ABSTRACT
Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodulatory lead compounds. For this purpose we examined whether chicken cathelicidin-2 (CATH-2)-derived peptides modulate the function and inflammatory response of avian immune cells. Using a chicken macrophage cell line (HD11) we found that full-length CATH-2 dose-dependently induced transcription of chemokines CXCLi2/IL-8, MCP-3 and CCLi4/RANTES, but not of pro-inflammatory cytokine IL-1ß. In addition, CATH-2 efficiently inhibited IL-1ß and nitric oxide production by HD11 cells induced by different sources of lipopolysaccharides (LPS). N-terminal truncated CATH-2 derived peptides maintained the capacity to selectively induce chemokine transcription, but despite their high LPS affinity several analogs lacked LPS-neutralizing capacity. Substitution of phenylalanine residues by tryptophan introduced endotoxin neutralization capacity in inactive truncated CATH-2 derived peptides. In contrast, amino acid substitution of phenylalanine by tyrosine abrogated endotoxin neutralization activity of CATH-2 analogs. These findings support a pivotal role for aromatic residues in peptide-mediated endotoxin neutralization by CATH-2 analogs and were shown to be independent of LPS affinity. The capacity to modulate chemokine production and dampen endotoxin-induced pro-inflammatory responses in chicken immune cells implicates that small CATH-2 based peptides could serve as leads for the design of CATH-2 based immunomodulatory anti-infectives.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Immunologic Factors/pharmacology , Peptide Fragments/pharmacology , Amino Acid Sequence , Amino Acid Substitution , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Antimicrobial Cationic Peptides/genetics , Cell Line , Chemokines/genetics , Chemokines/metabolism , Chickens , Cytokines/genetics , Cytokines/metabolism , Escherichia coli/drug effects , Gene Expression Regulation/drug effects , Immunologic Factors/chemistry , Inflammation Mediators/metabolism , L-Lactate Dehydrogenase/metabolism , Lipopolysaccharides , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Microbial Sensitivity Tests , Models, Molecular , Mutation , Nitric Oxide/biosynthesis , Peptide Fragments/chemistry , Protein Conformation , Reactive Oxygen Species/metabolism , Transcription, GeneticABSTRACT
BACKGROUND: Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. METHODS: Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. RESULTS: The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed. CONCLUSIONS: The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.
Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Molecular Diagnostic Techniques , Neutralization Tests/methods , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Viruses/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cell Line , Chlorocebus aethiops , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Hep G2 Cells , Humans , Molecular Sequence Data , Respiratory Syncytial Viruses/genetics , Vero Cells , Viral Plaque AssayABSTRACT
Zinc is an essential micronutrient for all living organisms. When facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation are not known. We present the identification of two closely related members of the Arabidopsis thaliana basic-region leucine-zipper (bZIP) transcription factor gene family, bZIP19 and bZIP23, that regulate the adaptation to low zinc supply. They were identified, in a yeast-one-hybrid screening, to associate to promoter regions of the zinc deficiency-induced ZIP4 gene of the Zrt- and Irt-related protein (ZIP) family of metal transporters. Although mutation of only one of the bZIP genes hardly affects plants, we show that the bzip19 bzip23 double mutant is hypersensitive to zinc deficiency. Unlike the wild type, the bzip19 bzip23 mutant is unable to induce the expression of a small set of genes that constitutes the primary response to zinc deficiency, comprising additional ZIP metal transporter genes. This set of target genes is characterized by the presence of one or more copies of a 10-bp imperfect palindrome in their promoter region, to which both bZIP proteins can bind. The bZIP19 and bZIP23 transcription factors, their target genes, and the characteristic cis zinc deficiency response elements they can bind to are conserved in higher plants. These findings are a significant step forward to unravel the molecular mechanism of zinc homeostasis in plants, allowing the improvement of zinc bio-fortification to alleviate human nutrition problems and phytoremediation strategies to clean contaminated soils.
