A PAMP-triggered MAPK cascade inhibits phosphatidylinositol 4,5-bisphosphate production by PIP5K6 in Arabidopsis thaliana.

The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen-activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen-associated molecular pattern (PAMP) flg22. Promoter-ß-glucuronidase analyses and quantitative real-time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6-targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4-phosphate 5-kinase activity in mesophyll protoplasts, indicating that the flg22-activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post-translational control of PIP5K6 activity. PtdIns(4,5)P2 -dependent endocytosis of FM 4-64, PIN2 and the NADPH-oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD-endocytosis was correlated with enhanced ROS production. We conclude that MPK6-mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception.

Identification and molecular characterization of peroxiredoxin 2 in grass carp (Ctenopharyngodon idella).

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H2O2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses.

Acetalated Dextran Microparticles for Codelivery of STING and TLR7/8 Agonists.

Vaccines are the most effective tool for preventing infectious diseases; however, subunit vaccines, considered the safest type, suffer from poor immunogenicity and require adjuvants to create a strong and sustained immune response. As adjuvants, pathogen-associated molecular patterns (PAMPs) offer potent immunostimulatory properties and defined mechanisms of action through their cognate pattern recognition receptors (PRRs). Their activity can be further enhanced through combining two or more PAMPs, particularly those that activate multiple immune signaling pathways. However, the cytosolic localization of many PRRs requires intracellular delivery of PAMPs for optimal biological activity, which is particularly true of the stimulator of interferon genes (STING) PRR. Using acetalated dextran (Ace-DEX) microparticles (MPs) encapsulating STING agonist 3'3'-cyclic GMP-AMP (cGAMP) combined with soluble PAMPS, we screened the effect of codelivery of adjuvants using primary mouse bone marrow derived dendritic cells (BMDCs). We identified that codelivery of cGAMP MPs and soluble Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) elicited the broadest cytokine response. cGAMP and R848 were then coencapsulated within Ace-DEX MPs via electrospray. Using the model antigen ovalbumin, we observed that Ace-DEX MPs coencapsulating cGAMP and R848 (cGAMP/R848 Ace-DEX MPs) induced antigen-specific cellular immunity, and a balanced Th1/Th2 humoral response that was greater than cGAMP Ace-DEX MPs alone and PAMPs delivered in separate MPs. These data indicate that polymeric Ace-DEX MPs loaded with STING and TLR7/8 agonists represent a potent cellular and humoral vaccine adjuvant.

Two randomized, double-blind, placebo-controlled, dose-escalation phase 1 studies evaluating BTH1677, a 1, 3-1,6 beta glucan pathogen associated molecular pattern, in healthy volunteer subjects.

BACKGROUND: BTH1677 is a beta glucan pathogen associated molecular pattern (PAMP) currently being investigated as a novel cancer therapy. Here, the initial safety and pharmacokinetic (PK) results of BTH1677 in healthy subjects are reported. SUBJECTS AND METHODS: In the Phase 1a single-dosing study, subjects were randomized (3:1 per cohort) to a single intravenous (i.v.) infusion of BTH1677 at 0.5, 1, 2, 4, or 6 mg/kg or placebo, respectively. In the Phase 1b multi-dosing study, subjects were randomized (3:1 per cohort) to 7 daily i.v. infusions of BTH1677 at 1, 2, or 4 mg/kg or placebo, respectively. Safety and PK non-compartmental analyses were performed. RESULTS: Thirty-six subjects (N = 24 Phase 1a; N = 12 Phase 1b) were randomized to treatment. No deaths or serious adverse events occurred in either study. Mild or moderate adverse events (AEs) occurred in 67% of BTH1677-treated subjects in both studies. Treatment-related AEs (occurring in ≥10% of subjects) included dyspnea, flushing, headache, nausea, paraesthesia, and rash in Phase 1a and conjunctivitis and headache in Phase 1b. BTH1677 serum concentration was linear with dose. Clearance, serum elimination half-life (t1/2) and volume of distribution (Vss) were BTH1677 dose-independent. In Phase 1b, area under the curve, t1/2, and Vss values were larger at steady state on days 6-30 versus day 0. CONCLUSIONS: BTH1677 was well tolerated after single doses up to 6 mg/kg and after 7 daily doses up to 4 mg/kg.

Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish.

BACKGROUND: Although the responses to many pathogen associated molecular patterns (PAMPs) in cell cultures and extracted organs are well characterized, there is little known of transcriptome responses to PAMPs in whole organisms. To characterize this in detail, we have performed RNAseq analysis of responses of zebrafish embryos to injection of PAMPs in the caudal vein at one hour after exposure. We have compared two ligands that in mammals have been shown to specifically activate the TLR2 and TLR5 receptors: Pam3CSK4 and flagellin, respectively. RESULTS: We identified a group of 80 common genes that respond with high stringency selection to stimulations with both PAMPs, which included several well-known immune marker genes such as il1b and tnfa. Surprisingly, we also identified sets of 48 and 42 genes that specifically respond to either Pam3CSK4 or flagellin, respectively, after a comparative filtering approach. Remarkably, in the Pam3CSK4 specific set, there was a set of transcription factors with more than 2 fold-change, as confirmed by qPCR analyses, including cebpb, fosb, nr4a1 and egr3. We also showed that the regulation of the Pam3CSK4 and flagellin specifically responding sets is inhibited by knockdown of tlr2 or tlr5, respectively. CONCLUSIONS: Our studies show that Pam3CSK4 and flagellin can stimulate the Tlr2 and Tlr5 signaling pathways leading to common and specific responses in the zebrafish embryo system.

Transdermal electroosmotic flow generated by a porous microneedle array patch.

A microneedle array is an attractive option for a minimally invasive means to break through the skin barrier for efficient transdermal drug delivery. Here, we report the applications of solid polymer-based ion-conductive porous microneedles (PMN) containing interconnected micropores for improving iontophoresis, which is a technique of enhancing transdermal molecular transport by a direct current through the skin. The PMN modified with a charged hydrogel brings three innovative advantages in iontophoresis at once: (1) lowering the transdermal resistance by low-invasive puncture of the highly resistive stratum corneum, (2) transporting of larger molecules through the interconnected micropores, and (3) generating electroosmotic flow (EOF). In particular, the PMN-generated EOF greatly enhances the transdermal molecular penetration or extraction, similarly to the flow induced by external pressure. The enhanced efficiencies of the EOF-assisted delivery of a model drug (dextran) and of the extraction of glucose are demonstrated using a pig skin sample. Furthermore, the powering of the PMN-based transdermal EOF system by a built-in enzymatic biobattery (fructose / O2 battery) is also demonstrated as a possible totally organic iontophoresis patch.

Dogs with osteosarcoma have altered pro- and anti-inflammatory cytokine profiles.

BACKGROUND: Current advances in immunotherapy are an exciting area of study in canine osteosarcoma (OSA). The objective of this study was to determine the immune response in dogs with osteosarcoma by measuring stimulated leukocyte production of tumor necrosis factor (TNF), interleukin (IL)-6, IL-10 and TNF and IL-6 to IL-10 ratios. METHODS: Whole blood was collected from dogs with osteosarcoma receiving non-steroidal anti-inflammatory drugs (NSAIDs, n = 11), dogs with osteosarcoma not receiving NSAIDs (n = 14) and healthy dogs (n = 5). RESULTS: No difference in TNF production was found among healthy and OSA dogs regardless of NSAID administration following stimulation with lipopolysaccharide (LPS) (p = .410), lipoteichoic acid (LTA) (p = .693) or PBS (p = .120). Leukocyte IL-6 production was greater in all dogs with OSA after stimulation with LPS (p = .015), LTA (p = .014) and PBS (p = .034) with no difference between OSA dogs receiving NSAIDs and those not. No differences in IL-10 were found among healthy controls and dogs with OSA regardless of NSAID use. There was no difference among groups for LPS-stimulated TNF to IL-10 ratios (p = .407). For LTA-stimulated leukocytes, the TNF to IL-10 ratio was lower in dogs with OSA than in healthy dogs (p = .031) with no difference between OSA NSAID dogs compared to OSA non-NSAID dogs (p = .059). No differences were found in LPS (p = .310)- or LTA (p = .265)-stimulated leukocyte IL-6 to IL-10 production ratios among groups. CONCLUSIONS: Dogs with osteosarcoma have an altered pro- and anti-inflammatory immunologic profile compared to healthy dogs regardless of NSAID use. Further study is indicated to determine the potential prognostic and therapeutic implications of these findings.