Assessment of Estrogenic Endocrine-Disrupting Chemical Actions in the Brain Using in Vivo Somatic Gene Transfer

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element–thymidine kinase–luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17β-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 μg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo

Startle responding in the context of visceral pain

This study aimed to investigate affective modulation of eye blink startle by aversive visceral stimulation. Startle blink EMG responses were measured in 31 healthy participants receiving painful, intermittent balloon distentions in the distal esophagus during 4 blocks (positive, negative, neutral or no pictures), and compared with startles during 3 ‘safe’ blocks without esophageal stimulations (positive, negative or neutral emotional pictures). Women showed enhanced startle during blocks with distentions (as compared with ‘safe’ blocks), both when the balloon was in inflated and deflated states, suggesting that fear and/or expectations may have played a role. Men's startle did not differ between distention and non-distention blocks. In this particular study context affective picture viewing did not further impose any effect on startle eye blink responses. The current results may contribute to a better understanding of emotional reactions to aversive interoceptive stimulation

Earth Virtualization Engines (EVE)

To manage Earth in the Anthropocene, new tools, new institutions, and new forms of international cooperation will be required. Earth Virtualization Engines is proposed as an international federation of centers of excellence to empower all people to respond to the immense and urgent challenges posed by climate change

Earth Virtualization Engines (EVE)

To manage Earth in the Anthropocene, new tools, new institutions, and new forms of international cooperation will be required. Earth Virtualization Engines is proposed as an international federation of centers of excellence to empower all people to respond to the immense and urgent challenges posed by climate change

Earth Virtualization Engines (EVE)

To manage Earth in the Anthropocene, new tools, new institutions, and new forms of international cooperation will be required. Earth Virtualization Engines is proposed as an international federation of centers of excellence to empower all people to respond to the immense and urgent challenges posed by climate change

KSHV Targeted Therapy: An Update on Inhibitors of Viral Lytic Replication

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing

Inhibitors of y-herpesvirus replication and their mode of action

Since the discovery of Epstein-Barr virus (EBV) and Kaposi s sarcoma-associated herpesvirus (KSHV), 50 and 20 years ago, respectively, little progress has been made towards therapies directed against these oncogenic viruses. A primary infection with EBV may be associated with infectious mononucleosis when it occurs in adolescence or early adulthood. The severity in symptoms related to infectious mononucleosis varies greatly between individuals and, in some cases, major complications require hospitalization. Furthermore, EBV and KSHV are the causative agents of a number of human malignancies and proliferative diseases, such as Burkitt s lymphoma and nasopharyngeal carcinoma for EBV and Kaposi s sarcoma for KSHV. The current treatment modalities for EBV- and KSHV-related diseases lack efficiency, and virus-directed therapies are limited. Despite the inhibition of EBV and KSHV lytic replication by several antiviral agents that target the viral DNA polymerase, these drugs have no efficacy against a latent EBV or KSHV infection in tumor cells. Because of the unclear role of antiviral therapy in the prevention or treatment of virally-induced diseases, randomized controlled clinical trials are needed to determine the true efficacy of these drugs in different clinical settings. Today, no agents have been approved for use against EBV and KSHV, and therefore, new studies are needed with regard to selective drugs against gammaherpesviruses.In this thesis, we aimed to search for potent and selective antiviral drugs against EBV and KSHV. This includes in particular the detailed study of the mechanism of action of several new nucleoside analogs, i.e. HDVD, KAY-2-41 and KAH-39-149. Also, no data were available on the molecular basis of drug-resistance in EBV and KSHV. To address this topic, we characterized animal gammaherpesvirus mutants as an approach to unravel the mechanisms of drug-resistance.In the first part of this work, we evaluated the inhibitory effect of various compounds against gammaherpesvirus replication. In addition to nucleoside analogs currently available for treatment of herpesvirus infections (e.g. acyclovir, penciclovir, ganciclovir and brivudin), we examined the anti-gammaherpesvirus potency and selectivity of structurally-related 5-substituted uridine (CVDU, IDU, EDU, etc.) and cytidine [FIAC and (E)-CVDC] analogs. This study highlighted variations in the antiviral activity of nucleoside derivatives against EBV and KSHV. Additionally, discrepancies in the spectrum of activity was seen among human and closely-related animal gammaherpesviruses, i.e. herpesvirus saimiri (HVS), murine gammaherpesvirus 68 (MHV-68) and rhesus rhadinovirus (RRV). The selectivity of nucleoside analogs is determined by the efficiency of the viral thymidine kinase (TK) or protein kinase (PK) to phosphorylate, and consequently activate these nucleoside analogs in gammaherpesvirus replicating cells. Selection of HVS and MHV-68 mutants under pressure of acyclovir and brivudin, and characterization of their drug-susceptibility profile to various nucleoside analogs, underlined the differences existing in substrate recognition among the viral TK and PK of gammaherpesviruses.L-dioxolane uracil nucleosides were previously described as a potent class of anti-EBV agents. The novel analog HDVD, was found to be a selective inhibitor of not only EBV replication, but also of KSHV, HVS, MHV-68 and herpes simplex virus type 1 (HSV-1). To gain more insights into the mode of action of HDVD against herpesviruses, in vitro selected HSV-1, HVS and MHV-68 mutants were characterized. They displayed distinct mutations in the viral TK, resulting in the loss of HDVD activity. Hence, we proved that the compound was activated by the viral TK. HDVD and its prodrug demonstrated also efficacy in a small animal model, in which they were shown to reduce viral replication in the lungs of MHV-68-infected mice. A distinct spectrum of anti-herpesvirus activity was determined for two novel thiothymidine derivatives, KAY-2-41 and KAH-39-149. These compounds were particularly active against EBV replication, in addition to their inhibitory effect against HSV and varicella-zoster virus. While KAY-2-41- and KAH-39-149-resistant herpesvirus strains (HSV-1, MHV-68 and HVS) harbored mutations in the viral TK, we were able to prove that their activity was also dependent on phosphorylation by cytosolic TK1. The efficacy of both thionucleosides in mice against MHV-68 acute infection was particularly promising for KAH-39-149, which showed similar in vivo potency as HPMPC and HPMP-5-azaC, two acyclic nucleoside phosphonate (ANP) derivatives.In the second part of our research, we focused on the activity of nucleotide analogs against gammaherpesviruses and their interactions with the viral DNA polymerase. We reported the in vitro potency of drugs belonging to the class of ANPs of which HPMP-5-azaC, 3-deaza-HPMPA and their cyclic derivatives emerged as highly potent anti-gammaherpesvirus agents. In this chapter, we also described an interesting observation regarding reduced activities of cyclic prodrugs of ANPs against the EBV P3HR-1 strain. Moreover, characterization of HVS and MHV-68 mutants selected under pressure of HPMPC, HPMP-5-azaC, HPMPO-DAPy, and PMEA, as well as the pyrophosphate analog foscarnet, allowed us to link viral DNA polymerase mutations with a particular cross-resistance profile to various nucleoside and nucleotide analogs. In the urge to develop novel and potent anti-gammaherpesvirus agents, our studies showed the potential of distinct classes of nucleos(t)ide derivatives for further development as selective therapeutics against EBV and/or KSHV. Our findings underlined the requirement of appropriate surrogate viruses and cell culture systems for antiviral studies related to EBV and KSHV. We initiated drug-resistance studies with HVS and MHV-68 mutants and provided significant data on the mechanisms of action of new nucleoside analogs and the drug-enzyme interactions of inhibitors of gammaherpesvirus replication.status: publishe

KSHV targeted therapy: An update on inhibitors of viral lytic replication

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.status: publishe

Spectrum of activity and mechanisms of resistance of various nucleoside derivatives against gammaherpesviruses

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-β-d-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase.status: publishe