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications. PMID:26621184

  3. A peptide tetramer Tk-tPN induces tolerance of cardiac allografting by conversion of type 1 to type 2 immune responses via the Toll-like receptor 2 signal-promoted activation of the MCP1 gene.

    PubMed

    Li, Zuoqing; Yang, Neng; Zhou, Ling; Gu, Peng; Wang, Hui; Zhou, Yun; Zhou, Peijun; Lu, Liming; Chou, Kuang-Yen

    2016-03-01

    The plant protein trichosanthin (Tk) and its derived peptide tetramer Tk-tPN have been shown to stimulate the type 2 immune responses for treating autoimmune disease. This work explores the possibility of using Tk-tPN as a non-toxic immunosuppressant to induce transplantation tolerance using the mechanisms by which T-cell-mediated immune responses are transferred from type 1 to type 2 through innate immunity-related pathways. Immunocytes and cytokine secretions involved in the mouse cardiac allografting model with Tk-tPN treatment were characterized. Identification of critical genes and analysis of their functions through Toll-like receptor (TLR) -initiated signalling and the possible epigenetic changes were performed. Mean survival times of the cardiac allografts were delayed from 7.7 ± 0.3 days (control) to 22.7 ± 3.9 days (P < 0.01) or 79.1 ± 19.2 days (P < 0.0001) when Tk-tPN was introduced into the recipients alone or together with rapamycin, respectively. The grafting tolerance was donor-specific. The secretion pattern of the type 1 cytokine/transcription factor (IL-2(+) IFN-γ(+) T-bet(+)), which is responsible for the acute graft rejection, was shifted to the type 2 factor (IL-4(+) IL-10(+) Gata3+), together with a selective expansion of the IL-4/IL-10-producing CD8+ CD28- regulatory T-cell subset. A TLR2-initiated high expression of chemokine gene MCP1 was detectable simultaneously. Epigenetically Tk/Tk-tPN could also acetylate the histone H3K9 of MCP1 promoter to skew the immunity towards T helper type 2 responses. Tk/Tk-tPN is therefore capable of down-regulating the type 1 response-dominant rejection of cardiac allografts by evoking type 2 immunity through the activation of a TLR2-initiated signalling pathway and MCP1 gene to expand the IL-4/IL-10-secreting CD8+ CD28- regulatory T cells. Tk-tPN could be a promising novel immunosuppressant to induce tolerance in allotransplantation. PMID:26694804

  4. Synthetic Aperture Radar Imaging on a Cuda-Enabled Mobile Platform

    NASA Astrophysics Data System (ADS)

    Fatica, M.; Philllips, E.

    2014-12-01

    This talk will present the details of a Synthetic Aperture Radar (SAR) imaging on the smallest CUDA-capable platform available, the Jetson TK1. The results indicate that GPU accelerated embedded platforms have considerable potential for this type of workload and in conjunction with low power consumption, light weight and standard programming tools, could open new horizons in the embedded space.

  5. Bacterial Thymidine Kinase as a Non-Invasive Imaging Reporter for Mycobacterium tuberculosis in Live Animals

    PubMed Central

    Davis, Stephanie L.; Be, Nicholas A.; Lamichhane, Gyanu; Nimmagadda, Sridhar; Pomper, Martin G.; Bishai, William R.; Jain, Sanjay K.

    2009-01-01

    Background Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis. Methodology/Principal Findings We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 - a strong constitutive mycobacterial promoter. [125I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis Phsp60 TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis Phsp60 TK strain actively accumulated [125I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis Phsp60 TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested. Conclusion We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research. PMID:19606217

  6. Anti-herpes simplex virus activity of alkaloids isolated from Stephania cepharantha.

    PubMed

    Nawawi, A; Ma, C; Nakamura, N; Hattori, M; Kurokawa, M; Shiraki, K; Kashiwaba, N; Ono, M

    1999-03-01

    By screening water and MeOH extracts of 30 Chinese medicinal plants for their anti-herpes simplex virus (HSV)-1 activity, a MeOH extract of the root tubers of Stephania cepharantha HAYATA showed the most potent activity on the plaque reduction assay with an IC50 value of 18.0 microg/ml. Of 49 alkaloids isolated from the MeOH extract, 17 alkaloids were found to be active against HSV-1, including 13 bisbenzylisoquinoline, 1 protoberberine, 2 morphinane and 1 proaporphine alkaloids, while benzylisoquinoline and hasubanane alkaloids were inactive. Although N-methylcrotsparine was active against HSV-1, as well as HSV-1 thymidine kinase deficient (acyclovir resistant type, HSV-1 TK-) and HSV-2 (IC50 values of 8.3, 7.7 and 6.7 microg/ml, respectively), it was cytotoxic. FK-3000 was found to be the most active against HSV-1, HSV-1 TK- and HSV-2 (IC50 values of 7.8, 9.9 and 8.7 microg/ml) with in vitro therapeutic indices of 90, 71 and 81, respectively. FK-3000 was found to be a promising candidate as an anti-HSV agent against HSV-1, acyclovir (ACV) resistant-type HSV-1 and HSV-2. PMID:10220283

  7. Development of novel epidermal growth receptor-basedradiopharmaceuticals: Imaging agents for breast cancer

    SciTech Connect

    Van Brocklin, Henry F.

    2001-09-25

    The goal of this research was to develop epidermal growthfactor receptor (EGFR) nuclear medicine breast cancer imaging agents. Ourapproach was to synthesize small molecule inhibitors of the EGFR tyrosinekinase (tk) suitable for labeling with single photon or positron-emittingradioisotopes and evaluate the imaging potential of these new molecules.We have synthesized and fully characterized 22 quinazoline compounds. Allcompounds inhibit EGFR tk phosphorylation activity in the nanomolarrange. All compounds tested exhibited specificity for the EGFR tk versusthe ErbB2 and ErbB4 tyrosine kinases. A radiometric binding assay usingan iodine-125 labeled quinazoline was developed to determine the affinityof the quinazolines for the EGFR tk ATP binding site. The affinitiesranged from 0.4-51 nM. The octanol/water partition coefficients (Log P;lipophilicity) of the new compounds ranged from 2.2-5.5. Six compoundshave been labeled with fluorine-18. Biodistribution in EGFRoverexpressing tumor bearing mice demonstrated tumor uptake buthighlighted delivery and metabolism issues. The 2-fluoro quinazoline wasnot metabolized in an in vitro hepatocyte study. From this work a breadthof agent characteristics was created establishing the foundation forfuture research toward the optimal EGFR imaging agent.

  8. Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

    PubMed Central

    2014-01-01

    Background Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. Results We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. Conclusion Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required. PMID:24484525

  9. Application of speckle image correlation for real-time assessment of metabolic activity in herpes virus-infected cells

    NASA Astrophysics Data System (ADS)

    Vladimirov, A. P.; Malygin, A. S.; Mikhailova, J. A.; Borodin, E. M.; Bakharev, A. A.; Poryvayeva, A. P.

    2014-09-01

    Earlier we reported developing a speckle interferometry technique and a device designed to assess the metabolic activity of a cell monolayer cultivated on a glass substrate. This paper aimed at upgrading the technique and studying its potential for real-time assessment of herpes virus development process. Speckle dynamics was recorded in the image plane of intact and virus-infected cell monolayer. HLE-3, L-41 and Vero cells were chosen as research targets. Herpes simplex virus-1-(HSV-1)- infected cell cultures were studied. For 24 h we recorded the digital value of optical signal I in one pixel and parameter η characterizing change in the distribution of the optical signal on 10 × 10-pixel areas. The coefficient of multiple determination calculated by η time dependences for three intact cell cultures equals 0.94. It was demonstrated that the activity parameters are significantly different for intact and virus-infected cells. The difference of η value for intact and HSV-1-infected cells is detectable 10 minutes from the experiment start.

  10. Image

    Energy Science and Technology Software Center (ESTSC)

    2007-08-31

    The computer side of the IMAGE project consists of a collection of Perl scripts that perform a variety of tasks; scripts are available to insert, update and delete data from the underlying Oracle database, download data from NCBI's Genbank and other sources, and generate data files for download by interested parties. Web scripts make up the tracking interface, and various tools available on the project web-site (image.llnl.gov) that provide a search interface to the database.

  11. Detection of K-ras Mutations in Predicting Efficacy of Epidermal Growth Factor Receptor Tyrosine Kinase (EGFR-TK) Inhibitor in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Li, Ze; Liu, Xue-Wei; Chi, Zhao-Cheng; Sun, Bao-Sheng; Cheng, Ying; Cheng, Long-Wei

    2015-01-01

    Epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors are useful in treating different advanced human cancers; however, their clinical efficacy varies. This study detected K-ras mutations to predict the efficacy of EGFR-TK inhibitor cetuximab treatment on Chinese patients with metastatic colorectal cancer (mCRC). A total of 87 patients with metastatic colorectal cancer were treated with cetuximab for 2-16 months, in combination with chemotherapy between August 2008 and July 2012, and tissue samples were used to detect K-ras mutations. The data showed that K-ras mutation occurred in 27/87 (31%). The objective response rates and disease control rate in K-ras wild type and mutant patients were 42% (25/60) versus 11% (3/27) (p<0.05) and 60% (36/60) versus 26% (7/27) (p<0.05), respectively. Patients with the wild-type K-ras had significantly higher median survival times and progression-free survival, than patients with mutated K-ras (21 months versus 17 months, p=0.017; 10 months versus 6 months, p=0.6). These findings suggest that a high frequency of K-ras mutations occurs in Chinese mCRC patients and that K-ras mutation is required to select patients for eligibility for cetuximab therapy. Further prospective studies using a large sample size are needed to confirm these preliminary findings. PMID:25950441

  12. Images.

    ERIC Educational Resources Information Center

    Barr, Catherine, Ed.

    1997-01-01

    The theme of this month's issue is "Images"--from early paintings and statuary to computer-generated design. Resources on the theme include Web sites, CD-ROMs and software, videos, books, and others. A page of reproducible activities is also provided. Features include photojournalism, inspirational Web sites, art history, pop art, and myths. (AEF)

  13. Fialuridine Induces Acute Liver Failure in Chimeric TK-NOG Mice: A Model for Detecting Hepatic Drug Toxicity Prior to Human Testing

    PubMed Central

    Xu, Dan; Nishimura, Toshi; Nishimura, Sachiko; Zhang, Haili; Zheng, Ming; Guo, Ying-Ying; Masek, Marylin; Michie, Sara A.; Glenn, Jeffrey; Peltz, Gary

    2014-01-01

    Background Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. Methods and Findings Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. Conclusions FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in

  14. Ciprofloxacin-induced G2 arrest and apoptosis in TK6 lymphoblastoid cells is not dependent on DNA double-strand break formation

    PubMed Central

    Smart, Daniel J.; Halicka, H. Dorota; Traganos, Frank; Darzynkiewicz, Zbigniew; Williams, Gary M.

    2008-01-01

    Drugs developed for the treatment of conditions other than neoplasia can also show promise as potential antitumor agents. The fluoroquinolone antibiotic ciprofloxacin (CPFX) is known to modulate cycle cell progression and apoptosis in cancer cells, and is thought to induce DNA double-strand breaks (DSBs) via topoisomerase II (topo II) inhibition and stabilized cleavage complex (SCC) formation. DSBs trigger Ser-139 phosphorylation of histone H2AX (γH2AX) by PI-3-like kinases including ATM; γH2AX can serve as a marker of DNA damage when measured in situ using immunocytochemistry and flow cytometry. The aim of the present study was to investigate the relationship between CPFX-mediated DNA damage and induction of apoptosis in human lymphoblastoid cells and phytohaemagglutinin (PHA)-stimulated lymphocytes (Lymphs). Treatment of TK6 cells (wild-type p53) with 100 µg/ml CPFX for 2-10 h produced no increase in γH2AX; to the contrary, its level in S phase cells was reduced at 10 h compared to controls. Nevertheless, stabilization of topo IIα, ATM Ser-1981 phosphorylation and G2 arrest was observed in TK6 cells exposed to CPFX for ≥4 h. However, following 24 h treatment, γH2AX was dramatically increased in a sub-population of cells indicating the onset of apoptosis (confirmed by presence of activated caspase 3). CPFX had a similar lack of effect on induction of γH2AX at early time points in WTK1 and NH32 cells (devoid of functional p53) and proliferating Lymphs, however, induction of apoptosis was less pronounced than in TK6 cells. Formation of SCC and activation of ATM (but lack of γH2AX induction) indicates topo II-mediated chromatin or DNA changes in the absence of DSBs; ATM activation apparently triggers the G2M checkpoint leading to G2 arrest. The subsequent induction of apoptosis appears to be facilitated by functional p53. CPFX may therefore have a potential use as a chemotherapeutic agent in the treatment of lymphoblast-derived cancer. PMID:18059176

  15. Characterization of ATI, TK and IFN-alpha/betaR genes in the genome of the BeAn 58058 virus, a naturally attenuated wild Orthopoxvirus.

    PubMed

    Marques, J T; Trindade, G D; Da Fonseca, F G; Dos Santos, J R; Bonjardim, C A; Ferreira, P C; Kroon, E G

    2001-12-01

    The lack of knowledge about the natural host of Vaccinia virus (VV) along with the description of human infections caused by poxviruses after smallpox eradication has increased the need to characterize poxviruses isolated from the wild. Moreover, in the past years poxviruses have been widely studied as potential vaccination tools, with the discovery of several genes implicated in the evasion of the host immune response involved in virus pathogenesis. Among them, an Interferon (IFN)-binding protein was identified in the supernatant of VV strain WR infected cells coded by the B18R gene. It was shown that many other Orthopoxviruses also encode and express this soluble receptor although some VV strains such as Lister and modified Ankara, which were less reactogenic vaccines, do not. The BeAn 58058 virus (BAV) has been recently characterized and proposed to be an Orthopoxvirus. BAV was also shown to be less virulent in animal models than VV Lister. Here we report the identification of an IFN-alpha/betaR gene in the BAV genome with 99% of sequence identity with the VVWR B18R gene. The identified gene encodes a B18R-like IFN binding protein as demonstrated by its capacity to inhibit the IFN-mediated protection of VERO cells against EMC virus. In order to better characterize the virus we have searched for the A type inclusion body (ATI) gene currently used in the classification of Orthopoxviruses but did not detect it in the BAV genome. We have also sequenced the BAV thymidine kinase (TK) gene, a poxvirus-conserved gene, which, as expected, showed high homology with the TK gene of other poxviruses. Phylogenetic trees were constructed based on sequences of the IFN-alpha/betaR and TK genes from several poxviruses and in both cases BAV was placed in the same cluster as other VV strains. These observations strengthened the hypothesis that this virus is a variant of the VV vaccine used in Brazil. However the explanation for the BAV lack of virulence remains to be discovered

  16. Recombinant herpes simplex virus type 1 strains with targeted mutations relevant for aciclovir susceptibility

    PubMed Central

    Brunnemann, Anne-Kathrin; Liermann, Kristin; Deinhardt-Emmer, Stefanie; Maschkowitz, Gregor; Pohlmann, Anja; Sodeik, Beate; Fickenscher, Helmut; Sauerbrei, Andreas; Krumbholz, Andi

    2016-01-01

    Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17+ that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1. PMID:27426251

  17. Recombinant herpes simplex virus type 1 strains with targeted mutations relevant for aciclovir susceptibility.

    PubMed

    Brunnemann, Anne-Kathrin; Liermann, Kristin; Deinhardt-Emmer, Stefanie; Maschkowitz, Gregor; Pohlmann, Anja; Sodeik, Beate; Fickenscher, Helmut; Sauerbrei, Andreas; Krumbholz, Andi

    2016-01-01

    Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17(+) that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1. PMID:27426251

  18. Thyroid hormone-dependent epigenetic suppression of herpes simplex virus-1 gene expression and viral replication in differentiated neuroendocrine cells.

    PubMed

    Figliozzi, Robert W; Chen, Feng; Balish, Matthew; Ajavon, Amakoe; Hsia, S Victor

    2014-11-15

    A global HSV-1 gene repression occurs during latency in sensory neurons where most viral gene transcriptions are suppressed. The molecular mechanisms of gene silencing and how stress factors trigger the reactivation are not well understood. Thyroid hormones are known to be altered due to stress, and with its nuclear receptor impart transcriptional repression or activation depending upon the hormone level. Therefore we hypothesized that triiodothyronine (T3) treatment of infected differentiated neuron like cells would reduce the ability of HSV-1 to produce viral progeny compared to untreated infected cells. Previously we identified putative thyroid hormone receptor elements (TREs) within the promoter regions of HSV-1 thymidine kinase (TK) and other key genes. Searching for a human cell line that can model neuronal HSV-1 infection, we performed HSV-1 infection experiments on differentiated human neuroendocrine cells, LNCaP. Upon androgen deprivation these cells undergo complete differentiation and exhibit neuronal-like morphology and physiology. These cells were readily infected by our HSV-1 recombinant virus, expressing GFP and maintaining many processes iconic of dendritic morphology. Our results demonstrated that differentiated LNCaP cells produced suppressive effects on HSV-1 gene expression and replication compared to its undifferentiated counterpart and T3 treatment has further decreased the viral plaque counts compared to untreated cells. Upon washout of the T3 viral plaque counts were restored, indicating an increase of viral replication. The qRT-PCR experiments using primers for TK showed reduced expression under T3 treatment. ChIP assays using a panel of antibodies for H3 lysine 9 epigenetic marks showed increased repressive marks on the promoter regions of TK. In conclusion we have demonstrated a T3 mediated quiescent infection in differentiated LNCaP cells that has potential to mimic latent infection. In this HSV-1 infection model thyroid hormone

  19. Thyroid Hormone-dependent Epigenetic Suppression of Herpes Simplex Virus-1 Gene Expression and Viral Replication in Differentiated Neuroendocrine Cells

    PubMed Central

    Figliozzi, Robert W.; Chen, Feng; Balish, Matthew; Ajavon, Amakoe; Hsia, S. Victor

    2014-01-01

    A global HSV-1 gene repression occurs during latency in sensory neurons where most viral gene transcriptions are suppressed. The molecular mechanisms of gene silencing and how stress factors trigger the reactivation are not well understood. Thyroid hormones are known to be altered due to stress, and with its nuclear receptor impart transcriptional repression or activation depending upon the hormone level. Therefore we hypothesized that triiodothyronine (T3) treatment of infected differentiated neuron like cells would reduce the ability of HSV-1 to produce viral progeny compared to untreated infected cells. Previously we identified putative thyroid hormone receptor elements (TREs) within the promoter regions of HSV-1 thymidine kinase (TK) and other key genes. Searching for a human cell line that can model neuronal HSV-1 infection, we performed HSV-1 infection experiments on differentiated human neuroendocrine cells, LNCaP. Upon androgen deprivation these cells undergo complete differentiation and exhibit neuronal-like morphology and physiology. These cells were readily infected by our HSV-1 recombinant virus, expressing GFP and maintaining many processes iconic of dendritic morphology. Our results demonstrated that differentiated LNCaP cells produced suppressive effects on HSV-1 gene expression and replication compared to its undifferentiated counterpart and T3 treatment have further decreased the viral plaque counts compared to untreated cells. Upon washout of the T3 viral plaque counts were restored, indicating an increase of viral replication. The qRT-PCR experiments using primers for TK showed reduced expression under T3 treatment. ChIP assays using a panel of antibodies for H3 lysine 9 epigenetic marks showed increased repressive marks on the promoter regions of TK. In conclusion we have demonstrated a T3 mediated quiescent infection in differentiated LNCaP cells that has potential to mimic latent infection. In this HSV-1 infection model thyroid hormone

  20. Intraprostatic distribution and long-term follow-up after AdV-tk immunotherapy as neoadjuvant to surgery in patients with prostate cancer.

    PubMed

    Rojas-Martínez, A; Manzanera, A G; Sukin, S W; Esteban-María, J; González-Guerrero, J F; Gomez-Guerra, L; Garza-Guajardo, R; Flores-Gutiérrez, J P; Elizondo Riojas, G; Delgado-Enciso, I; Ortiz-López, R; Aguilar, L K; Butler, E B; Barrera-Saldaña, H A; Aguilar-Cordova, E

    2013-11-01

    A phase I-II study to evaluate gene-mediated cytotoxic immunotherapy in newly diagnosed prostate cancer before radical prostatectomy was conducted in Monterrey, Mexico. First, to investigate delivery of adenovirus to the prostate, fluorescently labeled vector was injected into fresh prostatectomy specimens and distribution was visually analyzed. The optimal volume and site instillation was then used for transrectal ultrasound guided intraprostatic injection in 10 patients with adenocarcinoma scheduled for radical prostatectomy. Each received two apical and two basal 0.5 ml injections of AdV-tk for a total of 1 × 10(11) vp followed by 14 days of prodrug. Nine patients continued to tumor resection: six high risk, one intermediate and two low risk. In vivo vector distribution was analyzed from the resected tissue of four patients. Patients were monitored for tumor progression and acute and long-term safety. For vector delivery, two apical and two basal injections of 0.5 ml led to optimal organ-wide distribution ex vivo and in vivo. Cytotoxicity was evidenced by transient rise in PSA and tumor histology. There were no significant adverse events deemed related to the treatment and no late toxicities after median follow-up of 11.3 years. All six high-risk patients had positive surgical margins and one had seminal vesicle involvement. Despite slow PSA rise post surgery in three of these patients, none developed metastases. The intermediate- and low-risk patients had complete resections and none have progressed. In conclusion, in vivo transrectal ultrasound guided instillation of an adenoviral vector into four sites in the prostate was practical as an outpatient procedure, well tolerated and led to distribution throughout the intraprostatic tumor mass. AdV-tk demonstrated no significant acute or late toxicities. Trends in PSA and disease progression conveyed the possibility of a sustained immune response against residual disease. PMID:24052127

  1. Intraprostatic distribution and long term follow-up after AdV-tk immunotherapy as neoadjuvant to surgery in patients with prostate cancer

    PubMed Central

    Rojas-Martínez, Augusto; Manzanera, Andrea G.; Sukin, Steven W.; Esteban-María, Jacinto; González-Guerrero, Juan Francisco; Gomez-Guerra, Lauro; Garza-Guajardo, Raquel; Flores-Gutiérrez, Juan Pablo; Riojas, Guillermo Elizondo; Delgado-Enciso, Iván; Ortiz-López, Rocío; Aguilar, Laura K.; Butler, E. Brian; Barrera-Saldaña, Hugo A.; Aguilar-Cordova, Estuardo

    2013-01-01

    A phase I-II study to evaluate gene mediated cytotoxic immunotherapy in newly diagnosed prostate cancer before radical prostatectomy was conducted in Monterrey, Mexico. Methods To investigate delivery of adenovirus to the prostate, fluorescently labeled vector was injected into fresh prostatectomy specimens and distribution visually analyzed. The optimal volume and site instillation was then used for transrectal ultrasound guided intraprostatic injection in 10 patients with adenocarcinoma scheduled for radical prostatectomy. Each received 2-apical and 2-basal 0.5 ml injections of AdV-tk for a total of 1×1011 vp followed by 14 days of prodrug. Nine patients continued to tumor resection: 6 high-risk, 1 intermediate and 2 low-risk. In-vivo vector distribution was analyzed from resected tissue of four patients. Patients were monitored for tumor progression and acute and long-term safety. Results Two apical and two basal injections of 0.5ml led to optimal organ-wide distribution of an adenoviral vector ex-vivo and in-vivo. Cytotoxicity was evidenced by transient rise in PSA and tumor histology. There were no significant adverse events deemed related to the treatment and no late toxicities after median follow up of 11.3 years. All six high-risk patients had positive surgical margins and one had seminal vesicle involvement. Despite slow PSA rise post-surgery in 3 of these patients, none developed metastases. The intermediate and low-risk patients had complete resections and none have progressed. Conclusion In-vivo transrectal ultrasound guided instillation of an adenoviral vector into four sites in the prostate was practical as an outpatient procedure, well tolerated and led to distribution throughout the intraprostatic tumor mass. AdV-tk demonstrated no significant acute or late toxicities. Trends in PSA and disease progression conveyed the possibility of a sustained immune response against residual disease. PMID:24052127

  2. Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and αVβ3 Integrin Antibody in Human Hepatocellular Carcinoma HepG2 Cells

    PubMed Central

    Li, Jiale; Zhou, Ping; Li, Lan; Zhang, Yan; Shao, Yang; Tang, Li; Tian, Shuangming

    2016-01-01

    Objective Hepatocellular carcinoma (HCC), mostly derived from hepatitis or cirrhosisis, is one of the most common types of liver cancer. T-cell mediated immune response elicited by CD/TK double suicide gene has shown a substantial antitumor effect in HCC. Integrin αVβ3 over expresssion has been suggested to regulate the biology behavior of HCC. In this study, we investigated the strategy of incorporating CD/TK double suicide gene and anti-αVβ3 integrin monoclonal antibodies into cationic microbubbles (CMBsαvβ3), and evaluated its killing effect in HCC cells. Methods To improve the transfection efficiency of targeted CD/TK double suicide gene, we adopted cationic microbubbles (CMBs), a cationic delivery agent with enhanced DNA-carrying capacity. The ultrasound and high speed shearing method was used to prepare the non-targeting cationic microbubbles (CMBs). Using the biotin-avidin bridge method, αVβ3 integrin antibody was conjugated to CMBs, and CMBsαvβ3 was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBsαvβ3 was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBsαvβ3 were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBsαvβ3 and CMBs to HCC HepG2 and normal L-02 cells were compared using rosette formation assay. To detect EGFP fluorescence and examine the transfection efficiencies of CMBsαvβ3 and CMBs in HCC cells, fluorescence microscope and contrast-enhanced sonography were adopted. mRNA and protein level of CD/TK gene were detected by RT-PCR and Western blot, respectively. To evaluate the anti-tumor effect of CMBsαvβ3, HCC cells with CMBsαvβ3 were exposed to 5-flurocytosine / ganciclovir (5-FC/GCV). Then, cell cycle distribution after treatment were detected by PI staining and flow cytometry. Apoptotic cells death were detected by optical microscope and assessed by MTT assay and TUNEL

  3. In Vivo Image Analysis of BoHV-4-Based Vector in Mice

    PubMed Central

    Franceschi, Valentina; Stellari, Fabio Franco; Mangia, Carlo; Jacca, Sarah; Lavrentiadou, Sophia; Cavirani, Sandro; Heikenwalder, Mathias; Donofrio, Gaetano

    2014-01-01

    Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver. PMID:24752229

  4. In vivo image analysis of BoHV-4-based vector in mice.

    PubMed

    Franceschi, Valentina; Stellari, Fabio Franco; Mangia, Carlo; Jacca, Sarah; Lavrentiadou, Sophia; Cavirani, Sandro; Heikenwalder, Mathias; Donofrio, Gaetano

    2014-01-01

    Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver. PMID:24752229

  5. N3-substituted thymidine bioconjugates for cancer therapy and imaging

    PubMed Central

    Khalil, Ahmed; Ishita, Keisuke; Ali, Tehane; Tjarks, Werner

    2013-01-01

    The compound class of 3-carboranyl thymidine analogues (3CTAs) are boron delivery agents for boron neutron capture therapy (BNCT), a binary treatment modality for cancer. Presumably, these compounds accumulate selectively in tumor cells via intracellular trapping, which is mediated by hTK1. Favorable in vivo biodistribution profiles of 3CTAs led to promising results in preclinical BNCT of rats with intracerebral brain tumors. This review presents an overview on the design, synthesis, and biological evaluation of first- and second-generation 3CTAs. Boronated nucleosides developed prior to 3CTAs for BNCT and non-boronated N3-substituted thymidine conjugates for other areas of cancer therapy and imaging are also described. In addition, basic features of carborane clusters, which are used as boron moieties in the design and synthesis of 3CTAs, and the biological and structural features of TK1-like enzymes, which are the molecular targets of 3CTAs, are discussed. PMID:23617430

  6. 3,19-isopropylideneandrographolide suppresses early gene expression of drug-resistant and wild type herpes simplex viruses.

    PubMed

    Kongyingyoes, Bunkerd; Priengprom, Thongkoon; Pientong, Chamsai; Aromdee, Chantana; Suebsasana, Supawadee; Ekalaksananan, Tipaya

    2016-08-01

    A diterpenoid lactone, 3,19-isopropylideneandrographolide (IPAD) compound isolated from Andrographis paniculata (Burm. f.) Nees, has been reported to inhibit herpes simplex virus type 1 (HSV-1) infection at the post-entry step. To identify the molecular target of IPAD, this study characterized the inhibitory effect of IPAD on infection of Vero cells by HSV-1, HSV-2 and a drug-resistant (DR) HSV-1 strain ACGr4 (acyclovir-resistant and thymidine kinase (TK)-deficient). Viral production, gene and protein expression were determined using plaque assays, quantitative RT-PCR and western blotting, respectively. The results showed that IPAD inhibited HSV-1, HSV-2 and DR-HSV-1 infections at 6-12 h post-infection, a time that corresponded with E gene expression. IPAD completely suppressed ICP8 transcription and translation as well as DNA replication and gD expression in the three strains tested, while acyclovir suppressed transcription and translation of UL30 and gD of HSV-2, HSV-1, but had no effect on DR-HSV-1. These results showed that IPAD has a different molecular target from acyclovir and might therefore be an alternative drug for HSV-1 and HSV-2 wild types and DR-HSV-1 strains. PMID:27424493

  7. Dual-modality imaging demonstrates the enhanced antitumoral effect of herpes simplex virus-thymidine kinase/ganciclovir plus gemcitabine combination therapy on cholangiocarcinoma

    PubMed Central

    WANG, JIANFENG; LI, ANG; JIN, MEI; ZHANG, FAN; LI, XIAOLING

    2016-01-01

    Herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) therapy is one of the most promising therapeutic strategies for the treatment of cholangiocarcinoma, which is the second most common hepatobiliary cancer. The aim of the present study was to evaluate the enhanced therapeutic effects of HSV-TK/GCV with gemcitabine on cholangiocarcinoma. QBC939 cholangiocarcinoma cells and mouse models of cholangiocarcinoma (established via tumor xenografts) received one of the following treatments: i) Gemcitabine therapy (3 µg/ml); ii) HSV-TK/GCV monotherapy; iii) HSV-TK/GCV + gemcitabine; and iv) control group, treated with phosphate-buffered saline. Cell proliferation was quantified using MTT assay and post-treatment tumor alterations were monitored using ultrasound imaging and optical imaging. For the in vitro experiments, the MTT assays demonstrated that the relative cell viabilities in the gene therapy, gemcitabine and gemcitabine + gene groups were 70.37±9.07, 52.64±8.28 and 34.21±6.63%, respectively. For the in vivo experiments, optical imaging indicated significantly decreased optical signals in the combination therapy group, as compared with the gemcitabine and gemcitabine + gene groups (1.68±0.74 vs. 2.27±0.58 and 2.87±0.82, respectively; Р<0.05). As demonstrated by ultrasound imaging, reduced tumor volumes were detected in the combination therapy group, as compared with the three control groups (114.32±17.17 vs. 159±23.74, 201.63±19.26 and 298.23±36.1 mm3, respectively; P<0.05). The results of the present study demonstrated that gemcitabine enhances the antitumoral effects of HSV-TK/GCV on cholangiocarcinoma, which may provide a novel therapeutic strategy for the management and treatment of cholangiocarcinoma using gemcitabine and gene therapy. PMID:27347037

  8. Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.

    PubMed Central

    Zhang, F; Cole, C N

    1987-01-01

    Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal. Images PMID:2823124

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Oncolytic HSV-1 Infection of Tumors Induces Angiogenesis and Upregulates CYR61

    PubMed Central

    Kurozumi, Kazuhiko; Hardcastle, Jayson; Thakur, Roopa; Shroll, Joshua; Nowicki, Michal; Otsuki, Akihiro; Chiocca, E Antonio; Kaur, Balveen

    2009-01-01

    Oncolytic viral therapy is under evaluation for toxicity and efficacy in clinical trials relating to several different tumors. We report a significant increase in the angiogenic index of oncolytic virus (OV)-treated glioma-matrigel implants (2.83-fold, P < 0.02). In a rat intracranial glioma model, large tumors from OV-treated animals were significantly more angiogenic than the phosphate-buffered saline (PBS)-treated control tumors (OV: 101 ± 21.6; PBS: 19.8 ± 10; P = 0.0037). Transcript profiling of OV-treated tumors revealed dysregulation of several transcripts involved in glioma angiogenesis. OV-mediated induction of CYR61 gene expression (8.94-fold, P = 0.001) correlated significantly with the presence of OV in tumor tissue in vivo (R = 0.7, P < 0.001). Further, induction of CYR61 mRNA and protein were confirmed in multiple human cancer cell lines and primary human tumor-derived cells in vitro, and in tumor lysate and cerebrospinal fluid (CSF) in vivo. Finally, we show that treatment of glioma cells with Cilengitide, known to counter CYR61-induced integrin activation, significantly suppressed the proangiogenic effect of OV treatment of gliomas (P < 0.05). PMID:18545226

  11. Enhanced Antitumor Efficacy of Vasculostatin (Vstat120) Expressing Oncolytic HSV-1

    PubMed Central

    Hardcastle, Jayson; Kurozumi, Kazuhiko; Dmitrieva, Nina; Sayers, Martin P; Ahmad, Sarwat; Waterman, Peter; Weissleder, Ralph; Chiocca, E Antonio; Kaur, Balveen

    2009-01-01

    Oncolytic viral (OV) therapy is a promising therapeutic modality for brain tumors. Vasculostatin (Vstat120) is the cleaved and secreted extracellular fragment of brain-specific angiogenesis inhibitor 1 (BAI1), a brain-specific receptor. To date, the therapeutic efficacy of Vstat120 delivery into established tumors has not been investigated. Here we tested the therapeutic efficacy of combining Vstat120 gene delivery in conjunction with OV therapy. We constructed RAMBO (Rapid Antiangiogenesis Mediated By Oncolytic virus), which expresses Vstat120 under the control of the herpes simplex virus (HSV) IE4/5 promoter. Secreted Vstat120 was detected as soon as 4 hours postinfection in vitro and was retained for up to 13 days after OV therapy in subcutaneous tumors. RAMBO-produced Vstat120 efficiently inhibited endothelial cell migration and tube formation in vitro (P = 0.0005 and P = 0.0184, respectively) and inhibited angiogenesis (P = 0.007) in vivo. There was a significant suppression of intracranial and subcutaneous glioma growth in mice treated with RAMBO compared to the control virus, HSVQ (P = 0.0021 and P < 0.05, respectively). Statistically significant reduction in tumor vascular volume fraction (VVF) and microvessel density (MVD) was observed in tumors treated with RAMBO. This is the first study to report the antitumor effects of Vstat120 delivery into established tumors and supports the further development of RAMBO as a possible cancer therapy. PMID:19844198

  12. Anti HSV-1 flavonoid derivatives tethered with houttuynin from Houttuynia cordata.

    PubMed

    Chen, Shao-Dan; Li, Ting; Gao, Hao; Zhu, Qin-Chang; Lu, Chuan-Jian; Wu, Hong-Ling; Peng, Tao; Yao, Xin-Sheng

    2013-12-01

    This paper reports the phytochemical investigation of the 50% aq. EtOH extract of Houttuynia cordata, an effective TCM and functional food in China, which led to the isolation of 17 flavonoids including four new ones. The four new compounds were flavonoid derivatives tethered with houttuynin (3-oxododecanal). Each of the new compounds was obtained as a pair of inseparable diasteriomeric epimers due to the chiral carbon of hemiketal at C-3″. This phenomenon is rooted in the ring-chain tautomerism of the hemiketal functional group in solution, which was proved by dynamic NMR experiments. The new compounds 1-4 displayed inhibitory activities against herpes simplex virus 1, with respective IC50 values of 38.46, 14.10, 62.00 and 70.76 µM, which was associated with the medicinal functions of H. cordata. PMID:24288290

  13. Role of ND10 nuclear bodies in the chromatin repression of HSV-1.

    PubMed

    Gu, Haidong; Zheng, Yi

    2016-01-01

    Herpes simplex virus (HSV) is a neurotropic virus that establishes lifelong latent infection in human ganglion sensory neurons. This unique life cycle necessitates an intimate relation between the host defenses and virus counteractions over the long course of infection. Two important aspects of host anti-viral defense, nuclear substructure restriction and epigenetic chromatin regulation, have been intensively studied in the recent years. Upon viral DNA entering the nucleus, components of discrete nuclear bodies termed nuclear domain 10 (ND10), converge at viral DNA and place restrictions on viral gene expression. Meanwhile the infected cell mobilizes its histones and histone-associated repressors to force the viral DNA into nucleosome-like structures and also represses viral transcription. Both anti-viral strategies are negated by various HSV countermeasures. One HSV gene transactivator, infected cell protein 0 (ICP0), is a key player in antagonizing both the ND10 restriction and chromatin repression. On one hand, ICP0 uses its E3 ubiquitin ligase activity to target major ND10 components for proteasome-dependent degradation and thereafter disrupts the ND10 nuclear bodies. On the other hand, ICP0 participates in de-repressing the HSV chromatin by changing histone composition or modification and therefore activates viral transcription. Involvement of a single viral protein in two seemingly different pathways suggests that there is coordination in host anti-viral defense mechanisms and also cooperation in viral counteraction strategies. In this review, we summarize recent advances in understanding the role of chromatin regulation and ND10 dynamics in both lytic and latent HSV infection. We focus on the new observations showing that ND10 nuclear bodies play a critical role in cellular chromatin regulation. We intend to find the connections between the two major anti-viral defense pathways, chromatin remodeling and ND10 structure, in order to achieve a better understanding of how host orchestrates a concerted defense and how HSV adapts with and overcomes the host immunity. PMID:27048561

  14. Ocular infection with herpes simplex virus (HSV-1) in vitamin A-deficient and control rats.

    PubMed

    Nauss, K M; Anderson, C A; Conner, M W; Newberne, P M

    1985-10-01

    An experimental model was developed for studying ocular infections with herpes simplex virus (HSV) type 1 in vitamin A-deficient (-A) and pair-fed control (+A) rats. The severity and course of the disease was evaluated by clinical examination, slit lamp biomicroscopy and histopathologic observations. Experimental animals were in good health and were infected in the early stages of vitamin deficiency (either prior to or at the beginning of the weight plateau). In all trials the onset of herpetic keratitis was more rapid and the clinical disease more severe in -A rats compared to +A controls. Mean slit lamp scores (which assessed the severity of the corneal disease) increased from 3 to 10 d after infection and were higher (P less than 0.002) in -A rats at all time points and doses of virus tested. The inflammatory response in the cornea and uveal tract of -A rats was significantly higher than that of +A animals. Since ocular HSV disease is a common cause of blindness, the availability of a rat model should be valuable in studies of the role of nutritional factors in host susceptibility and response to viral challenge. Mild vitamin A deficiency increased the severity of experimental corneal HSV infections and resulted in a high incidence of epithelial ulceration and necrosis. PMID:2995622

  15. Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles

    NASA Technical Reports Server (NTRS)

    Evans, Helen H.; Horng, Min-Fen; Ricanati, Marlene; Diaz-Insua, M.; Jordan, Robert; Schwartz, Jeffrey L.

    2003-01-01

    The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.

  16. IMAGEP - A FORTRAN ALGORITHM FOR DIGITAL IMAGE PROCESSING

    NASA Technical Reports Server (NTRS)

    Roth, D. J.

    1994-01-01

    IMAGEP is a FORTRAN computer algorithm containing various image processing, analysis, and enhancement functions. It is a keyboard-driven program organized into nine subroutines. Within the subroutines are other routines, also, selected via keyboard. Some of the functions performed by IMAGEP include digitization, storage and retrieval of images; image enhancement by contrast expansion, addition and subtraction, magnification, inversion, and bit shifting; display and movement of cursor; display of grey level histogram of image; and display of the variation of grey level intensity as a function of image position. This algorithm has possible scientific, industrial, and biomedical applications in material flaw studies, steel and ore analysis, and pathology, respectively. IMAGEP is written in VAX FORTRAN for DEC VAX series computers running VMS. The program requires the use of a Grinnell 274 image processor which can be obtained from Mark McCloud Associates, Campbell, CA. An object library of the required GMR series software is included on the distribution media. IMAGEP requires 1Mb of RAM for execution. The standard distribution medium for this program is a 1600 BPI 9track magnetic tape in VAX FILES-11 format. It is also available on a TK50 tape cartridge in VAX FILES-11 format. This program was developed in 1991. DEC, VAX, VMS, and TK50 are trademarks of Digital Equipment Corporation.

  17. Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population.

    PubMed

    Caruso, M; Klatzmann, D

    1992-01-01

    We have stably expressed in CD4+ lymphoid cells the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene under the control of the human immunodeficiency virus (HIV) promoter and transactivation response element sequences. Upon HIV infection these regulatory sequences were transactivated, switching on high-level expression of HSV1-TK. This in turn caused the death of HIV-infected cells when they were cultured in the presence of acyclovir, a nucleoside analog that becomes toxic after phosphorylation by HSV1-TK. The elimination of HIV-infected cells resulted in the arrest of HIV spreading in the culture. Complete protection of HSV1-TK-expressing cells was obtained using acyclovir concentrations that are commonly detected in the plasma of patients treated for HSV1 infection. Thus, expression of this DNA construct generates a pool of CD4+ booby-trapped cells that, as a population, are resistant to HIV infection. Our data provide a rationale for the use of suicide genes in the design of gene therapy of HIV infection. PMID:1346066

  18. Combination of PEI-Mn0.5Zn0.5Fe2O4 nanoparticles and pHsp 70-HSV-TK/GCV with magnet-induced heating for treatment of hepatoma

    PubMed Central

    Tang, Qiusha; Lu, Mudan; Chen, Daozhen; Liu, Peidang

    2015-01-01

    Background To explore a new combination of thermal treatment and gene therapy for hepatoma, a heat-inducible herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy system was developed in which thermal energy generated by Mn0.5Zn0.5Fe2O4 nanoparticles (MZF-NPs) under an alternating magnetic field was used to activate gene expression. Methods First, a recombinant eukaryotic plasmid, pHsp 70-HSV-TK, was constructed as a target gene for therapy. This recombinant plasmid was used to transfect SMMC-7721 hepatoma cells and the gene expression was evaluated. Magnet-induced heating was then applied to cells to assess the antihepatoma effects of the polyethylenimine (PEI)-MZF-NPs/pHsp 70-HSV-TK/GCV complex, in vitro and in vivo. Results The results showed that cells were successfully transfected with pHsp 70-HSV-TK and that expression levels of HSV-TK remained stable. Both in vitro and in vivo results indicated that the combination of gene therapy and heat treatment resulted in better therapeutic effects than heating-alone group. The rates of apoptosis and necrosis in the combined treatment group were 49.0% and 7.21%, respectively. The rate of inhibition of cell proliferation in the combined treatment group was significantly higher (87.5%) than that in the heating-alone group (65.8%; P<0.01). The tumor volume and mass inhibition rates of the combined treatment group were 91.3% and 87.91%, respectively, and were significantly higher than the corresponding rates of the heating-alone group (70.41% and 57.14%; P<0.01). The expression levels of Stat3 and Bcl-xL messenger RNA and p-Stat3 and Bcl-xL protein in the combined treatment group were significantly lower than those in the other groups (P<0.01). The expression levels of Bax messenger RNA and protein in the recombinant plasmid group were significantly higher than those in the other groups (P<0.01). Conclusion It can therefore be concluded that the combined application of heat treatment and gene therapy

  19. Application of the TGx-28.65 transcriptomic biomarker to classify genotoxic and non-genotoxic chemicals in human TK6 cells in the presence of rat liver S9.

    PubMed

    Yauk, Carole L; Buick, Julie K; Williams, Andrew; Swartz, Carol D; Recio, Leslie; Li, Heng-Hong; Fornace, Albert J; Thomson, Errol M; Aubrecht, Jiri

    2016-05-01

    In vitro transcriptional signatures that predict toxicities can facilitate chemical screening. We previously developed a transcriptomic biomarker (known as TGx-28.65) for classifying agents as genotoxic (DNA damaging) and non-genotoxic in human lymphoblastoid TK6 cells. Because TK6 cells do not express cytochrome P450s, we confirmed accurate classification by the biomarker in cells co-exposed to 1% 5,6 benzoflavone/phenobarbital-induced rat liver S9 for metabolic activation. However, chemicals may require different types of S9 for activation. Here we investigated the response of TK6 cells to higher percentages of Aroclor-, benzoflavone/phenobarbital-, or ethanol-induced rat liver S9 to expand TGx-28.65 biomarker applicability. Transcriptional profiles were derived 3 to 4 hr following a 4 hr co-exposure of TK6 cells to test chemicals and S9. Preliminary studies established that 10% Aroclor- and 5% ethanol-induced S9 alone did not induce the TGx-28.65 biomarker genes. Seven genotoxic and two non-genotoxic chemicals (and concurrent solvent and positive controls) were then tested with one of the S9s (selected based on cell survival and micronucleus induction). Relative survival and micronucleus frequency was assessed by flow cytometry in cells 20 hr post-exposure. Genotoxic/non-genotoxic chemicals were accurately classified using the different S9s. One technical replicate of cells co-treated with dexamethasone and 10% Aroclor-induced S9 was falsely classified as genotoxic, suggesting caution in using high S9 concentrations. Even low concentrations of genotoxic chemicals (those not causing cytotoxicity) were correctly classified, demonstrating that TGx-28.65 is a sensitive biomarker of genotoxicity. A meta-analysis of datasets from 13 chemicals supports that different S9s can be used in TK6 cells, without impairing classification using the TGx-28.65 biomarker. Environ. Mol. Mutagen. 57:243-260, 2016. © 2016 Her Majesty the Queen in Right of Canada. Environmental and

  20. γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9.

    PubMed

    Bernacki, Derek T; Bryce, Steven M; Bemis, Jeffrey C; Kirkland, David; Dertinger, Stephen D

    2016-08-01

    Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry-based assay (MultiFlow™ DNA Damage Kit-p53, γH2AX, Phospho-Histone H3) can distinguish direct-acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. []: Environ Mol Mutagen 57:171-189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk-away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen. 57:546-558, 2016. © 2016 Wiley Periodicals, Inc. PMID:27364561

  1. The Use of 14C-FIAU to Predict Bacterial Thymidine Kinase Presence: Implications for Radiolabeled FIAU Bacterial Imaging

    PubMed Central

    Peterson Kristin, L; Reid William, C; Freeman Alexandra, F; Holland Steven, M; Roderic, Pettigrew; Gharib Ahmed, M; Hammoud Dima, A

    2013-01-01

    Currently available infectious disease imaging techniques cannot differentiate between infection and sterile inflammation or between different types of infections. Recently, radiolabeled FIAU was found to be a substrate for the thymidine kinase (TK) enzyme of multiple pathogenic bacteria, leading to its translational use in the imaging of bacterial infections. Patients with immunodeficiencies, however, are susceptible to a different group of pathogenic bacteria when compared to immunocompetent subjects. In this study, we wanted to predict the usefulness of radiolabeled FIAU in the detection of bacterial infections commonly occurring in patients with immunodeficiencies, in vitro, prior to attempting in vivo imaging with 124I-FIAU-PET. Methods We obtained representative strains of bacterial pathogens isolated from actual patients with genetic immunodeficiencies. We evaluated the bacterial susceptibility of different strains to the effect of incubation with FIAU, which would implicate the presence of the thymidine kinase (TK) enzyme. We also incubated the bacteria with 14C-FIAU and consequently measured its rate of incorporation in the bacterial DNA using a liquid scintillation counter. Results Unlike the other bacterial strains, the growth of Pseudomonas aeruginosa was not halted by FIAU at any concentration. All the tested clinical isolates demonstrated different levels of 14C-FIAU uptake, except for P. aeruginosa. Conclusion Radiolabeled FIAU has been successful in delineating bacterial infections, both in preclinical and pilot translational studies. In patients with immunodeficiencies, Pseudomonas infections are commonly encountered and are usually difficult to differentiate from fungal infections. The use of radiolabeled FIAU for in vivo imaging of those patients, however, would not be useful, considering the apparent lack of TK enzyme in Pseudomonas. One has to keep in mind that not all pathogenic bacteria possess the TK enzyme and as such will not all retain

  2. Integrity assessment report of tanks TK-101 and TK-102

    SciTech Connect

    MCSHANE, D.S.

    1999-08-25

    This Integrity Assessment Report (IAR) is prepared by Fluor Daniel Northwest (FDNW) for Waste Management Federal Services of Hanford, Inc., (WMH), the operations contractor; Fluor Daniel Hanford (FDH), the Hanford Site Manager; and the U. S. Department of Energy (DOE), the system owner. This IAR addresses the evaluation of Tanks 101 and 102 and other existing components located in the 219-S Waste Handling Facility. This report will be included in the Part B Permit for the 2226 Laboratory and is a portion of the integrity assessment of the overall 222-5 Laboratory radioactive liquid waste disposal system. This IAR is prepared in accordance with WAC 173-303, Dangerous Waste Regulations; Section 640(2), ''Assessment of Existing Tank Systems Integrity .''

  3. The therapeutic effect of PEI-Mn0.5Zn0.5Fe2O4 nanoparticles/pEgr1-HSV-TK/GCV associated with radiation and magnet-induced heating on hepatoma

    NASA Astrophysics Data System (ADS)

    Lin, Mei; Huang, Junxing; Zhang, Jia; Wang, Li; Xiao, Wei; Yu, Hong; Li, Yuntao; Li, Hongbo; Yuan, Chenyan; Hou, Xinxin; Zhang, Hao; Zhang, Dongsheng

    2013-01-01

    Comprehensive therapy based on the integration of hyperthermia, radiation, gene therapy and chemotherapy is a promising area of study in cancer treatment. Using PEI-Mn0.5Zn0.5Fe2O4 nanoparticles (PEI-MZF-NPs) as a gene transfer vector, the authors transfected self-prepared pEgr1-HSV-TK into HepG2 cells and measured the expression of the exogenous gene HSV-TK by RT-PCR. The results showed that HSV-TK was successfully transfected into HepG2 cells and the expression levels of HSV-TK remained stable. Besides, PEI-MZF-NPs were used as magnetic media for thermotherapy to treat hepatoma by magnet-induced heating, combined with radiation-gene therapy. Both in vitro and in vivo results suggest that this combined treatment with gene, radiation and heating has a better therapeutic effect than any of them alone. The apoptotic rate and necrotic rate of the combined treatment group was 51.84% and 15.45%, respectively. In contrast, it was only 20.55% and 6.80% in the radiation-gene group, 7.49% and 3.62% in the radiation-alone group, 15.23% and 7.90% in the heating-alone group, and only 3.52% and 2.16% in the blank control group. The inhibition rate of cell proliferation (88.5%) of the combined treatment group was significantly higher than that of the radiation-gene group (59.5%), radiation-alone group (37.6%) and heating-alone group (60.6%). The tumor volume and mass inhibition rate of the combined treatment group was 94.45% and 93.38%, respectively, significantly higher than 41.28% and 33.58% of the radiation-alone group, 60.76% and 52.18% of the radiation-gene group, 79.91% and 77.40% of the heating-alone group. It is therefore concluded that this combined application of heating, radiation and gene therapy has a good synergistic and complementary effect and PEI-MZF-NPs can act as a novel non-viral gene vector and magnetic induction medium, which offers a viable approach for the treatment of cancer.

  4. Inhibition of TNFα-induced iNOS expression in HSV-tk transduced 9L glioblastoma cell lines by Marasmius oreades substances through NF-κB- and MAPK-dependent mechanisms.

    PubMed

    Ruimi, Nili; Petrova, Roumyana D; Agbaria, Riad; Sussan, Sherbel; Wasser, Solomon P; Reznick, Abraham Z; Mahajna, Jamal

    2010-12-01

    Nitric oxide (NO) is a gaseous, radical molecule that plays a role in various physiological processes. Previously, we reported that transduction of murine colon cancer cells (MC38) with herpes simplex virus thymidine kinase (HSV-tk) gene resulted in a significant over-expression of cyclooxygenase-2 (COX-2) and activation of NF-kB pathway. In this study we show that TNFα, but not LPS, was significantly able to stimulate the production of NO in HSV-tk transduced 9L glioblastoma cell lines, mediated by the up-regulation of iNOS transcript and iNOS protein. The TNFα-induced up-regulation of iNOS expression was mediated by MAPK and NF-κB signaling pathways as revealed by using selective pharmaceutical inhibitors. A culture liquid extract of the edible and medicinal mushroom Marasmius oreades that was previously shown to inhibit iNOS expression in MCF-7 was utilized to prepare fractions and evaluate their ability to affect TNFα-induced iNOS expression in HSV tk transduced 9L cell lines. While most of the tested fractions were shown to inhibit TNFα-induced iNOS expression, they targeted different signaling pathways in a selective fashion. Here, we report that fraction SiSiF1 interfered with IKBα phosphorylation and consequently interfered with NF-κB activation pathway. SiSiF1 showed minimal interference with the phosphorylation of p38 and JNK proteins. In contrast, fraction SiSiF3 selectively inhibited the phosphorylation of p38 and fractions SiSiF4 and SiSiF5 selectively inhibited the phosphorylation of JNK with no observed effect against IKBα and p38 phosphorylation. Our data illustrate the complexity of iNOS regulation in HSV tk transduced 9L cell lines and also the richness of natural products with bioactive substances that may act synergistically through different signaling pathways to affect iNOS gene expression. PMID:20224909

  5. Synthesis of 5-Fluoroalkylated Pyrimidine Nucleosides via Negishi Cross-Coupling

    PubMed Central

    Chacko, Ann-Marie; Qu, Wenchao

    2014-01-01

    5-fluoroalkylated pyrimidine nucleosides (1) have potential as therapeutic agents and molecular imaging agents targeting HSV1-tk suicide gene therapy. Thus, straightforward preparation of 5-fluoroalkylated nucleoside derivatives has been developed. Reported herein are the first examples of Pd-catalyzed Negishi cross-coupling of 3-N-benzoyl-3′,5′-di-O-benzoyl-5-iodo-2′-deoxyuridine (2a) and 3-N-benzoyl-3′,5′-di-O-benzoyl-5-iodo-2′-deoxy-2′-fluoroarabinouridine (2b) with unactivated Csp3 fluoroalkylzinc bromides. This paper demonstrates the first synthesis of six 5-fluoroalkyl-2′-deoxy pyrimidine nucleoside derivatives with three to five methylene-chain lengths (5). Furthermore, this methodology has been extended to create a series of thirteen 5-alkyl substituted nucleosides, including the target nucleosides 5 and 5-silyloxypropyl and 5-cyanobutyl derivatives. PMID:18522415

  6. Sequence Analysis of Herpes Simplex Virus 1 Thymidine Kinase and DNA Polymerase Genes from over 300 Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral Resistance.

    PubMed

    Schmidt, Susanne; Bohn-Wippert, Kathrin; Schlattmann, Peter; Zell, Roland; Sauerbrei, Andreas

    2015-08-01

    A total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All HSV-1 isolates were analyzed genotypically for nonsynonymous mutations in the thymidine kinase (TK) and DNA polymerase (Pol) genes. The resistance phenotype against acyclovir (ACV) and/or foscarnet (FOS) was examined in the case of novel, unclear, or resistance-related mutations. Twenty-six novel natural polymorphisms could be detected in the TK gene and 69 in the DNA Pol gene. Furthermore, three novel resistance-associated mutations (two in the TK gene and one in the DNA Pol gene) were analyzed, and eight known but hitherto unclear amino acid substitutions (two encoded in TK and six in the DNA Pol gene) could be clarified. Between 1973 and 2014, the distribution of amino acid changes related to the natural gene polymorphisms of TK and DNA Pol remained largely stable. Resistance to ACV was confirmed phenotypically for 16 isolates, and resistance to ACV plus FOS was confirmed for 1 isolate. Acyclovir-resistant strains were observed from the year 1995 onwards, predominantly in immunosuppressed patients, especially those with stem cell transplantation, and the number of ACV-resistant strains increased during the last 2 decades. The data confirm the strong genetic variability among HIV-1 isolates, which is more pronounced in the DNA Pol gene than in the TK gene, and will facilitate considerably the rapid genotypic diagnosis of HSV-1 resistance. PMID:26055375

  7. Database on natural polymorphisms and resistance-related non-synonymous mutations in thymidine kinase and DNA polymerase genes of herpes simplex virus types 1 and 2.

    PubMed

    Sauerbrei, Andreas; Bohn-Wippert, Kathrin; Kaspar, Marisa; Krumbholz, Andi; Karrasch, Matthias; Zell, Roland

    2016-01-01

    The use of genotypic resistance testing of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is increasing because the rapid availability of results significantly improves the treatment of severe infections, especially in immunocompromised patients. However, an essential precondition is a broad knowledge of natural polymorphisms and resistance-associated mutations in the thymidine kinase (TK) and DNA polymerase (pol) genes, of which the DNA polymerase (Pol) enzyme is targeted by the highly effective antiviral drugs in clinical use. Thus, this review presents a database of all non-synonymous mutations of TK and DNA pol genes of HSV-1 and HSV-2 whose association with resistance or natural gene polymorphism has been clarified by phenotypic and/or functional assays. In addition, the laboratory methods for verifying natural polymorphisms or resistance mutations are summarized. This database can help considerably to facilitate the interpretation of genotypic resistance findings in clinical HSV-1 and HSV-2 strains. PMID:26433780

  8. IMAGES, IMAGES, IMAGES

    SciTech Connect

    Marcus, A.

    1980-07-01

    The role of images of information (charts, diagrams, maps, and symbols) for effective presentation of facts and concepts is expanding dramatically because of advances in computer graphics technology, increasingly hetero-lingual, hetero-cultural world target populations of information providers, the urgent need to convey more efficiently vast amounts of information, the broadening population of (non-expert) computer users, the decrease of available time for reading texts and for decision making, and the general level of literacy. A coalition of visual performance experts, human engineering specialists, computer scientists, and graphic designers/artists is required to resolve human factors aspects of images of information. The need for, nature of, and benefits of interdisciplinary effort are discussed. The results of an interdisciplinary collaboration are demonstrated in a product for visualizing complex information about global energy interdependence. An invited panel will respond to the presentation.

  9. Prevalence of Intrathecal Acyclovir Resistant Virus in Herpes Simplex Encephalitis Patients.

    PubMed

    Mitterreiter, Johanna G; Titulaer, Maarten J; van Nierop, Gijsbert P; van Kampen, Jeroen J A; Aron, Georgina I; Osterhaus, Albert D M E; Verjans, Georges M G M; Ouwendijk, Werner J D

    2016-01-01

    Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR-associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real-time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients. Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients. Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV-sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients. PMID:27171421

  10. Prevalence of Intrathecal Acyclovir Resistant Virus in Herpes Simplex Encephalitis Patients

    PubMed Central

    Mitterreiter, Johanna G.; Titulaer, Maarten J.; van Nierop, Gijsbert P.; van Kampen, Jeroen J. A.; Aron, Georgina I.; Osterhaus, Albert D. M. E.; Verjans, Georges M. G. M.; Ouwendijk, Werner J. D.

    2016-01-01

    Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR–associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real-time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients. Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients. Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV-sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients. PMID:27171421

  11. Evidence for a novel regulatory pathway for herpes simplex virus gene expression in trigeminal ganglion neurons.

    PubMed Central

    Kosz-Vnenchak, M; Jacobson, J; Coen, D M; Knipe, D M

    1993-01-01

    Thymidine kinase (TK)-negative (TK-) mutant strains of herpes simplex virus type 1 (HSV-1) show reduced expression of alpha and beta viral genes during acute infection of trigeminal ganglion neurons following corneal infection (M. Kosz-Vnenchak, D. M. Coen, and D. M. Knipe, J. Virol. 64:5396-5402, 1990). It was surprising that a defect in a beta gene product would lead to decreased alpha and beta gene expression, given the regulatory pathways demonstrated for HSV infection of cultured cells. In this study, we have examined viral gene expression during reactivation from latent infection in explanted trigeminal ganglion tissue. In explant reactivation studies with wild-type virus, we observed viral productive gene expression over the first 48 h of explant incubation occurring in a temporal order (alpha, beta, gamma) similar to that in cultured cells. This occurred predominantly in latency-associated transcript-positive neurons but was limited to a fraction of these cells. In contrast, TK- mutant viruses showed greatly reduced alpha and beta gene expression upon explant of latently infected trigeminal ganglion tissue. An inhibitor of viral TK or an inhibitor of viral DNA polymerase greatly decreased viral lytic gene expression in trigeminal ganglion tissue latently infected with wild-type virus and explanted in culture. These results indicate that the regulatory mechanisms governing HSV gene expression are different in trigeminal ganglion neurons and cultured cells. We present a new model for viral gene expression in trigeminal ganglion neurons with implications for the nature of the decision process between latent infection and productive infection by HSV. Images PMID:8394454

  12. Details of ssDNA annealing revealed by an HSV-1 ICP8–ssDNA binary complex

    PubMed Central

    Tolun, Gökhan; Makhov, Alexander M.; Ludtke, Steven J.; Griffith, Jack D.

    2013-01-01

    Infected cell protein 8 (ICP8) from herpes simplex virus 1 was first identified as a single-strand (ss) DNA-binding protein. It is essential for, and abundant during, viral replication. Studies in vitro have shown that ICP8 stimulates model replication reactions, catalyzes annealing of complementary ssDNAs and, in combination with UL12 exonuclease, will catalyze ssDNA annealing homologous recombination. DNA annealing and strand transfer occurs within large oligomeric filaments of ssDNA-bound ICP8. We present the first 3D reconstruction of a novel ICP8–ssDNA complex, which seems to be the basic unit of the DNA annealing machine. The reconstructed volume consists of two nonameric rings containing ssDNA stacked on top of each other, corresponding to a molecular weight of 2.3 MDa. Fitting of the ICP8 crystal structure suggests a mechanism for the annealing reaction catalyzed by ICP8, which is most likely a general mechanism for protein-driven DNA annealing. PMID:23605044

  13. Induction of apoptosis accelerates reactivation of latent HSV-1 in ganglionic organ cultures and replication in cell cultures

    PubMed Central

    Du, Te; Zhou, Guoying; Roizman, Bernard

    2012-01-01

    Herpes simplex viruses replicate at the portal of entry into the body and are transported retrograde to sensory neurons in which they can establish a silent, latent infection characterized by the expression of a noncoding latency-associated transcript and a set of microRNAs. At the portal of entry into the body and in cell culture a viral protein, VP16, recruits cellular proteins that initiate a sequential derepression of several kinetic classes of viral genes. Earlier studies have shown that upon reactivation of latent virus in ganglionic organ cultures all genes are derepressed at once, thus obviating the need for VP16 to initiate sequential derepression of viral genes. One hypothesis that could explain the data is that the massive reactivation of all classes of viral genes is the consequence of activation of an apoptotic pathway. Here we show that two proapoptotic drugs, dexamethasone and 2[[3-(2,3-dichlorophenoxy)propyl]amino]-ethanol, each accelerates viral gene expression in ganglionic organ cultures. We also show that in cultured cells apoptosis induced by dexamethasone accelerates viral gene expression and accumulation of infectious virus. The results are surprising in light of the relatively large number of viral proteins that independently block apoptosis induced by viral gene products or exogenous agents. The results suggest that the virus may rely on apoptosis to exit from latency but that apoptosis may be detrimental for virus replication or spread at the portal of entry into the body. PMID:22908263

  14. Crystal Structure of the HSV-1 Fc Receptor Bound to Fc Reveals a Mechanism for Antibody Bipolar Bridging

    SciTech Connect

    Sprague, E.R.; Wang, C.; Baker, D.; Bjorkman, P.J.; /Caltech /Howard Hughes Med. Inst.

    2007-08-08

    Herpes simplex virus type-1 expresses a heterodimeric Fc receptor, gE-gI, on the surfaces of virions and infected cells that binds the Fc region of host immunoglobulin G and is implicated in the cell-to-cell spread of virus. gE-gI binds immunoglobulin G at the basic pH of the cell surface and releases it at the acidic pH of lysosomes, consistent with a role in facilitating the degradation of antiviral antibodies. Here we identify the C-terminal domain of the gE ectodomain (CgE) as the minimal Fc-binding domain and present a 1.78-{angstrom} CgE structure. A 5-{angstrom} gE-gI/Fc crystal structure, which was independently verified by a theoretical prediction method, reveals that CgE binds Fc at the C{sub H}2-C{sub H}3 interface, the binding site for several mammalian and bacterial Fc-binding proteins. The structure identifies interface histidines that may confer pH-dependent binding and regions of CgE implicated in cell-to-cell spread of virus. The ternary organization of the gE-gI/Fc complex is compatible with antibody bipolar bridging, which can interfere with the antiviral immune response.

  15. Monitoring metabolic responses to chemotherapy in single cells and tumors using nanostructure-initiator mass spectrometry (NIMS) imaging

    PubMed Central

    2013-01-01

    Background Tissue imaging of treatment-induced metabolic changes is useful for optimizing cancer therapies, but commonly used methods require trade-offs between assay sensitivity and spatial resolution. Nanostructure-Initiator Mass Spectrometry imaging (NIMS) permits quantitative co-localization of drugs and treatment response biomarkers in cells and tissues with relatively high resolution. The present feasibility studies use NIMS to monitor phosphorylation of 3′-deoxy-3′-fluorothymidine (FLT) to FLT-MP in lymphoma cells and solid tumors as an indicator of drug exposure and pharmacodynamic responses. Methods NIMS analytical sensitivity and spatial resolution were examined in cultured Burkitt’s lymphoma cells treated briefly with Rapamycin or FLT. Sample aliquots were dispersed on NIMS surfaces for single cell imaging and metabolic profiling, or extracted in parallel for LC-MS/MS analysis. Docetaxel-induced changes in FLT metabolism were also monitored in tissues and tissue extracts from mice bearing drug-sensitive tumor xenografts. To correct for variations in FLT disposition, the ratio of FLT-MP to FLT was used as a measure of TK1 thymidine kinase activity in NIMS images. TK1 and tumor-specific luciferase were measured in adjacent tissue sections using immuno-fluorescence microscopy. Results NIMS and LC-MS/MS yielded consistent results. FLT, FLT-MP, and Rapamycin were readily detected at the single cell level using NIMS. Rapid changes in endogenous metabolism were detected in drug-treated cells, and rapid accumulation of FLT-MP was seen in most, but not all imaged cells. FLT-MP accumulation in xenograft tumors was shown to be sensitive to Docetaxel treatment, and TK1 immunoreactivity co-localized with tumor-specific antigens in xenograft tumors, supporting a role for xenograft-derived TK1 activity in tumor FLT metabolism. Conclusions NIMS is suitable for monitoring drug exposure and metabolite biotransformation with essentially single cell resolution, and

  16. Hypertools in image and volume visualization

    SciTech Connect

    Bossart, P.L.

    1996-06-17

    This paper describes our experience in image and volume visualization at the Lawrence Livermore National Laboratory. After an introduction on visualization issues, we present a new software approach to the analysis and visualization of images and volumes. The efficiency of the visualization process is improved by letting the user combine small and reusable applications by the means of a machine-independent interpreted language such as Tcl/Tk. These hypertools can communicate with each other over a network, which has a direct impact on the design of graphical interfaces. We first describe the implementation of a flexible gray-scale image widget that can handle large data sets, provides complete control of the color palette and allows for manual and semi-interactive segmentation. This visualization tool can be embedded in a data-flow image processing environment to assess the quality of acquisition, preprocessing and filtering of raw data. This approach combines the simplicity of visual programming with the power of a high-level interpreted language. We show how hypertools can be used in surface and volume rendering and how they increase the interaction efficiency by performing complex or tedious tasks automatically. One biomedical application is presented.

  17. Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes

    PubMed Central

    Moroz, Maxim A.; Zhang, Hanwen; Lee, Jason; Moroz, Ekaterina; Zurita, Juan; Shenker, Larissa; Serganova, Inna; Blasberg, Ronald; Ponomarev, Vladimir

    2015-01-01

    Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. Methods Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodiumiodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 105 to 3 × 106 reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (back-ground) was measured. Results The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-18F-fluorobenzylguanidine (18F-MFBG) reporter system could detect less than 1 × 105 T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/124I-iodide reporter system could detect approximately 1 × 106 T cells; 124I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2′-18F-fluoro-5-ethyl-1-β-D-arabinofuranosyluracil (18F-FEAU) and hsvTK/18F-FEAU reporter systems detected approximately 3 × 105 T cells

  18. Antiherpesvirus Activities of Two Novel 4′-Thiothymidine Derivatives, KAY-2-41 and KAH-39-149, Are Dependent on Viral and Cellular Thymidine Kinases

    PubMed Central

    Coen, Natacha; Duraffour, Sophie; Haraguchi, Kazuhiro; Balzarini, Jan; van den Oord, Joost J.; Snoeck, Robert

    2014-01-01

    The emergence of drug-resistant herpesviruses represents a significant problem in clinical practice, primarily in immunocompromised patients. Furthermore, effective antiviral therapies against gammaherpesvirus-associated diseases are lacking. Here, we present two thiothymidine derivatives, KAY-2-41 and KAH-39-149, with different spectra of antiviral activity from those of the reference antiherpetic drugs, showing inhibitory activities against herpes simplex virus, varicella-zoster virus (VZV), and particularly against Epstein-Barr virus, with high selectivity in vitro. While KAY-2-41- and KAH-39-149-resistant herpesviruses were found to harbor mutations in the viral thymidine kinase (TK), these mutations conferred only low levels of resistance to these drugs but high levels to other TK-dependent drugs. Also, antiviral assays in HeLa TK-deficient cells showed a lack of KAY-2-41 and KAH-39-149 activities against herpes simplex virus 1 (HSV-1) and HSV-2 TK-deficient mutants. Furthermore, enzymatic TK assays showed the ability of HSV-1 TK, VZV TK, and cellular TK1 and TK2 to recognize and phosphorylate KAY-2-41 and KAH-39-149. These results demonstrate that the compounds depend on both viral and host TKs to exert antiviral activity. Additionally, the antiviral efficacy of KAH-39-149 proved to be superior to that of KAY-2-41 in a mouse model of gammaherpesvirus infection, highlighting the potential of this class of antiviral agents for further development as selective therapeutics against Epstein-Barr virus. PMID:24820089

  19. 5-Iodo-2'-deoxy-L-uridine and (E)-5-(2-bromovinyl)-2'-deoxy-L-uridine: selective phosphorylation by herpes simplex virus type 1 thymidine kinase, antiherpetic activity, and cytotoxicity studies.

    PubMed

    Spadari, S; Ciarrocchi, G; Focher, F; Verri, A; Maga, G; Arcamone, F; Iafrate, E; Manzini, S; Garbesi, A; Tondelli, L

    1995-06-01

    5-Iodo-2'-deoxy-L-uridine (L-IdU) and (E)-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU) have been prepared and found to inhibit herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) with activities comparable to those of their analogs with the natural D-sugar configuration. The mechanism of inhibition is purely competitive for L-IdU (Ki = 0.24 microM) and mixed-type for L-BVdU (Ki = 0.13 microM). High performance liquid chromatographic analysis of the reaction products demonstrated that the viral enzyme phosphorylates both L-enantiomers to their corresponding monophosphates with efficiency comparable to that for D-enantiomers. Neither L-enantiomer inhibits the human cytosolic TK. In contrast to their D-enantiomers, L-IdU and L-BVdU have no effect on human thymidylate synthase, either in HeLa cells or in TK-deficient HeLa cells transformed with the HSV-1 TK gene. Both L-enantiomers (i) have no effect on HeLa cell growth, (ii) are 1000-fold less cytotoxic toward TK-deficient HeLa cells transformed with the HSV-1 TK gene than are their D-enantiomers, (iii) in contrast to their D-enantiomers, are fully resistant to hydrolysis by nucleoside phosphorylase, and, (iv) in spite of their much lower cytotoxicity, most probably due to the very low affinity of L-BVdU monophosphate and L-IdU monophosphate for thymidylate synthase, are only 1 or 2 orders of magnitude less potent than their D-enantiomers in inhibiting viral growth, with potency comparable to that of acyclovir. PMID:7603465

  20. Treatment of human papillomavirus (HPV) type 16-infected cells using herpes simplex virus type 1 thymidine kinase-mediated gene therapy transcriptionally regulated by the HPV E2 protein.

    PubMed

    Sethi, Neerja; Palefsky, Joel

    2003-01-01

    Human papillomavirus type 16 (HPV-16) is associated with development of anogenital squamous cell cancers (SCCs) and their precursor, intraepithelial neoplasia (IN). Few approaches to the treatment of IN to prevent SCC are targeted specifically to HPV. We have designed an HPV-specific therapy using the herpes simplex virus type 1 thymidine kinase (HSV-1 TK) gene driven by an HPV-specific promoter in the HPV-16 long control region (LCR) (nucleotide 7450-nucleotide 104), which is regulated by the HPV E2 protein. Expression of the HSV-1 TK gene is designed to render HPV-infected cells sensitive to the prodrugs ganciclovir (GCV) and acyclovir (ACV). To assess the E2 specificity of gene expression driven by the HPV-16 LCR, we measured luciferase expression in HPV-positive and HPV-negative cell lines. Significant induction of luciferase activity was observed in HPV-positive cells when compared with four different HPV-negative epithelial cell lines. Cotransfection of an HPV-negative cell line, MDCK, with an HPV-16 E2-expressing plasmid resulted in 15- to 20-fold induction of luciferase activity, suggesting specific activation by E2 protein. A plasmid expressing the HSV-1 TK gene driven by the LCR was transfected into CaSki and SiHa cells. Treatment of transfected cells with either GCV or ACV (20-30 microg/ml) for 6-10 days resulted in 80-95% cell death. Cell death was progressive, dose dependent, and mediated by apoptosis. These results suggest that direct gene transfer of the HSV-1 TK gene into HPV-16-infected cells expressing E2 protein, accompanied by treatment with either GCV or ACV, may be a clinically feasible therapeutic strategy. PMID:12573058

  1. Image Gallery

    MedlinePlus

    ... R S T U V W X Y Z Image Gallery Share: The Image Gallery contains high-quality digital photographs available from ... Select a category below to view additional thumbnail images. Images are available for direct download in 2 ...

  2. Cancer Imaging

    MedlinePlus

    ... I/II Trials CIP ARRA-Funded Clinical Trials Informatics The Cancer Imaging Archive TCGA Imaging Genomics Quantitative Imaging Network LIDC-IDRI Imaging Informatics Resources News & Events News and Announcements Events – Meetings ...

  3. Pronounced antitumor effects and tumor radiosensitization of double suicide gene therapy.

    PubMed

    Rogulski, K R; Zhang, K; Kolozsvary, A; Kim, J H; Freytag, S O

    1997-11-01

    The efficacy of HSV-1 thymidine kinase (TK) and Escherichia coli cytosine deaminase (CD) suicide gene therapies as cancer treatments are currently being examined in humans. We demonstrated previously that compared to single suicide gene therapy, greater levels of targeted cytotoxicity and radiosensitization can be achieved in vitro by genetically modifying tumor cells to express CD and HSV-1 TK concomitantly, as a fusion protein. In the present study, the efficacy of the combined double suicide gene therapy/radiotherapy approach was examined in vivo. Nude mice were injected either s.c. or i.m. with 9L gliosarcoma cells expressing an E. coli CD/HSV-1 TK fusion gene. Double suicide gene therapy using 5-fluorocytosine (500 mg/kg) and ganciclovir (30 mg/kg) proved to be markedly better at delaying tumor growth and achieving a tumor cure than single suicide gene therapy, which used 5-fluorocytosine or ganciclovir administered independently. Importantly, double suicide gene therapy was highly effective against large experimental tumors (>2 cm3), reducing tumor volume an average of 99% and producing a 40% tumor cure. Moreover, double suicide gene therapy profoundly potentiated the antitumor effects of radiation. The results indicate that double suicide gene therapy, particularly when coupled with radiotherapy, may represent a highly effective means of eradicating tumors. PMID:9815600

  4. (18)F-FLT PET imaging of cellular proliferation in pancreatic cancer.

    PubMed

    Lamarca, Angela; Asselin, Marie-Claude; Manoharan, Prakash; McNamara, Mairéad G; Trigonis, Ioannis; Hubner, Richard; Saleem, Azeem; Valle, Juan W

    2016-03-01

    Pancreatic ductal adenocarcinoma is known for its poor prognosis. Since the development of computerized tomography, magnetic resonance and endoscopic ultrasound, novel imaging techniques have struggled to get established in the management of patients diagnosed with pancreatic adenocarcinoma for several reasons. Thus, imaging assessment of pancreatic cancer remains a field with scope for further improvement. In contrast to cross-sectional anatomical imaging methods, molecular imaging modalities such as positron emission tomography (PET) can provide information on tumour function. Particularly, tumour proliferation may be assessed by measurement of intracellular thymidine kinase 1 (TK1) activity level using thymidine analogues radiolabelled with a positron emitter for use with PET. This approach, has been widely explored with [(18)F]-fluoro-3'-deoxy-3'-l-fluorothymidine ((18)F-FLT) PET. This manuscript reviews the rationale and physiology behind (18)F-FLT PET imaging, with special focus on pancreatic cancer and other gastrointestinal malignancies. Potential benefit and challenges of this imaging technique for diagnosis, staging and assessment of treatment response in abdominal malignancies are discussed. PMID:26778585

  5. Imaging medical imaging

    NASA Astrophysics Data System (ADS)

    Journeau, P.

    2015-03-01

    This paper presents progress on imaging the research field of Imaging Informatics, mapped as the clustering of its communities together with their main results by applying a process to produce a dynamical image of the interactions between their results and their common object(s) of research. The basic side draws from a fundamental research on the concept of dimensions and projective space spanning several streams of research about three-dimensional perceptivity and re-cognition and on their relation and reduction to spatial dimensionality. The application results in an N-dimensional mapping in Bio-Medical Imaging, with dimensions such as inflammatory activity, MRI acquisition sequencing, spatial resolution (voxel size), spatiotemporal dimension inferred, toxicity, depth penetration, sensitivity, temporal resolution, wave length, imaging duration, etc. Each field is represented through the projection of papers' and projects' `discriminating' quantitative results onto the specific N-dimensional hypercube of relevant measurement axes, such as listed above and before reduction. Past published differentiating results are represented as red stars, achieved unpublished results as purple spots and projects at diverse progress advancement levels as blue pie slices. The goal of the mapping is to show the dynamics of the trajectories of the field in its own experimental frame and their direction, speed and other characteristics. We conclude with an invitation to participate and show a sample mapping of the dynamics of the community and a tentative predictive model from community contribution.

  6. Image Calibration

    NASA Technical Reports Server (NTRS)

    Peay, Christopher S.; Palacios, David M.

    2011-01-01

    Calibrate_Image calibrates images obtained from focal plane arrays so that the output image more accurately represents the observed scene. The function takes as input a degraded image along with a flat field image and a dark frame image produced by the focal plane array and outputs a corrected image. The three most prominent sources of image degradation are corrected for: dark current accumulation, gain non-uniformity across the focal plane array, and hot and/or dead pixels in the array. In the corrected output image the dark current is subtracted, the gain variation is equalized, and values for hot and dead pixels are estimated, using bicubic interpolation techniques.

  7. Indexing Images.

    ERIC Educational Resources Information Center

    Rasmussen, Edie M.

    1997-01-01

    Focuses on access to digital image collections by means of manual and automatic indexing. Contains six sections: (1) Studies of Image Systems and their Use; (2) Approaches to Indexing Images; (3) Image Attributes; (4) Concept-Based Indexing; (5) Content-Based Indexing; and (6) Browsing in Image Retrieval. Contains 105 references. (AEF)

  8. Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

    PubMed Central

    Haffey, M L; Novotny, J; Bruccoleri, R E; Carroll, R D; Stevens, J T; Matthews, J T

    1990-01-01

    The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity. Images PMID:2168983

  9. Image intensification

    SciTech Connect

    Csorba, I.P.

    1989-01-01

    These proceedings discuss the papers on image intensification. The topics discussed are : High speed optical detector tube technology; image tube camera technology; microchannel plate technology; high resolution x-ray imaging device; and process and evaluation techniques.

  10. BamI, KpnI, and SalI restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: occurrence of heterogeneities in defined regions of the viral DNA.

    PubMed Central

    Locker, H; Frenkel, N

    1979-01-01

    We present the locations of the cleavage sites for the BamI, KpnI, and SalI restriction endonucleases within the DNA molecules of herpes simplex virus type 1 (HSV-1) strains Justin and F. These restriction enzymes cleave the HSV-1 DNA at many sites, producing relatively small fragments which should prove useful in future studies of HSV-1 gene structure and function. The mapping data revealed the occurrence of heterogeneity within three regions of the viral genome including (i) the region spanning map coordinates 0.74--0.76, (ii) the ends of the large (L) DNA component, and (iii) the junction between the large (L) and the small (S) components. The heterogeneity in the ends of L and the S-L junctions of HSV-1 (Justin) and HSV-1 (F) DNAs was grossly similar to that previously reported to occur in the ends of L and the S-L junctions of the HSV-1 (KOS) DNA (M. J. Wagner and W. C. Summers, J. Virol. 27:374--387, 1978). Thus, cleavage of these regions with restriction endonucleases yielded sets of minor fragments differing in size by constant increments. However, the various strains of HSV-1 differed with respect to the numbers, size increments, and relative molarities of the various minor fragments, suggesting that the parameters of the heterogeneity are inherited in the structural makeup of the HSV-1 genome. The strain dependence of the pattern of heterogeneity can be most easily explained in terms of variable sizes of the terminally reiterated a sequence, contained in the DNA molecules of these three strains of HSV-1. Images PMID:228068

  11. Monitoring and quantitative assessment of tumor burden using in vivo bioluminescence imaging

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Chi; Hwang, Jeng-Jong; Ting, Gann; Tseng, Yun-Long; Wang, Shyh-Jen; Whang-Peng, Jaqueline

    2007-02-01

    In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating tumor growth. In this study, the kinetic of tumor growth has been assessed in C26 colon carcinoma bearing BALB/c mouse model. The ability of BLI to noninvasively quantitate the growth of subcutaneous tumors transplanted with C26 cells genetically engineered to stably express firefly luciferase and herpes simplex virus type-1 thymidine kinase (C26/ tk-luc). A good correlation ( R2=0.998) of photon emission to the cell number was found in vitro. Tumor burden and tumor volume were monitored in vivo over time by quantitation of photon emission using Xenogen IVIS 50 and standard external caliper measurement, respectively. At various time intervals, tumor-bearing mice were imaged to determine the correlation of in vivo BLI to tumor volume. However, a correlation of BLI to tumor volume was observed when tumor volume was smaller than 1000 mm 3 ( R2=0.907). γ Scintigraphy combined with [ 131I]FIAU was another imaging modality used for verifying the previous results. In conclusion, this study showed that bioluminescence imaging is a powerful and quantitative tool for the direct assay to monitor tumor growth in vivo. The dual reporter genes transfected tumor-bearing animal model can be applied in the evaluation of the efficacy of new developed anti-cancer drugs.

  12. BIODISTRIBUTION AND PET IMAGING OF [18F]-FLUOROADENOSINE DERIVATIVES

    PubMed Central

    Alauddin, Mian M.; Shahinian, Antranik; Park, Ryan; Tohme, Michael; Fissekis, John D.; Conti, Peter S.

    2007-01-01

    Introduction: Many fluorinated analogues of adenosine nucleoside have been synthesized and studied as potential antitumor and antiviral agents. Earlier we reported radiosynthesis of 2′-deoxy-2′-[18F]fluoro-1-β-D-arabinofuranosyl-adenine ([18F]-FAA) and 3′-deoxy-3′-[18F]fluoro-1-β-D-xylofuranosyl-adenine ([18F]FXA). Now we report their in vivo studies including blood clearance, biodistribution and micro-PET imaging in tumor-bearing nude mice. Methods: Tumors were grown in six weeks old athymic nude mice (Harlan, Indianapolis, IN) by inoculation of HT-29 cells, wild type cells in the left flank and transduced cells with HSV-tk on the right flank. When the tumor was about 1 cm in size, animals were injected with these radiotracers for in vivo studies, including blood clearance, micro-PET imaging and biodistribution. Results: Uptake of [18F]FAA in tumor was 3.3-fold higher than blood, with highest uptake in the spleen. Maximum uptake of [18F]FXA was observed in the heart compared to other organs. There was no tumor uptake of [18F]FXA. Biodistribution results were supported by micro-PET images, which also showed very high uptake of [18F]FAA in spleen and visualization of tumors, and high uptake of [18F]FXA in the heart. Conclusion: These results suggest that [18F]FAA may be useful for tumor imaging, while [18F]FXA may have potential as a heart imaging agent with PET. PMID:17383576

  13. Replication-competent adenovirus-mediated suicide gene therapy with radiation in a preclinical model of pancreatic cancer.

    PubMed

    Freytag, Svend O; Barton, Kenneth N; Brown, Stephen L; Narra, Vinod; Zhang, Yingshu; Tyson, Don; Nall, Colleen; Lu, Mei; Ajlouni, Munther; Movsas, Benjamin; Kim, Jae Ho

    2007-09-01

    In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer. PMID:17551507

  14. Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging

    PubMed Central

    Santulli-Marotto, Sandra; Gervais, Alexis; Fisher, Jamie; Strake, Brandy; Ogden, Carol Anne; Riveley, Chelsea; Giles-Komar, Jill

    2015-01-01

    Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the “professional phagocyte” of the body, are responsible for efficient, non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity in vivo. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways. PMID:26674639

  15. Spread of HSV-1 to the mouse eye after inoculation in the skin of the snout requires an intact nerve supply to the inoculation site.

    PubMed

    Shimeld, C; Dyson, H; Lewkowicz-Moss, S; Hill, T J; Blyth, W A; Easty, D L

    1987-01-01

    Infection of the eye following inoculation of herpes simplex virus on the skin of the snout was monitored using slit lamp examination of the eye, isolation of virus from eyewashings and identification of virus antigens in whole corneal epithelial sheets by peroxidase-antiperoxidase staining. Infection of the eye was prevented by removing a section of the sensory nerves which supply the inoculation site. This provided evidence that spread from the skin of the snout to the eye occurred via the nerves. PMID:3030659

  16. Production of Bioactive Soluble Interleukin-15 in Complex with Interleukin-15 Receptor Alpha from a Conditionally-Replicating Oncolytic HSV-1

    PubMed Central

    Gaston, David C.; Odom, Carl I.; Li, Li; Markert, James M.; Roth, Justin C.; Cassady, Kevin A.; Whitley, Richard J.; Parker, Jacqueline N.

    2013-01-01

    Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8+ T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x107 plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models. PMID:24312353

  17. Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.

    PubMed

    Rupesh, Kanchi Ravi; Smith, Aaron; Boehmer, Paul E

    2014-11-01

    We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1μM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner. PMID:25449284

  18. Evaluation of the immune response to Anaplasma marginale MSP5 protein using a HSV-1 amplicon vector system or recombinant protein.

    PubMed

    Palacios, Carlos; Torioni de Echaide, Susana; Mattion, Nora

    2014-12-01

    Anaplasma marginale is an intraerythrocytic vector-borne infectious agent of cattle. Immunization with the current vaccine, based on parasitized erythrocytes with live Anaplasma centrale, shows some constraints and confers partial protection, suggesting the feasibility for the development of new generation of vaccines. The aim of the present study was to assess the effect of sequential immunization of BALB/c mice, with herpesvirus amplicon vector-based vaccines combined with protein-based vaccines, on the quality of the immune response against the major surface protein 5 of A. marginale. The highest antibody titers against MSP5 were elicited in mice that received two doses of adjuvanted recombinant protein (p < 0.0001). Mice treated with a heterologous prime-boost strategy generated sustained antibody titers at least up to 200 days, and a higher specific cellular response. The results presented here showed that sequential immunization with HSV-based vectors and purified antigen enhances the quality of the immune response against A. marginale. PMID:25458492

  19. A DNA vaccine encoding VP22 of herpes simplex virus type I (HSV-1) and OprF confers enhanced protection from Pseudomonas aeruginosa in mice.

    PubMed

    Yu, Xian; Wang, Yan; Xia, Yifan; Zhang, Lijuan; Yang, Qin; Lei, Jun

    2016-08-17

    Pseudomonas aeruginosa antimicrobial resistance is a major therapeutic challenge. DNA vaccination is an attractive approach for antigen-specific immunotherapy against P. aeruginosa. We explored the feasibility of employing Herpes simplex virus type 1 tegument protein, VP22, as a molecular tool to enhance the immunogenicity of an OprF DNA vaccine against P. aeruginosa. Recombinant DNA vaccines, pVAX1-OprF, pVAX1-OprF-VP22 (encoding a n-OprF-VP22-c fusion protein) and pVAX1-VP22-OprF (encoding a n-VP22-OprF-c fusion protein) were constructed. The humoral and cellular immune responses and immune protective effects of these DNA vaccines in mice were evaluated. In this report, we showed that vaccination with pVAX1-OprF-VP22 induced higher levels of IgG titer, T cell proliferation rate. It also provided better immune protection against the P. aeruginosa challenge when compared to that induced by pVAX1-OprF or pVAX1-VP22-OprF DNA vaccines. Molecular mechanistic analyses indicated vaccination with pVAX1-OprF-VP22 triggered immune responses characterized by a preferential increase in antigen specific IgG2a and IFN-γ in mice, indicating Th1 polarization. We concluded that VP22 is a potent stimulatory molecular tool for DNA vaccination when fused to the carboxyl end of OprF gene. Our study provides a novel strategy for prevention and treatment of P. aeruginosa infection. PMID:27449680

  20. Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay

    PubMed Central

    Rupesh, Kanchi Ravi; Smith, Aaron; Boehmer, Paul E.

    2014-01-01

    We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1 °C for (dT)5 to a maximum of 9 °C with oligomers ≥10 nucleotides, with an apparent Kd of <1 µM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9 °C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner. PMID:25449284

  1. Difference in Incidence of Spontaneous Mutations between Herpes Simplex Virus Types 1 and 2

    PubMed Central

    Sarisky, Robert T.; Nguyen, Tammy T.; Duffy, Karen E.; Wittrock, Robert J.; Leary, Jeffry J.

    2000-01-01

    Spontaneous mutations within the herpes simplex virus (HSV) genome are introduced by errors during DNA replication. Indicative of the inherent mutation rate of HSV DNA replication, heterogeneous HSV populations containing both acyclovir (ACV)-resistant and ACV-sensitive viruses occur naturally in both clinical isolates and laboratory stocks. Wild-type, laboratory-adapted HSV type 1 (HSV-1) strains KOS and Cl101 reportedly accumulate spontaneous ACV-resistant mutations at a frequency of approximately six to eight mutants per 104 plaque-forming viruses (U. B. Dasgupta and W. C. Summers, Proc. Natl. Acad. Sci. USA 75:2378–2381, 1978; J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. T. Gelep, and P. A. Schaffer, Virology 132:26–37, 1984). Typically, these resistance mutations map to the thymidine kinase (TK) gene and render the virus TK deficient. To examine this process more closely, a plating efficiency assay was used to determine whether the frequencies of naturally occurring mutations in populations of the laboratory strains HSV-1 SC16, HSV-2 SB5, and HSV-2 333 grown in MRC-5 cells were similar when scored for resistance to penciclovir (PCV) and ACV. Our results indicate that (i) HSV mutants resistant to PCV and those resistant to ACV accumulate at approximately equal frequencies during replication in cell culture, (ii) the spontaneous mutation frequency for the HSV-1 strain SC16 is similar to that previously reported for HSV-1 laboratory strains KOS and Cl101, and (iii) spontaneous mutations in the laboratory HSV-2 strains examined were 9- to 16-fold more frequent than those in the HSV-1 strain SC16. These observations were confirmed and extended for a group of eight clinical isolates in which the HSV-2 mutation frequency was approximately 30 times higher than that for HSV-1 isolates. In conclusion, our results indicate that the frequencies of naturally occurring, or spontaneous, HSV mutants resistant to PCV and those resistant to

  2. Diagnostic Imaging

    MedlinePlus

    Diagnostic imaging lets doctors look inside your body for clues about a medical condition. A variety of machines and ... and activities inside your body. The type of imaging your doctor uses depends on your symptoms and ...

  3. Medical Imaging.

    ERIC Educational Resources Information Center

    Barker, M. C. J.

    1996-01-01

    Discusses four main types of medical imaging (x-ray, radionuclide, ultrasound, and magnetic resonance) and considers their relative merits. Describes important recent and possible future developments in image processing. (Author/MKR)

  4. Neonatal herpes simplex virus type-1 central nervous system disease with acute retinal necrosis.

    PubMed

    Fong, Choong Yi; Aye, Aye Mya Min; Peyman, Mohammadreza; Nor, Norazlin Kamal; Visvaraja, Subrayan; Tajunisah, Iqbal; Ong, Lai Choo

    2014-04-01

    We report a case of neonatal herpes simplex virus (HSV)-1 central nervous system disease with bilateral acute retinal necrosis (ARN). An infant was presented at 17 days of age with focal seizures. Cerebrospinal fluid polymerase chain reaction was positive for HSV-1 and brain magnetic resonance imaging showed cerebritis. While receiving intravenous acyclovir therapy, the infant developed ARN with vitreous fluid polymerase chain reaction positive for HSV-1 necessitating intravitreal foscarnet therapy. This is the first reported neonatal ARN secondary to HSV-1 and the first ARN case presenting without external ocular or cutaneous signs. Our report highlights that infants with neonatal HSV central nervous system disease should undergo a thorough ophthalmological evaluation to facilitate prompt diagnosis and immediate treatment of this rapidly progressive sight-threatening disease. PMID:24378951

  5. Proof Image

    ERIC Educational Resources Information Center

    Kidron, Ivy; Dreyfus, Tommy

    2014-01-01

    The emergence of a proof image is often an important stage in a learner's construction of a proof. In this paper, we introduce, characterize, and exemplify the notion of proof image. We also investigate how proof images emerge. Our approach starts from the learner's efforts to construct a justification without (or before) attempting any…

  6. Image alignment

    DOEpatents

    Dowell, Larry Jonathan

    2014-04-22

    Disclosed is a method and device for aligning at least two digital images. An embodiment may use frequency-domain transforms of small tiles created from each image to identify substantially similar, "distinguishing" features within each of the images, and then align the images together based on the location of the distinguishing features. To accomplish this, an embodiment may create equal sized tile sub-images for each image. A "key" for each tile may be created by performing a frequency-domain transform calculation on each tile. A information-distance difference between each possible pair of tiles on each image may be calculated to identify distinguishing features. From analysis of the information-distance differences of the pairs of tiles, a subset of tiles with high discrimination metrics in relation to other tiles may be located for each image. The subset of distinguishing tiles for each image may then be compared to locate tiles with substantially similar keys and/or information-distance metrics to other tiles of other images. Once similar tiles are located for each image, the images may be aligned in relation to the identified similar tiles.

  7. Canonical Images

    ERIC Educational Resources Information Center

    Hewitt, Dave

    2007-01-01

    In this article, the author offers two well-known mathematical images--that of a dot moving around a circle; and that of the tens chart--and considers their power for developing mathematical thinking. In his opinion, these images each contain the essence of a particular topic of mathematics. They are contrasting images in the sense that they deal…

  8. Image tubes

    SciTech Connect

    Csorba, I.P.

    1985-01-01

    This text provides a wealth of valuable, hard-to-find data on electron optics, imaging, and image intensification systems. The author explains details of image tube theory, design, construction, and components. He includes material on the design and operation of camera tubes, power components, and secondary electron emitters, as well as data on photomultiplier tubes and electron guns.

  9. Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

    PubMed

    Parashurama, Natesh; Ahn, Byeong-Cheol; Ziv, Keren; Ito, Ken; Paulmurugan, Ramasamy; Willmann, Jürgen K; Chung, Jaehoon; Ikeno, Fumiaki; Swanson, Julia C; Merk, Denis R; Lyons, Jennifer K; Yerushalmi, David; Teramoto, Tomohiko; Kosuge, Hisanori; Dao, Catherine N; Ray, Pritha; Patel, Manishkumar; Chang, Ya-Fang; Mahmoudi, Morteza; Cohen, Jeff Eric; Goldstone, Andrew Brooks; Habte, Frezghi; Bhaumik, Srabani; Yaghoubi, Shahriar; Robbins, Robert C; Dash, Rajesh; Yang, Phillip C; Brinton, Todd J; Yock, Paul G; McConnell, Michael V; Gambhir, Sanjiv S

    2016-09-01

    Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article. PMID:27308957

  10. Platinum(II) and Palladium(II) Complexes of Pyridine-2-Carbaldehyde Thiosemicarbazone as Alternative Antiherpes Simplex Virus Agents

    PubMed Central

    Kovala-Demertzi, D.; Varadinova, T.; Genova, P.; Souza, P.; Demertzis, M. A.

    2007-01-01

    The cytotoxicity and the antivirus activity of Pd(II) and Pt(II) complexes with pyridine-2-carbaldehyde thiosemicarbazone (HFoTsc) against HSV replication were evaluated on four HSV strains—two wt strains Victoria (HSV-1) and BJA (HSV-2) and two ACVR mutants with different tk gene mutations R-100 (TKA, HSV-1) and PU (TKN, HSV-2). The experiments were performed on continuous MDBK cells and four HSV 1 and HSV 2 strains were used, two sensitive to acyclovir and two resistant mutants. The five complexes of HFoTsc, [Pt(FoTsc)Cl], [Pt(FoTsc)(H2FoTsc)]Cl2, [Pt(FoTsc)2], [Pd(FoTsc)(H2FoTsc)]Cl2, and [Pd(FoTsc)2], were found to be effective inhibitors of HSV replication. The most promising, active, and selective anti-HSV agent was found to be complex [Pt(FoTsc)(H2FoTsc)]Cl2. This complex could be useful in the treatment of HSV infections, since it is resistant to ACV mutants. PCR study of immediate early 300 bp ReIV Us1 region reveals that the complex [Pt(FoTsc)(H2FoTsc)]Cl2 specifically suppressed wt HSV-1 genome 2 hours after the infection, not inducing apoptosis/necrosis on the 8 hours after virus infection. The target was found to be most probably the viral, instead of the host cell DNA. PMID:17541481

  11. Molecular imaging to target transplanted muscle progenitor cells.

    PubMed

    Gutpell, Kelly; McGirr, Rebecca; Hoffman, Lisa

    2013-01-01

    Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse). Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living

  12. Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

    SciTech Connect

    Zhang, Jufeng; Wang, Zhanli; Wei, Fang; Qiu, Wei; Zhang, Liangren; Huang, Qian . E-mail: qhuang@sjtu.edu.cn

    2007-08-17

    Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.

  13. Antitumor effects of non-replicative herpes simplex vectors expressing antiangiogenic proteins and thymidine kinase on Lewis lung carcinoma establishment and growth.

    PubMed

    Berto, E; Bozac, A; Volpi, I; Lanzoni, I; Vasquez, F; Melara, N; Manservigi, R; Marconi, P

    2007-09-01

    There is growing evidence that combinations of antiangiogenic proteins with other antineoplastic treatments such as chemo- or radiotherapy and suicide genes-mediated tumor cytotoxicity lead to synergistic effects. In the present work, we tested the activity of two non-replicative herpes simplex virus (HSV)-1-based vectors, encoding human endostatin::angiostatin or endostatin::kringle5 fusion proteins in combination with HSV-1 thymidine kinase (TK) molecule, on endothelial cells (ECs) and Lewis lung carcinoma (LLC) cells. We observed a significant reduction of the in vitro growth, migration and tube formation by primary ECs upon direct infection with the two recombinant vectors or cultivation with conditioned media obtained from the vector-infected LLC cells. Moreover, direct cytotoxic effect of HSV-1 TK on both LLC and ECs was demonstrated. We then tested the vectors in vivo in two experimental settings, that is, LLC tumor growth or establishment, in C57BL/6 mice. The treatment of pre-established subcutaneous tumors with the recombinant vectors with ganciclovir (GCV) induced a significant reduction of tumor growth rate, while the in vitro infection of LLC cells with the antiangiogenic vectors before their implantation in mice flanks, either in presence or absence of GCV, completely abolished the tumor establishment. PMID:17557110

  14. Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27.

    PubMed Central

    Panagiotidis, C A; Lium, E K; Silverstein, S J

    1997-01-01

    The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4. PMID:8995681

  15. Optimized thymidylate kinase assay, based on enzymatically synthesized 5-(/sup 125/I)iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases

    SciTech Connect

    Karlstroem, A.R.G.; Gronowitz, J.S.

    1987-05-01

    The biological synthesis and purification of 5-(/sup 125/I)iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.

  16. Imaging Biomarkers or Biomarker Imaging?

    PubMed Central

    Mitterhauser, Markus; Wadsak, Wolfgang

    2014-01-01

    Since biomarker imaging is traditionally understood as imaging of molecular probes, we highly recommend to avoid any confusion with the previously defined term “imaging biomarkers” and, therefore, only use “molecular probe imaging (MPI)” in that context. Molecular probes (MPs) comprise all kinds of molecules administered to an organism which inherently carry a signalling moiety. This review highlights the basic concepts and differences of molecular probe imaging using specific biomarkers. In particular, PET radiopharmaceuticals are discussed in more detail. Specific radiochemical and radiopharmacological aspects as well as some legal issues are presented. PMID:24967536

  17. Adenoviral-mediated Gene Transfer into the Canine Brain In Vivo

    PubMed Central

    Candolfi, Marianela; Kroeger, Kurt M.; Pluhar, G. Elizabeth; Bergeron, Josee; Puntel, Mariana; Curtin, James F.; McNiel, Elizabeth A.; Freese, Andrew B.; Ohlfest, John R.; Moore, Peter; Lowenstein, Pedro R.; Castro, Maria G.

    2007-01-01

    OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding β-galactosidase (βGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding βGal (Ad-βGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, βGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm2 area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials. PMID:17228266

  18. Indirect Imaging

    NASA Astrophysics Data System (ADS)

    Kundu, Mukul R.

    This book is the Proceedings of an International Symposium held in Sydney, Australia, August 30-September 2, 1983. The meeting was sponsored by the International Union of Radio Science and the International Astronomical Union.Indirect imaging is based upon the principle of determining the actual form of brightness distribution in a complex case by Fourier synthesis, using information derived from a large number of Fourier components. The main topic of the symposium was how to get the best images from data obtained from telescopes and other similar imaging instruments. Although the meeting was dominated by radio astronomers, with the consequent dominance of discussion of indirect imaging in the radio domain, there were quite a few participants from other disciplines. Thus there were some excellent discussions on optical imaging and medical imaging.

  19. Image Processing

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Electronic Imagery, Inc.'s ImageScale Plus software, developed through a Small Business Innovation Research (SBIR) contract with Kennedy Space Flight Center for use on space shuttle Orbiter in 1991, enables astronauts to conduct image processing, prepare electronic still camera images in orbit, display them and downlink images to ground based scientists for evaluation. Electronic Imagery, Inc.'s ImageCount, a spin-off product of ImageScale Plus, is used to count trees in Florida orange groves. Other applications include x-ray and MRI imagery, textile designs and special effects for movies. As of 1/28/98, company could not be located, therefore contact/product information is no longer valid.

  20. Medical imaging

    SciTech Connect

    Chapman, D.

    1996-09-01

    There are a number of medically related imaging programs at synchrotron facilities around the world. The most advanced of these are the dual energy transvenous coronary angiography imaging programs, which have progressed to human imaging for some years. The NSLS facility will be discussed and patient images from recent sessions from the NSLS and HASYLAB will be presented. The effort at the Photon Factory and Accumulator Ring will also be briefly covered, as well as future plans for the new facilities. Emphasis will be on the new aspects of these imaging programs; this includes imaging with a peripheral venous injection of the iodine contrast agent, imaging at three photon energies, and the potential of a hospital-based compact source. Other medical programs to be discussed, are the multiple energy computed tomography (MECT) project at the NSLS and plans for a MECT program at the ESRF. Recently, experiments performed at the NSLS to image mammography phantoms using monochromatic beam have produced very promising results. This program will be discussed as well as some new results from imaging a phantom using a thin Laue crystal analyzer after the object to eliminate scatter onto the detector. {copyright} {ital 1996 American Institute of Physics.}

  1. Image barcodes

    NASA Astrophysics Data System (ADS)

    Damera-Venkata, Niranjan; Yen, Jonathan

    2003-01-01

    A Visually significant two-dimensional barcode (VSB) developed by Shaked et. al. is a method used to design an information carrying two-dimensional barcode, which has the appearance of a given graphical entity such as a company logo. The encoding and decoding of information using the VSB, uses a base image with very few graylevels (typically only two). This typically requires the image histogram to be bi-modal. For continuous-tone images such as digital photographs of individuals, the representation of tone or "shades of gray" is not only important to obtain a pleasing rendition of the face, but in most cases, the VSB renders these images unrecognizable due to its inability to represent true gray-tone variations. This paper extends the concept of a VSB to an image bar code (IBC). We enable the encoding and subsequent decoding of information embedded in the hardcopy version of continuous-tone base-images such as those acquired with a digital camera. The encoding-decoding process is modeled by robust data transmission through a noisy print-scan channel that is explicitly modeled. The IBC supports a high information capacity that differentiates it from common hardcopy watermarks. The reason for the improved image quality over the VSB is a joint encoding/halftoning strategy based on a modified version of block error diffusion. Encoder stability, image quality vs. information capacity tradeoffs and decoding issues with and without explicit knowledge of the base-image are discussed.

  2. Scientific Images

    MedlinePlus

    ... Financial Planning Organizations Clinical Trials Research Alzheimer's Disease ... | Alzheimer’s Disease Mechanisms and Processes The medical illustration images available below may be downloaded in ...

  3. Body Imaging

    NASA Technical Reports Server (NTRS)

    1990-01-01

    Magnetic Resonance Imaging (MRI) and Computer-aided Tomography (CT) images are often complementary. In most cases, MRI is good for viewing soft tissue but not bone, while CT images are good for bone but not always good for soft tissue discrimination. Physicians and engineers in the Department of Radiology at the University of Michigan Hospitals are developing a technique for combining the best features of MRI and CT scans to increase the accuracy of discriminating one type of body tissue from another. One of their research tools is a computer program called HICAP. The program can be used to distinguish between healthy and diseased tissue in body images.

  4. Multispectral imaging and image processing

    NASA Astrophysics Data System (ADS)

    Klein, Julie

    2014-02-01

    The color accuracy of conventional RGB cameras is not sufficient for many color-critical applications. One of these applications, namely the measurement of color defects in yarns, is why Prof. Til Aach and the Institute of Image Processing and Computer Vision (RWTH Aachen University, Germany) started off with multispectral imaging. The first acquisition device was a camera using a monochrome sensor and seven bandpass color filters positioned sequentially in front of it. The camera allowed sampling the visible wavelength range more accurately and reconstructing the spectra for each acquired image position. An overview will be given over several optical and imaging aspects of the multispectral camera that have been investigated. For instance, optical aberrations caused by filters and camera lens deteriorate the quality of captured multispectral images. The different aberrations were analyzed thoroughly and compensated based on models for the optical elements and the imaging chain by utilizing image processing. With this compensation, geometrical distortions disappear and sharpness is enhanced, without reducing the color accuracy of multispectral images. Strong foundations in multispectral imaging were laid and a fruitful cooperation was initiated with Prof. Bernhard Hill. Current research topics like stereo multispectral imaging and goniometric multispectral measure- ments that are further explored with his expertise will also be presented in this work.

  5. Image Tool

    SciTech Connect

    Baker, S.A.; Gardner, S.D.; Rogers, M.L.; Sanders, F.; Tunnell, T.W.

    2001-01-01

    ImageTool is a software package developed at Bechtel Nevada, Los Alamos Operations. This team has developed a set of analysis tools, in the form of image processing software used to evaluate camera calibration data. Performance measures are used to identify capabilities and limitations of a camera system, while establishing a means for comparing systems. The camera evaluations are designed to provide system performance, camera comparison and system modeling information. This program is used to evaluate digital camera images. ImageTool provides basic image restoration and analysis features along with a special set of camera evaluation tools which are used to standardize camera system characterizations. This process is started with the acquisition of a well-defined set of calibration images. Image processing algorithms provide a consistent means of evaluating the camera calibration data. Performance measures in the areas of sensitivity, noise, and resolution are used as a basis for comparing camera systems and evaluating experimental system performance. Camera systems begin with a charge-coupled device (CCD) camera and optical relay system and may incorporate image intensifiers, electro-static image tubes, or electron bombarded charge-coupled devices (EBCCDs). Electro-optical components provide fast shuttering and/or optical gain to camera systems. Camera types evaluated include gated intensified cameras and multi-frame cameras used in applications ranging from X-ray radiography to visible and infrared imaging. It is valuable to evaluate the performance of a camera system in order to determine if a particular system meets experimental requirements. In this paper we highlight the processing features of ImageTool.

  6. Blurred Image

    ERIC Educational Resources Information Center

    Conde, Maryse

    1975-01-01

    The growing influence of Western culture has greatly affected African women's status and image in the traditional society. Working women are confronted with the dilemma of preserving family traditions while changing their behavior and image to become members of the labor force. (MR)

  7. Image fusion

    NASA Technical Reports Server (NTRS)

    Pavel, M.

    1993-01-01

    The topics covered include the following: a system overview of the basic components of a system designed to improve the ability of a pilot to fly through low-visibility conditions such as fog; the role of visual sciences; fusion issues; sensor characterization; sources of information; image processing; and image fusion.

  8. Cerenkov imaging.

    PubMed

    Das, Sudeep; Thorek, Daniel L J; Grimm, Jan

    2014-01-01

    Cerenkov luminescence (CL) has been used recently in a plethora of medical applications like imaging and therapy with clinically relevant medical isotopes. The range of medical isotopes used is fairly large and expanding. The generation of in vivo light is useful since it circumvents depth limitations for excitation light. Cerenkov luminescence imaging (CLI) is much cheaper in terms of infrastructure than positron emission tomography (PET) and is particularly useful for imaging of superficial structures. Imaging can basically be done using a sensitive camera optimized for low-light conditions, and it has a better resolution than any other nuclear imaging modality. CLI has been shown to effectively diagnose disease with regularly used PET isotope ((18)F-FDG) in clinical setting. Cerenkov luminescence tomography, Cerenkov luminescence endoscopy, and intraoperative Cerenkov imaging have also been explored with positive conclusions expanding the current range of applications. Cerenkov has also been used to improve PET imaging resolution since the source of both is the radioisotope being used. Smart imaging agents have been designed based on modulation of the Cerenkov signal using small molecules and nanoparticles giving better insight of the tumor biology. PMID:25287690

  9. Imaging Atherosclerosis.

    PubMed

    Tarkin, Jason M; Dweck, Marc R; Evans, Nicholas R; Takx, Richard A P; Brown, Adam J; Tawakol, Ahmed; Fayad, Zahi A; Rudd, James H F

    2016-02-19

    Advances in atherosclerosis imaging technology and research have provided a range of diagnostic tools to characterize high-risk plaque in vivo; however, these important vascular imaging methods additionally promise great scientific and translational applications beyond this quest. When combined with conventional anatomic- and hemodynamic-based assessments of disease severity, cross-sectional multimodal imaging incorporating molecular probes and other novel noninvasive techniques can add detailed interrogation of plaque composition, activity, and overall disease burden. In the catheterization laboratory, intravascular imaging provides unparalleled access to the world beneath the plaque surface, allowing tissue characterization and measurement of cap thickness with micrometer spatial resolution. Atherosclerosis imaging captures key data that reveal snapshots into underlying biology, which can test our understanding of fundamental research questions and shape our approach toward patient management. Imaging can also be used to quantify response to therapeutic interventions and ultimately help predict cardiovascular risk. Although there are undeniable barriers to clinical translation, many of these hold-ups might soon be surpassed by rapidly evolving innovations to improve image acquisition, coregistration, motion correction, and reduce radiation exposure. This article provides a comprehensive review of current and experimental atherosclerosis imaging methods and their uses in research and potential for translation to the clinic. PMID:26892971

  10. Image reconstruction

    SciTech Connect

    Defrise, Michel; Gullberg, Grant T.

    2006-04-05

    We give an overview of the role of Physics in Medicine andBiology in development of tomographic reconstruction algorithms. We focuson imaging modalities involving ionizing radiation, CT, PET and SPECT,and cover a wide spectrum of reconstruction problems, starting withclassical 2D tomogra tomography in the 1970s up to 4D and 5D problemsinvolving dynamic imaging of moving organs.

  11. Cerenkov Imaging

    PubMed Central

    Das, Sudeep; Thorek, Daniel L.J.; Grimm, Jan

    2014-01-01

    Cerenkov luminescence (CL) has been used recently in a plethora of medical applications like imaging and therapy with clinically relevant medical isotopes. The range of medical isotopes used is fairly large and expanding. The generation of in vivo light is useful since it circumvents depth limitations for excitation light. Cerenkov luminescence imaging (CLI) is much cheaper in terms of infrastructure than positron emission tomography (PET) and is particularly useful for imaging of superficial structures. Imaging can basically be done using a sensitive camera optimized for low-light conditions, and it has a better resolution than any other nuclear imaging modality. CLI has been shown to effectively diagnose disease with regularly used PET isotope (18F-FDG) in clinical setting. Cerenkov luminescence tomography, Cerenkov luminescence endoscopy, and intraoperative Cerenkov imaging have also been explored with positive conclusions expanding the current range of applications. Cerenkov has also been used to improve PET imaging resolution since the source of both is the radioisotope being used. Smart imaging agents have been designed based on modulation of the Cerenkov signal using small molecules and nanoparticles giving better insight of the tumor biology. PMID:25287690

  12. Piramal Imaging.

    PubMed

    Dinkelborg, Ludger

    2015-08-01

    Piramal Imaging, a division of Piramal Enterprises Ltd, is a global radiopharmaceutical company that is actively developing novel PET radiotracers for use in molecular imaging. The company focuses on developing innovative products that improve early detection and characterization of chronic and life-threatening diseases, leading to better therapeutic outcomes and improved quality of life. PMID:26295720

  13. Imaging Genetics

    ERIC Educational Resources Information Center

    Munoz, Karen E.; Hyde, Luke W.; Hariri, Ahmad R.

    2009-01-01

    Imaging genetics is an experimental strategy that integrates molecular genetics and neuroimaging technology to examine biological mechanisms that mediate differences in behavior and the risks for psychiatric disorder. The basic principles in imaging genetics and the development of the field are discussed.

  14. Imaging Atherosclerosis

    PubMed Central

    Tarkin, Jason M.; Dweck, Marc R.; Evans, Nicholas R.; Takx, Richard A.P.; Brown, Adam J.; Tawakol, Ahmed; Fayad, Zahi A.

    2016-01-01

    Advances in atherosclerosis imaging technology and research have provided a range of diagnostic tools to characterize high-risk plaque in vivo; however, these important vascular imaging methods additionally promise great scientific and translational applications beyond this quest. When combined with conventional anatomic- and hemodynamic-based assessments of disease severity, cross-sectional multimodal imaging incorporating molecular probes and other novel noninvasive techniques can add detailed interrogation of plaque composition, activity, and overall disease burden. In the catheterization laboratory, intravascular imaging provides unparalleled access to the world beneath the plaque surface, allowing tissue characterization and measurement of cap thickness with micrometer spatial resolution. Atherosclerosis imaging captures key data that reveal snapshots into underlying biology, which can test our understanding of fundamental research questions and shape our approach toward patient management. Imaging can also be used to quantify response to therapeutic interventions and ultimately help predict cardiovascular risk. Although there are undeniable barriers to clinical translation, many of these hold-ups might soon be surpassed by rapidly evolving innovations to improve image acquisition, coregistration, motion correction, and reduce radiation exposure. This article provides a comprehensive review of current and experimental atherosclerosis imaging methods and their uses in research and potential for translation to the clinic. PMID:26892971

  15. Retinal Imaging and Image Analysis

    PubMed Central

    Abràmoff, Michael D.; Garvin, Mona K.; Sonka, Milan

    2011-01-01

    Many important eye diseases as well as systemic diseases manifest themselves in the retina. While a number of other anatomical structures contribute to the process of vision, this review focuses on retinal imaging and image analysis. Following a brief overview of the most prevalent causes of blindness in the industrialized world that includes age-related macular degeneration, diabetic retinopathy, and glaucoma, the review is devoted to retinal imaging and image analysis methods and their clinical implications. Methods for 2-D fundus imaging and techniques for 3-D optical coherence tomography (OCT) imaging are reviewed. Special attention is given to quantitative techniques for analysis of fundus photographs with a focus on clinically relevant assessment of retinal vasculature, identification of retinal lesions, assessment of optic nerve head (ONH) shape, building retinal atlases, and to automated methods for population screening for retinal diseases. A separate section is devoted to 3-D analysis of OCT images, describing methods for segmentation and analysis of retinal layers, retinal vasculature, and 2-D/3-D detection of symptomatic exudate-associated derangements, as well as to OCT-based analysis of ONH morphology and shape. Throughout the paper, aspects of image acquisition, image analysis, and clinical relevance are treated together considering their mutually interlinked relationships. PMID:21743764

  16. Anti-(herpes simplex virus) activity of 4'-thio-2'-deoxyuridines: a biochemical investigation for viral and cellular target enzymes.

    PubMed Central

    Verri, A; Focher, F; Duncombe, R J; Basnak, I; Walker, R T; Coe, P L; de Clercq, E; Andrei, G; Snoeck, R; Balzarini, J; Spadari, S

    2000-01-01

    The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4'-thio-2'-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20-600 times) than that shown by the corresponding 4'-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4'-thio-2'-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4'-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4'-thio-2'-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4'-oxy- and 4'-thio-2'-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4'-thio-2'-deoxyuridines are better inhibitors than their 4'-oxy counterparts of [(3)H]thymidine incorporation in HSV-1-infected cells; (vi) 4'-thio-2'-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4'-thio-2'-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4'-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase. PMID:11023816

  17. Natural and semisynthetic diterpenoids with antiviral and immunomodulatory activities block the ERK signaling pathway.

    PubMed

    Bueno, Carlos Alberto; Michelini, Flavia Mariana; Pertino, Mariano Walter; Gómez, Catalina Arredondo; Schmeda-Hirschmann, Guillermo; Alché, Laura Edith

    2015-10-01

    The pathogenesis of many viral infections lies on the damage caused by the immune response against the virus. Current antiviral drugs do not act on the inflammatory component of the disease. Thus, new compounds that inhibit both viral multiplication and the immunopathology elicited by the virus are an approach that should be considered. In the present study, we identified two jatropholones (2A and 5B) and one carnosic acid derivative (9C) that significantly inhibited multiplication of TK+ and TK- strains of HSV-1 in Vero cells. Compounds 2A, 5B and 9C also prevented HSV-1- and TLRs-induced inflammatory response in cultivated murine macrophages. In macrophages infected with HSV-1, the inhibitory effect of compounds 2A, 5B and 9C on TNF-α and IL-6 production could be associated with the block of ERK pathway, whereas NF-κB pathway was not hampered by any of the compounds. Besides, 2A, 5B and 9C also inhibited ERK pathway and reduced TNF-α production in macrophages stimulated with TLR2, TLR4 or TLR9 agonists and were able to hinder IL-6 secretion after activation with TLR2 or TLR4, but not with TLR9. The immunomodulatory effect of 2A, 5B and 9C in macrophages infected with HSV-1 may be a consequence of the inhibition of ERK pathway activated by TLRs. The availability of compounds with both antiviral and immunomodulatory properties which affect TLR signaling pathways might be a useful strategy to control the progress of virus-induced disease. PMID:25528328

  18. Raman Imaging

    NASA Astrophysics Data System (ADS)

    Stewart, Shona; Priore, Ryan J.; Nelson, Matthew P.; Treado, Patrick J.

    2012-07-01

    The past decade has seen an enormous increase in the number and breadth of imaging techniques developed for analysis in many industries, including pharmaceuticals, food, and especially biomedicine. Rather than accept single-dimensional forms of information, users now demand multidimensional assessment of samples. High specificity and the need for little or no sample preparation make Raman imaging a highly attractive analytical technique and provide motivation for continuing advances in its supporting technology and utilization. This review discusses the current tools employed in Raman imaging, the recent advances, and the major applications in this ever-growing analytical field.

  19. Medical Imaging.

    ERIC Educational Resources Information Center

    Jaffe, C. Carl

    1982-01-01

    Describes principle imaging techniques, their applications, and their limitations in terms of diagnostic capability and possible adverse biological effects. Techniques include film radiography, computed tomography, nuclear medicine, positron emission tomography (PET), ultrasonography, nuclear magnetic resonance, and digital radiography. PET has…

  20. Body Imaging

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The high-tech art of digital signal processing (DSP) was pioneered at NASA's Jet Propulsion Laboratory (JPL) in the mid-1960s for use in the Apollo Lunar Landing Program. Designed to computer enhance pictures of the Moon, this technology became the basis for the Landsat Earth resources satellites and subsequently has been incorporated into a broad range of Earthbound medical and diagnostic tools. DSP is employed in advanced body imaging techniques including Computer-Aided Tomography, also known as CT and CATScan, and Magnetic Resonance Imaging (MRI). CT images are collected by irradiating a thin slice of the body with a fan-shaped x-ray beam from a number of directions around the body's perimeter. A tomographic (slice-like) picture is reconstructed from these multiple views by a computer. MRI employs a magnetic field and radio waves, rather than x-rays, to create images. In this photograph, a patient undergoes an open MRI.

  1. Body Imaging

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The high-tech art of digital signal processing (DSP) was pioneered at NASA's Jet Propulsion Laboratory (JPL) in the mid-1960s for use in the Apollo Lunar Landing Program. Designed to computer enhance pictures of the Moon, this technology became the basis for the Landsat Earth resources satellites and subsequently has been incorporated into a broad range of Earthbound medical and diagnostic tools. DSP is employed in advanced body imaging techniques including Computer-Aided Tomography, also known as CT and CATScan, and Magnetic Resonance Imaging (MRI). CT images are collected by irradiating a thin slice of the body with a fan-shaped x-ray beam from a number of directions around the body's perimeter. A tomographic (slice-like) picture is reconstructed from these multiple views by a computer. MRI employs a magnetic field and radio waves, rather than x-rays, to create images.

  2. EPR imaging

    NASA Astrophysics Data System (ADS)

    Eaton, Sandra S.; Eaton, Gareth R.

    Useful EPR imaging has been achieved using simple gradient coils on a standard spectrometer. Resolution of less than 1 mm is possible without deconvolution of the resulting spectra. Examples are presented using DPPH and nitroxyl radicals.

  3. Imaging Immunosenescence

    PubMed Central

    Qian, Feng; Montgomery, Ruth R.

    2016-01-01

    Summary To demonstrate effects of aging visually requires a robust technique that can reproducibly detect small differences in efficiency or kinetics between groups. Investigators of aging will greatly appreciate the benefits of Amnis ImageStream technology (www.amnis.com/), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging. Imagestream is quantitative, reproducible, feasible with limited samples, and it facilitates in-depth examination of cellular mechanisms between cohorts of samples. PMID:26420711

  4. Imaging System

    NASA Technical Reports Server (NTRS)

    1995-01-01

    The 1100C Virtual Window is based on technology developed under NASA Small Business Innovation (SBIR) contracts to Ames Research Center. For example, under one contract Dimension Technologies, Inc. developed a large autostereoscopic display for scientific visualization applications. The Virtual Window employs an innovative illumination system to deliver the depth and color of true 3D imaging. Its applications include surgery and Magnetic Resonance Imaging scans, viewing for teleoperated robots, training, and in aviation cockpit displays.

  5. Diagnostic imaging.

    PubMed

    Morris, Peter; Perkins, Alan

    2012-04-21

    Physical techniques have always had a key role in medicine, and the second half of the 20th century in particular saw a revolution in medical diagnostic techniques with the development of key imaging instruments: x-ray imaging and emission tomography (nuclear imaging and PET), MRI, and ultrasound. These techniques use the full width of the electromagnetic spectrum, from gamma rays to radio waves, and sound. In most cases, the development of a medical imaging device was opportunistic; many scientists in physics laboratories were experimenting with simple x-ray images within the first year of the discovery of such rays, the development of the cyclotron and later nuclear reactors created the opportunity for nuclear medicine, and one of the co-inventors of MRI was initially attempting to develop an alternative to x-ray diffraction for the analysis of crystal structures. What all these techniques have in common is the brilliant insight of a few pioneering physical scientists and engineers who had the tenacity to develop their inventions, followed by a series of technical innovations that enabled the full diagnostic potential of these instruments to be realised. In this report, we focus on the key part played by these scientists and engineers and the new imaging instruments and diagnostic procedures that they developed. By bringing the key developments and applications together we hope to show the true legacy of physics and engineering in diagnostic medicine. PMID:22516558

  6. Stellar Imager

    NASA Technical Reports Server (NTRS)

    Carpenter, Kenneth

    2007-01-01

    The Stellar Imager (SI) is one of NASA's "Vision Missions" - concepts for future, space-based, strategic missions that could enormously increase our capabilities for observing the Cosmos. SI is designed as a UV/Optical Interferometer which will enable 0.1 milli-arcsecond (mas) spectral imaging of stellar surfaces and, via asteroseismology, stellar interiors and of the Universe in general. The ultra-sharp images of the Stellar Imager will revolutionize our view of many dynamic astrophysical processes by transforming point sources into extended sources, and snapshots into evolving views. SI, with a characteristic angular resolution of 0.1 milli-arcseconds at 2000 Angstroms, represents an advance in image detail of several hundred times over that provided by the Hubble Space Telescope. The Stellar Imager will zoom in on what today-with few exceptions - we only know as point sources, revealing processes never before seen, thus providing a tool as fundamental to astrophysics as the microscope is to the study of life on Earth. SI's science focuses on the role of magnetism in the Universe, particularly on magnetic activity on the surfaces of stars like the Sun. It's prime goal is to enable long-term forecasting of solar activity and the space weather that it drives, in support of the Living With a Star program in the Exploration Era. SI will also revolutionize our understanding of the formation of planetary systems, of the habitability and climatology of distant planets, and of many magneto-hydrodynamically controlled processes in the Universe. Stellar Imager is included as a "Flagship and Landmark Discovery Mission" in the 2005 Sun Solar System Connection (SSSC) Roadmap and as a candidate for a "Pathways to Life Observatory" in the Exploration of the Universe Division (EUD) Roadmap (May, 2005) and as such is a candidate mission for the 2025-2030 timeframe. An artist's drawing of the current "baseline" concept for SI is presented.

  7. Activating the Expression of Human K-rasG12D Stimulates Oncogenic Transformation in Transgenic Goat Fetal Fibroblast Cells

    PubMed Central

    Gong, Jianhua; Wang, Zhongde; Polejaeva, Irina; Salgia, Ravi; Kao, Chien-Min; Chen, Chin-Tu; Chen, Guangchun; Chen, Liaohai

    2014-01-01

    Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established. PMID:24594684

  8. Imaging infection.

    PubMed

    Ketai, Loren; Jordan, Kirk; Busby, Katrina H

    2015-06-01

    Thoracic imaging is widely used to detect lower respiratory tract infections, identify their complications, and aid in differentiating infectious from noninfectious thoracic disease. Less commonly, the combination of imaging findings and a clinical setting can favor infection with a specific organism. This confluence can occur in cases of bronchiectatic nontuberculous mycobacterial infections in immune-competent hosts, invasive fungal disease among neutropenic patients, Pneumocystis jiroveci pneumonia in patients with AIDS, and in cytomegalovirus infections in patients with recent hematopoietic cell transplantation. These specific diagnoses often depend on computed tomography scanning rather than chest radiography alone. PMID:26024600

  9. Brain Imaging

    PubMed Central

    Racine, Eric; Bar-Ilan, Ofek; Illes, Judy

    2007-01-01

    Advances in neuroscience are increasingly intersecting with issues of ethical, legal, and social interest. This study is an analysis of press coverage of an advanced technology for brain imaging, functional magnetic resonance imaging, that has gained significant public visibility over the past ten years. Discussion of issues of scientific validity and interpretation dominated over ethical content in both the popular and specialized press. Coverage of research on higher order cognitive phenomena specifically attributed broad personal and societal meaning to neuroimages. The authors conclude that neuroscience provides an ideal model for exploring science communication and ethics in a multicultural context. PMID:17330151

  10. Image sets for satellite image processing systems

    NASA Astrophysics Data System (ADS)

    Peterson, Michael R.; Horner, Toby; Temple, Asael

    2011-06-01

    The development of novel image processing algorithms requires a diverse and relevant set of training images to ensure the general applicability of such algorithms for their required tasks. Images must be appropriately chosen for the algorithm's intended applications. Image processing algorithms often employ the discrete wavelet transform (DWT) algorithm to provide efficient compression and near-perfect reconstruction of image data. Defense applications often require the transmission of images and video across noisy or low-bandwidth channels. Unfortunately, the DWT algorithm's performance deteriorates in the presence of noise. Evolutionary algorithms are often able to train image filters that outperform DWT filters in noisy environments. Here, we present and evaluate two image sets suitable for the training of such filters for satellite and unmanned aerial vehicle imagery applications. We demonstrate the use of the first image set as a training platform for evolutionary algorithms that optimize discrete wavelet transform (DWT)-based image transform filters for satellite image compression. We evaluate the suitability of each image as a training image during optimization. Each image is ranked according to its suitability as a training image and its difficulty as a test image. The second image set provides a test-bed for holdout validation of trained image filters. These images are used to independently verify that trained filters will provide strong performance on unseen satellite images. Collectively, these image sets are suitable for the development of image processing algorithms for satellite and reconnaissance imagery applications.

  11. Imaging sciences workshop

    SciTech Connect

    Candy, J.V.

    1994-11-15

    This workshop on the Imaging Sciences sponsored by Lawrence Livermore National Laboratory contains short abstracts/articles submitted by speakers. The topic areas covered include the following: Astronomical Imaging; biomedical imaging; vision/image display; imaging hardware; imaging software; Acoustic/oceanic imaging; microwave/acoustic imaging; computed tomography; physical imaging; imaging algorithms. Selected papers are indexed separately for inclusion in the Energy Science and Technology Database.

  12. [Endometrial imaging].

    PubMed

    Lemercier, E; Genevois, A; Dacher, J N; Benozio, M; Descargues, G; Marpeau, L

    2000-12-01

    The diagnostic value of endovaginal sonography in benign or malignant endometrial pathology is high, increased by sonohysterography. Sonohysterography is useful in the diagnosis of endometrial thickness and to determine further investigations. MRI is accurate in the uterine adenomyosis diagnosis and is the imaging modality of choice for the preoperative endometrial cancer staging. PMID:11173754

  13. Inner Image

    ERIC Educational Resources Information Center

    Mollhagen, Nancy

    2004-01-01

    In this article, the author states that she has always loved self portraits but most teenagers do not enjoy looking too closely at their own faces in an effort to replicate them. Thanks to a new digital camera, she was able to use this new technology to inspire students to take a closer look at their inner image. Prior to the self-portrait…

  14. Forest Imaging

    NASA Technical Reports Server (NTRS)

    1992-01-01

    NASA's Technology Applications Center, with other government and academic agencies, provided technology for improved resources management to the Cibola National Forest. Landsat satellite images enabled vegetation over a large area to be classified for purposes of timber analysis, wildlife habitat, range measurement and development of general vegetation maps.

  15. Photoacoustic imaging platforms for multimodal imaging

    PubMed Central

    2015-01-01

    Photoacoustic (PA) imaging is a hybrid biomedical imaging method that exploits both acoustical Epub ahead of print and optical properties and can provide both functional and structural information. Therefore, PA imaging can complement other imaging methods, such as ultrasound imaging, fluorescence imaging, optical coherence tomography, and multi-photon microscopy. This article reviews techniques that integrate PA with the above imaging methods and describes their applications. PMID:25754364

  16. Death and Proliferation Time Course of Stem Cells Transplanted in the Myocardium

    PubMed Central

    Qiao, Hui; Surti, Suleman; Choi, Seok Rye; Raju, Karthik; Zhang, Hualei; Ponde, Datta E.; Kung, Hank F.; Karp, Joel; Zhou, Rong

    2010-01-01

    Purpose Noninvasive positron emission tomography (PET) imaging of reporter gene is combined with quantitative real-time polymerase reverse transcription (RT-PCR) method to study the time course of death and proliferation of stem cells transplanted in the myocardium. Methods Male murine embryonic stem cells (ESCs) were stably transfected with a mutant version of herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter gene; 5×106 such cells were injected into the myocardium of female athymic rats. While the transplanted cells was monitored by in vivo 9-(4-[F-18]fluoro-3-hydroxymethylbutyl)guanine ([F-18]FHBG) PET imaging of the heart, their absolute number was estimated by RT-PCR from hearts harvested at 3–5 h, 24 h, days 4, 7, and 14 after transplantation. Results (1) Forty percent of injected cells were retained in the heart while majority of injected cells were lost within a few hours after injection. Cell death was peaked at 24 h when 18% of donor cells retained in the heart were dead. (2) The substantial cell loss was reversed by significant proliferation of ESCs. This led to the recovery of cell number to 3.4 million (70% of injected dose) at day 4 and first visual observation of in vivo [F-18] signal in the heart. (3) A robust correlation (R2=0.9) between percent of injected dose per gram of tissue derived from in vivo PET signal and the number of donor cells estimated by RT-PCR was revealed. Conclusions The time course of transplanted stem cells surviving in the heart reveals a process of substantial cell loss within 24 h of injection and subsequent recovery of cell number through proliferation. Such proliferation can be noninvasively monitored by reporter gene imaging. PMID:19459013

  17. Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro.

    PubMed Central

    McKnight, S L; Gavis, E R

    1980-01-01

    When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5' flanking and intragenic sequences are deleted. The other set is deleted at the 3' end of the gene. By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5' and 3' termini of the HSV tk gene. Images PMID:6258155

  18. Imaging bolometer

    DOEpatents

    Wurden, G.A.

    1999-01-19

    Radiation-hard, steady-state imaging bolometer is disclosed. A bolometer employing infrared (IR) imaging of a segmented-matrix absorber of plasma radiation in a cooled-pinhole camera geometry is described. The bolometer design parameters are determined by modeling the temperature of the foils from which the absorbing matrix is fabricated by using a two-dimensional time-dependent solution of the heat conduction equation. The resulting design will give a steady-state bolometry capability, with approximately 100 Hz time resolution, while simultaneously providing hundreds of channels of spatial information. No wiring harnesses will be required, as the temperature-rise data will be measured via an IR camera. The resulting spatial data may be used to tomographically investigate the profile of plasmas. 2 figs.

  19. Imaging bolometer

    DOEpatents

    Wurden, Glen A.

    1999-01-01

    Radiation-hard, steady-state imaging bolometer. A bolometer employing infrared (IR) imaging of a segmented-matrix absorber of plasma radiation in a cooled-pinhole camera geometry is described. The bolometer design parameters are determined by modeling the temperature of the foils from which the absorbing matrix is fabricated by using a two-dimensional time-dependent solution of the heat conduction equation. The resulting design will give a steady-state bolometry capability, with approximately 100 Hz time resolution, while simultaneously providing hundreds of channels of spatial information. No wiring harnesses will be required, as the temperature-rise data will be measured via an IR camera. The resulting spatial data may be used to tomographically investigate the profile of plasmas.

  20. Brain imaging

    SciTech Connect

    Bradshaw, J.R.

    1989-01-01

    This book presents a survey of the various imaging tools with examples of the different diseases shown best with each modality. It includes 100 case presentations covering the gamut of brain diseases. These examples are grouped according to the clinical presentation of the patient: headache, acute headache, sudden unilateral weakness, unilateral weakness of gradual onset, speech disorders, seizures, pituitary and parasellar lesions, sensory disorders, posterior fossa and cranial nerve disorders, dementia, and congenital lesions.

  1. Phenotypic and genotypic characterization of clinical isolates of herpes simplex virus resistant to aciclovir.

    PubMed

    Harris, Wendy; Collins, Peter; Fenton, Rob J; Snowden, Wendy; Sowa, Mike; Darby, Graham

    2003-06-01

    A panel of 10 clinical isolates of herpes simplex virus (HSV) deficient in the expression of thymidine kinase (TK) and phenotypically resistant to aciclovir was characterized. Sequence analysis revealed a variety of mutations in TK (nucleotide substitutions, insertions and deletions), most of which resulted in truncated TK polypeptides. In line with previous reports, the most common mutation was a single G insertion in the 'G-string' motif. One HSV-1 isolate and two HSV-2 isolates appeared to encode full-length polypeptides and, in each case, an amino acid substitution likely to be responsible for the phenotype was identified. Pathogenicity was determined using a zosteriform model of HSV infection in BALB/c mice. The majority of isolates appeared to show impaired growth at the inoculation site compared with wild-type virus. They also showed poor replication in the peripheral nervous system and little evidence of zosteriform spread. One exception was isolate 4, which had a double G insertion in the G-string but, nevertheless, exhibited zosteriform spread. These studies confirmed that TK-deficient viruses display a range of neurovirulence with respect to latency and zosteriform spread. These results are discussed in the light of previous experience with TK-deficient viruses. PMID:12771406

  2. Magnetic Imaging

    NASA Astrophysics Data System (ADS)

    Petford-Long, A. K.

    Spin-transport effects, such as giant magnetoresistance, rely on the fact that there is a difference in scattering between the spin-up and spin-down electrons in a ferromagnetic material. The degree to which each electron channel is scattered depends on the magnetisation direction within the material, and thus on the local magnetic domain structure. It is therefore of importance when analysing spin-transport devices to understand their magnetic domain structure, both as a bulk property and locally. The aim of this chapter is to review a number of the techniques currently used to image magnetic domain structure in materials. Although a considerable amount of information about the magnetic properties and behaviour of a piece of material, for example a thin ferromagnetic film, can be obtained from bulk magnetometry measurements, it is often extremely useful to image the magnetic domain structure of the film and thus gain information about its magnetic properties at a local level. The various magnetic imaging techniques yet to be described can be extended, by the application of in-situ magnetic fields which allow not only the magnetic domains but also the magnetisation reversal process to be followed in real-time.

  3. Expression and characterization of the thymidine kinase gene of African swine fever virus.

    PubMed Central

    Martin Hernandez, A M; Tabares, E

    1991-01-01

    The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus. Images PMID:1987368

  4. Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

    PubMed Central

    Lieberman, H B; Lin, P F; Yeh, D B; Ruddle, F H

    1988-01-01

    We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed. Images PMID:3244356

  5. Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame.

    PubMed Central

    Shelton, L S; Pensiero, M N; Jenkins, F J

    1990-01-01

    The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between map units 0.527 and 0.552. We have identified and characterized the UL37 protein product in HSV-1-infected cells. The presence of the UL37 protein was detected by using a polyclonal rabbit antiserum directed against an in vitro-translated product derived from an in vitro-transcribed UL37 mRNA. The UL37 open reading frame encodes for a protein with an apparent molecular mass of 120 kDa in HSV-1-infected cells; the protein's mass was assigned on the basis of its migration in sodium dodecyl sulfate-polyacrylamide gels. The UL37 protein is not present at detectable levels in purified HSV-1 virions, suggesting that it is not a structural protein. Analysis of time course experiments and experiments using DNA synthesis inhibitors demonstrated that the UL37 protein is expressed prior to the onset of viral DNA synthesis, reaching maximum levels late in infection, classifying it as a gamma 1 gene. Elution of HSV-1-infected cell proteins from single-stranded DNA agarose columns by using a linear KCl gradient demonstrated that the UL37 protein elutes from this matrix at a salt concentration similar to that observed for ICP8, the major HSV-1 DNA-binding protein. In addition, computer-assisted analysis revealed a potential ATP-binding domain in the predicted UL37 amino acid sequence. On the basis of the kinetics of appearance and DNA-binding properties, we hypothesize that UL37 represents a newly recognized HSV-1 DNA-binding protein that may be involved in late events in viral replication. Images PMID:2173782

  6. Infection of peripheral blood mononuclear cells by herpes simplex and Epstein-Barr viruses. Differential induction of interleukin 6 and tumor necrosis factor-alpha.

    PubMed Central

    Gosselin, J; Flamand, L; D'Addario, M; Hiscott, J; Menezes, J

    1992-01-01

    Infection by herpesviruses can result in profound immunosuppressive or immunomodulatory effects. However, no significant information is available on the effect of such infections on the production of immunoregulatory cytokines. We studied the kinetics of production of two monocyte-derived cytokines, interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF alpha), induced by Epstein-Barr virus (EBV) and herpes simplex virus type 1 (HSV-1) in peripheral blood mononuclear cell cultures and in fractionated cell populations. We observed that, when compared to HSV-1, EBV is a stronger inducer of IL-6. In EBV-infected cultures, IL-6 protein was detected at day 1 postinfection and gradually increased with time. In contrast, lower amounts of IL-6 were detected 5 d postinfection in HSV-1-infected cultures. HSV-1-infected cultures secreted significant amounts of TNF alpha protein after 5 d of culture and reached a maximal level of production at day 7, whereas EBV inhibited TNF alpha production. In fractionated cell populations, monocytic cells were found to be the main source of IL-6 synthesis after EBV or HSV-1 infection. However, TNF alpha synthesis in HSV-1-infected cultures was from both B and monocytic cells. By using the polymerase chain reaction technique we show that, after infection by these two herpesviruses, differences in cytokine gene products are also observed at the transcriptional level. These observations demonstrate that EBV and HSV-1 exert differential effects on IL-6 and TNF alpha gene transcription and on the resulting protein secretion in human mononuclear blood cells. Images PMID:1318324

  7. Relationship between herpes simplex virus-1-specific antibody titers and cortical brain damage in Alzheimer’s disease and amnestic mild cognitive impairment

    PubMed Central

    Baglio, Francesca; Agostini, Simone; Agostini, Monia Cabinio; Laganà, Maria M.; Hernis, Ambra; Margaritella, Nicolò; Guerini, Franca R.; Zanzottera, Milena; Nemni, Raffaello; Clerici, Mario

    2014-01-01

    Alzheimer’s disease (AD) is a multifactorial disease with a still barely understood etiology. Herpes simplex virus 1 (HSV-1) has long been suspected to play a role in the pathogenesis of AD because of its neurotropism, high rate of infection in the general population, and life-long persistence in neuronal cells, particularly in the same brain regions that are usually altered in AD. The goal of this study was to evaluate HSV-1-specific humoral immune responses in patients with a diagnosis of either AD or amnestic mild cognitive impairment (aMCI), and to verify the possible relation between HSV-1-specific antibody (Ab) titers and cortical damage; results were compared to those obtained in a group of healthy controls (HC). HSV-1 serum IgG titers were measured in 225 subjects (83 AD, 68 aMCI, and 74 HC). HSV-specific Ab avidity and cortical gray matter volumes analyzed by magnetic resonance imaging (MRI) were evaluated as well in a subgroup of these individuals (44 AD, 23 aMCI, and 26 HC). Results showed that, whereas HSV-1 seroprevalence and IgG avidity were comparable in the three groups, increased Ab titers (p < 0.001) were detected in AD and aMCI compared to HC. Positive significant correlations were detected in AD patients alone between HSV-1 IgG titers and cortical volumes in orbitofrontal (region of interest, ROI1 RSp0.56; p = 0.0001) and bilateral temporal cortices (ROI2 RSp0.57; p < 0.0001; ROI3 RSp0.48; p = 0.001); no correlations could be detected between IgG avidity and MRI parameters. Results herein suggest that a strong HSV-1-specific humoral response could be protective toward AD-associated cortical damage. PMID:25360113

  8. Molecular Imaging of Induced Pluripotent Stem Cell Immunogenicity with In Vivo Development in Ischemic Myocardium

    PubMed Central

    Wang, Haibin; Zhou, Jin; Zhao, Mengge; Lin, Qiuxia; Wang, Yan; Li, Junjie; Li, Dexue; Du, Zhiyan; Yao, Anning; Cao, Feng; Wang, Changyong

    2013-01-01

    Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy. PMID:23840453

  9. Image Editing Via Searching Source Image

    NASA Astrophysics Data System (ADS)

    Yu, Han; Deng, Liang-Jian

    Image editing has important applications by changing the image texture, illumination, target location, etc. As an important application of Poisson equation, Poisson image editing processes images on the gradient domain and has been applied to seamless clone, selection editing, image denoising, etc. In this paper, we present a new application of Poisson image editing, which is based on searching source image. The main feature of the new application is all modifying information comes from the source image. Experimental results show that the proposed application performs well.

  10. Image Processor

    NASA Technical Reports Server (NTRS)

    1989-01-01

    Texas Instruments Programmable Remapper is a research tool used to determine how to best utilize the part of a patient's visual field still usable by mapping onto his field of vision with manipulated imagery. It is an offshoot of a NASA program for speeding up, improving the accuracy of pattern recognition in video imagery. The Remapper enables an image to be "pushed around" so more of it falls into the functional portions in the retina of a low vision person. It works at video rates, and researchers hope to significantly reduce its size and cost, creating a wearable prosthesis for visually impaired people.

  11. Multiscale image restoration for photon imaging systems

    NASA Astrophysics Data System (ADS)

    Jammal, Ghada; Bijaoui, Albert

    1999-05-01

    Nuclear medicine imaging is a widely used commercial imaging modality which relies on photon detection as the basis of image formation. As a diagnosis tool, it is unique in that it documents organ function and structure. It is a way to gather information that may be otherwise unavailable or require surgery. Practical limitations on imaging time and the amount of activity that can be administered safely to patients are serious impediments to substantial further improvements in nuclear medicine imaging. Hence, improvements of image quality via optimized image processing represent a significant opportunity to advance the state-of-the-art int his field. We present in this paper a new multiscale image restoration method that is concerned with eliminating one of the major sources of error in nuclear medicine imaging, namely Poisson noise, which degrades images in both quantitative and qualitative senses and hinders image analysis and interpretation. The paper then quantitatively evaluates the performances of the proposed method.

  12. Digital image processing.

    PubMed

    Seeram, Euclid

    2004-01-01

    Digital image processing is now commonplace in radiology, nuclear medicine and sonography. This article outlines underlying principles and concepts of digital image processing. After completing this article, readers should be able to: List the limitations of film-based imaging. Identify major components of a digital imaging system. Describe the history and application areas of digital image processing. Discuss image representation and the fundamentals of digital image processing. Outline digital image processing techniques and processing operations used in selected imaging modalities. Explain the basic concepts and visualization tools used in 3-D and virtual reality imaging. Recognize medical imaging informatics as a new area of specialization for radiologic technologists. PMID:15352557

  13. Scrotal imaging

    PubMed Central

    Studniarek, Michał; Modzelewska, Elza

    2015-01-01

    Pathological lesions within the scrotum are relatively rare in imaging except for ultrasonography. The diseases presented in the paper are usually found in men at the age of 15–45, i.e. men of reproductive age, and therefore they are worth attention. Scrotal ultrasound in infertile individuals should be conducted on a routine basis owing to the fact that pathological scrotal lesions are frequently detected in this population. Malignant testicular cancers are the most common neoplasms in men at the age of 20–40. Ultrasound imaging is the method of choice characterized by the sensitivity of nearly 100% in the differentiation between intratesticular and extratesticular lesions. In the case of doubtful lesions that are not classified for intra-operative verification, nuclear magnetic resonance is applied. Computed tomography, however, is performed to monitor the progression of a neoplastic disease, in pelvic trauma with scrotal injury as well as in rare cases of scrotal hernias involving the ureters or a fragment of the urinary bladder. PMID:26674847

  14. Geospatial Analysis Using Remote Sensing Images: Case Studies of Zonguldak Test Field

    NASA Astrophysics Data System (ADS)

    Bayık, Çağlar; Topan, Hüseyin; Özendi, Mustafa; Oruç, Murat; Cam, Ali; Abdikan, Saygın

    2016-06-01

    Inclined topographies are one of the most challenging problems for geospatial analysis of air-borne and space-borne imageries. However, flat areas are mostly misleading to exhibit the real performance. For this reason, researchers generally require a study area which includes mountainous topography and various land cover and land use types. Zonguldak and its vicinity is a very suitable test site for performance investigation of remote sensing systems due to the fact that it contains different land use types such as dense forest, river, sea, urban area; different structures such as open pit mining operations, thermal power plant; and its mountainous structure. In this paper, we reviewed more than 120 proceeding papers and journal articles about geospatial analysis that are performed on the test field of Zonguldak and its surroundings. Geospatial analysis performed with imageries include elimination of systematic geometric errors, 2/3D georeferencing accuracy assessment, DEM and DSM generation and validation, ortho-image production, evaluation of information content, image classification, automatic feature extraction and object recognition, pan-sharpening, land use and land cover change analysis and deformation monitoring. In these applications many optical satellite images are used i.e. ASTER, Bilsat-1, IKONOS, IRS-1C, KOMPSAT-1, KVR-1000, Landsat-3-5-7, Orbview-3, QuickBird, Pleiades, SPOT-5, TK-350, RADARSAT-1, WorldView-1-2; as well as radar data i.e. JERS-1, Envisat ASAR, TerraSAR-X, ALOS PALSAR and SRTM. These studies are performed by Departments of Geomatics Engineering at Bülent Ecevit University, at İstanbul Technical University, at Yıldız Technical University, and Institute of Photogrammetry and GeoInformation at Leibniz University Hannover. These studies are financially supported by TÜBİTAK (Turkey), the Universities, ESA, Airbus DS, ERSDAC (Japan) and Jülich Research Centre (Germany).

  15. Eos visible imagers

    NASA Technical Reports Server (NTRS)

    Barnes, W. L.

    1990-01-01

    Some of the proposed Earth Observing System (Eos) optical imagers are examined. These imagers include: moderate resolution imaging spectrometer (MODIS); geoscience laser ranging system (GLRS); high resolution imaging spectrometer (HIRIS); the intermediate thermal infrared spectrometer (ITIR); multi-angle imaging spectrometer (MISR); earth observing scanning polarimeter (EOSP); and the lightening imaging sensor (LIS).

  16. Speckle imaging algorithms for planetary imaging

    SciTech Connect

    Johansson, E.

    1994-11-15

    I will discuss the speckle imaging algorithms used to process images of the impact sites of the collision of comet Shoemaker-Levy 9 with Jupiter. The algorithms use a phase retrieval process based on the average bispectrum of the speckle image data. High resolution images are produced by estimating the Fourier magnitude and Fourier phase of the image separately, then combining them and inverse transforming to achieve the final result. I will show raw speckle image data and high-resolution image reconstructions from our recent experiment at Lick Observatory.

  17. Image catalogs.

    PubMed

    Gomoll, Andreas H; Thornhill, Thomas S

    2004-04-01

    The advent of digital photography and radiography allows documentation of interesting clinical findings with unprecedented ease, and many orthopaedic surgeons have taken extensive advantage of this opportunity to create large digital libraries of clinical results. However, this leaves surgeons with a rapidly increasing volume of data to store and organize; therefore, a system for archiving, locating, and managing images, radiographs, and digital slide presentations has become a crucial need in most orthopaedic groups and practices. However, many surgical groups and practices are not familiar with the computer technology available to initiate such systems. In this review, we discuss several software solutions currently on the market to address the specific needs of orthopaedic surgeons, and as a practical example, discuss a system that is in place in the Department of Orthopaedic Surgery at our institution. Overall, depending on the individual circumstances of each institution, there are various options that meet different technologic and financial requirements. PMID:15123922

  18. Medical Imaging System

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The MD Image System, a true-color image processing system that serves as a diagnostic aid and tool for storage and distribution of images, was developed by Medical Image Management Systems, Huntsville, AL, as a "spinoff from a spinoff." The original spinoff, Geostar 8800, developed by Crystal Image Technologies, Huntsville, incorporates advanced UNIX versions of ELAS (developed by NASA's Earth Resources Laboratory for analysis of Landsat images) for general purpose image processing. The MD Image System is an application of this technology to a medical system that aids in the diagnosis of cancer, and can accept, store and analyze images from other sources such as Magnetic Resonance Imaging.

  19. Herpes simplex virus type 1 and 2 intracellular p40: type-specific and cross-reactive antigenic determinants on peptides generated by partial proteolysis.

    PubMed Central

    Heilman, C J; Zweig, M; Hampar, B

    1981-01-01

    Intracellular p40 is a class of protein ranging in molecular weight from 39,000 to 45,000 that is immunoprecipitated from herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell extracts by mouse monoclonal antibodies or guinea pig antisera against HSV-1 and HSV-2 nucleocapsid p40. Analysis by a two-dimensional gel system showed that HSV-1 and HSV-2 intracellular p40 each consisted of three major components. However, these HSV-1 and HSV-2 proteins differed in charge and size. Analysis of Staphylococcus aureus V8 protease partial digests by two-dimensional gel electrophoresis indicated that none of the peptides of HSV-1 and HSV-2 intracellular p40 were identical. Immunoprecipitation of the partial digest products of intracellular p40-1 and p40-2 with homologous and heterologous guinea pig antisera resulted in the precipitation of various combinations of peptides indicating the presence of either type-specific or cross-reactive antigenic determinants. Images PMID:6172597

  20. Activity investigation of pinostrobin towards herpes simplex virus-1 as determined by atomic force microscopy.

    PubMed

    Wu, Nan; Kong, Yu; Zu, Yuangang; Fu, Yujie; Liu, Zhiguo; Meng, Ronghua; Liu, Xia; Efferth, Thomas

    2011-01-15

    In the present study, the antiviral activity of pinostrobin towards herpes simplex virus-1 (HSV-1) was investigated by MTT assay and atomic force microscopy. Pinostrobin can inhibit HSV-1 replication with 50% effective concentration (EC(50)) of 22.71 ± 1.72 μg/ml. MTT assay showed HSV-1 was significantly inhibited when pretreated with pinostrobin, with the inhibition of 85.69 ± 2.59%. Significant changes in morphology and size of HSV-1 were observed by atomic force microscopy (AFM) in response to pinostrobin treatment. AFM topography and phase images showed that with increasing time, the envelope was shedded and damaged, finally leading to virus inactivation. With increasing concentration, pinostrobin caused a gradual leakage, also contributing to breakage of the envelope and virus inactivation. Treatment effect of oral pinostrobin in vivo showed that pinostrobin (50mg/kg/dose) possesses definite therapeutical effect in the development of lesion score. In general, the results showed that AFM represents a powerful technique for the investigation of morphology and size of HSV-1 treated by antiviral agents. AFM is applicable to study chemically induced morphological changes at the nanometer level. PMID:20739162

  1. Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides.

    PubMed

    Kumar, R; Rai, D; Sharma, S K; Saffran, H A; Blush, R; Tyrrell, D L

    2001-10-11

    A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes

  2. Medical imaging.

    PubMed Central

    Kreel, L.

    1991-01-01

    There is now a wide choice of medical imaging to show both focal and diffuse pathologies in various organs. Conventional radiology with plain films, fluoroscopy and contrast medium have many advantages, being readily available with low-cost apparatus and a familiarity that almost leads to contempt. The use of plain films in chest disease and in trauma does not need emphasizing, yet there are still too many occasions when the answer obtainable from a plain radiograph has not been available. The film may have been mislaid, or the examination was not requested, or the radiograph had been misinterpreted. The converse is also quite common. Examinations are performed that add nothing to patient management, such as skull films when CT will in any case be requested or views of the internal auditory meatus and heal pad thickness in acromegaly, to quote some examples. Other issues are more complicated. Should the patient who clinically has gall-bladder disease have more than a plain film that shows gall-stones? If the answer is yes, then why request a plain film if sonography will in any case be required to 'exclude' other pathologies especially of the liver or pancreas? But then should cholecystography, CT or scintigraphy be added for confirmation? Quite clearly there will be individual circumstances to indicate further imaging after sonography but in the vast majority of patients little or no extra information will be added. Statistics on accuracy and specificity will, in the case of gall-bladder pathology, vary widely if adenomyomatosis is considered by some to be a cause of symptoms or if sonographic examinations 'after fatty meals' are performed. The arguments for or against routine contrast urography rather than sonography are similar but the possibility of contrast reactions and the need to limit ionizing radiation must be borne in mind. These diagnostic strategies are also being influenced by their cost and availability; purely pragmatic considerations are not

  3. The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

    PubMed Central

    Robbins, A K; Dorney, D J; Wathen, M W; Whealy, M E; Gold, C; Watson, R J; Holland, L E; Weed, S D; Levine, M; Glorioso, J C

    1987-01-01

    known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history. Images PMID:3039163

  4. scikit-image: image processing in Python.

    PubMed

    van der Walt, Stéfan; Schönberger, Johannes L; Nunez-Iglesias, Juan; Boulogne, François; Warner, Joshua D; Yager, Neil; Gouillart, Emmanuelle; Yu, Tony

    2014-01-01

    scikit-image is an image processing library that implements algorithms and utilities for use in research, education and industry applications. It is released under the liberal Modified BSD open source license, provides a well-documented API in the Python programming language, and is developed by an active, international team of collaborators. In this paper we highlight the advantages of open source to achieve the goals of the scikit-image library, and we showcase several real-world image processing applications that use scikit-image. More information can be found on the project homepage, http://scikit-image.org. PMID:25024921

  5. scikit-image: image processing in Python

    PubMed Central

    Schönberger, Johannes L.; Nunez-Iglesias, Juan; Boulogne, François; Warner, Joshua D.; Yager, Neil; Gouillart, Emmanuelle; Yu, Tony

    2014-01-01

    scikit-image is an image processing library that implements algorithms and utilities for use in research, education and industry applications. It is released under the liberal Modified BSD open source license, provides a well-documented API in the Python programming language, and is developed by an active, international team of collaborators. In this paper we highlight the advantages of open source to achieve the goals of the scikit-image library, and we showcase several real-world image processing applications that use scikit-image. More information can be found on the project homepage, http://scikit-image.org. PMID:25024921

  6. [Medical image enhancement: Sharpening].

    PubMed

    Kats, L; Vered, M

    2015-04-01

    Most digital imaging systems provide opportunities for image enhancement operations. These are applied to improve the original image and to make the image more appealing visually. One possible means of enhancing digital radiographic image is sharpening. The purpose of sharpening filters is to improve image quality by removing noise or edge enhancement. Sharpening filters may make the radiographic images subjectively more appealing. But during this process, important radiographic features may disappear while artifacts that simulate pathological process might be generated. Therefore, it is of utmost importance for dentists to be familiar with and aware of the use of image enhancement operations, provided by medical digital imaging programs. PMID:26255429

  7. X-Ray Imaging

    MedlinePlus

    ... Brain Surgery Imaging Clinical Trials Basics Patient Information X-Ray Imaging Print This Page X-ray imaging is perhaps the most familiar type of imaging. Images produced by X-rays are due to the different absorption rates of ...

  8. Split image optical display

    DOEpatents

    Veligdan, James T.

    2007-05-29

    A video image is displayed from an optical panel by splitting the image into a plurality of image components, and then projecting the image components through corresponding portions of the panel to collectively form the image. Depth of the display is correspondingly reduced.

  9. Split image optical display

    DOEpatents

    Veligdan, James T.

    2005-05-31

    A video image is displayed from an optical panel by splitting the image into a plurality of image components, and then projecting the image components through corresponding portions of the panel to collectively form the image. Depth of the display is correspondingly reduced.

  10. Terahertz wave reciprocal imaging

    NASA Astrophysics Data System (ADS)

    Xu, Jingzhou; Zhang, X.-C.

    2006-04-01

    A reciprocal imaging technology with an encoding/decoding image readout method allows a single detector (such as a heterodyne detector) to produce a two dimensional (2D) image simultaneously. Applying it in a pulsed terahertz imaging system could create a 2D terahertz image with 100pixels per frame which produces the same signal to noise ratio as a signal spot measurement.

  11. Smart Image Enhancement Process

    NASA Technical Reports Server (NTRS)

    Jobson, Daniel J. (Inventor); Rahman, Zia-ur (Inventor); Woodell, Glenn A. (Inventor)

    2012-01-01

    Contrast and lightness measures are used to first classify the image as being one of non-turbid and turbid. If turbid, the original image is enhanced to generate a first enhanced image. If non-turbid, the original image is classified in terms of a merged contrast/lightness score based on the contrast and lightness measures. The non-turbid image is enhanced to generate a second enhanced image when a poor contrast/lightness score is associated therewith. When the second enhanced image has a poor contrast/lightness score associated therewith, this image is enhanced to generate a third enhanced image. A sharpness measure is computed for one image that is selected from (i) the non-turbid image, (ii) the first enhanced image, (iii) the second enhanced image when a good contrast/lightness score is associated therewith, and (iv) the third enhanced image. If the selected image is not-sharp, it is sharpened to generate a sharpened image. The final image is selected from the selected image and the sharpened image.

  12. An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

    PubMed Central

    Conner, J; Cooper, J; Furlong, J; Clements, J B

    1992-01-01

    We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication. Images PMID:1331536

  13. Intensive Pharmacological Immunosuppression Allows for Repetitive Liver Gene Transfer With Recombinant Adenovirus in Nonhuman Primates

    PubMed Central

    Fontanellas, Antonio; Hervás-Stubbs, Sandra; Mauleón, Itsaso; Dubrot, Juan; Mancheño, Uxua; Collantes, María; Sampedro, Ana; Unzu, Carmen; Alfaro, Carlos; Palazón, Asis; Smerdou, Cristian; Benito, Alberto; Prieto, Jesús; Peñuelas, Iván; Melero, Ignacio

    2010-01-01

    Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector. PMID:20087317

  14. An improved strategy for the synthesis of [18F]-labeled arabinofuranosyl nuclosides

    PubMed Central

    Zhang, Hanwen; Cantorias, Melchor V.; Pillarsetty, NagaVaraKishore; Burnazi, Eva M.; Cai, Shangde; Lewis, Jason S.

    2012-01-01

    The expression of the herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene can be imaged efficaciously using a variety of 2′-[18F]fluoro-2′-deoxy-1-b-D-arabinofuranosyl-uracil derivatives [[18F]-FXAU, X= I(iodo), E(ethyl), and M(methyl)]. However, the application of these derivatives in clinical and translational studies has been impeded by their complicated and long syntheses (3–5 h). To remedy these issues, in the study at hand we have investigated whether microwave or combined catalysts could facilitate the coupling reaction between sugar and nucleobase and, further, have probed the feasibility of establishing a novel approach for [18F]-FXAU synthesis. We have demonstrated that the rate of the trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed coupling reaction between the 2-deoxy-sugar and uracil derivatives at 90°C can be significantly accelerated by microwave-driven heating or by the addition of Lewis acid catalyst (SnCl4). Further, we have observed that the stability of the α- and β-anomers of [18F]-FXAU derivatives differs during the hydrolysis step. Using the microwave-driven heating approach, overall decay-corrected radiochemical yields of 19–27% were achieved for [18F]-FXAU in 120 min at a specific activity of >22 MBq/nmol (595 Ci/mmol). Ultimately, we believe that these high yielding syntheses of [18F]-FIAU, [18F]-FMAU and [18F]-FEAU will facilitate routine production for clinical applications. PMID:22819195

  15. What Is an Image?

    ERIC Educational Resources Information Center

    Gerber, Andrew J.; Peterson, Bradley S.

    2008-01-01

    The article helps to understand the interpretation of an image by presenting as to what constitutes an image. A common feature in all images is the basic physical structure that can be described with a common set of terms.

  16. Registration Of SAR Images With Multisensor Images

    NASA Technical Reports Server (NTRS)

    Evans, Diane L.; Burnette, Charles F.; Van Zyl, Jakob J.

    1993-01-01

    Semiautomated technique intended primarily to facilitate registration of polarimetric synthetic-aperture-radar (SAR) images with other images of same or partly overlapping terrain while preserving polarization information conveyed by SAR data. Technique generally applicable in sense one or both of images to be registered with each other generated by polarimetric or nonpolarimetric SAR, infrared radiometry, conventional photography, or any other applicable sensing method.

  17. Far Ultraviolet Imaging from the Image Spacecraft

    NASA Technical Reports Server (NTRS)

    Mende, S. B.; Heetderks, H.; Frey, H. U.; Lampton, M.; Geller, S. P.; Stock, J. M.; Abiad, R.; Siegmund, O. H. W.; Tremsin, A. S.; Habraken, S.

    2000-01-01

    Direct imaging of the magnetosphere by the IMAGE spacecraft will be supplemented by observation of the global aurora. The IMAGE satellite instrument complement includes three Far Ultraviolet (FUV) instruments. The Wideband Imaging Camera (WIC) will provide broad band ultraviolet images of the aurora for maximum spatial and temporal resolution by imaging the LBH N2 bands of the aurora. The Spectrographic Imager (SI), a novel form of monochromatic imager, will image the aurora, filtered by wavelength. The proton-induced component of the aurora will be imaged separately by measuring the Doppler-shifted Lyman-a. Finally, the GEO instrument will observe the distribution of the geocoronal emission to obtain the neutral background density source for charge exchange in the magnetosphere. The FUV instrument complement looks radially outward from the rotating IMAGE satellite and, therefore, it spends only a short time observing the aurora and the Earth during each spin. To maximize photon collection efficiency and use efficiently the short time available for exposures the FUV auroral imagers WIC and SI both have wide fields of view and take data continuously as the auroral region proceeds through the field of view. To minimize data volume, the set of multiple images are electronically co-added by suitably shifting each image to compensate for the spacecraft rotation. In order to minimize resolution loss, the images have to be distort ion-corrected in real time. The distortion correction is accomplished using high speed look up tables that are pre-generated by least square fitting to polynomial functions by the on-orbit processor. The instruments were calibrated individually while on stationary platforms, mostly in vacuum chambers. Extensive ground-based testing was performed with visible and near UV simulators mounted on a rotating platform to emulate their performance on a rotating spacecraft.

  18. Hadamard transform imager and imaging spectrometer.

    PubMed

    Swift, R D; Wattson, R B; Decker, J A; Paganetti, R; Harwit, M

    1976-06-01

    An imager and a spectrometric imager, which achieve multiplexing by the use of binary optical encoding masks, have been built and tested. The masks are based on orthogonal, pseudorandom digital codes derived from Hadamard matrices. The spatial (and/or spectral) data are therefore obtained in the form of a Hadamard transform of the spatial (and/or spectral) scene; computer algorithms are used to decode the data and reconstruct images of the original scene. The hardware, algorithms processing and display facility are described. A number of spatial and spatial/spectral images, obtained in the laboratory, are presented. We present an analysis of the situations for which the multiplex advantage may be gained and of the limitations of the technique. Potential applications of the spectrometric imager are discussed. The spectrometric imager is covered by U.S. Patent 3,720,469 assigned to Spectral Imaging Inc., Concord, Mass. PMID:20165224

  19. Radar image analysis utilizing junctive image metamorphosis

    NASA Astrophysics Data System (ADS)

    Krueger, Peter G.; Gouge, Sally B.; Gouge, Jim O.

    1998-09-01

    A feasibility study was initiated to investigate the ability of algorithms developed for medical sonogram image analysis, to be trained for extraction of cartographic information from synthetic aperture radar imagery. BioComputer Research Inc. has applied proprietary `junctive image metamorphosis' algorithms to cancer cell recognition and identification in ultrasound prostate images. These algorithms have been shown to support automatic radar image feature detection and identification. Training set images were used to develop determinants for representative point, line and area features, which were used on test images to identify and localize the features of interest. The software is computationally conservative; operating on a PC platform in real time. The algorithms are robust; having applicability to be trained for feature recognition on any digital imagery, not just those formed from reflected energy, such as sonograms and radar images. Applications include land mass characterization, feature identification, target recognition, and change detection.

  20. Image management research

    NASA Technical Reports Server (NTRS)

    Watson, Andrew B.

    1988-01-01

    Two types of research issues are involved in image management systems with space station applications: image processing research and image perception research. The image processing issues are the traditional ones of digitizing, coding, compressing, storing, analyzing, and displaying, but with a new emphasis on the constraints imposed by the human perceiver. Two image coding algorithms have been developed that may increase the efficiency of image management systems (IMS). Image perception research involves a study of the theoretical and practical aspects of visual perception of electronically displayed images. Issues include how rapidly a user can search through a library of images, how to make this search more efficient, and how to present images in terms of resolution and split screens. Other issues include optimal interface to an IMS and how to code images in a way that is optimal for the human perceiver. A test-bed within which such issues can be addressed has been designed.

  1. Imaging Sciences Workshop Proceedings

    SciTech Connect

    Candy, J.V.

    1996-11-21

    This report contains the proceedings of the Imaging Sciences Workshop sponsored by C.A.S.LS., the Center for Advanced Signal & Image Sciences. The Center, established primarily to provide a forum where researchers can freely exchange ideas on the signal and image sciences in a comfortable intellectual environment, has grown over the last two years with the opening of a Reference Library (located in Building 272). The Technical Program for the 1996 Workshop include a variety of efforts in the Imaging Sciences including applications in the Microwave Imaging, highlighted by the Micro-Impulse Radar (MIR) system invented at LLNL, as well as other applications in this area. Special sessions organized by various individuals in Speech, Acoustic Ocean Imaging, Radar Ocean Imaging, Ultrasonic Imaging, and Optical Imaging discuss various applica- tions of real world problems. For the more theoretical, sessions on Imaging Algorithms and Computed Tomography were organized as well as for the more pragmatic featuring a session on Imaging Systems.

  2. Modern Brain Tumor Imaging

    PubMed Central

    Barajas, Ramon F.; Cha, Soonmee

    2015-01-01

    The imaging and clinical management of patients with brain tumor continue to evolve over time and now heavily rely on physiologic imaging in addition to high-resolution structural imaging. Imaging remains a powerful noninvasive tool to positively impact the management of patients with brain tumor. This article provides an overview of the current state-of-the art clinical brain tumor imaging. In this review, we discuss general magnetic resonance (MR) imaging methods and their application to the diagnosis of, treatment planning and navigation, and disease monitoring in patients with brain tumor. We review the strengths, limitations, and pitfalls of structural imaging, diffusion-weighted imaging techniques, MR spectroscopy, perfusion imaging, positron emission tomography/MR, and functional imaging. Overall this review provides a basis for understudying the role of modern imaging in the care of brain tumor patients. PMID:25977902

  3. Multiscale Image Processing of Solar Image Data

    NASA Astrophysics Data System (ADS)

    Young, C.; Myers, D. C.

    2001-12-01

    It is often said that the blessing and curse of solar physics is too much data. Solar missions such as Yohkoh, SOHO and TRACE have shown us the Sun with amazing clarity but have also increased the amount of highly complex data. We have improved our view of the Sun yet we have not improved our analysis techniques. The standard techniques used for analysis of solar images generally consist of observing the evolution of features in a sequence of byte scaled images or a sequence of byte scaled difference images. The determination of features and structures in the images are done qualitatively by the observer. There is little quantitative and objective analysis done with these images. Many advances in image processing techniques have occured in the past decade. Many of these methods are possibly suited for solar image analysis. Multiscale/Multiresolution methods are perhaps the most promising. These methods have been used to formulate the human ability to view and comprehend phenomena on different scales. So these techniques could be used to quantitify the imaging processing done by the observers eyes and brains. In this work we present several applications of multiscale techniques applied to solar image data. Specifically, we discuss uses of the wavelet, curvelet, and related transforms to define a multiresolution support for EIT, LASCO and TRACE images.

  4. Image processor development with synthetic images

    NASA Astrophysics Data System (ADS)

    Guivens, Norman R., Jr.; Henshaw, Philip D.

    1992-03-01

    Many impressive developments in image simulation technology have led to extensive use of synthetic images in the motion picture industry for special effects and animation, and also in applications such as aircraft flight simulators. Although these images appear correct to the human eye, they generally are not suitable for development of image processing and machine vision applications because the logarithmic response of the human eye does not match the linear response of most electronic detectors. Synthetic images must accurately represent the effects which are present in detected images, whether produced by the source(s) of illumination, the scene itself, the medium through which the sensor is viewing the scene, the sensor system, or electronic circuits between the detector array and the processing system if they are to be useful for development and analysis of image processing (and machine vision) systems. Recent developments have led to the use of laser sensors for various machine vision applications including collision avoidance, wire detection and avoidance, intrusion detection, and underwater imaging systems. With recent developments in low cost laser systems, the use of these sensors for numerous applications relating to machine vision is likely to continue to expand for the foreseeable future. SPARTA's work in the area of image synthesis began with the development of a coherent laser radar simulation running on IBM and compatible personal computers, and has since branched into modeling of incoherent active and passive systems as well. SPARTA's current optical imaging sensor simulation, SENSORSIM, is written in ANSI standard FORTRAN '77 to ensure portability.

  5. Imaging systems and applications.

    PubMed

    Bennett, Gisele; Catrysse, Peter B; Farrell, Joyce E; Fowler, Boyd; Mait, Joseph N

    2012-02-01

    Imaging systems are used in consumer, medical, and military applications. Designing, developing, and building imaging systems requires a multidisciplinary approach. This issue features current research in imaging systems that ranges from fundamental theories to novel applications. Although the papers collected are diverse, their unique compilation provides a systems perspective to imaging. PMID:22307134

  6. Adolescence and Body Image.

    ERIC Educational Resources Information Center

    Weinshenker, Naomi

    2002-01-01

    Discusses body image among adolescents, explaining that today's adolescents are more prone to body image distortions and dissatisfaction than ever and examining the historical context; how self-image develops; normative discontent; body image distortions; body dysmorphic disorder (BDD); vulnerability of boys (muscle dysmorphia); who is at risk;…

  7. Imaging the temporomandibular joint

    SciTech Connect

    Katzberg, R.W.; Manzione, J.V.; Westesson, P.L.

    1988-01-01

    This book encompasses all imaging modalities as they apply to the Temporomandibular Joint and its disorders. The volume employs correlative line drawings to elaborate on diagnostic images. It helps teach methods of TMJ imaging and describes findings identified by different imaging modalities to both radiologists and dental clinicians.

  8. Image-Processing Educator

    NASA Technical Reports Server (NTRS)

    Gunther, F. J.

    1986-01-01

    Apple Image-Processing Educator (AIPE) explores ability of microcomputers to provide personalized computer-assisted instruction (CAI) in digital image processing of remotely sensed images. AIPE is "proof-of-concept" system, not polished production system. User-friendly prompts provide access to explanations of common features of digital image processing and of sample programs that implement these features.

  9. Seismic Imaging and Monitoring

    SciTech Connect

    Huang, Lianjie

    2012-07-09

    I give an overview of LANL's capability in seismic imaging and monitoring. I present some seismic imaging and monitoring results, including imaging of complex structures, subsalt imaging of Gulf of Mexico, fault/fracture zone imaging for geothermal exploration at the Jemez pueblo, time-lapse imaging of a walkway vertical seismic profiling data for monitoring CO{sub 2} inject at SACROC, and microseismic event locations for monitoring CO{sub 2} injection at Aneth. These examples demonstrate LANL's high-resolution and high-fidelity seismic imaging and monitoring capabilities.

  10. Ultrasound Imaging System Video

    NASA Technical Reports Server (NTRS)

    2002-01-01

    In this video, astronaut Peggy Whitson uses the Human Research Facility (HRF) Ultrasound Imaging System in the Destiny Laboratory of the International Space Station (ISS) to image her own heart. The Ultrasound Imaging System provides three-dimension image enlargement of the heart and other organs, muscles, and blood vessels. It is capable of high resolution imaging in a wide range of applications, both research and diagnostic, such as Echocardiography (ultrasound of the heart), abdominal, vascular, gynecological, muscle, tendon, and transcranial ultrasound.

  11. Automatic digital image registration

    NASA Technical Reports Server (NTRS)

    Goshtasby, A.; Jain, A. K.; Enslin, W. R.

    1982-01-01

    This paper introduces a general procedure for automatic registration of two images which may have translational, rotational, and scaling differences. This procedure involves (1) segmentation of the images, (2) isolation of dominant objects from the images, (3) determination of corresponding objects in the two images, and (4) estimation of transformation parameters using the center of gravities of objects as control points. An example is given which uses this technique to register two images which have translational, rotational, and scaling differences.

  12. Image Enhancement, Image Quality, and Noise

    NASA Technical Reports Server (NTRS)

    Rahman, Zia-ur; Jobson, Daniel J.; Woodell, Glenn A.; Hines, Glenn D.

    2005-01-01

    The Multiscale Retinex With Color Restoration (MSRCR) is a non-linear image enhancement algorithm that provides simultaneous dynamic range compression, color constancy and rendition. The overall impact is to brighten up areas of poor contrast/lightness but not at the expense of saturating areas of good contrast/brightness. The downside is that with the poor signal-to-noise ratio that most image acquisition devices have in dark regions, noise can also be greatly enhanced thus affecting overall image quality. In this paper, we will discuss the impact of the MSRCR on the overall quality of an enhanced image as a function of the strength of shadows in an image, and as a function of the root-mean-square (RMS) signal-to-noise (SNR) ratio of the image.

  13. Image registration method for medical image sequences

    DOEpatents

    Gee, Timothy F.; Goddard, James S.

    2013-03-26

    Image registration of low contrast image sequences is provided. In one aspect, a desired region of an image is automatically segmented and only the desired region is registered. Active contours and adaptive thresholding of intensity or edge information may be used to segment the desired regions. A transform function is defined to register the segmented region, and sub-pixel information may be determined using one or more interpolation methods.

  14. Image processing software for imaging spectrometry

    NASA Technical Reports Server (NTRS)

    Mazer, Alan S.; Martin, Miki; Lee, Meemong; Solomon, Jerry E.

    1988-01-01

    The paper presents a software system, Spectral Analysis Manager (SPAM), which has been specifically designed and implemented to provide the exploratory analysis tools necessary for imaging spectrometer data, using only modest computational resources. The basic design objectives are described as well as the major algorithms designed or adapted for high-dimensional images. Included in a discussion of system implementation are interactive data display, statistical analysis, image segmentation and spectral matching, and mixture analysis.

  15. Parallel MR Imaging

    PubMed Central

    Deshmane, Anagha; Gulani, Vikas; Griswold, Mark A.; Seiberlich, Nicole

    2015-01-01

    Parallel imaging is a robust method for accelerating the acquisition of magnetic resonance imaging (MRI) data, and has made possible many new applications of MR imaging. Parallel imaging works by acquiring a reduced amount of k-space data with an array of receiver coils. These undersampled data can be acquired more quickly, but the undersampling leads to aliased images. One of several parallel imaging algorithms can then be used to reconstruct artifact-free images from either the aliased images (SENSE-type reconstruction) or from the under-sampled data (GRAPPA-type reconstruction). The advantages of parallel imaging in a clinical setting include faster image acquisition, which can be used, for instance, to shorten breath-hold times resulting in fewer motion-corrupted examinations. In this article the basic concepts behind parallel imaging are introduced. The relationship between undersampling and aliasing is discussed and two commonly used parallel imaging methods, SENSE and GRAPPA, are explained in detail. Examples of artifacts arising from parallel imaging are shown and ways to detect and mitigate these artifacts are described. Finally, several current applications of parallel imaging are presented and recent advancements and promising research in parallel imaging are briefly reviewed. PMID:22696125

  16. IMAGES: An interactive image processing system

    NASA Technical Reports Server (NTRS)

    Jensen, J. R.

    1981-01-01

    The IMAGES interactive image processing system was created specifically for undergraduate remote sensing education in geography. The system is interactive, relatively inexpensive to operate, almost hardware independent, and responsive to numerous users at one time in a time-sharing mode. Most important, it provides a medium whereby theoretical remote sensing principles discussed in lecture may be reinforced in laboratory as students perform computer-assisted image processing. In addition to its use in academic and short course environments, the system has also been used extensively to conduct basic image processing research. The flow of information through the system is discussed including an overview of the programs.

  17. Fast image decompression for telebrowsing of images

    NASA Technical Reports Server (NTRS)

    Miaou, Shaou-Gang; Tou, Julius T.

    1993-01-01

    Progressive image transmission (PIT) is often used to reduce the transmission time of an image telebrowsing system. A side effect of the PIT is the increase of computational complexity at the viewer's site. This effect is more serious in transform domain techniques than in other techniques. Recent attempts to reduce the side effect are futile as they create another side effect, namely, the discontinuous and unpleasant image build-up. Based on a practical assumption that image blocks to be inverse transformed are generally sparse, this paper presents a method to minimize both side effects simultaneously.