Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar L.) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar L.) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3 to 6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.

Background: Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means. Methods: By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection. Results: We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication. Conclusion: Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

The importance of the Atlantic salmon peritoneal cavity B cell response: Local IgM secreting cells are predominant upon Piscirickettsia salmonis infection.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

Cellulose Nanofibril Formulations Incorporating a Low-Molecular-Weight Alginate Oligosaccharide Modify Bacterial Biofilm Development.

Cellulose nanofibrils (CNFs) from wood pulp are a renewable material possessing advantages for biomedical applications because of their customizable porosity, mechanical strength, translucency, and environmental biodegradability. Here, we investigated the growth of multispecies wound biofilms on CNF formulated as aerogels and films incorporating the low-molecular-weight alginate oligosaccharide OligoG CF-5/20 to evaluate their structural and antimicrobial properties. Overnight microbial cultures were adjusted to 2.8 × 109 colony-forming units (cfu) mL-1 in Mueller Hinton broth and growth rates of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus 1061A monitored for 24 h in CNF dispersions sterilized by γ-irradiation. Two CNF formulations were prepared (20 g m-2) with CNF as air-dried films or freeze-dried aerogels, with or without incorporation of an antimicrobial alginate oligosaccharide (OligoG CF-5/20) as a surface coating or bionanocomposite, respectively. The materials were structurally characterized by scanning electron microscopy (SEM) and laser profilometry (LP). The antimicrobial properties of the formulations were assessed using single- and mixed-species biofilms grown on the materials and analyzed using LIVE/DEAD staining with confocal laser scanning microscopy (CLSM) and COMSTAT software. OligoG-CNF suspensions significantly decreased the growth of both bacterial strains at OligoG concentrations >2.58% (P < 0.05). SEM showed that aerogel-OligoG bionanocomposite formulations had a more open three-dimensional structure, whereas LP showed that film formulations coated with OligoG were significantly smoother than untreated films or films incorporating PEG400 as a plasticizer (P < 0.05). CLSM of biofilms grown on films incorporating OligoG demonstrated altered biofilm architecture, with reduced biomass and decreased cell viability. The OligoG-CNF formulations as aerogels or films both inhibited pyocyanin production (P < 0.05). These novel CNF formulations or bionanocomposites were able to modify bacterial growth, biofilm development, and virulence factor production in vitro. These data support the potential of OligoG and CNF bionanocomposites for use in biomedical applications where prevention of infection or biofilm growth is required.

Ultrapure Wood Nanocellulose-Assessments of Coagulation and Initial Inflammation Potential.

Using a lepirudin-based human whole blood model, we evaluated the initial inflammatory and coagulation responses of dense and porous ultrapure (<50 endotoxin units/grams) cellulose nanofibrils (CNF), of carboxylated grade. The CNF was compared to the wound dressing AquaCel because it is a potential wound-healing material. The porous CNF aerogels induced the strongest coagulation potential measured as prothrombin factor 1.2 (PTF1.2). AquaCel induced the strongest complement response by terminal complement complex (TCC) and surface C3c. All materials activated leukocytes CD11b, while the levels of only 3 of 27 cytokines were significantly changed, limited to (i) an elevation of the monocyte chemoattractant protein-1 (MCP-1/CCL) by the CNF aerogel, (ii) a reduction of eosinophil chemotactic proteins (eotaxin/CCL11) by the CNF aerogel, and (iii) a reduction of platelet-derived growth factor BB (PDGF-BB) by all CNF materials. In conclusion, the CNF materials and AquaCel differently activate coagulation, complement, and cytokines, improving the selection possibilities in various treatment situations of wound healing.

Mechanical characteristics of nanocellulose-PEG bionanocomposite wound dressings in wet conditions.

Wood nanocellulose has been proposed for wound dressing applications partly based on its capability to form translucent films with good liquid absorption capabilities. Such properties are adequate for non-healing and chronic wounds where adequate management of exudates is a requirement. In addition, the translucency will allow to follow the wound development without the necessity to remove the dressing from the wound. Understanding the mechanical properties of nanocellulose films and dressings are also most important for tailoring optimizing wound dressing structures with adequate strength, conformability, porosity and exudate management. Mechanical properties are usually assessed in standard conditions (50% relative humidity, RH), which is not relevant in a wound management situation. In this study we have assessed the mechanical properties of three nanocellulose grades varying in the degree of nanofibrillation. The effect of nanofibrillation and of polyethylene glycol (PEG) addition, on the tensile strength, elongation and elastic modulus were assessed after 24h in water and in phosphate-buffered saline (PBS). The results reveal the behavior of the nanocellulose dressings after wetting and shed light into the development of mechanical properties in environments, which are relevant from a wound management point of view.

The interaction of wood nanocellulose dressings and the wound pathogen P. aeruginosa.

Chronic wounds pose an increasingly significant worldwide economic burden (over £1 billion per annum in the UK alone). With the escalation in global obesity and diabetes, chronic wounds will increasingly be a significant cause of morbidity and mortality. Cellulose nanofibrils (CNF) are highly versatile and can be tailored with specific physical properties to produce an assortment of three-dimensional structures (hydrogels, aerogels or films), for subsequent utilization as wound dressing materials. Growth curves using CNF (diameter <20nm) in suspension demonstrated an interesting dose-dependent inhibition of bacterial growth. In addition, analysis of biofilm formation (Pseudomonas aeruginosa PAO1) on nanocellulose aerogels (20g/m2) revealed significantly less biofilm biomass with decreasing aerogel porosity and surface roughness. Importantly, virulence factor production by P. aeruginosa in the presence of nanocellulose materials, quantified for the first time, was unaffected (p>0.05) over 24h. These data demonstrate the potential of nanocellulose materials in the development of novel dressings that may afford significant clinical potential.

Producing ultrapure wood cellulose nanofibrils and evaluating the cytotoxicity using human skin cells.

Wood cellulose nanofibrils (CNF) have been suggested as a potential wound healing material, but its utilization is limited by FDA requirements regarding endotoxin levels. In this study a method using sodium hydroxide followed by TEMPO mediated oxidation was developed to produce ultrapure cellulose nanofibrils, with an endotoxin level of 45 endotoxin units/g (EU/g) cellulose. Scanning transmission electron microscopy (S(T)EM) revealed a highly nanofibrillated structure (lateral width of 3.7±1.3nm). Assessment of cytotoxicity and metabolic activity on Normal Human Dermal Fibroblasts and Human Epidermal Keratinocytes was done. CNF-dispersion of 50µg/ml did not affect the cells. CNF-aerogels induced a reduction of metabolic activity by the fibroblasts and keratinocytes, but no significant cell death. Cytokine profiling revealed no induction of the 27 cytokines tested upon exposure to CNF. The moisture-holding capacity of aerogels was relatively high (∼7500%), compared to a commercially available wound dressing (∼2500%), indicating that the CNF material is promising as dressing material for management of wounds with a moderate to high amount of exudate.