Endothelial Senescence and Its Impact on Angiogenesis in Alzheimer's Disease.

Endothelial cells are constantly exposed to environmental stress factors that, above a certain threshold, trigger cellular senescence and apoptosis. The altered vascular function affects new vessel formation and endothelial fitness, contributing to the progression of age-related diseases. This narrative review highlights the complex interplay between senescence, oxidative stress, extracellular vesicles, and the extracellular matrix and emphasizes the crucial role of angiogenesis in aging and Alzheimer's disease. The interaction between the vascular and nervous systems is essential for the development of a healthy brain, especially since neurons are exceptionally dependent on nutrients carried by the blood. Therefore, anomalies in the delicate balance between pro- and antiangiogenic factors and the consequences of disrupted angiogenesis, such as misalignment, vascular leakage and disturbed blood flow, are responsible for neurodegeneration. The implications of altered non-productive angiogenesis in Alzheimer's disease due to dysregulated Delta-Notch and VEGF signaling are further explored. Additionally, potential therapeutic strategies such as exercise and caloric restriction to modulate angiogenesis and vascular aging and to mitigate the associated debilitating symptoms are discussed. Moreover, both the roles of extracellular vesicles in stress-induced senescence and as an early detection marker for Alzheimer's disease are considered. The intricate relationship between endothelial senescence and angiogenesis provides valuable insights into the mechanisms underlying angiogenesis-related disorders and opens avenues for future research and therapeutic interventions.

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble ß-Glucan.

ß-Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.

Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli.

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.

Serum Levels of Incretin Hormones - GLP-1 and GIP in Patients with Type 1 Diabetes Mellitus.

BACKGROUND: The glucagon-like peptide-1 (GLP-1) and the glucose- dependent Insulinotropic peptide (GIP) are natural incretin hormones, which are secreted respectively by the L- and K-cells of the intestinal mucosa in response to the physiological gastrointestinal glucose absorption. In patients with type 2 diabetes mellitus, the incretin effect is reduced, whereas the results in type 1 diabetes mellitus (T1DM) are heterogeneous, in some patients normal incretin response is observed. AIM: Comparative analysis of the basal serum levels of the incretin hormones GLP-1 and GIP in patients with type 1 DM and in individuals without carbohydrate disorders. MATERIALS AND METHODS: The study included 27 patients with diagnosed T1DM and a control group of 39 individuals without carbohydrate disorders. All participants in the study were subjected to the following clinical measurements and laboratory tests - height, weight, bioimpedance analysis of body composition, fasting blood sugar (BS 0'), postprandial blood sugar (PPBS), glycated haemoglobin (HbA1c) in T1DM patients, total cholesterol (TC), HDL cholesterol (HDL chol), triglycerides (TG), transaminase (AST and ALT), basal serum levels of GLP-1 and GIP. RESULTS: The serum levels of GIP in the patients with type T1DM were significantly higher, compared to the individuals without carbohydrate disorders (P<0.05), while there was no statistically significant difference in the GLP-1 levels. CONCLUSION: The significantly higher GIP levels and the similar GLP-1 levels in our patients with type 1 DM, compared to the individuals without carbohydrate disorders, support the hypothesis of intact incretin effect in this type of diabetes mellitus Key Words: Glucagon-like peptide-1, Glucose-dependent insulinotropic peptide, Type 1 diabetes mellitus.

CpGs Induce Differentiation of Atlantic Salmon Mononuclear Phagocytes Into Cells With Dendritic Morphology and a Proinflammatory Transcriptional Profile but an Exhausted Allostimulatory Activity.

Due to their ability to present foreign antigens and prime naïve T cells, macrophages, and dendritic cells (DCs) are referred to as professional antigen-presenting cells (APCs). Although activated macrophages may function as APCs, these cells are particularly effective at directly engaging pathogens through phagocytosis, and production of antimicrobial compounds. On the other hand, DCs possess superb antigen-presenting and costimulatory capacity and they are essential for commencement and regulation of adaptive immune responses. In in vitro models, development of mature mammalian DCs from monocytes requires sequential exposure to growth factors (including GM-CSF and IL-4) and proinflammatory stimuli such as toll-like receptor (TLR) ligands. Currently, except for IL-4/13, neither orthologs nor functional analogs of the growth factors which are essential for the differentiation of mammalian DCs (including GM-CSF and FLT3) have been identified in teleosts and data about differentiation of piscine APCs is scant. In the present study, primary salmon mononuclear phagocytes (MPs) stimulated in vitro for 5-7 days with a B-class CpG oligodeoxynucleotides (ODN 2006PS) underwent morphological differentiation and developed "dendritic" morphology, characterized by long, branching pseudopodia. Transcriptional profiling showed that these cells expressed high levels of proinflammatory mediators characteristic for M1 polarized MPs. However, the cells treated with CpGs for 7 days downregulated their surface MHCII molecules as well as their capacity to endocytose ovalbumin and exhibited attenuated allostimulatory activity. This concurred with transcriptional downregulation of costimulatory CD80/86 and upregulation of inhibitory CD274 (B7-H1) genes. Despite their exhausted allostimulatory activity, these cells were still responsive to re-stimulation with gardiquimod (a TLR7/8 ligand) and further upregulated a wide array of immune genes including proinflammatory mediators such as intereukin-1 beta and tumor necrosis factor. Overall, the presented data highlight the disparate effects TLR ligands may have on the proinflammatory status of APCs, on one side, and their antigen-presenting/costimulatory functions, on the other. These findings also indicate that despite the poor phylogenetic conservation of the growth factors involved in the differentiation of DCs, some of the processes that orchestrate the development and the differentiation of professional APCs are conserved between teleosts in mammals.

Stimulation of exosome release by extracellular DNA is conserved across multiple cell types.

Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30-100 nm) and their composition which reflects their endo-lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide-induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll-like receptor 9-negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B-II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome-related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA - their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions.

Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

Functional conservation of suppressors of cytokine signaling proteins between teleosts and mammals: Atlantic salmon SOCS1 binds to JAK/STAT family members and suppresses type I and II IFN signaling.

Suppressor of cytokine signaling (SOCS) proteins are crucially involved in the control of inflammatory responses through their impact on various signaling pathways including the JAK/STAT pathway. Although all SOCS protein family members are identified in teleost fish, their functional properties in non-mammalian vertebrates have not been extensively studied. To gain further insight into SOCS functions in bony fish, we have identified and characterized the Atlantic salmon (Salmo salar) SOCS1, SOCS2 and CISH genes. These genes exhibited sequence conservation with their mammalian counterparts and they were ubiquitously expressed. SOCS1 in mammalian species has been recognized as a key negative regulator of interferon (IFN) signaling and recent data for the two model fish Tetraodon (Tetraodon nigroviridis) and zebrafish (Danio rerio) suggest that these functions are conserved from teleost to mammals. In agreement with this we here demonstrate a strong negative regulatory activity of salmon SOCS1 on type I and type II IFN signaling, while SOCS2a and b and CISH only moderately affected IFN responses. SOCS1 also inhibited IFNγ-induced nuclear localization of STAT1 and a direct interaction between SOCS1 and STAT1 and between SOCS1 and the Tyk2 kinase was found. Using SOCS1 mutants lacking either the KIR domain or the ESS, SH2 and SOCS box domains showed that all domains affected the ability of SOCS1 to inhibit IFN-mediated signaling. These results are the first to demonstrate that SOCS1 is a potent inhibitor of IFN-mediated JAK-STAT signaling in teleost fish.

The Atlantic salmon protein tyrosine kinase Tyk2: molecular cloning, modulation of expression and function.

Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-α/ß binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto-phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overexpressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway.

CpG- and LPS-activated MAPK signaling in in vitro cultured salmon (Salmo salar) mononuclear phagocytes.

The Mitogen-activated protein kinases (MAPK) are involved in transmitting intracellular signals downstream of diverse cell surface receptors and mediate the response to ligands such as growth factors, hormones and cytokines. In addition, MAPK are critically involved in the innate immune response to pathogen-derived substances, commonly referred to as pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS) and bacterial DNA rich in CpG dinucleotides. Currently, a great deal of knowledge is available about the involvement of MAPK in the innate immune response to PAMPs in mammals; however, little is known about the role of the different MAPK classes in the immune response to PAMPs in lower vertebrates. In the current study, p38 phosphorylation was induced by CpG oligonucleotides (ODNs) and LPS in primary salmon mononuclear phagocytes. Pre-treatment of the cells with a p38 inhibitor (SB203580) blocked the PAMP-induced p38 activity and suppressed the upregulation of most of the CpG- and LPS-induced transcripts highlighting the role of this kinase in the salmon innate immune response to PAMPs. In contrast to p38, the phosphorylation of extracellular signal-regulated kinase (ERK), a MAPK involved primarily in response to mitogens, was high in resting cells and, surprisingly, incubation with both CpG and control ODNs downregulated the phospho-ERK levels independently of p38 activation. The basal phospho-ERK level and the CpG-inducible p38 phosphorylation were greatly influenced by the length of in vitro incubation. The basal phospho-ERK level increased gradually throughout a 5-day culture period and was PI3K-dependent as demonstrated by its sensitivity to Wortmannin suggesting it is influenced by growth factors. Overall these data indicate that both basal and PAMP-induced activity of MAPKs might be greatly influenced by the differentiation status of salmon mononuclear phagocytes.

Homing of Antigen-Presenting Cells in Head Kidney and Spleen - Salmon Head Kidney Hosts Diverse APC Types.

Lymph nodes and spleen are major organs where mammalian antigen-presenting cells (APCs) initiate and orchestrate Ag-specific immune responses. Unlike mammals, teleosts lack lymph nodes and an interesting question is whether alternative organs may serve as sites for antigen presentation in teleosts. In the current study, fluorescent ovalbumin (Ova) and CpG oligonucleotides (ODNs) injected intra-abdominally were detected in significant numbers of salmon head kidney (HK) MHCII+ cells over a period of 2 weeks while in spleen the percentage of these was transient and declined from day 1 post injection. In vitro studies further shed light on the properties of the diverse MHCII+ cell types found in HK. The ultrastructure of a subpopulation of MHCII+ cells with a high capacity to endocytose and process Ova indicated that these were able to perform constitutive macropinocytosis. Upon stimulation with CpG ODNs these cells upregulated CD86 and gave very high levels of TNF mRNA indicating that these are professional APCs, related to macrophages and dendritic cells (DCs). A subpopulation of HK granulocytes expressed high levels of surface MHCII and upon CpG stimulation upregulated most of the tested APC marker genes. Although these granulocytes expressed TNF weakly, they had relatively high basal levels of IL-1ß mRNA and the CpG stimulation upregulated IL-1ß, along with its signaling and decoy receptors, to the highest levels as compared to other HK cell types. Interestingly, the high expression of IL-1ß mRNA in the granulocytes correlated with a high autophagy flux as demonstrated by LC3-II conversion. Autophagy has recently been found to be implicated in IL-1ß processing and secretion and the presented data suggests that granulocytes of salmon, and perhaps other teleost species, may serve as a valuable model to study the involvement of autophagy in regulation of the vertebrate immune response.

Contact angle hysteresis and meniscus corrugation on randomly heterogeneous surfaces with mesa-type defects.

The results of a numerical study of the various characteristics of the static contact of a liquid meniscus with a flat but heterogeneous surface, consisting of two types of homogeneous materials, forming regularly and randomly distributed microscopic defects are presented. The solutions for the meniscus shape are obtained numerically using the full expression of the system free energy functional. The goal is to establish how the magnitude and the limits of the hysteresis interval of the equilibrium contact angle, the Cassie's angle, and the contact line (CL) roughness exponent are related to the parameters, characterizing the heterogeneous surface-the equilibrium contact angles on the two materials and their fractions. We compare the results of different ways of determining the averaged contact angle on heterogeneous surfaces. We study the spread of the CL corrugation along the liquid meniscus. We compare our results with the numerical results, obtained using linearized energy functional, and also with experimental results for the CL roughness exponent. The obtained results support the conclusion that some characteristics depends on the type (regular or random) of the heterogeneity pattern.

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.

CD40L--a costimulatory molecule involved in the maturation of antigen presenting cells in Atlantic salmon (Salmo salar).

The CD40L/CD40 signalling pathway is critically involved in the final stage of the maturation of DCs. This paper reports the identification and functional characterization of CD40L and CD40 from Atlantic salmon (Salmo salar). Salmon CD40L is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, while CD40 is a type I membrane-bound receptor with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The salmon CD40L and CD40 were widely expressed, particularly in immune tissues, and while CD40L expression was induced by in vitro stimulation of HKLs with PHA and ConA, CpG increased CD40 expression. A CD40L construct was overexpressed in the CHSE-214 cell line and co-cultivation of the CD40L-CHSE transfectants with HKL induced a rapid and long-lasting upregulation of important costimulatory molecules like CD40, CD83, B7-H1 and the cytokines IL-12p40, IL-10, IL-1ß and IFNs, which all are involved in T-helper cell responses. Furthermore, the CD40L transfected cells increased the percentage of HKLs expressing surface MHCIIß but unlike other APC maturation stimuli, like CpG, they did not reduce the capacity to internalise antigen. Our results provide the first evidence for the existence of a functional CD40L mediated costimulatory pathway in Atlantic salmon.

Immunoprotective activity of a Salmonid Alphavirus Vaccine: comparison of the immune responses induced by inactivated whole virus antigen formulations based on CpG class B oligonucleotides and poly I:C alone or combined with an oil adjuvant.

CpG oligonucleotides and polyinosinic:polycytidylic acid (poly I:C) are toll-like receptor (TLR) agonists that mimic the immunostimulatory properties of bacterial DNA and double-stranded viral RNA respectively, and which have exhibited potential to serve as vaccine adjuvants in previous experiments. Here, a combination of CpGs and poly I:C together with water- or oil-formulated Salmonid Alphavirus (SAV) antigen preparations has been used for a vaccine in Atlantic salmon and tested for protection in SAV challenge trial. The results demonstrate that vaccination with a high dose of the SAV antigen induced protection against challenge with SAV which correlated with production of neutralizing antibodies (NAbs). As the high antigen dose alone induced full protection, no beneficial effect from the addition of CpG and poly I:C could be observed. Nevertheless, these TLR ligands significantly enhanced the levels of NAbs in serum of vaccinated fish. Interestingly, gene expression analysis demonstrated that while addition of oil suppressed the CpG/poly I:C-induced expression of IFN-γ, the upregulation of IFNa1 was substantially enhanced. A low dose of the SAV antigen combined with oil did not induce any detectable levels of NAbs either with or without TLR ligands present, however the addition of CpG and poly I:C to the low SAV antigen dose formulation significantly enhanced the protection against SAV suggesting that CpG/poly I:C may have enhanced a cytotoxic response - a process which is dependent on the up-regulation of type I IFN. These results highlight the immunostimulatory properties of the tested TLR ligands and will serve as a ground for further, more detailed studies aimed to investigate their capacity to serve as adjuvants in vaccine formulations for Atlantic salmon.

MyD88 interacts with interferon regulatory factor (IRF) 3 and IRF7 in Atlantic salmon (Salmo salar): transgenic SsMyD88 modulates the IRF-induced type I interferon response and accumulates in aggresomes.

MyD88 is an intracellular adaptor protein that transmits signals downstream of immune receptors such as the IL-1 receptor and the majority of the known mammalian toll-like receptors. Homologs of MyD88 have been identified in many vertebrate species; however, the adaptor has been studied mostly in mammals, and little is known about its function in lower vertebrates. The results presented in the current paper demonstrate, for the first time, that the teleost MyD88, through its Toll/Interleukin-1 receptor domain, interacts with SsIRF3 and two SsIRF7 paralogs: transcription factors that are critically involved in the virus-induced IFN responses. The data further highlight the potential of transgenic SsMyD88 to modulate the IRF-induced type I IFN response as the adaptor synergized with SsIRF3 to activate IRF-E/IFN-stimulated response element-containing reporter gene constructs and endogenous myxovirus resistance homolog expression. Microscopy analyses demonstrated that, similar to mammalian MyD88, both endogenous and transgenic SsMyD88 accumulated in intracellular aggregates. However, unlike the endogenous SsMyD88 clusters, which co-localized with endocytosed CpGs and probably represented myddosomes, overexpressed SsMyD88 accumulated in aggresomes. Although these structures accumulated ubiquitinated proteins, they did not associate with the autophagosome markers p62 and light chain 3-like protein, indicating that they are most likely classical aggresomes rather than aggresome-like induced structures, aggregates of ubiquitinated proteins induced by toll-like receptor/MyD88 signaling in antigen-presenting cells. The significance of the accumulation of transgenic MyD88 in aggresomes is currently unknown; nevertheless it is tempting to speculate that it might represent a defense mechanism against the potentially harmful effects of excessive MyD88 signaling.

Genomic survey of early responses to viruses in Atlantic salmon, Salmo salar L.

Viral diseases are one of the main problems and risk factors in aquaculture. At present diseases are diagnosed by detection of pathogens and clinical symptoms. Identification of genes involved in early responses to viruses is important for better knowledge of antiviral defence and development of diagnostic tools. The aim of this study was to search for gene markers common for viral infections in Atlantic salmon based on microarray analyses of a wide range of samples. Gene expression profiles from fish and cell cultures infected with different viruses and treated with the synthetic double-stranded RNA poly(I:C) were compared in order to identify virus responsive genes (VRG). The list of VRG defined in this study contained 117 genes with known or unidentified functions. Several genes, including the most highly ranked one (receptor transporting protein), had not been previously reported to be involved in antiviral defence. VRG were characterized by a rapid induction and low tissue specificity, and their expression levels were related to the viral load. Immunofluorescence analyses of proteins encoded by VRG in cardiac tissue of salmon with the viral disease cardiomyopathy syndrome (CMS) revealed a common expression pattern. In head kidney leukocytes VRG showed comparable or equal responses to CpG and poly(I:C), which mimic respectively bacterial DNA and viral RNA. Most VRG showed highly correlated expression with interferon-a (IFNa). Sequence comparison of salmon VRG with those from other species gave an understanding of the evolution of these genes, which showed a remarkably rapid sequence divergence in comparison with the entire proteome. VRG emerged both before and after separation of teleosts and tetrapods, and among genes found exclusively in fish species there were members of several multigene families: tripartite motif proteins, gig1- and gig2-like proteins. Several VRG, including genes with unknown functions and orthologs to mammalian RNA helicase RIG-I and chemokine C-X-C type 10, were present in cyprinid and salmonid fish but not in the phylogenetically advanced orders, suggesting that they have been lost in the evolution of Teleostei. Apparently, a number of genes involved in antiviral responses in salmon have acquired different functional roles in higher vertebrates.

Formulations combining CpG containing oliogonucleotides and poly I:C enhance the magnitude of immune responses and protection against pancreas disease in Atlantic salmon.

Both CpG oligodeoxynucleotides and double-stranded RNA (poly I:C) have documented effects as treatments against several viral diseases in fish. However, as stand-alone treatments their effects have been modest. We have tested here whether CpG and poly I:C, alone or in combination induce protection against Salmonid Alphavirus (SAV), the causative agent of pancreas disease in Atlantic salmon. Our results revealed a significant reduction of viraemia 2 weeks after ip injection of the combined treatment and 1 week after challenge with SAV subtype 3, followed by reduced SAV induced heart pathology 3 weeks later. The SAV titers in blood samples from the combination group were lower as compared to single treatments with either CpG or poly I:C. Surprisingly, reduced SAV levels could also be found in fish as long as 7 weeks after receiving the combination treatment. The expression of IFNγ and to a lesser extent IFNa and Mx was up-regulated in head kidney and spleen 5 days after the fish had been treated with CpG and poly I:C. Furthermore, the complement factor C4 was depleted in serum 8 weeks post treatment, suggesting complement activation leading to C4 consumption. We hypothesize that the CpG/poly I:C-induced protection against SAV3 is mediated by mechanisms involving type I and type II IFN induced antiviral activity and complement mediated protective responses.

Asymptotic solutions for the relaxation of the contact line in the Wilhelmy-plate geometry: The contact line dissipation approach.

The relaxation of straight contact lines is considered in the context of the Wilhelmy-plate experiment: a homogeneous solid plate is moving vertically at constant velocity in a tank of liquid in the partial wetting regime. We apply the contact line dissipation approach to describe the quasistatic relaxation of the contact line toward the stationary state (below the entrainment transition). Asymptotic solutions are derived from the differential equations describing the capillary rise height and the contact angle relaxation for small initial deviations of the height from the final stationary value in the model considering the friction dissipation at the moving contact line, in the model considering the viscous flow dissipation in the wedge, and in the combined model taking into account both channels of dissipation. We find that for all models the time relaxation of the height and the cosine of the contact angle are given by sums of exponential functions up to a second order in the expansion of the small parameter. We analyze the implications which follow when only one dissipation channel is taken into account and compare them to the case when both dissipation channels are included. The asymptotic solutions are compared with experimental results and with numerically obtained solutions which are based on hydrodynamic approach in lubrication approximation with and without a correction factor for finite contact angles. The best description of the experimental data, based on multicriteria testing, is obtained with the combined contact line dissipation model which takes into account both channels of dissipation.

CpG-induced secretion of MHCIIbeta and exosomes from salmon (Salmo salar) APCs.

Major histocompatibility complex class II (MHCII) is encoded by polymorphic genes present in vertebrates and expressed predominately in leukocytes. Upon leukocyte differentiation, intracellular MHCII is dynamically redistributed within the cells and it is expressed at maximal levels on mature antigen presenting cells (APCs). In addition, APCs secrete MHCII within endosome-derived vesicles known as exosomes which possess diverse immunomodulatory properties. Genetic and biochemical data have confirmed that piscine leukocytes express the MHCII components as well as costimulatory molecules that are necessary for the function of APCs. However data concerning the biosynthesis and the distribution of the MHCII complex within leukocytes of lower vertebrates is scarce. The presented data demonstrates for the first time that salmon leukocytes secrete vesicles that contain exosomal markers and the abundance of MHCII indicates that these exosomes are released by APCs. The secretion was specifically induced by CpG stimulation in vitro and it was observed only in head kidney leukocytes but not in splenocyte cultures. Flow cytometry revealed that, unlike splenocytes, the majority of the MHCII-positive head kidney leukocytes were Ig-negative and a population of cells expressing high levels of surface MHCII underwent degranulation upon CpG stimulation suggesting that the MHCII-containing exosomes were derived from maturing salmon APCs. Gene expression analyses have further demonstrated that CpG-B, despite its relatively weak proinflammatory activity compared to LPS, induced expression of a larger group of genes involved in regulation of the adaptive immune response.