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Macrophage polarization is an important effector process in acute lung injury (ALI) induced by sepsis. MicroRNAs (miRNAs) have emerged as important players in regulating ALI process. Here, we showed that elevated microRNA‐23a‐3p (miR‐23a‐3p) promoted LPS‐induced macrophage polarization and ALI in mice, while inhibition of miR‐23a‐3p led to reduced macrophage response and ameliorated ALI inflammation. Mechanically, miR‐23a‐3p regulated macrophage M1 polarization through targeting polo‐like kinase 1 (PLK1). PLK1 was downregulated in LPS‐treated macrophages and ALI mouse lung tissues. Knockdown of PLK1 increased macrophage M1 polarization through promoting STAT1/STAT3 activation, while overexpression of PLK1 reduced macrophage immune response. Collectively, our results reveal a key miRNA regulon that regulates macrophage polarization for LPS‐induced immune response. [ABSTRACT FROM AUTHOR]

Japanese flounder (Paralichthys olivaceus) is one of the most economically important marine fish cultured in north Asia. Vibrio anguillarum is a severe bacterial pathogen to Japanese flounder and many other aquaculture species. In order to understand the immune response of flounder during bacterial infection, we systematically examined the transcriptome profiles of flounder spleen at three time points after V. anguillarum challenge. More than one billion high quality reads were obtained, approximately 80.70% of which were successfully mapped to the reference genome of flounder. A total of 6060, 4688 and 4235 differentially expressed genes (DEGs) were captured at 6, 12 and 24-h post-infection, respectively. The DEGs exhibited dynamic changes in expression and were assigned into four different profiles based on expression trend. GO and KEGG analysis showed that the DEGs were enriched in various immune-related terms, including response to stimulation, immune system and pathways of cytokine-cytokine receptor interaction, Jak-STAT signaling and Toll-like receptor signaling. Furthermore, a network of highly interactive DEGs involved in 11 immune-related pathways was detected by utilizing the weighted co-expressing network analysis (WGCNA). Accordingly, 26 hub genes were discovered that constituted an elaborate immune regulatory network and functioned mainly in pathogen recognition, antigen processing, and molecular signaling. The results of this study provided the first systematical transcriptome profile of flounder in association with V. anguillarum infection and can serve as a valuable resource of target genes for future studies on the molecular mechanisms underlying the immune defense of flounder against bacterial infection. • Vibrio anguillarum -induced transcriptome profiles of flounder were investigated. • A total of 8923 differentially expressed genes (DEGs) were found at 6, 12 and 24 hpi. • Co-expression networks of immune relevant DEGs were constructed. • Twenty-six hub genes with multiple interactions were identified. • The hub genes represented mainly genes involved in immune defense against pathogens. [ABSTRACT FROM AUTHOR]

Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense. [ABSTRACT FROM AUTHOR]

The imidazoquinoline compound R848 is a specific agonist of toll-like receptor (TLR) 7/TLR8 that has been used as an immunostimulant in humans against viral diseases. Although R848-induced immune response has been reported in teleost fish, the relevant mechanism is not clear. In this study, we investigated the antiviral potential and the signaling pathway of R848 in a model of Japanese flounder ( Paralichthys olivaceus ). We found that R848 was able to inhibit the replication of megalocytivirus, stimulated the proliferation of peripheral blood leukocytes (PBL), enhanced the expression of immune genes, and reduced apoptosis of PBL. When endosomal acidification was blocked by chloroquine (CQ), R848-mediated antiviral activity and immune response were significantly reduced. Likewise, inhibition of Myd88 activation markedly impaired the pro-proliferation and anti-apoptosis effect of R848. Cellular study showed that cultured founder cells treated with R848 exhibited augmented NF-κB activity, which, however, was dramatically reduced in the presence of CQ and Myd88 inhibitor. Furthermore, when NF-κB was inactivated, the effect of R848 on cell proliferation and apoptosis was significantly decreased. Taken together, these results indicate that R848 is an immunostimulant with antiviral property in a teleost species, and that the immune response of R848 is mediated by, most likely, TLR7/TLR8 signaling pathway, in which Myd88 and NK-κB play an essential role. [ABSTRACT FROM AUTHOR]

Inflammation is involved in the development and/or progression of diabetic nephropathy (DN). Curcumin has been reported for its anti-inflammation activity in DN. However, the mechanisms involved in the renoprotective effects of curcumin have not been clearly demonstrated. In this study, we hypothesized that curcumin affected high glucose (HG)-induced inflammation profiles in vivo and in vitro and then prevented renal injury in diabetic rats via reversing cav-1 Tyr 14 phosphorylation that influenced TLR4 activation. Streptozotocin (STZ)-induced diabetic rats received vehicle or curcumin for twelve weeks and podocytes were treated with HG in the presence or absence of curcumin in vitro. To further evaluate the effect of cav-1 phosphorylation at Tyr 14 on HG-induced podocyte inflammation response and TLR4 activation, a recombinant plasmid GFP-Cav-1 Y14F with a mutated phosphorylation site of cav-1, was transfected into cultured podocytes. In vivo, curcumin improved histological abnormalities and fibrosis of a diabetic kidney, inhibited renal inflammatory gene expression and reduced cav-1 phosphorylation at Tyr 14 and the expression of TLR4. Pretreatment of podocytes with curcumin reduced HG-stimulated production of proinflammatory cytokines, TLR4 and the phosphorylation of cav-1. But immunohistochemistry in rat kidney showed that the elevation of TLR4 expression is more evident in the renal interstitum than in the glomerulus where podocytes are located, and the possibility that the anti-inflammatory effects of curcumin on other cells in the kidney may be mediated through the same molecular pathways as in podocytes. Our study suggests that curcumin treatment ameliorates DN via inhibition of inflammatory gene expression by reversing caveolin-1 Tyr 14 phosphorylation that influenced TLR4 activation. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGF-β), interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF) are three of the commonly studied cytokines playing an important role in tumor initiation and progression. Besides their promotional effects on tumor progression, the three cytokines have immunosuppressive effects that facilitate tumor initiation and progression as well. Ganoderma lucidum polysaccharides ( Gl-PS) with multiple bioactivities may have the effect on B16F10 melanoma cells to induce stronger antitumor immune response that has been demonstrated. Gl-PS may have the suppressive effects on the production of these three cytokines, which has yet to be demonstrated. In this study, we tested these effects of Gl-PS by incubating Gl-PS with malignant tumor cells such as B16F10 cells, a melanoma cell line, and LA795 cells, a lung carcinoma cell line. RT-qPCR and enzyme-linked immunosorbent assay showed that the production of TGF-β1, IL-10, and VEGF in B16F10 melanoma cells and LA795 lung carcinoma cells was suppressed by Gl-PS at both mRNA and protein levels, suggesting that the suppression on production of TGF-β, IL-10, and VEGF in B16F10 melanoma cells and LA795 lung carcinoma cells by Gl-PS may contribute to the therapy on melanoma and lung carcinoma along with the induction of stronger antitumor immune response. [ABSTRACT FROM AUTHOR]

Cryptococcosis causes significant morbidity and mortality in the world. Pulmonary cryptococcosis is a kind of subacute or chronic pulmonary fungal disease. We present a case of pulmonary cryptococcosis with a trachea wall invasion-like malignant tumor in an immunocompetent patient and a literature review. The 44-year-old man, a nonsmoker, suffered from mild dyspnea and white sputum with intermittent blood streaks. A computed tomography (CT) scan of his chest showed two possibly malignant lesions in the right hilum and upper-right field of his lung, which have higher uptake values of fluorodeoxyglucose on positron emission tomography (PET)/CT. Lung biopsy pathology showed scattered fungal spores and positive periodic acid-Schiff (PAS) staining. The immune status and blood tumor markers were all normal in this patient. The titer of Cryptococcus antigen latex agglutination test was 1:1,280. Under fiberoptic bronchoscopy, a prominent new mass on the right wall of the trachea blocked most of the right main bronchus. To reduce the symptoms of airway obstruction, treatment by bronchoscopy, i.e. ablation and endotracheal stent, was used. As his symptoms were aggravated by the use of itraconazole, amphotericin B liposome was used as antifungal treatment. All these methods led to a better prognosis. We conclude that pulmonary cryptococcosis may mimic lung neoplasms radiologically and bronchoscopically, even in immunocompetent patients. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Tissue factor pathway inhibitor 1 (TFPI-1) is a Kunitz-type serine protease inhibitor that regulates the activation of tissue factor-induced coagulation. In teleosts, TFPI-1-like sequences have been found to exist in two species (Danio rerio and Cyprinus carpio); however, the potential function of fish TFPI-1 has not been investigated. In this study, we identified and analyzed a TFPI-1 homologue, SoTFPI-1, from red drum (Sciaenops ocellatus). The deduced amino acid sequence of SoTFPI-1 is 284 residues in length and contains three Kunitz domains, an acidic N-terminus, and a basic C-terminus. SoTFPI-1 shares 49.5% and 46.9% overall sequence identities with the TFPI-1 of D. rerio and C. carpio, respectively. Quantitative real time RT-PCR analysis showed that constitutive SoTFPI-1 expression occurred, in increasing order, in kidney, brain, liver, gill, blood, spleen, muscle, and heart. Bacterial infection and lipopolysaccharide exposure upregulated SoTFPI-1 expression in kidney in time-dependent manners. Recombinant SoTFPI-1 (rSoTFPI-1) purified from Escherichia coli exhibits not only serine protease inhibitor activity but also bactericidal activity in a manner that is independent of any host factors. A synthetic peptide, TO17, corresponding to the C-terminal basic region of SoTFPI-1 also possesses antibacterial effect that is more potent than that of the full-length rSoTFPI-1. Taken together, these results demonstrate that (i) SoTFPI-1 is a biologically active serine protease inhibitor endowed with bactericidal property; (ii) provide the first indication that teleost TFPI-1 is likely to be involved in anti-microbial infection and thus is linked to innate immune defense. [Copyright &y& Elsevier]

Background: Anti-PD-1 immunotherapy has emerged as an important therapeutic modality in advanced gastric cancer (GC). However, drug resistance frequently develops, limiting its effectiveness. Methods: The role of gastric cancer mesenchymal stem cells (GCMSCs) in anti-PD-1 resistance was evaluated in vivo in NPGCD34+ or NCGPBMC xenograft mouse model. In addition, we investigated CD8+T cell infiltration and effector function by spectral cytometry and IHC. The effects of GCMSCs conditional medium (GCMSC-CM) on GC cell lines were characterized at the level of the proteome, secretome using western blot, and ELISA assays. Results: We reported that GCMSCs mediated tolerance mechanisms contribute to tumor immunotherapy tolerance. GCMSC-CM attenuated the antitumor activity of PD-1 antibody and inhibited immune response in humanized mouse model. In GC cells under serum deprivation and hypoxia, GCMSC-CM promoted GC cells proliferation via upregulating PD-L1 expression. Mechanistically, GCMSC-derived IL-8 and AKT-mediated phosphorylation facilitated HK2 nuclear localization. Phosphorylated-HK2 promoted PD-L1 transcription by binding to HIF-1α. What is more, GCMSC-CM also induced lactate overproduction in GC cells in vitro and xenograft tumors in vivo, leading to impaired function of CD8+ T cells. Furthermore, CXCR1/2 receptor depletion, CXCR2 receptor antagonist AZD5069 and IL-8 neutralizing antibody application also significantly reversed GCMSCs mediated immunosuppression, restoring the antitumor capacity of PD-1 antibody. Conclusions: Our findings reveal that blocking GCMSCs-derived IL-8/CXCR2 pathway decreasing PD-L1 expression and lactate production, improving antitumor efficacy of anti-PD-1 immunotherapy, may be of value for the treatment of advanced gastric carcinoma. [ABSTRACT FROM AUTHOR]

Anti-lipopolysaccharide factor (ALF) is a type of antimicrobial peptides (AMPs) with a vital role in antimicrobial defense. Although a large amount of ALFs have been identified from neritic and fresh water crustacean species, no functional investigation of ALFs from deep-sea animals have been documented. In the present study, we characterized the immune function of an ALF molecule (named RspALF1) from the shrimp Rimicaris sp. residing in the deep-sea hydrothermal vent in Desmos, Manus Basin. RspALF1 shares 51.5%–62.4% overall sequence identities with known shrimp ALFs and contains the conserved LPS binding domain (LBD). Both recombinant RspALF1 (rRspALF1) and the LBD-derived peptide (ALF1P1) bound to the cell wall components of Gram-negative and Gram-positive bacteria and killed a wide range of bacteria, especially those from deep-sea hydrothermal field, by damaging bacterial cellular structures. The bactericidal activities of rRspALF1 and ALF1P1 were optimal and stably maintained from 4 °C to 37 °C, which is comparable to the ambient temperature range of the habitat of Rimicaris sp. In addition to bacteria, rRspALF1 and ALF1P1 also exhibited anti-fungal activity. rRspALF1 and ALF1P1 exhibited high killing efficiencies, which, in terms of MIC values, were ranged between 0.25 μM and 4 μM for bacteria and 4 μM–8 μM for fungi. When introduced in vivo , both rRspALF1 and ALF1P1 effectively inhibited bacterial infection in shrimp and reduced the dissemination of bacterial and viral pathogens in fish. Together, these results provide the first insight into the biological property of deep-sea ALF and indicate that RspALF1 very likely plays a significant role in immune defense by functioning as a highly effective antimicrobial with a broad target range. [ABSTRACT FROM AUTHOR]

In mammals, CD83 is a surface marker on mature dendritic cells and vital to lymphocyte activation. In teleost, studies on the function of CD83 are very limited. In this study, we examined the potential involvement of turbot ( Scophthalmus maximus ) CD83, SmCD83, in vaccine-induced immunity. For this purpose, turbot were immunized with pORF75, a DNA vaccine against megalocytivirus, in the presence or absence of pSmCD83, a plasmid that constitutively expresses SmCD83. Immune response and protection analysis showed that the presence of pSmCD83 significantly (i) enhanced the activation of head kidney macrophages (HKM) and immune gene expression, (ii) inhibited viral replication in fish tissues following megalocytivirus challenge and increased the survival of the vaccinated fish, and (iii) stimulated production of specific serum antibody and the cytotoxicity of peripheral blood leukocytes. To further examine the effect of SmCD83, pORF75 was administered into turbot in which SmCD83 was knocked down. Subsequent analysis showed that in fish with SmCD83 knockdown, vaccine-induced HKM activation and antibody production were severely reduced, and, consistently, the protectivity of pORF75 was drastically decreased. Taken together, these results indicate for the first time that teleost CD83 is required for the induction of protective immune response by DNA vaccine. [ABSTRACT FROM AUTHOR]

Polyinosinic:polycytidylic acid (poly(I:C)) is a ligand of toll-like receptor (TLR) 3 that has been used as an immunostimulant in humans and mice against viral diseases based on its ability to enhance innate and adapt immunity. Antiviral effect of poly(I:C) has also been observed in teleost, however, the underling mechanism is not clear. In this study, we investigated the potential and signaling mechanism of poly(I:C) as an antiviral agent in a model of Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus. We found that poly(I:C) exhibited strong antiviral activity and enhanced activation of head kidney macrophages and peripheral blood leukocytes. In vivo studies showed that (i) TLR3 as well as MDA5 knockdown reduced poly(I:C)-mediated immune response and antiviral activity to significant extents; (ii) when Myd88 was overexpressed in flounder, poly(I:C)-mediated antiviral activity was significantly decreased; (iii) when Myd88 was inactivated, the antiviral effect of poly(I:C) was significantly increased. Cellular study showed that (i) the NF-κB activity induced by poly(I:C) was upregulated in Myd88-overexpressing cells and unaffected in Myd88-inactivated cells; (ii) Myd88 overexpression inhibited and upregulated the expression of poly(I:C)-induced antiviral genes and inflammatory genes respectively; (iii) Myd88 inactivation enhanced the expression of the antiviral genes induced by poly(I:C). Taken together, these results indicate that poly(I:C) is an immunostimulant with antiviral potential, and that the immune response of poly(I:C) requires TLR3 and MDA5 and is negatively regulated by Myd88 in a manner not involving NK-κB. These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish. [ABSTRACT FROM AUTHOR]

NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Highlights: [•] Three CpG ODNs that inhibited megalocytivirus replication in flounder were selected. [•] ODN C7 was able to promote leukocyte and phagocyte activation. [•] Knockdown of toll-like receptor 9 expression blocked C7-mediated immune response. [•] C7 exhibited adjuvant property and augmented the protection of a DNA vaccine. [•] C7 enhanced the expression of vaccine-induced genes of innate and adaptive immunity. [ABSTRACT FROM AUTHOR]

Highlights: [•] SmLMWPTP is a low molecular weight protein tyrosine phosphatase from turbot. [•] rSmLMWPTP exhibited optimum tyrosine phosphatase activity at pH 5 and 50°C. [•] Peripheral blood leukocytes (PBL) infected by pathogen expressed and secreted SmLMWPTP. [•] rSmLMWPTP reduced the biological activity, gene expression, and phagocytosis of PBL. [•] Mutation of the P-loop sequence abolished the regulatory effect of SmLMWPTP on PBL. [ABSTRACT FROM AUTHOR]

Abstract: IκBα is a member of the NF-κB inhibitor family that inhibits NF-κB activity by sequestering NF-κB in an inactive form in the cytosol. Unlike mammalian IκBα, which has been extensively studied, very little is known about the function of fish IκBα. In this study, we identified and analyzed an IκBα homologue, CsIκBα from half-smooth tongue sole (Cynoglossus semilaevis), a marine flatfish with important economic value. The deduced amino acid sequence of CsIκBα contains 308 residues and shares 58–82% overall sequence identities with the IκBα of a number of teleosts. In silico analysis identified in CsIκBα conserved domains that in mammals are known to be involved in phosphorylation, ubiquitination, and degradation of IκBα. Quantitative real time RT-PCR detected constitutive expression of CsIκBα in gut, spleen, liver, gill, heart, brain, muscle, and kidney. Experimental challenge with a bacterial pathogen-induced significant inductions of CsIκBα expression in head and trunk kidney, which, however, were transient and much lower in magnitude than that of interleukin-1β. To examine the effect of unregulated overexpression of CsIκBα in a live fish model, tongue sole were administered via intramuscular injection with plasmid pCNCsIkBa, which constitutively expresses CsIκBα. PCR, RT-PCR, and immunohistochemistry analysis showed that pCNCsIkBa was able to translocate to internal tissues, where transcription and translation of the recombinant CsIκBα took place. Compared to control fish, fish administered with pCNCsIkBa were impaired in the ability to block bacterial dissemination and survival in kidney and exhibited significantly reduced expression of multiple immune genes. These results suggest the possible existence in tongue sole of a NF-κB–IκBα signaling pathway that is negatively regulated by CsIκBα and required for effective defense against bacterial infection. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is a Gram-negative bacterial pathogen and the etiological agent of a systematic fish disease called edwardsiellosis, which affects a wide range of marine and freshwater fish. E. tarda vaccines in various forms have been reported by a number of research groups; however, comparative studies on the immune mechanisms of these vaccines are lacking. In this report, we identified a new E. tarda vaccine candidate, Eta2, and analyzed in a comparative manner the immune response induced by Eta2 in two different forms: purified recombinant subunit vaccine and DNA vaccine. Eta2 is a protein of 178 residues and shares high levels of sequence identities with the OmpH family of outer membrane protein chaperones of several bacterial species. Recombinant Eta2 (rEta2) purified from Escherichia coli was highly protective against E. tarda challenge in a Japanese flounder (Paralichthys olivaceus) model and produced relative percent of survival rates of 83% and 78%, respectively, at 4- and 8-week post-vaccination (p.v.). Eta2 as a DNA vaccine in the form of plasmid pCEta2 also induced strong protective immunity at 4- and 8-week p.v. Immunological analysis indicated that (i) rEta2 and pCEta2 enhanced head kidney macrophage activation at 1- and, for pCEta2, 7-day p.v.; (ii) rEta2 and pCEta2 induced similar patterns of serum antibody production, however, the antibodies induced by rEta2 were of much higher levels and afforded stronger passive immunoprotection upon naïve flounder than those induced by pCEta2; (iii) both rEta2 and pCEta2 upregulated the expression of specific and nonspecific immune factors which include, in the case of pCEta2 but not rEta2, interferon, interferon-induced Mx protein, and CD8α; however, the induction patterns effected by rEta2 and pCEta2 were different. While high levels of interleukin 1β (IL-1β), natural killer cell enhancing factor, Mx, MHC Iα, and IgM inductions were observed in pCEta2-vaccinated fish, only IL-1β, complement C3, and IgM inductions were highly induced in rEta2-vaccinated fish. Taken together, these results indicate that both rEta2 and pCEta2 induce specific and nonspecific immunities, however, pCEta2 induces both B cell and T cell responses, whereas rEta2 induces mainly humoral response. [Copyright &y& Elsevier]

Abstract: In the present study, a 71.43kDa heat shock protein cDNA was cloned from Asiatic hard clam Meretrix meretrix. The cDNA was 2292bp, containing an open reading frame (ORF) of 1959bp, which encodes a protein of 652 amino acids with a theoretical molecular weight of 71.43kDa and an isoelectric point of 5.32. Based on the amino acid sequence analysis and phylogenetic analysis, this hsp70 cDNA is a member of cytoplasmic hsc70 (constitutive genes) subfamily in the hsp70 family, and is designated as MmeHsc71. Quantitative RT-PCR was carried out to compare the spatial and temporal expression patterns of MmeHsc71 in the mRNA level between control clams and Vibrio parahaemolyticus-infected clams. Spatially, MmeHsc71 mRNA was found in all tested tissues, including foot, hepatopancreas, mantle and gill. MmeHsc71 mRNA expression level in hepatopancreas and gill displayed a significant increase in vibrio-challenged clams at 24h post-infection compared to control clams (P <0.05). Temporally, there was a significant increase of MmeHsc71 mRNA level in hepathopancreas of vibrio-challenged clams compared to control clams at 6, 12, and 24h post-challenge, respectively. The result of quantitative immunofluorescence also indicated that there was obvious increase of MmeHsc71 in hepatopancreas of vibrio-challenged clams compared to control clams in protein level at 24h post-infection. The results suggested that MmeHsc71 may play an important role in mediating the immune responses of M. meretrix to bacterial challenge. [Copyright &y& Elsevier]

Abstract: Lysozyme is a muramidase that inflicts damage on bacterial cell wall by catalyzing the cleavage of the beta-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan. Lysozymes are classified into several types, one of which is the goose-type (g-type). In this study, we identified and analyzed a g-type lysozyme (SmLysG) from turbot Scophthalmus maximus. The deduced amino acid sequence of SmLysG contains 193 residues and is most closely related to that of the g-type lysozyme of Scophthalmus rhombus (94% overall identity). SmLysG possesses a Goose Egg White Lysozyme (GEWL) domain with conserved residues essential for catalytic activity. Recombinant SmLysG (rSmLysG) purified from yeast exhibits strong lysozyme activity against Micrococcus luteus. Enzyme assays showed that the optimal temperature and pH of rSmLysG are 30°C and pH 7.0, respectively. Substrate spectrum analysis indicated that rSmLysG inhibited the growth of a number of important fish pathogens of both Gram-negative and Gram-positive natures. SmLysG transcription was detected in multiple tissues and was upregulated in kidney and spleen by experimental challenges with lipopolysaccharide and bacterial pathogens that are, respectively, sensitive to and resistant against the lytic effect of rSmLysG. Comparative analysis showed that although bacterial infection also induced the expression of c-type lysozyme, the induction levels were much lower than those of SmLysG. Taken together, these results indicate that SmLysG is a functional g-type lysozyme with a wide working range and is involved in innate immune defense against general bacterial infection. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is a Gram-negative bacterium that can infect a wide variety of marine and freshwater fish and cause severe economic losses worldwide. With an aim to elucidate the virulence mechanism of E. tarda, we in this study cloned and analyzed the function of an iron-cofactored superoxide dismutase (FeSOD) from a pathogenic E. tarda strain TX01 isolated from diseased fish. FeSOD is 192-residue in length and contains domain structures that are conserved among iron/manganese superoxide dismutases. Recombinant FeSOD purified from Escherichia coli exhibits apparent superoxide dismutase activity. Quantitative real-time RT-PCR analysis indicated that FeSOD expression is significantly upregulated immediately following TX01 infection of Japanese flounder (Paralichthys olivaceus) head kidney (HK) macrophages and cultured FG cells. Compared to the wild type strain TX01, the FeSOD mutant strain TXSod is (i) more sensitive to H2O2-induced oxidative damage, (ii) less resistant against serum- and macrophage-mediated bacterial killing, (iii) significantly weakened in the ability to invade into FG cells and to disseminate in fish blood and liver, and (iv) deficient in blocking macrophage respiratory burst activity and production of reactive oxygen species. Furthermore, HK macrophages infected by TXSod exhibits significantly increased expression of inflammatory cytokines compared to macrophages infected by TX01. Taken together, these results indicate that FeSOD is a virulence factor that plays an important role in the pathogenicity of E. tarda by inhibiting macrophage-mediated innate immune response. [ABSTRACT FROM AUTHOR]

Abstract: C2 domains are protein structural modules found in many eukaryotic proteins involved in signal transduction, membrane trafficking, and immune defense. Most of the studied C2 domain-containing proteins are multi-domained in structure, in which the C2 domain is an independently folded motif and plays an essential role in calcium-dependent membrane-targeting. Although C2 domains isolated from intact proteins have been studied for biological functions, no study on natural proteins containing C2 domain only has been documented. In this study, we identified a Scophthalmus maximus protein SmC2P1 that is comprised of a single C2 domain and lacks any other apparent domain structures. The deduced amino acid sequence of SmC2P1 contains 129 residues and shares 36–38% identities with the C2 domains of the perforins of several fish species. Like typical C2 domains, SmC2P1 is predicted to organize into eight β-strands with a Ca2+-binding site located in inter-strand loops. SmC2P1 expression was detected, in deceasing order, in liver, spleen, blood, brain, muscle, kidney, gill, and heart. Experimental challenge of turbot with a bacterial pathogen significantly upregulated SmC2P1 expression in kidney in a time-dependent manner. Recombinant SmC2P1 purified from yeast exhibits no hemolytic activity but binds to pathogen-infected kidney lymphocytes in the presence of calcium. Furthermore, interaction of recombinant SmC2P1 with bacterium-infected lymphocytes reduced bacterial survival. These results indicate that SmC2P1 is a functional protein that is involved in host immune defense against bacterial infection. [Copyright &y& Elsevier]

Interleukin (IL)-34 is a relatively recently discovered cytokine with pleiotropic effects on various cellular activities, including immune response. In fish, the knowledge on the function of IL-34 is limited. In the present work, we investigated the function of Japanese flounder Paralichthys olivaceus IL-34 (PoIL-34) in association with inflammation and immune defense. PoIL-34 possesses the conserved structure of IL-34 superfamily and shares 21.52% sequence identity with murine IL-34. PoIL-34 expression was detected in a wide range of tissues of flounder, in particular intestine, and was regulated to a significant extent by bacterial infection in a time-dependent fashion. In vitro studies showed that recombinant PoIL-34 (rPoIL-34) bound peripheral blood leukocytes (PBLs) and promoted ROS production, acid phosphatase activity, and cellular resistance against bacterial infection. At the molecular level, rPoIL-34 enhanced the expressions of inflammatory cytokines and specific JAK and STAT genes. Similar stimulatory effects of rPoIL-34 were observed in vivo. When PoIL-34 was overexpressed in flounder, the expressions of pro- and anti-inflammatory mediators were significantly affected in a tissue-dependent manner, which correlated with an augmented ability of the fish to eliminate invading pathogens from tissues. Together, these results indicated that PoIL-34 regulated inflammatory response probably via specific JAK/STAT pathways and had a significant influence on the immune defense of flounder against bacterial infection. • PoIL-34 expression occurred in multi-tissues and responded to bacterial infection. • rPoIL-34 bound to and promoted the activation and bacterial resistance of PBLs. • rPoIL-34 regulated the expressions of inflammatory cytokines and specific JAK/STAT. • In vivo PoIL-34 overexpession affected the expressions of inflammatory cytokines. • PoIL-34 overexpession reduced bacterial dissemination in fish tissues. [ABSTRACT FROM AUTHOR]

Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are involved in many biological activities including immune defense against pathogens. In this study, we applied high-throughput sequencing technology to examine miRNAs in Japanese flounder (Paralichthys olivaceus) infected with Streptococcus iniae at different times. A total of 1038 miRNAs were identified, of which, 249 were novel miRNAs, and 81 showed differential expression (named DEmiRNAs) after S. iniae infection. Of the 81 DEmiRNAs identified, 34 and 58 occurred at 6 h and 24 h post-infection, respectively; most DEmiRNAs were strongly time-specific, and only 13.6% of the DEmiRNAs were shared between the two time points. A total of 9582 target genes were predicted for the 81 DEmiRNAs. The putative target genes were enriched in various GO and KEGG pathways of biological processes and molecular/cellular functions, in particular endocytosis, regulation of transcription, lysososme, and the signaling pathways of MAPK, ErbB, and AMPK. One of the DEmiRNAs, pol-3p-10740_175, was found to target dual specificity phosphatase 6 (Dusp6) and repress the expression of the latter. Transfection of flounder FG cells with pol-3p-10740_175 caused a significant inhibition on S. iniae invasion. The results of this study provided the first S. iniae -induced miRNA profile in Japanese flounder and indicated that flounder miRNAs play an important role in antibacterial immunity. • Streptococcus iniae infection altered the expression of 81 miRNAs in flounder. • There were 34 and 58 differentially expressed miRNAs at 6 and 24 hpi, respectively. • The putative target genes of the miRNAs were enriched in various GO/KEGG pathways. • One miRNA, pol-3p-10740_175, negatively regulated dual specificity phosphatase 6. • Overexpression of pol-3p-10740_175 significantly inhibited S. iniae invasion. [ABSTRACT FROM AUTHOR]

Bmal1 is the key component of the central molecular clock that mediates the circadian control of various biological activities including immune reactions. In fish, the role of Bmal1 in immunity is unclear. In this study, we examined the expressional regulation and potential role of Japanese flounder Bmal1 (named PoBmal1) in pathogen associated immune response. PoBmal1 shares 85% identity with its human counterpart and possesses the conserved helix-loop-helix domain and PAS domain. PoBmal1 transcript was distributed extensively in nine tissues and altered to significant extents during the infection of two bacterial pathogens. PoBmal1 , as well as IL-1β, IL-6, IL-8, and TNF-α, exhibited oscillating expression through the light-dark cycle in a tissue-dependent manner, and the expression patterns of the cytokines were largely similar to that of PoBmal1 , both being higher during the photophase. Knockdown of PoBmal1 reduced the expressions of two clock genes (Cry1 and Cry2) and IL-1β, IL-6, IL-8, and TNF-α in a time-dependent manner. During bacterial infection, fish with PoBmal1 knockdown exhibited significantly increased expression of inflammatory cytokines in a tissue- and time-dependent fashion, which largely correlated with significantly decreased bacterial dissemination in tissues. Taken together, our study revealed that flounder Bmal1 exerted a regulatory effect on inflammation response in a manner that was situation-dependent, and that Bmal1 had a significant impact on the outcome of bacterial infection. These results provided new insights into the immune function of fish Bmal1. • PoBmal1 expression occurred in 9 tissues and was regulated by bacterial infection. • PoBmal1 and inflammatory cytokine expressions oscillated similarly with light input. • PoBmal1 knockdown impaired the expressions of Cry and inflammatory cytokines. • PoBmal1 knockdown enhanced inflammatory cytokine expression during bacterial infection. • PoBmal1 knockdown reduced bacterial dissemination in fish tissues. [ABSTRACT FROM AUTHOR]

Highlights • Salmonella Typhimurium and S. Stanley are two most prevalent serogroup B serogroup to infect human, but differ in prevalence. • Genetically diverse S. Typhimurium and clonally disseminated S. Stanley differed in immune response with higher phagocytized number, lower intracellular survival, higher ROS production and higher inflammation cytokine IL-1β expression by dead bacteria for S. Stanley, that may lead low prevalence of S. Stanley. Abstract Salmonella Typhimurium and S. Stanley are the most prevalent serogroup B serovars to infect humans in Taiwan. The aim was to determine possible factors to influence the prevalence between S. Typhimurium and S. Stanley. Genotypes were determined by pulsed field gel electrophoresis (PFGE) analysis and the intracellular survival, phagocytosis, reactive oxygen species (ROS) production of human monocyte THP-1 cell and tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), and IL-1βexpression in peripheral blood CD14+ cells after infection were analyzed. 182 S. Stanley was clonal disseminated with main pulsotypes 2 from 2004 to 2007. Overall S. Typhimurium evolved more genotypes, while S. Stanley conserved in genotypes. Human blood CD14+ monocytes expressed TNF-α, IL-6 and IL-1β differently among serovars and bacterial conditions (live vs. killed). Live S. Stanley and S. Typhimurium suppressed the TNF-α and IL-6 expression compared to killed bacteria. However, live S. Typhimurium stimulated more IL-1β expression than the killed bacteria, but S. Stanley expressed similar IL-1β levels in both conditions. Furthermore, S. Stanley and S. Typhimurium differed in intracellular survival in the THP-1 cells, an early decrease for S. Stanley, not for S. Typhimurium. Additionally, higher reactive oxygen species (ROS) production in THP-1 cells was found agsinst S. Stanley infection, not found in S. Typhimurium. However, some isolates of S. Stanley could recover from early loss to become more in the monocytes than S. Typhimurium. Difference in phagocytized number, intracellular survival, ROS production and IL-1β expression may contribute to prevalence different between two serovars. [ABSTRACT FROM AUTHOR]

Viral infections frequently lead to the activation of host innate immune signaling pathways involved in the defense against invading pathogens. To ensure their survival, viruses have evolved sophisticated mechanisms to overcome the host immune responses. The present study demonstrated for the first time that infectious hematopoietic necrosis virus (IHNV) activated NF-κB pathway in fish cells. We further identified that the IHNV L protein could activate the NF-κB signaling pathway and that IHNV NV functioned as an inhibitor of NF-κB activation. Further results demonstrated that the NV protein blocked the degradation of the inhibitor of NF-κB (IκBα) and suppressed the SeV-induced NF-κB nuclear translocation. In conclusion, our study explored the functions of different IHNV proteins on NF-κB activation, and revealed a potential mechanism by which IHNV evades innate immune responses. [ABSTRACT FROM AUTHOR]

Intranasal vaccination is more potent than parenteral injection for the prevention of influenza. However, because the poor efficiency of antigen uptake across the nasal mucosa is a key issue, immunostimulatory adjuvants are essential for intranasal vaccines. The immunomodulator mannatide or polyactin (PA) has been used for the clinical treatment of impaired immunity in China, but its adjuvant effect on an inactivated trivalent influenza vaccine (ITIV) via intranasal vaccination is unclear. To explore the adjuvant effect of PA, an inactivated trivalent influenza virus with or without PA or MF59 was instilled intranasally once a week in BALB/c mice. Humoral immunity was assessed by both the ELISA and hemagglutination inhibition (HI) methods using antigen-specific antibodies. Splenic lymphocyte proliferation and the IFN-γ level were measured to evaluate cell-mediated immunity. The post-vaccination serum HI antibody geometric mean titers (GMTs) for the H1N1 and H3N2 strains, antigen-specific serum IgG and IgA GMTs, mucosal SIgA GMT, splenic lymphocyte proliferation, and IFN-γ were significantly increased in the high-dose PA-adjuvanted vaccine group. The seroconversion rate and the mucosal response for the H3N2 strain were significantly elevated after high-dose PA administration. These adjuvant effects of high-dose PA for the influenza vaccine were comparable with those of the MF59 adjuvant, and abnormal signs or pathological changes were not found in the evaluated organs. In conclusion, PA is a novel mucosal adjuvant for intranasal vaccination with the ITIV that has safe and effective mucosal adjuvanticity in mice and successfully induces both serum and mucosal antibody responses and a cell-mediated response. [ABSTRACT FROM AUTHOR]

Streptococcus iniae is a Gram-positive bacterium and a severe pathogen to a wide range of economically important fish species. In addition, S. iniae is also a zoonotic pathogen and can cause serious infections in humans. In this study, we identified from a pathogenic S. iniae strain a putative bacteriocin, Sil, and examined its biological activity. Sil is composed of 101 amino acid residues and shares 35.6% overall sequence identity with the lactococcin 972 of Lactococcus lactis. Immunoblot analysis showed that Sil was secreted by S. iniae into the extracellular milieu. Purified recombinant Sil (rSil) exhibited a dose-dependent inhibitory effect on the growth of Bacillus subtilis but had no impact on the growths of other 16 Gram-positive bacteria and 10 Gram-negative bacteria representing 23 different bacterial species. Treatment of rSil by heating at 50°C abolished the activity of rSil. rSil bound to the surface of B. subtilis but induced no killing of the target cells. Cellular study revealed that rSil interacted with turbot (Scophthalmus maximus) head kidney monocytes and inhibited the innate immune response of the cells, which led to enhanced cellular infection of S. iniae. Antibody blocking of the extracellular Sil produced by S. iniae significantly attenuated the infectivity of S. iniae. Consistent with these in vitro observations, in vivo study showed that administration of turbot with rSil prior to S. iniae infection significantly increased bacterial dissemination and colonization in fish tissues. Taken together, these results indicate that Sil is a novel virulence-associated bacteriostatic and an immunoregulator that promotes S. iniae infection by impairing the immune defense of host fish. [ABSTRACT FROM AUTHOR]

Highlights: [•] CsEBI3 expression was detected in multiple tissues and induced by pathogen infection. [•] CsEBI3 was secreted by peripheral blood leukocytes (PBL) upon bacterial stimulation. [•] rCsEBI3 enhanced the immune response and bacterial resistance of PBL. [•] Antibody blocking of CsEBI3 on PBL reduced cell resistance to bacterial invasion. [•] The immunoregulatory function of rCsEBI3 depends on the FN3 domain of the protein. [Copyright &y& Elsevier]

Highlights: [•] SoMIF is a macrophage migration inhibitory factor from red drum. [•] SoMIF was expressed in multiple tissues and induced by bacterial and viral infection. [•] SoMIF was secreted by head kidney (HK) leukocytes. [•] rSoMIF inhibited the migration of HK monocytes and lymphocytes. [•] Fish received rSoMIF showed enhanced monocyte activation and bacterial clearance. [Copyright &y& Elsevier]

Background: Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine. Methods: Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA). Results: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively. Conclusion: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndrome (MDS) is a group of progressive,clonal, neoplastic bone marrow disorders characterized by hematopoietic stem cell dysregulation and abnormalities in the immune system. Mesenchymal stem cells (MSC) appear to modulate the immune system at the very first step of the immune response through the inhibition of dendritic cells (DCs) differentiation and maturation. However, it is still unclear whether the effects of MSC on the development of DCs will be altered with disease state. In addition, it is not clear whether there are differences in the effects between low-risk and high-risk MDS-MSC on DCs development. In this study, our data confirm that MDS-MSC mediate a potent inhibition of DCs differentiation. Additionaly, MDS-MSC greatly alter DCs functions, including endocytosis, IL-12 secretion, their ability to inhibit T cell proliferation. Moreover, our results show that there are major differences in DCs development and function between low-risk and high-risk MDS-MSC. Compared to high-risk MDS-MSC, low-risk MDS-MSC is characterized by a poor ability to inhibit DCs differentiation and maturation; and correspondingly, less dysfunctional DC endocytosis, mildly decreased IL-12 secretion, and a reduction in DC-mediated inhibition of T cell proliferation. Finally, our results demonstrate that MDS-MSC derived TGF-β1 is largely responsible for the inhitory effects. These results elucidate the different immunoregulatory role of MSC in low-risk and high-risk MDS on DCs development, which may be important for understanding the pathogenesis of MDS and the development of novel immune therapies for the treatment of MDS. [ABSTRACT FROM AUTHOR]

Abstract: The effects of BCG heat shock protein 70 (BCG HSP70) gene transfection on tumorigenicity and immunogenicity of murine lymphocytic leukemia cell line (L1210) were studied. After HSP70 gene transfection, the tumor cells became strongly immunogenic and lost their tumorigenicity in syngeneic mice. It mainly exhibited that tumor growth was slow or without the formation of tumor, mean survival time of mice was significantly prolonged and a marked stimulating effect on L1210 specific Th1 cells detected by IFN-γ ELISPOT assay. Tumor-bearing mice treated with the L1210-HSP70 cells showed thorough coagulation necrosis and abundant CD8+ T lymphocyte infiltration. Meanwhile, as the tumor vaccine, the HSP70-transfected tumor cells could induce a protective immune response in vivo. It showed that the tumor growth was significantly inhibited, tumor diameter was markedly reduced and the survival time of tumor-bearing mice was further prolonged. Immunization with it also resulted in regression of the established L1210 tumor and prolonged survival time of mice. These results suggest that gene transfection of BCG HSP70 could effectively improve the immunogenicity of tumor cells and it may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy. [Copyright &y& Elsevier]

Abstract: Iridoviruses are large double-stranded DNA viruses with icosahedral capsid. The Iridoviridae family contains five genera, one of which is Megalocytivirus. Megalocytivirus has emerged in recent years as an important pathogen to a wide range of marine and freshwater fish. In this study, we aimed at developing effective genetic vaccines against megalocytivirus affecting farmed fish in China. For this purpose, we constructed seven DNA vaccines based on seven genes of rock bream iridovirus isolate 1 from China (RBIV-C1), a megalocytivirus with a host range that includes Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). The protective potentials of these vaccines were examined in a turbot model. The results showed that after vaccination via intramuscular injection, the vaccine plasmids were distributed in spleen, kidney, muscle, and liver, and transcription of the vaccine genes and production of the vaccine proteins were detected in these tissues. Following challenge with a lethal-dose of RBIV-C1, fish vaccinated with four of the seven DNA vaccines exhibited significantly higher levels of survival compared to control fish. Of these four protective DNA vaccines, pCN86, which is a plasmid that expresses an 86-residue viral protein, induced the highest protection. Immunological analysis showed that pCN86 was able to (i) stimulate the respiratory burst of head kidney macrophages at 14 d, 21 d, and 28 d post-vaccination, (ii) upregulate the expression of immune relevant genes involved in innate and adaptive immunity, and (iii) induce production of serum antibodies that, when incubated with RBIV-C1 before infection, significantly reduced viral loads in kidney and spleen following viral infection of turbot. Taken together, these results indicate that pCN86 is an effective DNA vaccine that may be used in the control of megalocytivirus-associated diseases in aquaculture. [Copyright &y& Elsevier]

Abstract: Heat shock protein (Hsp) 70 is a molecular chaperone that plays an important role in protein folding and transport. In addition, Hsp70 is also involved in regulation of innate and adaptive immune response. In this study, we examined the biological activity and the immunomodulatory property of an Hsp70 homologue, PoHsp70, from Japanese flounder (Paralichthys olivaceus). Recombinant PoHsp70 purified from Escherichia coli exhibits apparent ATPase activity; however, a mutant PoHsp70, PoHsp70M, that bears mutation of the ATPase-associated domain, was completely abolished in activity. Expression of PoHsp70 was upregulated in a time-dependent manner by vaccination of flounder with a DNA vaccine, pSia10, that expresses a Streptococcus iniae antigen, Sia10. To examine whether PoHsp70 possessed any adjuvant potential, the DNA vaccine plasmids pSia10Hsp70 and pSia10Hsp70M were constructed. pSia10Hsp70 co-expresses Sia10 and PoHsp70, while pSia10Hsp70M co-expresses Sia10 and PoHsp70M. Following vaccination of flounder, production of Sia10 plus PoHsp70 and Sia10 plus PoHsp70M was detected in pSia10Hsp70- and pSia10Hsp70M-vaccinated fish respectively. At one month post-vaccination, comparable levels of serum antibodies were detected in fish vaccinated with pSia10Hsp70, pSia10Hsp70M, and pSia10. Subsequent protection analysis showed that, following S. iniae challenge, pSia10Hsp70 induced a survival rate that was significantly higher than that induced by pSia10, while pSia10Hsp70M induced a survival rate similar to that induced by pSia10. These results indicate that PoHsp70 is an effective adjuvant and that the adjuvanticity of PoHsp70 requires the intrinsic ATPase activity. [Copyright &y& Elsevier]

Abstract: The immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that include various immunoregulatory receptors such as novel immune type receptors (NITRs), which are a family of diversified proteins found exclusively in bony fish. In this study, we identified and analyzed an IgSF protein, SoIgSF1, from red drum (Sciaenops ocellatus). SoIgSF1 is composed of 225 amino acid residues and moderately related to teleost NITRs. In silico analysis indicated that SoIgSF1 is a type I transmembrane glycoprotein and contains an N-terminal signal peptide sequence, a single extracellular immunoglobulin V domain, a transmembrane region, and a cytoplasmic region. However, unlike most NITRs, the cytoplasmic region of SoIgSF1 exhibits no consensus inhibitory or stimulatory signaling sequences but has two tyrosine-containing motifs that conform to the right-half sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM). Quantitative real time RT-PCR analysis showed that SoIgSF1 expression occurred mainly in immune organs and was drastically induced by viral and bacterial infection. Immunofluorescence microscopy indicated that viral infection of head kidney (HK) leukocytes induced surface expression of SoIgSF1, which was able to interact with antibodies against recombinant SoIgSF1. Antibody cross-linking of SoIgSF1 on HK leukocytes inhibited the expression of immune relevant genes and promoted viral and bacterial infection. Taken together, these results indicate that SoIgSF1, though lacking canonical intracellular signaling motifs, is involved in regulation of host immune response during pathogen infection possibly by functioning as a negative signaling receptor through a novel mechanism. [Copyright &y& Elsevier]

The major histocompatibility complex (MHC) plays an important role in immune response. Avian MHCs are not well characterized, only reporting highly compact Galliformes MHCs and extensively fragmented zebra finch MHC. We report the first genomic structure of an endangered Pelecaniformes (crested ibis) MHC containing 54 genes in three regions spanning ~500 kb. In contrast to the loose BG (26 loci within 265 kb) and Class I (11 within 150) genomic structures, the Core Region is condensed (17 within 85). Furthermore, this Region exhibits a COL11A2 gene, followed by four tandem MHC class II αβ dyads retaining two suites of anciently duplicated 'αβ' lineages. Thus, the crested ibis MHC structure is entirely different from the known avian MHC architectures but similar to that of mammalian MHCs, suggesting that the fundamental structure of ancestral avian class II MHCs should be 'COL11A2-IIαβ1-IIαβ2.' The gene structures, residue characteristics, and expression levels of the five class I genes reveal inter-locus functional divergence. However, phylogenetic analysis indicates that these five genes generate a well-supported intra-species clade, showing evidence for recent duplications. Our analyses suggest dramatic structural variation among avian MHC lineages, help elucidate avian MHC evolution, and provide a foundation for future conservation studies. [ABSTRACT FROM AUTHOR]

Abstract: CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC IIα, IL-1β, Mx, and MHC Iα. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. [Copyright &y& Elsevier]

Abstract: Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class IIα, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. [Copyright &y& Elsevier]

Abstract: Vibrio harveyi is an important aquaculture pathogen that can infect a number of fish species and marine invertebrates. A putative protease, Vhp1, was identified from a pathogenic V. harveyi strain isolated from diseased fish as a protein with secretion capacity. Vhp1 is 530 amino acids in length and shares high sequence identities with several extracellular serine proteases of the Vibrio species. In silico analysis identified a protease domain in Vhp1, which is preceded by a subtilisin-N domain and followed by a bacterial pre-peptidase C-terminal domain. Purified recombinant protein corresponding to the protease domain of Vhp1 exhibited apparent proteolytic activity that was relatively heat-stable and reached maximum at pH 8.0 and 50°C. The activity of purified recombinant Vhp1 protease was enhanced by Ca2+ and inhibited by Mn2+ and ethylenedinitrilotetraacetic acid. Cytotoxicity analyses indicated that recombinant Vhp1 protease was toxic to cultured Japanese flounder cells and could cause complete cell lysis. Immunoprotective analysis using Japanese flounder as an animal model showed that purified recombinant Vhp1 in the form of a denatured and proteolytically inactive protein was an effective subunit vaccine. To improve the vaccine potential of Vhp1, an Escherichia coli strain that expresses and secrets a cytotoxically impaired Vhp1 was constructed, which, when used as a live vaccine, afforded a high level of protection upon the vaccinated fish against lethal V. harveyi challenge. Taken together, these results demonstrate that Vhp1 is a cytotoxic protease and an effective vaccine candidate against V. harveyi infection. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunoprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. [Copyright &y& Elsevier]

Abstract: VhhP2 is an outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a subunit vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V. harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) IIα. A VhhP2 surface display system, in the form of the fish commensal strain PF3 harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5α/pJVP18, which expresses and surface-displays the VhhP2–Et18 chimera, proved to be an effective vaccine that can protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as E. tarda. [Copyright &y& Elsevier]

Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad range of hosts, including fish and mammals. In the present study, we used an advanced antibody array technology to identify the expression pattern of cytokines induced by E. tarda in a mouse infection model. In total, 31 and 24 differentially expressed cytokines (DECs) were identified in the plasma at 6 h and 24 h post-infection (hpi), respectively. The DECs were markedly enriched in the Gene Ontology (GO) terms associated with cell migration and response to chemokine and in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with immunity, diseases, and infection. Ten key DECs, including IL6 and TNF-α, were found to form extensive protein-protein interaction networks. IL6 was demonstrated to inhibit E. tarda infection and be required for E. tarda-induced inflammatory response. TNF-α also exerted an inhibitory effect on E. tarda infection, and knockdown of fish (Japanese flounder) TNF-α promoted E. tarda invasion in host cells. Together, the results of this study revealed a comprehensive profile of cytokines induced by E. tarda, thus adding new insights into the role of cytokine-associated immunity against bacterial infection and also providing the potential plasma biomarkers of E. tarda infection for future studies. [ABSTRACT FROM AUTHOR]

Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection. [ABSTRACT FROM AUTHOR]

Background: Emerging studies suggest that long non-coding RNAs (lncRNAs) play crucial roles in colorectal cancer (CRC). Here, we report a lncRNA, SATB2-AS1, which is specifically expressed in colorectal tissue and is significantly reduced in CRC. We systematically elucidated its functions and possible molecular mechanisms in CRC. Methods: LncRNA expression in CRC was analyzed by RNA-sequencing and RNA microarrays. The expression level of SATB2-AS1 in tissues was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH). The functional role of SATB2-AS1 in CRC was investigated by a series of in vivo and in vitro assays. RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), chromatin isolation by RNA purification (ChIRP), Bisulfite Sequencing PCR (BSP) and bioinformatics analysis were utilized to explore the potential mechanisms of SATB2-AS1. Results: SATB2-AS1 is specifically expressed in colorectal tissues and downregulated in CRC. Survival analysis indicates that decreased SATB2-AS1 expression is associated with poor survival. Functional experiments and bioinformatics analysis revealed that SATB2-AS1 inhibits CRC cell metastasis and regulates TH1-type chemokines expression and immune cell density in CRC. Mechanistically, SATB2-AS1 directly binds to WDR5 and GADD45A, cis-activating SATB2 (Special AT-rich binding protein 2) transcription via mediating histone H3 lysine 4 tri-methylation (H3K4me3) deposition and DNA demethylation of the promoter region of SATB2. Conclusions: This study reveals the functions of SATB2-AS1 in CRC tumorigenesis and progression, suggesting new biomarkers and therapeutic targets in CRC. [ABSTRACT FROM AUTHOR]