ZN148 Is a Modular Synthetic Metallo-ß-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Effects of dietary n-3 fatty acids on Toll-like receptor activation in primary leucocytes from Atlantic salmon (Salmo salar).

The shortage of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the international markets has led to increasing substitution of fish oil by plant oils in Atlantic salmon (Salmo salar) feed and thereby reducing the EPA and DHA content in salmon. However, the minimum required levels of these fatty acids in fish diets for securing fish health are unknown. Fish were fed with 0, 1 or 2% EPA or DHA alone or in combination of both over a period, growing from 50 to 400 g. Primary head kidney leucocytes were isolated and stimulated with Toll-like receptor (TLR) ligands to determine if EPA and DHA deficiency can affect expression of important immune genes and eicosanoid production. Several genes related to viral immune response did not vary between groups. However, there was a tendency that the high-level EPA and DHA groups expressed lower levels of IL-1ß in non-stimulated leucocytes. These leucocytes were also more responsive to the TLR ligands, inducing higher expression levels of IL-1ß and Mx1 after stimulation. The levels of prostaglandin E2 and leukotriene B4 in serum and media from stimulated leucocytes were lower in both low and high EPA and DHA groups. In conclusion, leucocytes from low EPA and DHA groups seemed to be less responsive towards immunostimulants, like TLR ligands, indicating that low levels or absence of dietary EPA and DHA may have immunosuppressive effects.

Stability of a Vesicular Stomatitis Virus-Vectored Ebola Vaccine.

The live attenuated vesicular stomatitis virus-vectored Ebola vaccine rVSV-ZEBOV is currently undergoing clinical trials in West Africa. The vaccine is to be stored at -70°C or less. Since maintaining the cold chain is challenging in rural areas, the rVSV-ZEBOV vaccine's short-term and long-term stability at different temperatures was examined. Different dilutions were tested since the optimal vaccine dosage had not yet been determined at the start of this experiment. The results demonstrate that the original vaccine formulation was stable for 1 week at 4°C and for 24 hours at 25°C. The stability of the vaccine was compromised by both high temperatures and dilution.

Short-term effect of bisphenol-a on oxidative stress responses in Atlantic salmon kidney cell line: a transcriptional study.

Bisphenol A (BPA) is regularly detected in aquatic ecosystems due to increased use of products based on polycarbonate plastics and epoxy resins. It migrates from these products directly into rivers and marine waters or indirectly through effluents from wastewater treatment plants and landfilled sites. BPA can affect aquatic organisms both chronically and acutely at sensitive live stages. Despite reports indicating harmful effects of BPA, little is known about its role in oxidative stress responses in fish. In this study, we investigated the transcriptional effect of BPA (0, 1, 10, 100 µM) on an Atlantic salmon kidney (ASK) cell line for 6 h and 24 h by monitoring expression of 11 genes: elongation factor 1-alpha (ef1a), 18S ribosomal RNA (18s), gluthation (gsh), superoxide dismutase (sod), thioredoxin (txd), Salmo salar oxidative stress-responsive serine-rich 1 (oxr), glucose-regulated protein 78 (grp78), heat shock protein 70 (hsp70), sequestosome1 (p62), interleukin-1 beta (il-1beta) and toll-like receptor 8 (tlr8). In general, only the 100 µM concentration treatment altered the mRNA expression. BPA down-regulated the expression of gsh and sod genes for both exposure-times while txd gene was the only down-regulated after 6-h exposure. The up-regulation of genes in the ASK cell line exposed for 6 h was only observed in il-1beta, while the 24-h exposure resulted in the up-regulation of oxr, tlr8, hsp70, p62 and il-1beta genes. The last three genes increased several fold compared to the others. The results showed that BPA exposure at 100 µM imposed oxidative stress on the ASK cell line and longer exposure time involved transcriptional responses of immune-related genes. This may indicate the possible role of BPA-associated oxidative stress in induction of inflammatory response in this macrophage-like cell type.

Reactive oxygen species and cytotoxicity in rainbow trout hepatocytes: effects of medium and incubation time.

This study evaluated the effects of exposure medium and culture age on intracellular reactive oxygen species (ROS) development and cytotoxicity in fish hepatocytes following exposure to copper (Cu). ROS was quantified using the fluorescent probes DHR 123 and CM-H2DCFDA following exposure to Cu in Leibovitz' medium (L-15) or Tris-buffered saline (TBS). Similarly, culture age effects were investigated using 1-, 2- and 4-day-old cultured hepatocytes by exposing them to Cu in TBS. The exposure in L-15 resulted in significantly higher ROS compared to TBS using CM-H2DCFDA, but not DHR 123. The age of the primary cultures significantly affected the development of ROS for both probes. None of the exposures caused cytotoxicity in the hepatocytes. The results showed that both factors may affect responses to stressors, and suggested that the use of a simple medium such as TBS may be preferable for some applications. It is also preferable to use 1-day-old primary hepatocyte cultures.

Effects of eicosapentaneoic acid on innate immune responses in an Atlantic salmon kidney cell line in vitro.

Studies of the interplay between metabolism and immunity, known as immunometabolism, is steadily transforming immunological research into new understandings of how environmental cues like diet are affecting innate and adaptive immune responses. The aim of this study was to explore antiviral transcriptomic responses under various levels of polyunsaturated fatty acid. Atlantic salmon kidney cells (ASK cell line) were incubated for one week in different levels of the unsaturated n-3 eicosapentaneoic acid (EPA) resulting in cellular levels ranging from 2-20% of total fatty acid. These cells were then stimulated with the viral mimic and interferon inducer poly I:C (30 ug/ml) for 24 hours before total RNA was isolated and sequenced for transcriptomic analyses. Up to 200 uM EPA had no detrimental effects on cell viability and induced very few transcriptional changes in these cells. However, in combination with poly I:C, our results shows that the level of EPA in the cellular membranes exert profound dose dependent effects of the transcriptional profiles induced by this treatment. Metabolic pathways like autophagy, apelin and VEGF signaling were attenuated by EPA whereas transcripts related to fatty acid metabolism, ferroptosis and the PPAR signaling pathways were upregulated. These results suggests that innate antiviral responses are heavily influenced by the fatty acid profile of salmonid cells and constitute another example of the strong linkage between general metabolic pathways and inflammatory responses.

Dermomyotome-derived endothelial cells migrate to the dorsal aorta to support hematopoietic stem cell emergence.

Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Here, we profile the transcriptome of the earliest detectable endothelial cells (ECs) during zebrafish embryogenesis to demonstrate that tissue-specific EC programs initiate much earlier than previously appreciated, by the end of gastrulation. Classic studies in the chick embryo showed that paraxial mesoderm generates a subset of somite-derived endothelial cells (SDECs) that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. We describe a conserved program in the zebrafish, where a rare population of endothelial precursors delaminates from the dermomyotome to incorporate exclusively into the developing dorsal aorta. Although SDECs lack hematopoietic potential, they act as a local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of ECs contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

Chitosan nanoparticle formulation attenuates poly (I:C) induced innate immune responses against inactivated virus vaccine in Atlantic salmon (Salmo salar).

Many vaccine formulations, in particular vaccines based on inactivated virus, needs adjuvants to boost immunogenicity. In aquaculture, mineral and plant oil are used as adjuvant in commercial vaccines, and the advent of oil-adjuvanted vaccines was crucial to aquaculture development. Nevertheless, some of these approved vaccines display suboptimal performance in the field compared to experimental conditions. Therefore, there is a need to improve adjuvants and delivery methods for fish vaccines against viruses. We used RNA sequencing of Atlantic salmon head kidney to analyse the difference in gene expression 24 h after injection of different experimental vaccine formulations. We compared five different formulations in addition to a PBS control: inactivated virus alone (group V), soluble poly (I:C) (group P), nanoparticles containing poly (I:C) (group N), soluble poly (I:C) + inactivated virus (group PV) and finally nanoparticles containing poly (I:C) + inactivated virus (group NV). Our results showed poly (I:C)'s ability as adjuvant and its capacity influence innate immune genes expression in Atlantic salmon. Soluble poly (I:C) upregulated multiple immune related genes and was more effective compared to poly (I:C) formulated into chitosan nanoparticles (more than 10 fold increase in differentially expressed genes, DEGs). However, inclusion of inactivated ISA virus in the nanoparticle vaccine, increased the number of DEGs fivefold suggesting a synergistic effect of adjuvant and antigen. Our results indicate that the way poly (I:C) is formulated and the presence of antigen is important for the magnitude of the innate immune response in Atlantic salmon.

In situ localisation of major histocompatibility complex class I and class II and CD8 positive cells in infectious salmon anaemia virus (ISAV)-infected Atlantic salmon.

It is assumed that the mobilisation of a strong cellular immune response is important for the survival of Atlantic salmon infected with infectious salmon anaemia virus (ISAV). In this study, the characterisation of immune cell populations in tissues of non-ISAV infected Atlantic salmon and during the early viraemia of ISAV was undertaken. Immunohistochemical investigations of spleen, head kidney and gills using monoclonal antibodies against recombinant proteins from MHC I, II and CD8 were performed on tissues from Atlantic salmon collected day 17 post-challenge in a cohabitant infection model. The localisations of MHC I and II in control salmon were consistent with previous reports but this study presents novel observations on the distribution of CD8 labelled cell populations in Atlantic salmon including the description of significant mucosal populations in the gills. The distribution of MHC I, MHC II and CD8 positive cell populations differed between control salmon and cohabitant salmon in the early stages of ISAV infection. The changes in MHC I labelled cells differed between organs in ISAV cohabitants but all investigated organs showed a decreased presence of MHC II labelled cells. Together with a clustering of CD8 labelled cells in the head kidney and a reduced presence of CD8 labelled cells in the gills, these observations support the early mobilisation of cellular immunity in the response of Atlantic salmon to ISAV infection. However, differences between the present study and the findings from studies investigating immune gene mRNA expression during ISAV infection suggest that viral strategies to interfere with protein expression and circumvent the host immune response could be operative in the early response to ISAV infection.

Kinetics of transcriptional response against poly (I:C) and infectious salmon anemia virus (ISAV) in Atlantic salmon kidney (ASK) cell line.

Vaccine adjuvants induce host innate immune responses improving long-lasting adaptive immunity against vaccine antigens. In vitro models can be used to compare these responses between adjuvants and the infection targeted by the vaccine. We utilized transcriptomic profiling of an Atlantic salmon cell line to compare innate immune responses against ISAV and an experimental viral vaccine adjuvant: poly (I:C). Induction of interferon and interferon induced genes were observed after both treatments, but often with different amplitude and kinetics. Using KEGG ortholog database and available software from Bioconductor we could specify a complete bioinformatic pipeline for analysis of transcriptomic data from Atlantic salmon, a feature not previously available. We have identified important differences in the transcriptional profile of Atlantic salmon cells exposed to viral infection and a viral vaccine adjuvant candidate, poly (I:C). This report increases our knowledge of viral host-pathogen interaction in salmon and to which extent these can be mimicked by adjuvant compounds.

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; effects of n-3 and n-9 fatty acids.

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.

Analysis of host- and strain-dependent cell death responses during infectious salmon anemia virus infection in vitro.

BACKGROUND: Infectious salmon anemia virus (ISAV) is an aquatic orthomyxovirus and the causative agent of infectious salmon anemia (ISA), a disease of great importance in the Atlantic salmon farming industry. In vitro, ISAV infection causes cytophatic effect (CPE) in cell lines from Atlantic salmon, leading to rounding and finally detachment of the cells from the substratum. In this study, we investigated the mode of cell death during in vitro ISAV infection in different Atlantic salmon cell lines, using four ISAV strains causing different mortality in vivo. RESULTS: The results show that caspase 3/7 activity increased during the course of infection in ASK and SHK-1 cells, infected cells showed increased surface expression of phosphatidylserine and increased PI uptake, compared to mock infected cells; and morphological alterations of the mitochondria were observed. Expression analysis of immune relevant genes revealed no correlation between in vivo mortality and expression, but good correlation in expression of interferon genes. CONCLUSION: Results from this study indicate that there is both strain and cell type dependent differences in the virus-host interaction during ISAV infection. This is important to bear in mind when extrapolating in vitro findings to the in vivo situation.

Interaction between dietary fatty acids and genotype on immune response in Atlantic salmon (Salmo salar) after vaccination: A transcriptome study.

A pivotal matter to aquaculture is the sourcing of sustainable resources as ingredients to aquafeeds. Levels of plant delivered oils as source of fatty acids (FA) in aquafeeds have reached around 70% resulting in reduced levels of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in salmon fillet composition. EPA and DHA can modulate inflammation and immune response, so it is crucial to understand how fish immune response is affected by low LC n-3 PUFA diet and if this diet can have a detrimental effect on vaccine response. Atlantic salmon (Salmo salar) can produce EPA/DHA from α-linolenic acid (ALA) and this endogenous capacity can be explored to develop families with higher tolerance to low LC n-3 PUFA diets. Here we analyze innate and adaptive immune response in Atlantic salmon to a commercial vaccine after being fed low levels of EPA and DHA, and we also compare three strains of salmon selected by their endogenous capacity of synthesizing LC- n-3 PUFA. A total of 2,890 differentially expressed genes (DEGs) were identified (p-value adjusted < 0.1) when comparing vaccinated fish against control non-vaccinated. Gene ontology (GO) and KEGG analysis with 442 up/downregulated genes revealed that most DEGs were both related to immune response as well as part of important immune related pathways, as "Toll-like receptor" and "Cytokine-Cytokine interaction". Adaptive response was also addressed by measuring antigen specific IgM, and titers were significantly higher than in the pre-immune fish at 62 days post-immunization. However, diet and strain had no/little effect on vaccine-specific IgM or innate immune responses. Atlantic salmon therefore display robustness in its response to vaccination even when feed low levels of LC n-3 PUFA.

Synthesis and biological evaluation of zinc chelating compounds as metallo-ß-lactamase inhibitors.

The syntheses of metallo-ß-lactamase inhibitors comprising chelating moieties, with varying zinc affinities, and peptides partly inspired from bacterial peptide sequences, have been undertaken. The zinc chelator strength was varied using the following chelators, arranged in order of ascending binding affinity: dipicolylamine (DPA, tridentate), dipicolyl-1,2,3-triazolylmethylamine (DPTA, tetradentate) dipicolyl ethylenediamine (DPED, tetradentate) and trispicolyl ethylenediamine (TPED, pentadentate). The chosen peptides were mainly based on the known sequence of the C-terminus of the bacterial peptidoglycan precursors. Biological evaluation on clinical bacterial isolates, harbouring either the NDM-1 or VIM-2 metallo-ß-lactamase, showed a clear relationship between the zinc chelator strength and restoration of meropenem activity. However, evaluation of toxicity on different cancer cell lines demonstrated a similar trend, and thus inclusion of the bacterial peptides did possess rather high toxicity towards eukaryotic cells.

Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells.

The aquatic orthomyxovirus infectious salmon anemia virus (ISAV) is an important pathogen for salmonid aquaculture, however little is known about protective and pathological host responses to infection. We have investigated intracellular responses during cytopathic ISAV infection in the macrophage-like Atlantic salmon kidney (ASK) cell line by microarray analysis (1.8k SFA2.0 immunochip) and a functional assay for glutathione. Gene transcription changed rapidly and consistently with time and with minor differences between two virus isolates. While several pro-inflammatory and antiviral immune genes were induced, genes involved in cell signaling and integrity were down-regulated, suggesting isolation of infected cells from cell-to-cell interaction and responses to external signals. Differential expression of genes regulating cell cycle and apoptosis implied opposite cues from host cell and virus. This was in pace with massive down-regulation of genes involved in biosynthesis and processing of nucleotides and nucleic acids. Significant down-regulation of several genes involved in metabolism of reactive oxygen species suggested increased oxidative stress, which was confirmed by a functional assay showing reduced levels of glutathione during infection. Testing of expression data against a microarray database containing diverse experiments revealed candidate marker genes for ISAV infection. Our findings provide novel insight into cellular host responses and determinants for acute cytopathic ISAV infection.

Antimicrobial Activity and Cytotoxicity of Ag(I) and Au(I) Pillarplexes.

The biological activity of four pillarplex compounds featuring different metals and anions was investigated. The toxicity of the compounds against four bacterial strains [Bacillus subtilis (ATCC6633), Staphylococcus aureus (ATCC6538), Escherichia coli (UVI isolate), Pseudomonas aeruginosa], one fungus (Candida albicans), and a human cell line (HepG2) was determined. Additionally, a UV-Vis titration study of the pillarplexes was carried out to check for stability depending on pH- and chloride concentration changes and evaluate the applicability in physiological media. All compounds are bioactive: the silver compounds showed higher activity against bacteria and fungi, and the corresponding gold pillarplexes were less toxic against human cells.

Synthesis and Preclinical Evaluation of TPA-Based Zinc Chelators as Metallo-ß-lactamase Inhibitors.

The rise of antimicrobial resistance (AMR) worldwide and the increasing spread of multi-drug-resistant organisms expressing metallo-ß-lactamases (MBL) require the development of efficient and clinically available MBL inhibitors. At present, no such inhibitor is available, and research is urgently needed to advance this field. We report herein the development, synthesis, and biological evaluation of chemical compounds based on the selective zinc chelator tris-picolylamine (TPA) that can restore the bactericidal activity of Meropenem (MEM) against Pseudomonas aeruginosa and Klebsiella pneumoniae expressing carbapenemases Verona integron-encoded metallo-ß-lactamase (VIM-2) and New Delhi metallo-ß-lactamase 1 (NDM-1), respectively. These adjuvants were prepared via standard chemical methods and evaluated in biological assays for potentiation of MEM against bacteria and toxicity (IC50) against HepG2 human liver carcinoma cells. One of the best compounds, 15, lowered the minimum inhibitory concentration (MIC) of MEM by a factor of 32-256 at 50 µM within all tested MBL-expressing clinical isolates and showed no activity toward serine carbapenemase expressing isolates. Biochemical assays with purified VIM-2 and NDM-1 and 15 resulted in inhibition kinetics with kinact/ KI of 12.5 min-1 mM-1 and 0.500 min-1 mM-1, respectively. The resistance frequency of 15 at 50 µM was in the range of 10-7 to 10-9. 15 showed good tolerance in HepG2 cells with an IC50 well above 100 µM, and an in vivo study in mice showed no acute toxic effects even at a dose of 128 mg/kg.

Cloning and expression analysis of an Atlantic salmon (Salmo salar L.) tapasin gene.

Loading of the major histocompatibility complex (MHC) class I molecule with peptide is mediated by the multimeric peptide-loading complex in the ER where the glycoprotein tapasin (TAPBP) is required for stabilization of the complex and for control of peptide loading onto MHC class I. To expand our knowledge on antigen presentation genes in Atlantic salmon, we isolated a full-length salmon tapasin cDNA sequence (Sasa-TAPBP). It encoded a 443 bp amino acid sequence with two N-glycosylation sites, two conserved mammalian tapasin signature motifs, two Ig superfamily (IgSf) domains, a transmembrane (TM) domain and an ER-retention KK motif at the C-terminal end, indicative of a similar function as mammalian tapasins. We analysed the regulation of Sasa-TAPBP under immunostimulatory conditions and found an mRNA-upregulation during early infectious salmon anemia virus (ISAV) infection and poly I:C stimulation in vivo and in vitro, in line with our previous findings for other MHC class I pathway genes.

Effects of dietary thia fatty acids on lipid composition, morphology and macrophage function of Atlantic salmon (Salmo salar L.) kidney.

High lipid levels are being used in modern salmonid diets to promote rapid growth; however there is a limiting supply of the traditional fish oils as the fish farming industry expands. One way to utilize the lipid sources better, could be to find ways to stimulate fatty acid (FA) oxidation so that Atlantic salmon use more energy for muscle growth and less for storage in perivisceral adipose tissue. We have previously shown that dietary inclusion of the thia FA tetradecylthioacetic acid (TTA) promoted hepatic beta-oxidation and reduced total body lipid levels. However, dietary TTA also had some negative effects, leading to accumulation of sulfone and sulfoxide metabolites of TTA in the kidney and increasing mortality rates, particularly at low water temperatures. Therefore we also wish to investigate the effects of TTA on kidney function at high and low temperatures, including some immune system parameters. The production of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) immunoreactive material from exogenously added arachidonic acid in isolated head kidney macrophages was affected by both diet and temperature. The phagocytic activity in these cells was reduced by DTA in the 12 degrees C group and there was significantly higher protein degradation in head kidney macrophages at 12 degrees C compared to 5 degrees C in all dietary groups. Interestingly, the incorporation of thia FAs in the kidney was higher at 5 degrees C (0.3% TTA and 0.6% DTA) than at 12 degrees C (0.1% TTA and 0.5% DTA). Additionally, there were lower levels of saturated FAs, while higher levels of polyunsaturated FAs (PUFAs) in the kidney of TTA fed fish at 5 degrees C. We also observed temperature-independent tubular dilatation and a reduction in the density of melanomacrophages of the kidney in salmon fed TTA. Nevertheless, the mRNA expression of some immune-relevant genes in head kidney tissue was not affected by TTA-inclusion in salmon diets. In conclusion, it is clear that 0.6% TTA-inclusion in the feed leads to changes in the kidney function particularly at low water temperatures.