Dietary flavonoid (-)epicatechin stimulates phosphatidylinositol 3-kinase-dependent anti-oxidant response element activity and up-regulates glutathione in cortical astrocytes

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 mu M) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

Effect of a symbiotic on the response to seasonal influenza vaccination is strongly influenced by degree of immunosenescence

Background Ageing increases risk of respiratory infections and impairs the response to influenza vaccination. Pre- and probiotics offer an opportunity to modulate anti-viral defenses and the response to vaccination via alteration of the gut microbiota. This study investigated the effect of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52486, combined with a prebiotic, gluco-oligosaccharide (B. longum + Gl-OS), on the response to seasonal influenza vaccination in young and older subjects in a double-blind, randomized controlled trial, taking into account the influence of immunosenescence markers at baseline. Results Vaccination resulted in a significant increase in total antibody titres, vaccine-specific IgA, IgM and IgG and seroprotection to all three subunits of the vaccine in both young and older subjects, and in general, the increases in young subjects were greater. There was little effect of the synbiotic, although it tended to reduce seroconversion to the Brisbane subunit of the vaccine and the vaccine-specific IgG response in older subjects. Immunological characterization revealed that older subjects randomized to the synbiotic had a significantly higher number of senescent (CD28-CD57+) helper T cells at baseline compared with those randomized to the placebo, and they also had significantly higher plasma levels of anti-CMV IgG and a greater tendency for CMV seropositivity. Moreover, higher numbers of CD28-CD57+ helper T cells were associated with failure to seroconvert to Brisbane, strongly suggesting that the subjects randomized to the synbiotic were already at a significant disadvantage in terms of likely ability to respond to the vaccine compared with those randomized to the placebo. Conclusions Ageing was associated with marked impairment of the antibody response to influenza vaccination in older subjects and the synbiotic failed to reverse this impairment. However, the older subjects randomized to the synbiotic were at a significant disadvantage due to a greater degree of immunosenscence at baseline compared with those randomized to the placebo. Thus, baseline differences in immunosenescence between the randomized groups are likely to have influenced the outcome of the intervention, highlighting the need for detailed immunological characterization of subjects prior to interventions.

1,8-cineol potentiates IRF3-mediated antiviral response in human stem cells and an ex vivo model of rhinosinusitis

Common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial super-infections resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential of 1,8-cineole for treating primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates Poly(I:C)-induced activity of the anti-viral transcription factor Interferon Regulatory Factor 3, while simultaneously reducing pro-inflammatory NF-κB-activity in human cell lines, inferior turbinate stem cells (ITSCs) and ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with Poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared to Poly(I:C) alone, whereas NF-κB-activity was reduced. Accordingly, 1,8-cineole- and Poly(I:C)-treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared to the Poly(I:C)-treated approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with LPS and 1,8-cineole compared to the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on pro-inflammatory NF-κB-signalling and may thus broaden its field of application.

Immunogenicity of the outer domain of a HIV-1 clade C gp120

Background: The possibility that a sub domain of a C clade HIV-1 gp120 could act as an effective immunogen was investigated. To do this, the outer domain ( OD) of gp120(CN54) was expressed and characterized in a construct marked by a re-introduced conformational epitope for MAb 2G12. The expressed sequence showed efficient epitope retention on the isolated ODCN54 suggesting authentic folding. To facilitate purification and subsequent immunogenicity ODCN54 was fused to the Fc domain of human IgGl. Mice were immunised with the resulting fusion proteins and also with gp120(CN54)-Fc and gp120 alone. Results: Fusion to Fc was found to stimulate antibody titre and Fc tagged ODCN54 was substantially more immunogenic than non-tagged gp120. Immunogenicity appeared the result of Fc facilitated antigen processing as immunisation with an Fc domain mutant that reduced binding to the FcR lead to a reduction in antibody titre when compared to the parental sequence. The breadth of the antibody response was assessed by serum reaction with five overlapping fragments of gp120(CN54) expressed as GST fusion proteins in bacteria. A predominant anti-inner domain and anti-V3C3 response was observed following immunisation with gp120(CN54)-Fc and an anti-V3C3 response to the ODCN54-Fc fusion. Conclusion: The outer domain of gp120(CN54) is correctly folded following expression as a C terminal fusion protein. Immunogenicity is substantial when targeted to antigen presenting cells but shows V3 dominance in the polyvalent response. The gp120 outer domain has potential as a candidate vaccine component.

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

Proteomic biomarkers of peripheral blood mononuclear cells obtained from postmenopausal women undergoing an intervention with soy isoflavones

Background: The incidence of cardiovascular diseases increases after menopause, and soy consumption is suggested to inhibit disease development. Objective: The objective was to identify biomarkers of response to a dietary supplementation with an isoflavone extract in postmenopausal women by proteome analysis of peripheral blood mononuclear cells. Design: The study with healthy postmenopausal woman was performed in a placebo-controlled sequential design. Peripheral mononuclear blood cells were collected from 10 volunteers after 8 wk of receiving daily 2 placebo cereal bars and after a subsequent 8 wk of intervention with 2 cereal bars each providing 25 mg of isoflavones. The proteome of the cells was visualized after 2-dimensional gel electrophoresis, and peptide mass fingerprinting served to identify proteins that by the intervention displayed altered protein concentrations. Results: Twenty-nine proteins were identified that showed significantly altered expression in the mononuclear blood cells under the soy-isoflavone intervention, including a variety of proteins involved in an antiinflammatory response. Heat shock protein 70 or a lymphocyte-specific protein phosphatase and proteins that promote increased fibrinolysis, such as a-enolase, were found at increased intensities, whereas those that mediate adhesion, migration, and proliferation of vascular smooth muscle cells, such as galectin-1, were found at reduced intensities after soy extract consumption. Conclusion: Protcome analysis identified in vivo markers that respond to a dietary intervention with isoflavone-enriched soy extract in postmenopausal women. The nature of the proteins identified suggests that soy isoflavones may increase the anti inflammatory response in blood mononuclear cells that might contribute to the atherosclerosis-preventive activities of a soy-rich diet.

Sesquiterpenoids lactones: benefits to plants and people

Sesquiterpenoids, and specifically sesquiterpene lactones from Asteraceae, may play a highly significant role in human health, both as part of a balanced diet and as pharmaceutical agents, due to their potential for the treatment of cardiovascular disease and cancer. This review highlights the role of sesquiterpene lactones endogenously in the plants that produce them, and explores mechanisms by which they interact in animal and human consumers of these plants. Several mechanisms are proposed for the reduction of inflammation and tumorigenesis at potentially achievable levels in humans. Plants can be classified by their specific array of produced sesquiterpene lactones, showing high levels of translational control. Studies of folk medicines implicate sesquiterpene lactones as the active ingredient in many treatments for other ailments such as diarrhea, burns, influenza, and neurodegradation. In addition to the anti-inflammatory response, sesquiterpene lactones have been found to sensitize tumor cells to conventional drug treatments. This review explores the varied ecological roles of sesquiterpenes in the plant producer, depending upon the plant and the compound. These include allelopathy with other plants, insects, and microbes, thereby causing behavioural or developmental modification to these secondary organisms to the benefit of the sesquiterpenoid producer. Some sesquiterpenoid lactones are antimicrobial, disrupting the cell wall of fungi and invasive bacteria, whereas others protect the plant from environmental stresses that would otherwise cause oxidative damage. Many of the compounds are effective due to their bitter flavor, which has obvious implications for human consumers. The implications of sesquiterpenoid lactone qualitiesfor future crop production are discussed.

Direct experimental evidence that early-life farm environment influences regulation of immune responses

Background: In mammals, early-life environmental variations appear to affect microbial colonization and therefore competent immune development, and exposure to farm environments in infants has been inversely correlated with allergy development. Modelling these effects using manipulation of neonatal rodents is difficult due to their dependency on the mother, but the relatively independent piglet is increasingly identified as a valuable translational model for humans. This study was designed to correlate immune regulation in piglets with early-life environment. Methods: Piglets were nursed by their mother on a commercial farm, while isolatorreared siblings were formula fed. Fluorescence immunohistology was used to quantify T-reg and effector T-cell populations in the intestinal lamina propria and the systemic response to food proteins was quantified by capture ELISA. Results: There was more CD4+ and CD4+CD25+ effector T-cell staining in the intestinal mucosa of the isolator-reared piglets compared with their farm-reared counterparts. In contrast, these isolator-reared piglets had a significantly reduced CD4+CD25+Foxp3+ regulatory T-cell population compared to farm-reared littermates, resulting in a significantly higher T-reg-to-effector ratio in the farm animals. Consistent with these findings, isolator-reared piglets had an increased serum IgG anti-soya response to novel dietary soya protein relative to farm-reared piglets. Conclusion: Here, we provide the first direct evidence, derived from intervention, that components of the early-life environment present on farms profoundly affects both local development of regulatory components of the mucosal immune system and immune responses to food proteins at weaning. We propose that neonatal piglets provide a tractable model which allows maternal and treatment effects to be statistically separated.

Endocannabinoids, FOXO and the metabolic syndrome: redox, function and tipping point--the view from two systems

The endocannabinoid system (ECS) was only 'discovered' in the 1990s. Since then, many new ligands have been identified, as well as many new intracellular targets--ranging from the PPARs, to mitochondria, to lipid rafts. It was thought that blocking the CB-1 receptor might reverse obesity and the metabolic syndrome. This was based on the idea that the ECS was dysfunctional in these conditions. This has met with limited success. The reason may be that the ECS is a homeostatic system, which integrates energy seeking and storage behaviour with resistance to oxidative stress. It could be viewed as having thrifty actions. Thriftiness is an innate property of life, which is programmed to a set point by both environment and genetics, resulting in an epigenotype perfectly adapted to its environment. This thrifty set point can be modulated by hormetic stimuli, such as exercise, cold and plant micronutrients. We have proposed that the physiological and protective insulin resistance that underlies thriftiness encapsulates something called 'redox thriftiness', whereby insulin resistance is determined by the ability to resist oxidative stress. Modern man has removed most hormetic stimuli and replaced them with a calorific sedentary lifestyle, leading to increased risk of metabolic inflexibility. We suggest that there is a tipping point where lipotoxicity in adipose and hepatic cells induces mild inflammation, which switches thrifty insulin resistance to inflammation-driven insulin resistance. To understand this, we propose that the metabolic syndrome could be seen from the viewpoint of the ECS, the mitochondrion and the FOXO group of transcription factors. FOXO has many thrifty actions, including increasing insulin resistance and appetite, suppressing oxidative stress and shifting the organism towards using fatty acids. In concert with factors such as PGC-1, they also modify mitochondrial function and biogenesis. Hence, the ECS and FOXO may interact at many points; one of which may be via intracellular redox signalling. As cannabinoids have been shown to modulate reactive oxygen species production, it is possible that they can upregulate anti-oxidant defences. This suggests they may have an 'endohormetic' signalling function. The tipping point into the metabolic syndrome may be the result of a chronic lack of hormetic stimuli (in particular, physical activity), and thus, stimulus for PGC-1, with a resultant reduction in mitochondrial function and a reduced lipid capacitance. This, in the context of a positive calorie environment, will result in increased visceral adipose tissue volume, abnormal ectopic fat content and systemic inflammation. This would worsen the inflammatory-driven pathological insulin resistance and inability to deal with lipids. The resultant oxidative stress may therefore drive a compensatory anti-oxidative response epitomised by the ECS and FOXO. Thus, although blocking the ECS (e.g. via rimonabant) may induce temporary weight loss, it may compromise long-term stress resistance. Clues about how to modulate the system more safely are emerging from observations that some polyphenols, such as resveratrol and possibly, some phytocannabinoids, can modulate mitochondrial function and might improve resistance to a modern lifestyle.

A new understanding of El Niño's impact over East Asia: dominance of the ENSO combination mode

Previous studies have shown that the Indo-Pacific atmospheric response to ENSO comprises two dominant modes of variability: a meridionally quasi-symmetric response (independent from the annual cycle) and an anti-symmetric response (arising from the nonlinear atmospheric interaction between ENSO variability and the annual cycle), referred to as the combination mode (C-Mode). This study demonstrates that the direct El Niño signal over the tropics is confined to the equatorial region and has no significant impact on the atmospheric response over East Asia. The El Niño-associated equatorial anomalies can be expanded towards off-equatorial regions by the C-Mode through ENSO’s interaction with the annual cycle. The C-Mode is the prime driver for the development of an anomalous low-level anticyclone over the western North Pacific (WNP) during the El Niño decay phase, which usually transports more moisture to East Asia and thereby causes more precipitation over southern China. We use an Atmospheric General Circulation Model that well reproduces the WNP anticyclonic anomalies when both El Niño sea surface temperature (SST) anomalies as well as the SST annual cycle are prescribed as boundary conditions. However, no significant WNP anticyclonic circulation anomaly appears during the El Niño decay phase when excluding the SST annual cycle. Our analyses of observational data and model experiments suggest that the annual cycle plays a key role in the East Asian climate anomalies associated with El Niño through their nonlinear atmospheric interaction. Hence, a realistic simulation of the annual cycle is crucial in order to correctly capture the ENSO-associated climate anomalies over East Asia.

Modulation of potassium channels inhibits bunyavirus infection

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

The effect of highly active antiretroviral therapy for HIV on the anti-HCV specific humoral immune response

The effect of highly active antiretroviral therapy (HAART) on HCV replication is controversial, with some studies reporting no effect and others increases, reductions and even clearances of HCV RNA after treatment. In this study, the effect of HAART was investigated on the titre of anti-HCV specific antibodies and on the relationship between these antibodies and HCV RNA level in a cohort of 24 patients with inherited bleeding disorders. A significant inverse correlation between antibodies to both total HCV proteins and HCV RNA (R = -0.42, P = 0.05) and between antibodies to HCV envelope glycoproteins and HCV RNA (R = -0.54, P = 0.01) was observed pre-HAART. The relationship disappeared or was obscured after therapy (R = 0.24, P = 0.30 and R = 0.16, P = 0.50, respectively). Thus, we show that HAART affects the HCV specific humoral immune responses without affecting the HCV RNA level. (C) 2004 Wiley-Liss, Inc.

Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.