Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.
We describe here a new ion mobility capable mass spectrometer which comprises a drift cell for mobility separation and a quadrapole time of flight mass spectrometer for mass analysis--the MoQTOF. A commercial QToF instrument (Micromass UK Ltd., Manchester, UK) has been modified by the inclusion of an additional chamber containing a drift cell and ancillary ion optics. The drift cell is 5.1 cm long made from a copper block and is mounted from a top hat flange in a chamber situated post source optics and prior to the quadapole analyzer. Details of this instrument are provided along with information about how it can be used to acquire mobilities of ions along with their mass to charge ratios. The MoQTOF is used to examine conformations of a series of antimicrobial peptides based on a beta-defensin template. In vivo, these cationic cystine-rich amphiphilic peptides are conformationally restrained by three or more disulfide bridges, although recent findings by several groups have cast doubt on the importance of canonical disulfide pairing to antimicrobial activities. By synthesizing a panel of variants to Defb14 (the murine orthologue of HBD3), we exploit ion mobility to distinguish conformational differences which arise due to disulfide formation and to the hydrophobicity of the peptide sequence. Our gas-phase results are interpreted in terms of the antimicrobial and chemotacic properties of beta-defensins, and this mass spectrometry based approach to discern structure may have a role in future design of novel antibiotics.
-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human -defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V ) of the -defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I ) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V . Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of -defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.
Eosinophils have previously been shown to accumulate around the tissue invasive (L3) stage of sheep gastrointestinal parasites in vivo. In this study, eosinophils obtained from mammary washes of sheep, were shown to immobilize and kill H. contortus larvae in vitro in the presence of antibody specific against a defined L3 surface antigen. Eosinophils obtained from sheep primed by repeated infusion of H. contortus larvae were more effective than eosinophils obtained after a single infusion of parasite extract in Fasciola hepatica infected ewes suggesting the former were activated in vivo. The level of larval immobilization in the presence of antibody was significantly increased when complement was added to cultures containing activated eosinophils. The addition of interleukin-5 to larval cultures containing antibody and complement resulted in a significant increase in larval immobilization with unactivated eosinophils suggesting that eosinophil effector function is enhanced following priming with this cytokine. Ultrastructural analysis of the eosinophil/larvae interaction at 6 h of incubation revealed degranulation of adhering eosinophils onto the surface of larvae. By 24 h of incubation, many larvae showed signs of damage and most eosinophils had degenerated. These results suggest that eosinophil-mediated killing may be an effector mechanism for the elimination of L3 H. contortus larvae in immune sheep.
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