Chicken cathelicidin-2-derived peptides with enhanced immunomodulatory and antibacterial activities against biological warfare agents.

Host defence peptides (HDPs) are considered to be excellent candidates for the development of novel therapeutic agents. Recently, it was demonstrated that the peptide C1-15, an N-terminal segment of chicken HDP cathelicidin-2, exhibits potent antibacterial activity while lacking cytotoxicity towards eukaryotic cells. In the present study, we report that C1-15 is active against bacteria such as Bacillus anthracis and Yersinia pestis that may potentially be used by bioterrorists. Substitution of single and multiple phenylalanine (Phe) residues to tryptophan (Trp) in C1-15 resulted in variants with improved antibacterial activity against B. anthracis and Y. pestis as well as decreased salt sensitivity. In addition, these peptides exhibited enhanced neutralisation of lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). The antibacterial and LPS-neutralising activities of these C1-15-derived peptides are exerted at concentrations far below the concentrations that are toxic to human PBMCs. Taken together, we show that Phe-->Trp substitutions in C1-15 variants enhances the antibacterial and LPS-neutralising activities against pathogenic bacteria, including those that may potentially be used as biological warfare agents.

Activation of Toll-like receptors and dendritic cells by a broad range of bacterial molecules.

Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, their capacity to activate TLRs and to affect DC maturation, cytokine production and T cell polarizing capacity were determined. Different bacterial species differed in their potency to affect these parameters. In general, on the DC level differences were found in the maturation-inducing capacity of gram-negative and gram-positive bacteria. Remarkably, these differences did not result in differential polarization of the T cell response. With respect to TLRs, TLR4 activation by pathogens correlated with their ability to induce DC maturation, while for TLR2 and TLR5 such a correlation was absent. Taken together, this study provides insight into qualitative differences and general effects of pathogen-derived molecules on dendritic cells.

The bradykinin B2 receptor in the early immune response against Listeria infection.

The endogenous danger signal bradykinin was recently found implicated in the development of immunity against parasites via dendritic cells. We here report an essential role of the B(2) (B(2)R) bradykinin receptor in the early immune response against Listeria infection. Mice deficient in B(2)R (B(2)R(-/-) mice) were shown to suffer from increased hepatic bacterial burden and concomitant dramatic weight loss during infection with Listeria monocytogenes. Levels of cytokines known to play a pivotal role in the early phase immune response against L. monocytogenes, IL-12p70 and IFN-gamma, were reduced in B(2)R(-/-) mice. To extend these findings to the human system, we show that bradykinin potentiates the production of IL-12p70 in human monocyte-derived dendritic cells. Thus, we show that bradykinin and the B(2)R play a role in early innate immune functions during bacterial infection.

Construction and biological activity of a full-length molecular clone of human Torque teno virus (TTV) genotype 6.

Torque teno virus (TTV) is a non-enveloped human virus with a circular negative-sense (approximately 3800 nucleotides) ssDNA genome. TTV resembles in genome organization the chicken anemia virus, the animal pathogen of the Circoviridae family, and is currently classified as a member of a new, floating genus, Anellovirus. Molecular and cell biological research on TTV has been restricted by the lack of permissive cell lines and functional, replication-competent plasmid clones. In order to examine the key biological activities (i.e. RNA transcription and DNA replication) of this still poorly characterized ssDNA virus, we cloned the full-length genome of TTV genotype 6 and transfected it into cells of several types. TTV mRNA transcription was detected by RT-PCR in all the cell types: KU812Ep6, Cos-1, 293, 293T, Chang liver, Huh7 and UT7/Epo-S1. Replicating TTV DNA was detected in the latter five cell types by a DpnI-based restriction enzyme method coupled with Southern analysis, a novel approach to assess TTV DNA replication. The replicating full-length clone, the cell lines found to support TTV replication, and the methods presented here will facilitate the elucidation of the molecular biology and the life cycle of this recently identified human virus.