Recurrent herpes simplex virus-1 infection induces hallmarks of neurodegeneration and cognitive deficits in mice.

Herpes simplex virus type 1 (HSV-1) is a DNA neurotropic virus, usually establishing latent infections in the trigeminal ganglia followed by periodic reactivations. Although numerous findings suggested potential links between HSV-1 and Alzheimer's disease (AD), a causal relation has not been demonstrated yet. Hence, we set up a model of recurrent HSV-1 infection in mice undergoing repeated cycles of viral reactivation. By virological and molecular analyses we found: i) HSV-1 spreading and replication in different brain regions after thermal stress-induced virus reactivations; ii) accumulation of AD hallmarks including amyloid-ß protein, tau hyperphosphorylation, and neuroinflammation markers (astrogliosis, IL-1ß and IL-6). Remarkably, the progressive accumulation of AD molecular biomarkers in neocortex and hippocampus of HSV-1 infected mice, triggered by repeated virus reactivations, correlated with increasing cognitive deficits becoming irreversible after seven cycles of reactivation. Collectively, our findings provide evidence that mild and recurrent HSV-1 infections in the central nervous system produce an AD-like phenotype and suggest that they are a risk factor for AD.

Glutathione increase by the n-butanoyl glutathione derivative (GSH-C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus.

During aging, glutathione (GSH) content declines and the immune system undergoes a deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is associated with GSH depletion, could weaken the host defenses against viral infections. We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the effect of the administration of the N-butanoyl GSH derivative (GSH-C4) on the response of aged mice infected with influenza A PR8/H1N1 virus was studied through the determination of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production, and Th1/Th2 cytokine profile. Old mice had lower GSH than young mice in organs. Also the gene expression of endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of proteins, that is, Nrf2 and PDI, was reduced. Following infection, GSH content remained low and neither infection nor GSH-C4 treatment affected Nrf2 expression. In contrast, PDI expression was upregulated during infection and appeared counterbalanced by GSH-C4. Moreover, the treatment with GSH-C4 increased GSH content in organs, reduced viral replication and induced a predominant Th1 response. In conclusion, GSH-C4 treatment could be used in the elderly to contrast influenza virus infection by inducing immune response, in particular the Th1 profile.

Differential Redox State Contributes to Sex Disparities in the Response to Influenza Virus Infection in Male and Female Mice.

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza.

Antiviral and Antioxidant Activity of a Hydroalcoholic Extract from Humulus lupulus L.

A hydroalcoholic extract from female inflorescences of Humulus lupulus L. (HOP extract) was evaluated for its anti-influenza activity. The ability of the extract to interfere with different phases of viral replication was assessed, as well as its effect on the intracellular redox state, being unbalanced versus the oxidative state in infected cells. The radical scavenging power, inhibition of lipoperoxidation, and ferric reducing activity were assayed as antioxidant mechanisms. A phytochemical characterization of the extract was also performed. We found that HOP extract significantly inhibited replication of various viral strains, at different time from infection. Viral replication was partly inhibited when virus was incubated with extract before infection, suggesting a direct effect on the virions. Since HOP extract was able to restore the reducing conditions of infected cells, by increasing glutathione content, its antiviral activity might be also due to an interference with redox-sensitive pathways required for viral replication. Accordingly, the extract exerted radical scavenging and reducing effects and inhibited lipoperoxidation and the tBOOH-induced cytotoxicity. At phytochemical analysis, different phenolics were identified, which altogether might contribute to HOP antiviral effect. In conclusion, our results highlighted anti-influenza and antioxidant properties of HOP extract, which encourage further in vivo studies to evaluate its possible application.

Regioselective IBX-Mediated Synthesis of Coumarin Derivatives with Antioxidant and Anti-influenza Activities.

Different catechol and pyrogallol derivatives have been synthesized by oxidation of coumarins with 2-iodoxybenzoic acid (IBX) in DMSO at 25 °C. A high regioselectivity was observed in accordance with the stability order of the incipient carbocation or radical benzylic-like intermediate. The oxidation was also effective in water under heterogeneous conditions by using IBX supported on polystyrene. The new derivatives showed improved antioxidant effects in the DPPH test and inhibitory activity against the influenza A/PR8/H1N1 virus. These data represent a new entry for highly oxidized coumarins showing an antiviral activity possibly based on the control of the intracellular redox value.

Glutathione Fine-Tunes the Innate Immune Response toward Antiviral Pathways in a Macrophage Cell Line Independently of Its Antioxidant Properties.

Glutathione (GSH), a major cellular antioxidant, is considered an inhibitor of the inflammatory response involving reactive oxygen species (ROS). However, evidence is largely based on experiments with exogenously added antioxidants/reducing agents or pro-oxidants. We show that depleting macrophages of 99% of GSH does not exacerbate the inflammatory gene expression profile in the RAW264 macrophage cell line or increase expression of inflammatory cytokines in response to the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS); only two small patterns of LPS-induced genes were sensitive to GSH depletion. One group, mapping to innate immunity and antiviral responses (Oas2, Oas3, Mx2, Irf7, Irf9, STAT1, il1b), required GSH for optimal induction. Consequently, GSH depletion prevented the LPS-induced activation of antiviral response and its inhibition of influenza virus infection. LPS induction of a second group of genes (Prdx1, Srxn1, Hmox1, GSH synthase, cysteine transporters), mapping to nrf2 and the oxidative stress response, was increased by GSH depletion. We conclude that the main function of endogenous GSH is not to limit inflammation but to fine-tune the innate immune response to infection.

MC1568 inhibits HDAC6/8 activity and influenza A virus replication in lung epithelial cells: role of Hsp90 acetylation.

AIM: Histone deacetylases (HDACs) regulate the life cycle of several viruses. We investigated the ability of different HDAC inhibitors, to interfere with influenza virus A/Puerto Rico/8/34/H1N1 (PR8 virus) replication in Madin-Darby canine kidney and NCI cells. RESULTS: 3-(5-(3-Fluorophenyl)-3-oxoprop-1-en-1-yl)-1-methyl-1H-pyrrol-2-yl)-N-hydroxyacrylamide (MC1568) inhibited HDAC6/8 activity and PR8 virus replication, with decreased expression of viral proteins and their mRNAs. Such an effect may be related to a decrease in intranuclear content of viral polymerases and, in turn, to an early acetylation of Hsp90, a major player in their nuclear import. Later, the virus itself induced Hsp90 acetylation, suggesting a differential and time-dependent role of acetylated proteins in virus replication. CONCLUSION: The inhibition of HDAC6/8 activity during early steps of PR8 virus replication could lead to novel anti-influenza strategy.

Validation of a Reversed-Phase High Performance Liquid Chromatography Method for the Simultaneous Analysis of Cysteine and Reduced Glutathione in Mouse Organs.

A depletion of reduced glutathione (GSH) has been observed in pathological conditions and in aging. Measuring GSH in tissues using mouse models is an excellent way to assess GSH depletion and the potential therapeutic efficacy of drugs used to maintain and/or restore cellular redox potential. A high performance liquid chromatography (HPLC) method for the simultaneous determination of GSH and cysteine (Cys) in mouse organs was validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and precolumn derivatization with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The required validation parameters, that are, selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, and stability, were studied for spleen, lymph nodes, pancreas, and brain. The results showed that the lower limits of quantification were 0.313 µM and 1.25 µM for Cys and GSH, respectively. Intraday and interday precisions were less than 11% and 14%, respectively, for both compounds. The mean extraction recoveries of Cys and GSH from all organs were more than 93% and 86%, respectively. Moreover, the stability of both analytes during sample preparation and storage was demonstrated. The method was accurate, reliable, consistent, and reproducible and it was useful to determine Cys and GSH in the organs of different mouse strains.

Herpes simplex virus type 1 infection in neurons leads to production and nuclear localization of APP intracellular domain (AICD): implications for Alzheimer's disease pathogenesis.

Several data indicate that neuronal infection with herpes simplex virus type 1 (HSV-1) causes biochemical alterations reminiscent of Alzheimer's disease (AD) phenotype. They include accumulation of amyloid-ß (Aß), which originates from the cleavage of amyloid precursor protein (APP), and hyperphosphorylation of tau protein, which leads to neurofibrillary tangle deposition. HSV-1 infection triggers APP processing and drives the production of several fragments including APP intracellular domain (AICD) that exerts transactivating properties. Herein, we analyzed the production and intracellular localization of AICD following HSV-1 infection in neurons. We also checked whether AICD induced the transcription of two target genes, neprilysin (nep) and glycogen synthase kinase 3ß (gsk3ß), whose products play a role in Aß clearance and tau phosphorylation, respectively. Our data indicate that HSV-1 led to the accumulation and nuclear translocation of AICD in neurons. Moreover, results from chromatin immunoprecipitation assay showed that AICD binds the promoter region of both nep and gsk3ß. Time course analysis of NEP and GSK3ß expression at both mRNA and protein levels demonstrated that they are differently modulated during infection. NEP expression and enzymatic activity were initially stimulated but, with the progression of infection, they were down-regulated. In contrast, GSK3ß expression remained nearly unchanged, but the analysis of its phosphorylation suggests that it was inactivated only at later stages of HSV-1 infection. Thus, our data demonstrate that HSV-1 infection induces early upstream events in the cell that may eventually lead to Aß deposition and tau hyperphosphorylation and further suggest HSV-1 as a possible risk factor for AD.

Influenza A virus infection of intestinal epithelial cells enhances the adhesion ability of Crohn's disease associated Escherichia coli strains.

Modifications of intestinal glycoreceptors expression, in particular CEACAM6, typically found in ileal Crohn's disease (CD), favor, among the commensal species of microbiota, the enrichment in Escherichia coli. Removal of protein glycosidic residues by neuraminidase, a sialidase typical of influenza virus, increases adhesion ability of Escherichia coli to Caco-2 intestinal cells. In this study we investigated whether influenza virus infection of human intestinal epithelial cells could influence the adhesiveness of different Escherichia coli strains isolated from CD patients by altering surface glycoreceptors. Influenza virus infection of intestinal cells increased exposure of galactose and mannose residues on the cell surface. In particular, glycoreceptors Thomsen-Friedenreich and CEACAM6 were over-expressed in influenza virus infected cells. In the same experimental conditions, a significant increase in bacterial adhesiveness was observed, independently of their own adhesive ability. The increase was reverted by treatment with anti-TF and anti-CEACAM6 antibodies. Interestingly, influenza virus was able to efficiently replicate in human primary intestinal cells leading to TF exposure. Finally, intestinal infected cells produced high levels of pro-inflammatory cytokines compared to control. Overall these data suggest that influenza virus infection, could constitute an additional risk factor in CD patients.

Intracellular redox state as target for anti-influenza therapy: are antioxidants always effective?

Influenza virus infections represent a big issue for public health since effective treatments are still lacking. In particular, the emergence of strains resistant to drugs limits the effectiveness of anti-influenza agents. For this reason, many efforts have been dedicated to the identification of new therapeutic strategies aimed at targeting the virus-host cell interactions. Oxidative stress is a characteristic of some viral infections including influenza. Because antioxidants defend cells from damage caused by reactive oxygen species induced by different stimuli including pathogens, they represent interesting molecules to fight infectious diseases. However, most of the available studies have found that these would-be panaceas could actually exacerbate the diseases they claim to prevent, and have thus revealed "the dark side" of these molecules. This review article discusses the latest opportunities and drawbacks of the antioxidants used in anti-influenza therapy and new perspectives.

Characterization of biofilms in drug-sensitive and drug-resistant strains of Candida albicans.

In this study, we investigated the biofilm formation in strains of Candida albicans susceptible (CO23) or resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). The effect of drug resistance on biofilm formation was investigated through the cell surface hydrophobicity and the mannan content. Moreover, biofilm formation was evaluated after 24, 48 and 72 hours with crystal violet assay, dry weight, as well as scanning electron microscopy. Our results showed an increase in hydrophobicity, polysaccharides content, metabolic activity and dry weight. Observation of sensitive and resistant strains confirmed the differences in cell morphology. Finally, the expression of genes involved in biofilm formation, such as HWP1 and EFG1, evaluated with relative real-time RT-PCR. Resistant strains proved to up- regulate the expression of HWP1. These results demonstrated the existence of important differences between drug-susceptible and drug-resistant strains biofilm of C. albicans.

The Environmental Pollutant Cadmium Promotes Influenza Virus Replication in MDCK Cells by Altering Their Redox State.

Cadmium (Cd) is a toxic heavy metal that is considered an environmental contaminant. Several sources of human exposure to Cd, including employment in primary metal industries, production of certain batteries, foods, soil and cigarette smoke, are known. Its inhalation has been related to different respiratory diseases and toxic effects, among which alterations of the physiological redox state in individuals exposed to the metal have been described. Host-cell redox changes characteristic of oxidative stress facilitate the progression of viral infection through different mechanisms. In this paper, we have demonstrated that pre-treatment with CdCl(2) of MDCK cells increased influenza virus replication in a dose-dependent manner. This phenomenon was related to increased viral protein expression (about 40% compared with untreated cells). The concentration of CdCl(2), able to raise the virus titer, also induced oxidative stress. The addition of two antioxidants, a glutathione (GSH) derivative or the GSH precursor, N-acetyl-L-cysteine, to Cd pre-treated and infected cells restored the intracellular redox state and significantly inhibited viral replication. In conclusion, our data demonstrate that Cd-induced oxidative stress directly increases the ability of influenza virus to replicate in the host-cell, thus suggesting that exposure to heavy metals, such as this, could be a risk factor for individuals exposed to a greater extent to the contaminant, resulting in increased severity of virus-induced respiratory diseases.

Synthesis of 2'-deoxy-1'-homo-N-nucleosides with anti-influenza activity by catalytic methyltrioxorhenium (MTO)/H2O2 oxyfunctionalization.

This paper describes a new route for the synthesis of 1'-homo-N-nucleoside derivatives by means of either methyltrioxorhenium (MTO) or supported MTO catalysts, with H(2)O(2) as the primary oxidant. Under these selective conditions, the oxyfunctionalization of the heterocyclic ring and the N heteroatom oxidation were operative processes, regardless of the type of substrate used, that is, purine or pyrimidine derivatives. In addition, the oxidation of 1'-homo-N-thionucleosides, showed the occurrence of site-specific oxidative nucleophilic substitutions of the heterocyclic ring. The MTO/H(2)O(2) system showed, in general, high reactivity under both homogeneous and heterogeneous conditions, affording the final products with high conversion values of substrates and from medium to high yields. Many of the novel 1'-homo-N-nucleoside analogues were active against the influenza A virus, without any cytotoxic effects, retaining their activity in both protected and unprotected forms.

Sex differences in the response to viral infections: TLR8 and TLR9 ligand stimulation induce higher IL10 production in males.

BACKGROUND: Susceptibility to viral infections as well as their severity are higher in men than in women. Heightened antiviral responses typical of women are effective for rapid virus clearance, but if excessively high or prolonged, can result in chronic/inflammatory pathologies. We investigated whether this variability could be in part attributable to differences in the response to the Toll-Like Receptors (TLR) more involved in the virus recognition. METHODS: Cytokine production by peripheral blood mononuclear cells (PBMCs) from male and female healthy donors after stimulation with Toll-like receptors (TLR) 3, 7, 8, 9 ligands or with viruses (influenza and Herpes-simplex-1) was evaluated. RESULTS: Compared to females, PBMCs from males produced not only lower amounts of IFN-α in response to TLR7 ligands but also higher amounts of the immunosuppressive cytokine IL10 after stimulation with TLR8 and TLR9 ligands or viruses. IL10 production after TLR9 ligands or HSV-1 stimulation was significantly related with plasma levels of sex hormones in both groups, whereas no correlation was found in cytokines produced following TLR7 and TLR8 stimulation. CONCLUSIONS: Given the role of an early production of IL10 by cells of innate immunity in modulating innate and adaptive immune response to viruses, we suggest that sex-related difference in its production following viral nucleic acid stimulation of TLRs may be involved in the sex-related variability in response to viral infections.

Effects of polyphenol compounds on influenza A virus replication and definition of their mechanism of action.

A set of polyphenol compounds was synthesized and assayed for their ability in inhibiting influenza A virus replication. A sub-set of them showed low toxicity. The best compounds within this sub-set were 4 and 6g, which inhibited the viral replication in a dose-dependent manner. The antiviral activity of these molecules was demonstrated to be caused by their interference with intracellular pathways exploited for viral replication: (1) MAP kinases controlling nuclear-cytoplasmic traffic of viral ribonucleoprotein complex; (2) redox-sensitive pathways, involved in maturation of viral hemagglutinin protein.

Intracellular redox signaling as therapeutic target for novel antiviral strategy.

Reactive oxygen and nitrogen species play complex roles in the physiological regulation of cell metabolism and in many disease processes as well, including viral infections. Viral replication occurs within living cells and is totally dependent on its host's biosynthetic machinery. Many intracellular signaling pathways exploited by viruses for their own replication are regulated by the oxidoreductive (redox) state of the host cell. Consequently, factors that alter the balance between reactive oxygen/nitrogen species and antioxidant molecules/enzymes-including metabolic conditions like malnutrition, obesity, and diabetes-can influence cells' susceptibility to viral infection, the efficiency of viral replication, and as a result the progression and severity of virus-induced diseases. This review examines the ways in which the host-cell redox state affect viral replication and the actual potential of antioxidants to combat viral infections.

Lipooligosaccharide from Bordetella pertussis induces mature human monocyte-derived dendritic cells and drives a Th2 biased response.

Bordetella pertussis has a distinctive cell wall lipooligosaccharide (LOS) that is released from the bacterium during bacterial division and killing. LOS directly participates in host-bacterial interactions, in particular influencing the dendritic cells' (DC) immune regulatory ability. We analyze LOS mediated toll-like receptor (TLR) activation and dissect the role played by LOS on human monocyte-derived (MD)DC functions and polarization of the host T cell response. LOS activates TLR4-dependent signaling and induces mature MDDC able to secrete IL-10. LOS-matured MDDC enhance allogeneic presentation and skew T helper (Th) cell polarization towards a Th2 phenotype. LOS protects MDDC from undergoing apoptosis, prolonging their longevity and their functions. Compared to Escherichia coli lipopolysaccharide (LPS), the classical DC maturation stimulus, LOS was a less efficient inducer of TLR4 signaling, MDDC maturation, IL-10 secretion and allogeneic T cell proliferation and it was not able to induce IL-12p70 production in MDDC. However, the MDDC apoptosis protection exerted by LOS and LPS were comparable. In conclusion, LOS treated MDDC are able to perform antigen presentation in a context that promotes licensing of Th2 effectors. Considering these properties, the use of LOS in the formulation of acellular pertussis vaccines to potentiate protective and adjuvant capacity should be taken into consideration.