Monitoring fish welfare has become a central issue for the fast-growing aquaculture industry, and finding proper biomarkers of stress, inflammation and infection is necessary for surveillance and documentation of fish health. In this study, a proteomic approach using mass spectrometry was applied to identify indicators of the acute response in Atlantic salmon blood plasma by comparing Aeromonas salmonicida subsp. salmonicida infected fish and non-infected controls. The antimicrobial proteins cathelicidin (CATH), L-plastin (Plastin-2, LCP1) and soluble toll-like receptor 5 (sTLR5) were uniquely or mainly identified in the plasma of infected fish. In addition, five immune-related proteins showed significantly increased expression in plasma of infected fish: haptoglobin, high affinity immunoglobulin Fc gamma receptor I (FcγR1, CD64), leucine-rich alpha 2 glycoprotein (LRG1), complement C4 (C4) and phospholipase A2 inhibitor 31 kDa subunit-like protein. However, various fibrinogen components, CD209 and CD44 antigen-like molecules decreased in infected fish. Selected biomarkers were further verified by Western blot analysis of plasma and real time PCR of spleen and liver, including CATH1, CATH2 and L-plastin. A significant increase of L-plastin occurred as early as 24 h after infection, and a CATH2 increase was observed from 72 h in plasma of infected fish. Real time PCR of selected genes confirmed increased transcription of CATH1 and CATH2. In addition, serum amyloid A mRNA significantly increased in liver and spleen after bacterial infection. However, transcription of L-plastin was not consistently induced in liver and spleen. The results of the present study reveal novel and promising biomarkers of the acute phase response and inflammation in Atlantic salmon.

Francisellosis in fish is caused by the facultative intracellular Gram-negative bacterial pathogens Francisella noatunensis ssp. noatunensis and Francisella orientalis. The disease is affecting both farmed and wild fish worldwide and no commercial vaccines are currently available. In this study, we tested isolated membrane vesicles (MVs) as possible vaccine candidates based on previous trials in zebrafish (Danio rerio) indicating promising vaccine efficacy. Here, the MV vaccine-candidates were tested in their natural hosts, Atlantic cod (Gadus morhua L.) and Nile tilapia (Oreochromis niloticus). Injection of MVs did not display any toxicity or other negative influence on the fish and gene expression analysis indicated an influence on the host immune response. However, unlike in other tested fish species, a protective immunity following vaccine application and immunization period could not be detected in the Atlantic cod or tilapia. Further in vivo studies are required to achieve a better understanding of the development of immunological memory in different fish species.

Collagen and glycosaminoglycans (GAGs) constituting the ECM may limit the space available and thus exclude macromolecules from a fraction of the interstitial fluid (IF) phase. This exclusion phenomenon is of importance for transcapillary fluid and solute exchange. The purpose of the study was to examine the range of interstitial exclusion in rat skin by using probes within a span of molecular weights and electrical charge and also to test if a change in interstitial composition, occurring as a consequence of aging, affected exclusion. To this end, we used a novel approach, involving the exact determination of albumin concentration and mass in IF and tissue eluate by HPLC and thereafter, expressing the corresponding numbers relative to albumin for a set of probe proteins assessed by quantitative proteomics. Albumin was excluded from 55 ± 4% ( n = 8) of the extracellular fluid phase. There was a highly significant, positive correlation between probe Stokes-Einstein (SE) radius and fractional excluded volume (V_EF), described by V_EF = 0.078·SE radius + 0.269 ( P < 0.001), and oppositely, a negative correlation between probe isoelectric point (pI) and exclusion for proteins with comparable size, V_EF = −0.036·pI + 0.719 ( P = 0.04). Aging resulted in a significant reduction in skin hydration and sulfated GAGs, a moderate increase in hyaluronan, and a corresponding, reduced V_EF for albumin and the other macromolecular probes. Our findings suggest that the changes in the ECM in aged skin may result in delayed adjustments of fluid perturbations and reduced ability for salt storage.

The use of hydrogel microparticles as granular hydrogels is an emerging approach in tissue engineering, where microparticles offer printability, injectability, and serve as scaffolds for cell culture. While culturing dermal fibroblasts for tissue engineering applications is well reported, there is limited research on culturing primary human pulmonary fibroblasts on granular hydrogels. In this study, we grafted RGD-peptides to alginate, formed microbeads, and investigated the ability of granular hydrogels to support the adhesion and growth of primary normal human dermal fibroblasts (NHDFs) and human pulmonary fibroblasts (HPFa). NHDFs adhered to linear RGD (linRGD)-alginate microbeads and spread on the bead surfaces with increased adhesion with increased peptide concentration (0.3-1.3 mM). In contrast, HPFa did not adhere to the linRGD-alginate microbeads. However, HPFa adhered and spread on flat linRGD-alginate gels, indicating that HPFa do respond to linRGD as an adhesion ligand. Supplementation of Mn²⁺ resulted in cell adhesion to linRGD-alginate microbeads. Enhanced adhesion and spreading of HPFa to RGD-alginate microbeads were observed when using cyclic RGD. Hence, RGD-alginate microbeads is a promising material for structuring primary human dermal and pulmonary fibroblasts, showing the relevance of using alginate microbeads as scaffolds for 3D cultures with fibroblasts.

Aims: The aim of this study was to develop a high-throughput robotic microtiter plate-based screening assay for Candida albicans, optimizing growth conditions to replicate the filamentous biofilm growth found in vivo, and subsequently, to demonstrate the assay by evaluating the effect of nutritional drinks alone and in combination with the antifungal amphotericin B (AmB). Methods and Results: Candida albicans cultured in a defined growth medium showed filamentous growth in microcolonies, mimicking the morphology of oral mucosal disease (oral candidiasis). Addition of nutrient drinks containing fruit juices, fish oil and whey protein to the medium resulted in changed morphology and promoted growth as free yeast cells and with weak biofilm structures. Minimum inhibitory concentration of AmB on the biofilms was 0.25 μg ml−1, and this was eightfold reduced (0.0038 μg ml−1) in the presence of the nutritional drinks. Conclusions: The established assay demonstrated applicability for screening of antifungal and anti-biofilm effects of bioactive substances on C. albicans biofilm with clinically relevant morphology. Significance and Impact of the Study: Candida albicans is the causative agent of the majority of fungal infections globally. The filamentous morphology of C. albicans and the ability to form biofilm are traits known to increase virulence and resistance towards antifungals. This study describes the development of a plate-based in vitro screening method mimicking the filamentous morphology of C. albicans found in vivo. The assay established can thus facilitate efficient antifungal drug discovery and development.