The Role of TLR4 in the Paclitaxel Effects on Neuronal Growth In Vitro

Paclitaxel (Pac) is an antitumor agent that is widely used for treatment of solid cancers. While being effective as a chemotherapeutic agent, Pac in high doses is neurotoxic, specifically targeting sensory innervations. In view of these toxic effects associated with conventional chemotherapy, decreasing the dose of Pac has been recently suggested as an alternative approach, which might limit neurotoxicity and immunosuppression. However, it remains unclear if low doses of Pac retain its neurotoxic properties or might exhibit unusual effects on neuronal cells. The goal of this study was to analyze the concentration-dependent effect of Pac on isolated and cultured DRG neuronal cells from wild-type and TLR4 knockout mice. Three different morphological parameters were analyzed: the number of neurons which developed neurites, the number of neurites per cell and the total length of neurites per cell. Our data demonstrate that low concentrations of Pac (0.1 nM and 0.5 nM) do not influence the neuronal growth in cultures in both wild type and TLR4 knockout mice. Higher concentrations of Pac (1-100 nM) had a significant effect on DRG neurons from wild type mice, affecting the number of neurons which developed neurites, number of neurites per cell, and the length of neurites. In DRG from TLR4 knockout mice high concentrations of Pac showed a similar effect on the number of neurons which developed neurites and the length of neurites. At the same time, the number of neurites per cell, indicating the process of growth cone initiation, was not affected by high concentrations of Pac. Thus, our data showed that Pac in high concentrations has a significant damaging effect on axonal growth and that this effect is partially mediated through TLR4 pathways. Low doses of Pac are devoid of neuronal toxicity and thus can be safely used in a chemomodulation mode. © 2013 Ustinova et al

Fenofibrate Reduces Mortality and Precludes Neurological Deficits in Survivors in Murine Model of Japanese Encephalitis Viral Infection

Background: Japanese encephalitis (JE), the most common form of viral encephalitis occurs periodically in endemic areas leading to high mortality and neurological deficits in survivors. It is caused by a flavivirus, Japanese encephalitis virus (JEV), which is transmitted to humans through mosquitoes. No effective cure exists for reducing mortality and morbidity caused by JEV infection, which is primarily due to excessive inflammatory response. Fenofibrate, a peroxisome proliferator-activated receptor-a (PPARa) agonist is known to resolve inflammation by repressing nuclear factor-kB (NF-kB) and enhancing transcription of anti-oxidant and anti-inflammatory genes. In addition, fenofibrate also up-regulates a class of proteins, cytochrome P4504Fs (Cyp4fs), which are involved in detoxification of the potent pro-inflammatory eicosanoid, leukotriene B4 (LTB4) to 20-hydroxy LTB4. Methodology/Principal Findings: The neuroprotective effect of fenofibrate was examined using in vitro (BV-2 microglial cell line) and in vivo (BALB/c mice) models of JEV infection. Mice were treated with fenofibrate for 2 or 4 days prior to JEV exposure. Pretreatment with fenofibrate for 4 but not 2 days reduced mortality by 80 % and brain LTB4 levels decreased concomitantly with the induction of Cyp4f15 and 4f18, which catalyze detoxification of LTB4 through hydroxylation. Expression of cytokines and chemokine decreased significantly as did microglial activation and replication of the JEV virus. Conclusions/Significance: Fenofibrate confers neuroprotection against Japanese encephalitis, in vivo, in mouse model o

Tannic Acid Modified Silver Nanoparticles Show Antiviral Activity in Herpes Simplex Virus Type 2 Infection

The interaction between silver nanoparticles and herpesviruses is attracting great interest due to their antiviral activity and possibility to use as microbicides for oral and anogenital herpes. In this work, we demonstrate that tannic acid modified silver nanoparticles sized 13 nm, 33 nm and 46 nm are capable of reducing HSV-2 infectivity both in vitro and in vivo. The antiviral activity of tannic acid modified silver nanoparticles was size-related, required direct interaction and blocked virus attachment, penetration and further spread. All tested tannic acid modified silver nanoparticles reduced both infection and inflammatory reaction in the mouse model of HSV-2 infection when used at infection or for a post-infection treatment. Smaller-sized nanoparticles induced production of cytokines and chemokines important for anti-viral response. The corresponding control buffers with tannic acid showed inferior antiviral effects in vitro and were ineffective in blocking in vivo infection. Our results show that tannic acid modified silver nanoparticles are good candidates for microbicides used in treatment of herpesvirus infections.This work was supported by the Polish National Science Centre grant No. 2011/03/B/NZ6/04878 (for MK) and Centre for Preclinical Research and Technology (CePT) Project No. POIG.02.02.00-14-024/08-0 (for MG and MD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Herpes simplex virus and the lexicon of latency and reactivation: a call for defining terms and building an integrated collective framework [version 1; referees: 2 approved]

The field of herpes simplex virus (HSV) latency and reactivation has been marked by controversy, which is not unexpected considering the complexities of the biology involved. While controversy is an important tool for digging to the bottom of difficult issues, we propose that unproductive conflict in the field arises in part from poorly defined terminology and the need for a collective framework. The uses of advanced global molecular and next-generation sequencing approaches and an increasing array of in vitro model systems have provided new molecular-level insights into HSV latency and reactivation, with the promise of expanding our concepts of these processes. However, our current framework and language are inadequate to effectively integrate new data streams into the established theories. In this brief perspective, we look back into the past to examine when and how the lexicon of HSV latency and reactivation arose in the literature and its evolution. We propose to open a dialogue among investigators for the purpose of updating and clearly defining terms used to describe these processes and to build a collective integrated framework to move our field forward

De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia

<div><p>The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system.</p></div

The VP16 promoter contains a region required for efficient exit from the default latent state.

<p>Mice were infected on scarified corneas with 2x10⁵ pfu of (A) 17LATpLacZ or (B) 17VP16πRR+LATpLacZ. At 44 hrs pi TG were harvested and processed for whole ganglion histochemical detection of b-gal activity (blue) followed by immunohistochemical detection of viral proteins (brown) as detailed in methods. Each bar represents the number of positive neurons in a TG. Blue vertical bars are neurons positive for only b-gal, brown bars are those positive for only viral proteins, and blue and brown checkered bars are neurons positive for both. There were significantly more neurons containing viral proteins in 17LATpLacZ infected group, about 33% of the total labeled neurons vs. 2% of the total in the 17VP16πRR+LATpLacZ infected group (p<0.0001, Student’s t-test).</p

Quantification of the number of neurons expressing the lytic and/or the latent transcriptional program markers.

<p>A) Quantification of neurons positive for viral protein at 40 hrs pi. This experiment included 17LATpVP16R, the genomic rescue of 17LATpVP16, as well as 17LATpICP0 and two mutants made on the LAT null background parent 17AH (17AHLATpLacZ and 17AHLATpICP0). There was no significant difference in the number of neurons positive for viral proteins [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref011" target="_blank">11</a>] in whole TG except for the TG infected with 17LATpVP16, which contained significantly more positive neurons (p<0.0004, Fisher’s exact test). B) Quantification of neurons positive for viral protein, LATp activity, or both at 46 hrs pi. Each point represents the number of positive neurons in a TG. C) TG were fixed and processed for the simultaneous detection of b-gal activity (blue neurons:red arrows) and viral protein expression (brown neurons:black arrows). Shown are representative photomicrographs of whole ganglia at the same magnification. The number of neurons in TG expressing either viral proteins (lytic), LacZ from the LAT promoter (“latent”), or both (lytic+“latent”) were enumerated (shown in B).</p

Entry into lytic infection in TG neurons is regulated by sequences in the VP16 promoter.

<p>A) Sequence of the proximal VP16 promoter. Putative regulatory sites identified previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref083" target="_blank">83</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref084" target="_blank">84</a>] are color coded and labeled above the sequence. Potential sites known to confer reciprocal regulation to neuronal genes are underlined and in red. The boxed nucleotides were altered as detailed in methods. B) Replication kinetics on eyes and in TG on days 2 through 10 pi is shown as the area under the curve (AUC). Tissues from 3 mice were examined for each time point. 1 = 17syn+, 2a, b, c are three independently derived isolates of the VP16 promoter mutant 17VP16pπRR and 3 = the genomically restored isolate 17VP16pπRR-R. C) Mice were infected on the cornea with the VP16 promoter mutant 17VP16pπRR or its genomically restored isolate 17VP16pπRR-R. At 72 hrs pi tissues were harvested and processed for the whole tissue immunohistochemical detection of VP16 and the number of VP16 positive neurons enumerated. The data are shown as a scattergram with each point representing the number of positive neurons in an individual TG. The difference between the groups was significant (p<0.0001, Student’s t-test). D) Representative photomicrographs of corneas stained for VP16 protein which is seen as a brown precipitate in characteristic lesions on the corneal surface (arrows). E) Representative photomicrographs of sectioned trigeminal ganglia infected with 17VP16pπRR-R stained immunohistochemically for VP16 (purple precipitate). Numerous cells including neurons identifiable by their large size, morphology and axonal tracts are positive in the TG infected with 17VP16pπRR-R the genomically restored isolate which is not different than WT 17syn+ (white arrows). F) Rare VP16 positive cells in the 17VP16pπRR infected TG were detected and did not appear to be neurons. One such area is boxed and shown enlarged in an inset micrograph. This region appears to be a small focus of positive support cells.</p

Quantification of viral lytic and acute stage latency gene transcription in individual neurons at very early times pi.

<p>A. Relative promoter strength was assayed by transfection of rabbit skin cells with the constructs employed to generate the viral promoter/reporter mutants and analysis of the amount of b-gal determined with a CPRG assay as detailed in methods. Transfection efficiencies were normalized by including a renilla luciferase plasmid in the assay. Lane 1 = LATpLacZ, 2 = VP16pLacZ, 3 = ICP0pLacZ. The LAT promoter was the weakest and the levels of expression of this construct were set to one. Where indicated, a VP16 expressing plasmid was included in the transfections. Each bar represents the average of 3 transfection experiments of 3 wells each. B. Photomicrographs of TG from mice infected with17LATpLacZ processed for LATp activity and viral protein expression. At 22 hrs pi, expression was restricted to the LATp. A 36 hrs, viral proteins are expressed but restricted to a subset of neurons that are marked by LATp activity. C. At the indicated times pi, eyes and TG were harvested. Unsectioned TG were processed for the histochemical detection of b-gal activity and IHC detection of viral proteins as detailed previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref004" target="_blank">4</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref011" target="_blank">11</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref048" target="_blank">48</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref054" target="_blank">54</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref078" target="_blank">78</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref081" target="_blank">81</a>]. Bars represent the number of neurons positive in an individual TG. The number of TG positive over the number tested in each group is indicated. All TG tested from mice infected with 17LATpLacZ contained one or more neurons positive for b-gal activity at 22 hrs pi, whereas no TG infected with the lytic stage promoter reporter viruses were positive (p≤0.0001, ANOVA). At 36 hrs pi a subset of ganglia examined contained neurons positive for both b-gal and viral protein. The number of ganglia positive and the number of positive neurons in the TG was not different between groups (p≥0.5, ANOVA). Importantly, all neurons positive for viral protein were also positive for LAT promoter activity in the 17LATpLacZ group.</p