Activation of a G protein-coupled receptor by its endogenous ligand triggers the innate immune response of Caenorhabditis elegans.

Immune defenses are triggered by microbe-associated molecular patterns or as a result of damage to host cells. The elicitors of immune responses in the nematode Caenorhabditis elegans are unclear. Using a genome-wide RNA-mediated interference (RNAi) screen, we identified the G protein-coupled receptor (GPCR) DCAR-1 as being required for the response to fungal infection and wounding. DCAR-1 acted in the epidermis to regulate the expression of antimicrobial peptides via a conserved p38 mitogen-activated protein kinase pathway. Through targeted metabolomics analysis we identified the tyrosine derivative 4-hydroxyphenyllactic acid (HPLA) as an endogenous ligand. Our findings reveal DCAR-1 and its cognate ligand HPLA to be triggers of the epidermal innate immune response in C. elegans and highlight the ancient role of GPCRs in host defense.

Reactive oxygen species metabolism and phenylpropanoid pathway involved in disease resistance against Penicillium expansum in apple fruit induced by ϵ-poly-l-lysine.

BACKGROUND: Blue mould caused by Penicillium expansum comprises a notable disease of apple fruit during storage. ϵ-Poly-l-lysine (PL) consists of ϵ-amino and α-hydroxyl and has been used in food preservation. In the present study, apple fruits (cv. Fuji) were used to investigate the effects of PL dipping treatment, at different concentrations of PL, on the lesion diameter of fruit inoculated with P. expansum, aiming to screen the optimal concentration for controlling blue mould. The effects of PL at the optimal concentration on reactive oxygen species (ROS) metabolism and the phenylpropanoid pathway were also investigated. RESULTS: The results indicated that 25, 50, 100 and 200 µL L-1 PL treatment significantly decreased the lesion diameter in apple fruit inoculated with P. expansum and the smallest lesion diameter was determined for 50 µL L-1 PL-treated fruits. The results also indicated that 50 µL L-1 PL treatment increased the hydrogen peroxide content and the activities of enzymes involved in ROS metabolism, including superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and peroxidase in apple fruit. The activity of phenylalanine ammonia-lyase and the contents of lignin, total phenolic compounds and flavonoids were also enhanced by PL treatment. CONCLUSION: The disease resistance to P. expansum in apple fruits enhanced by PL treatment is related to activating ROS metabolism and the phenylpropanoid pathway and the accumulation of antifungal compounds. © 2018 Society of Chemical Industry.

Exogenous nitric oxide induces disease resistance against Monilinia fructicola through activating the phenylpropanoid pathway in peach fruit.

BACKGROUND: Nitric oxide (NO) is a multifunctional signaling molecule involved in plant-induced resistance to disease. The present study aimed to investigate the relationship between disease resistance induced by NO and the phenylpropanoid pathway in peach fruit. The present study investigated the effect of NO on the main enzymes and metabolites of the phenylpropanoid pathway of harvested peach, which are probably related to disease resistance against Monilinia fructicola. RESULTS: The results showed that treatment with 15 µmol L-1 NO significantly (P < 0.05) enhanced the activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, 4-coumaroyl-CoA ligase, chalcone synthase and chalcone isomerase and the expression of their genes. Furthermore, NO treatment significantly (P < 0.05) increased the contents of total phenolics, flavonoids and lignin over the entire storage period and maintained higher total anthocyanin, phenolic acid and anthocyanin contents during the earlier storage period. CONCLUSION: These results suggest that NO treatment could activate the phenylpropanoid pathway to enhance the activity of related enzymes and the contents of phenylpropanoid metabolites in peach to improve disease resistance and prevent pathogenic invasion. © 2016 Society of Chemical Industry.

Administration of Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and Avian Beta Defensin as Adjuvants in Inactivated Inclusion Body Hepatitis Virus and its Hexon Protein-Based Experimental Vaccine Formulations in Chickens.

Inclusion body hepatitis (IBH) is one of the major infectious diseases adversely affecting the poultry industry of the United States and Canada. Currently, no effective and safe vaccine is available for the control of IBH virus (IBHV) infection in chickens. However, based on the excellent safety and immunogenic profiles of experimental veterinary vaccines developed with the use of new generation adjuvants, we hypothesized that characterization of vaccine formulations containing inactivated IBHV or its capsid protein hexon as antigens, along with poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and avian beta defensin 2 (ABD2) as vaccine adjuvants, will be helpful in development of an effective and safe vaccine formulation for IBH. Our data demonstrated that experimental administration of vaccine formulations containing inactivated IBHV and a mixture of PCEP with or without ABD2 as an adjuvant induced significantly higher antibody responses compared with other vaccine formulations, while hexon protein-based vaccine formulations showed relatively lower levels of antibody responses. Thus, a vaccine formulation containing inactivated IBHV with PCEP or a mixture of PCEP and ABD2 (with a reduced dosage of PCEP) as an adjuvant may serve as a potential vaccine candidate. However, in order to overcome the risks associated with whole virus inactivated vaccines, characterization of additional viral capsid proteins, including fiber protein and penton of IBHV along with hexon protein in combination with more new generation adjuvants, will be helpful in further improvements of vaccines against IBHV infection.

Terpinen-4-ol Enhances Disease Resistance of Postharvest Strawberry Fruit More Effectively than Tea Tree Oil by Activating the Phenylpropanoid Metabolism Pathway.

This study aimed to reveal the effects and possible mechanism of terpinen-4-ol, the main component of tea tree oil (TTO), on the disease resistance of strawberry fruit. When the effects of TTO and its components were compared on the decay development in fruit inoculated with Botrytis cinerea after treatment, strawberry treated with terpinen-4-ol showed the lowest disease incidence (44.4%) after 48 h and also the smallest lesion diameter during the whole storage. This indicates that terpinen-4-ol induces the highest disease resistance in strawberry compared with TTO and other components. Untargeted metabolomic analysis showed that terpinen-4-ol treatment strongly activated phenylpropanoid biosynthesis and flavonoid metabolism pathway by increasing the accumulation of cinnamaldehyde, coniferyl aldehyde, naringenin, taxifolin, quercetin, and quercitrin in fruit at 12 h after treatment. In addition, terpinen-4-ol treatment also caused the accumulation of total phenolics and lignin by enhancing activities and relative gene expression of key enzymes in the phenylpropanoid metabolism pathway. These results suggest that terpinen-4-ol, as the key component of TTO, is the most important contributor to the effectiveness of TTO in improving disease resistance of strawberry fruit through activating the phenylpropanoid metabolism pathway.

Innate immune response profiles in pigs injected with vaccine adjuvants polydi(sodium carboxylatoethylphenoxy)phosphazene (PCEP) and Emulsigen.

Vaccines are formulated with adjuvants to enhance or direct antigen-specific immune responses against pathogens. However, the mechanisms of action (MOA) of adjuvants are not well understood and are under-investigated in large animal species. We have previously reported that injection of mice induced innate immune responses as indicated by increased cell recruitment and cytokine production at the site of injection with polyphosphazene (PCEP) adjuvant. In the present study, we evaluated whether PCEP induced similar innate immune responses in pigs. Piglets were injected with either PCEP or Emulsigen intradermally (I.D.) and the local cellular infiltration and cytokine production were evaluated at the site of injection and the draining lymph nodes. PCEP induced infiltration of macrophages, T and B cells, leucocytes and necrotic debris at the site of injection as well as PCEP-induced leucocyte infiltration in the draining lymph nodes. Emulsigen induced diffuse infiltration of leucocytes, macrophages, and lymphocytes at the site of injection as well as at the draining lymph nodes. PCEP induced significant production of interleukin IL-1ß, and IL-13 at the site of injection and IL-1ß, and IL-6 at the draining lymph nodes. Emulsigen promoted the production of IL-1ß, IL-6, and IL-12 at the site of injection but not in the draining lymph nodes. No cytokines were detected in blood after injection of either adjuvant. Together, our data indicate that in pigs, the adjuvants PCEP and Emulsigen stimulate early innate immune responses at the injection site by creating an immunocompetent environment that may contribute to increased immunogenicity of the co-administered antigens.

The microbial metabolite desaminotyrosine protects from influenza through type I interferon.

The microbiota is known to modulate the host response to influenza infection through as-yet-unclear mechanisms. We hypothesized that components of the microbiota exert effects through type I interferon (IFN), a hypothesis supported by analysis of influenza in a gain-of-function genetic mouse model. Here we show that a microbially associated metabolite, desaminotyrosine (DAT), protects from influenza through augmentation of type I IFN signaling and diminution of lung immunopathology. A specific human-associated gut microbe, Clostridium orbiscindens, produced DAT and rescued antibiotic-treated influenza-infected mice. DAT protected the host by priming the amplification loop of type I IFN signaling. These findings show that specific components of the enteric microbiota have distal effects on responses to lethal infections through modulation of type I IFN.

Response of immune response genes to adjuvants poly [di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP), CpG oligodeoxynucleotide and emulsigen at intradermal injection site in pigs.

Understanding the mechanisms by which adjuvants mediate their effects provide critical information on how innate immunity influences the development of adaptive immunity. Despite being a critical vaccine component, the mechanisms by which adjuvants mediate their effects are not fully understood and this is especially true when they are used in large animals. This lack of understanding limits our ability to design effective vaccines. In the present study, we administered polyphosphazene (PCEP), CpG oligodeoxynucleotides (CpG), emulsigen or saline via an intradermal injection into pigs and assessed the impact on the expression of reported 'adjuvant response genes' over time. CpG induced a strong upregulation of the chemokine CXL10 several 'Interferon Response Genes', as well as TNFα, and IL-10, and a down-regulation of IL-17 genes. Emulsigen upregulated expression of chemokines CCL2 and CCL5, proinflammatory cytokines IL-6 and TNFα, as well as TLR9, and several IFN response genes. PCEP induced the expression of chemokine CCL2 and proinflammatory cytokine IL-6. These results suggest that emulsigen and CpG may promote recruitment of innate immune cells and Th1 type cytokine production but that PCEP may promote a Th-2 type immune response through the induction of IL-6, an inducer of B cell activity and differentiation.

Loxoprofen sodium induces the production of complement C5a in human serum.

Basophil activation test (BAT) is an in vitro allergy test that is useful to identify allergens that cause IgE-dependent allergies. The test has been used to detect not only food allergies and allergies caused by environmental factors but also to detect drug hypersensitivity, which has been known to include IgE-independent reactions. In our preliminary studies in which BAT was applied to detect hypersensitivity of loxoprofen, a non-steroidal anti-inflammatory drug (NSAID), conventional BAT with incubation for 30min did not show basophil activation by means of increased CD203c expression. In this study, we extended the incubation time to 24h on the basis of the hypothesis that loxoprofen indirectly activates basophils. Basophils from healthy control donors as well as allergic patients showed up-regulation of CD203c after incubation with loxoprofen for 24h. Activation was induced using loxoprofen-treated serum. Proteomic and pharmacologic analyses revealed that serum incubation with loxoprofen generated an active complement component C5a, which induced CD203c expression via binding to the C5a receptor on basophils. Because C3a production was also detected after incubation for 24h, loxoprofen is likely to stimulate the complement classical pathway. Our findings suggest that the complement activation is involved in drug hypersensitivity and the suppression of this activation may contribute to the elimination of false positive of BAT for drug allergies.

In-vitro and in-vivo immunomodulatory effects of syringin.

Syringin was found to possess immunomodulatory activity by which it inhibited the in-vitro immunohaemolysis of antibody-coated sheep erythrocytes by guinea-pig serum through suppression of C3-convertase of the classical complement. In this study, we examined its in-vitro and in-vivo activity on tumour necrosis factor (TNF)-alpha and nitric oxide (NO) production, CD4+ T cell and CD8+ cytotoxic T cell (CTLL-2) proliferation, and croton oil-, arachidonic acid- and fluorescein-isothiocynate (FITC)-induced mouse ear oedema model. Syringin significantly inhibited both TNF-alpha production from lipopolysaccharide (LPS)-stimulated RAW264.7 cells and CD8+ T cell (CTLL-2) proliferation in a dose-dependent manner, whereas neither NO production nor CD4+ T cell proliferation were blocked even by high concentrations of syringin. In the invivo experiments, syringin also significantly suppressed FITC-induced ear oedema in mice but not the ear oedema induced by croton or arachidonic acid. These results suggest that syringin may be implicated as an immunomodulator having an anti-allergic effect rather than an anti-inflammatory effect. The anti-allergic effect of syringin seems to be due, in part, to inhibition of TNF-alpha production and cytotoxic T cell proliferation.

Insulin autoimmune syndrome caused by an adhesive skin patch containing loxoprofen-sodium.

A 62-year-old woman complained of repeated hypoglycemic events. A 75 g oral glucose tolerance test (75 gOGTT) showed a marked increase in the plasma insulin level and impaired glucose tolerance. The patient exhibited a high titer of plasma anti-insulin autoantibodies. Her diagnosis was insulin autoimmune syndrome (IAS). Following the cessation of loxoprofen-sodium (LOXs), she experienced no further hypoglycemic episodes. However, the hypoglycemic attacks recurred following the accidental readministration of LOXs in an adhesive skin patch. Considering the changes in the titer of anti-insulin autoantibodies, the repeated 75 gOGTT and the repeated Scatchard analysis, we determined LOXs to be the cause of the IAS and evaluated the characteristics of the autoantibodies.

Synthesis, properties, and biological activity of poly[di(sodium carboxylatoethylphenoxy)phosphazene].

A new water-soluble polyphosphazene polyelectrolyte containing carboxylate functionalities, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) was synthesized via reaction of macromolecular substitution. The polymer was characterized using (1)H, (31)P NMR, and gel permeation chromatography with multiangle laser light scattering detection. PCEP was shown to undergo hydrolytic degradation in aqueous solutions, as indicated by the decrease in the molecular weight and the release of side groups. A series of incompletely substituted copolymers of PCEP containing varying amounts of residual chlorine atoms was also prepared. The rate of degradation for such copolymers increased with the rise in the content of chlorine atoms. In vivo studies demonstrated high potency of PCEP as a vaccine immunoadjuvant. The new polyphosphazene was also shown to be capable of forming microspheres in aqueous solutions via reactions of ionic complexation with physiologically occurring amines, such as spermine.

Immunochemical detection of protein dityrosine in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody.

Dityrosine is one of the specific biomarkers for proteinoxidation. We prepared an antibody specific for proteindityrosine using a dimer of 3-p-(hydroxyphenyl)propionic acid (di-HP) as a hapten. Three clones (A8, G6, and 1C3) were obtained, and the antibody from the A8 clone reacted with the di-HP-conjugated protein but not with a dityrosine conjugate. The others (G6 and 1C3 clones) recognized both the di-HP and dityrosine conjugates. The antibodies reacted with peptidyl dityrosine, derived from Thr-Tyr-Ser, rather than the free dityrosine. The reactivity of the latter two antibodies with lens proteins oxidized by incubation with H(2)O(2)/Cu was in good accordance with the formation of the dityrosine-like fluorescence. Using the obtained monoclonal antibody, the immunopositive staining of atherosclerotic lesions in Apo E-deficient mice was confirmed by an immunohistochemical technique.