CpG oligonucleotides bind TLR9 and RRM-Containing proteins in Atlantic Salmon (Salmo salar)

Background: Bacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates. Results: In the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes. Conclusion: The presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA

IPNV with high and low virulence: host immune responses and viral mutations during infection

<p>Abstract</p> <p>Background</p> <p>Infectious pancreatic necrosis virus (IPNV) is an aquatic member of the <it>Birnaviridae </it>family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR), which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described.</p> <p>Methods</p> <p>In this study we compared two field isolates of IPNV (NFH-Ar and NFH-El). The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney.</p> <p>Results</p> <p>Significant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i.) was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin) was observed at 13 d p.i. (NFH-Ar) and 29 d p.i. (both isolates).</p> <p>Conclusions</p> <p>Mortality and expression levels of the immune genes were directly related to the rate of viral replication, which was in turn associated with sequences of viral genes. Rapid changes in the viral genome that dramatically reduced virus proliferation might indicate a higher susceptibility to protective mechanism employed by the host. Disease outbreak and mortality depend on a delicate balance between host defence, regulation of signalling cascades and virus genomic properties.</p

Beta-glucan-loaded nanofiber dressing improves wound healing in diabetic mice

The increased prevalence of chronic wounds requires novel treatment options. The aim of this study was to develop a beta-glucan (βG)-loaded nanofiber wound dressing. Nanofibers were prepared using the needle-free Nanospider™ technology, an electrospinning method which enables the production of nanofibers at an industrial scale. The βG was selected as active ingredient based on its confirmed wound healing potential in both animals and humans. Hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO) were included as copolymers. Rheological profiles of spinning solutions containing HPMC, PEO, βG, ethanol and water, were optimized. The nanofiber formation was confirmed by Field Emission Scanning Electron Microscopy (FE-SEM), and both nanofibers with (βG-nanofibers) or without βG (NoβG-nanofibers) were evaluated by their swelling index and FT-IR spectroscopy. The formulations, active ingredient and excipients were tested for their possible in vitro toxicity in keratinocytes. Finally, the wound healing potential of the nanofibers was tested in externally induced excisional wounds in male diabetic db/db mice. Three different doses of βG-nanofibers and the βG-free, NoβG-nanofibers, were evaluated for their in vivo wound healing efficacy. All nanofiber-treatments provided improved wound healing as compared to the negative control (water). All βG-nanofiber treated groups exhibited significantly improved wound healing as compared to the NoβG-nanofiber treated group, indicating the potential of βG-nanofibers as wound dressing