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Characterization of peptides loaded into MHC class I in IHNV infection of rainbow trout (Oncorhynchus mykiss)
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Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
characterised by down-regulation of purine biosynthesis and salvage pathways in both groups. Together with the suppression of tyrosinase, this implied modulation of the storage and transport of melanin. Remodeling of calcified tissues (scale resorption), supported by a drop in several bone markers, was more pronounced in fish fed E. Transfer of sugar residues during glycoprotein and glycolipid biosynthesis was also more negatively affected by E. On the other hand, immunosuppressive mode of T action before the onset of infection was evidenced by the suppression of genes coding for important inflammatory mediators, such as prostaglandin D synthase and several chemokines. Most informative skin responses were seen during infection with the younger parasite stages revealing most promising candidates of protection among Einduced responses. Mechanisms of resistance in this group largely relied on polarisation towards the Th1/Th17 type of inflammatory responses, aptly supported by the induction of numerous genes with important roles in these pathways (IL-17, nitric oxide synthase and Pu.2, to name but a few). Concerted increase in the expression of collagens implied thickening of skin in the T-group as one of the major responses to lice while higher level of tight junction claudin-10 suggested strengthening of cellto-cell contacts. A number of muscle-related transcripts that could be involved in the expulsion of parasites by contraction had higher expression in the T- compared to the E-group. These expression patterns are well characterized aspects of the Th2-type of inflammatory responses in mammals. Induction of the canonical Th2 type cytokine, IL-11, further corroborates this conclusion. Finally, similar responses elicited by both hormones deserve attention, as two increasingly better characterized fish antimicrobial proteins showed highly similar expression profiles in both treatment groups. * Corresponding author. E-mail address: [email protected] (S. Skugor)
O-457. Penaeus monodon serpins inhibit prophenoloxidase activation K. Somboonwiwat 1, *, N. Wetsaphan 1, S. Mingmongkol 1, P. Jaree 1, S. Tharntada 2, A. Tassanakajon 1, V. Rimphanitchayakit 1. 1 Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; 2 Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand
Abstract Serine proteinase inhibitors (Serpins) function as the regulator of the proteinase in various biological processes such as blood coagulation, fibrinolysis, inflammation, and immune responses. In the black tiger shrimp (Penaeus monodon), a few serpins have been identified but their functions have not been characterized. PmSERPINB3 and PmSERPIN3 were further investigated for the roles in regulating prophenoloxidase system. The full-length cDNA of these genes were identified and the genomic structure study showed that they were intronless. The recombinant proteins of PmSERPINB3 (rPmSERPINB3) and PmSERPIN3 (rPmSERPIN6) were over-produced in Escherichia coli system and used for antibody production as well as inhibitory activity assay. Immunofluorescence revealed that PmSERPINB3 and PmSERPIN3 were produced in shrimp hemocyte. The purified rPmSERPINB3 and rPmSERPIN3 strongly inhibited subtilisin and partially inhibited chymotrypsin, trypsin and elastase. Moreover, they exhibited inhibitory activity on prophenoloxidase activation. They reduced prophenoloxidase activation when added to the LPS-induced hemocyte lysate supernatant. Moreover, injecting rPmSERPIN3 into shrimp reduced the bacterial clearance efficacy of shrimp. According to these evidences, these shrimp serpins might play roles in regulating the prophenoloxidase activation pathway in shrimp. * Corresponding author. E-mail address: [email protected] (K. Somboonwiwat)
O-401. Characterization of peptides loaded into MHC class I in IHNV infection of rainbow trout (Oncorhynchus mykiss) V. Soto Lampe 1, F. Takizawa 2, M. Keller 1, U. Fischer 1,*. 1 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald, Germany; 2 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, PA, USA
Abstract Cytotoxic T lymphocytes (CTL) have proven to execute efficient adaptive immune responses to virus infections of higher vertebrates. Also in fish, adaptive cell-mediated cytotoxicity against virus infected cells has been demonstrated by functional assays. Moreover, the existence of CTLs could be confirmed by gene expression profiling of the corresponding effectors cells, and more recently using antibodies against their population specific surface marker CD8. Virus infected target cells are recognized through binding of the CTLs T cell receptor (TCR) to short antigenic peptides derived from processed viral proteins that are loaded into major histocompatibility complex (MHC) class I molecules. The interaction between effector CTLs and virus infected target cells triggers clonal expansion of antigen-specific CTLs in an MHC class I restricted fashion, meaning that the TCRs of a certain individual do only bind to the host's body own but not to another MHC class I. In vitro, such binding can only be achieved if the donor of CTLs bears the same MHC class I allele as the virus infected target cell. The identification of MHC class I loaded peptides as potential CTL epitopes is crucial for the understanding of antiviral immune responses and for vaccine development. In order to predict potential viral peptides that are involved in MHC class I restricted presentation during infection of rainbow trout with infectious haematopoietic necrosis virus (IHNV) a computational prediction system has been used. For this, the full length protein sequence of the rainbow trout MHC class I allele UBA*150101, as well as of the glycoprotein of IHNV was uploaded resulting in the prediction of several nonapeptides of different binding intensities. In parallel, the extracellular heavy chain domain of UBA*150101 and of the rainbow trout b2-microglobulin (b2m) were expressed in E. coli. The recombinant proteins were then incubated in the presence of the predicted synthetic nonapeptides and tested for the formation of properly refolded peptideUBA*150101-b2m complexes by size exclusion chromatography followed by PAGE and Western blot analysis. Those peptides that have induced proper complexing will be employed for preparation of fluorescent labelled MHC class I tetramers necessary for the recognition, isolation and characterization of antigen-specific T cells induced by viral infection or vaccination. * Corresponding author. E-mail address: uwe.fischer@fli.bund.de (U. Fischer)
O-277. Distinct roles of interferon A, B, C and D in antiviral immunity of Atlantic salmon revealed by differences in antiviral activity and cell dependent expression T. Svingerud x, T. Solstad x, B. Sun, M.L.J. Nyrud, O. Kileng, L. GreinerTollersrud, B. Robertsen*. Norwegian College of Fishery Science, University of Tromsø, 9037 Tromsø, Norway Abstract Antiviral activities and expression properties of the 2-cysteine-containing type I IFNs IFNa and IFNd and the 4-cysteine-containing IFNb and IFNc in antiviral immunity of Atlantic salmon were compared in this work. While IFNa and IFNc showed similar antiviral activities and ability to induce antiviral genes, IFNb was less active and IFNd showed no activity. Expression of IFNs was compared by treatment of cells or fish with the dsRNA poly (I:C), which induces IFNs via the viral RNA receptors MDA5,
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
characterised by down-regulation of purine biosynthesis and salvage pathways in both groups. Together with the suppression of tyrosinase, this implied modulation of the storage and transport of melanin. Remodeling of calcified tissues (scale resorption), supported by a drop in several bone markers, was more pronounced in fish fed E. Transfer of sugar residues during glycoprotein and glycolipid biosynthesis was also more negatively affected by E. On the other hand, immunosuppressive mode of T action before the onset of infection was evidenced by the suppression of genes coding for important inflammatory mediators, such as prostaglandin D synthase and several chemokines. Most informative skin responses were seen during infection with the younger parasite stages revealing most promising candidates of protection among Einduced responses. Mechanisms of resistance in this group largely relied on polarisation towards the Th1/Th17 type of inflammatory responses, aptly supported by the induction of numerous genes with important roles in these pathways (IL-17, nitric oxide synthase and Pu.2, to name but a few). Concerted increase in the expression of collagens implied thickening of skin in the T-group as one of the major responses to lice while higher level of tight junction claudin-10 suggested strengthening of cellto-cell contacts. A number of muscle-related transcripts that could be involved in the expulsion of parasites by contraction had higher expression in the T- compared to the E-group. These expression patterns are well characterized aspects of the Th2-type of inflammatory responses in mammals. Induction of the canonical Th2 type cytokine, IL-11, further corroborates this conclusion. Finally, similar responses elicited by both hormones deserve attention, as two increasingly better characterized fish antimicrobial proteins showed highly similar expression profiles in both treatment groups. * Corresponding author. E-mail address: [email protected] (S. Skugor)
O-457. Penaeus monodon serpins inhibit prophenoloxidase activation K. Somboonwiwat 1, *, N. Wetsaphan 1, S. Mingmongkol 1, P. Jaree 1, S. Tharntada 2, A. Tassanakajon 1, V. Rimphanitchayakit 1. 1 Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; 2 Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand
Abstract Serine proteinase inhibitors (Serpins) function as the regulator of the proteinase in various biological processes such as blood coagulation, fibrinolysis, inflammation, and immune responses. In the black tiger shrimp (Penaeus monodon), a few serpins have been identified but their functions have not been characterized. PmSERPINB3 and PmSERPIN3 were further investigated for the roles in regulating prophenoloxidase system. The full-length cDNA of these genes were identified and the genomic structure study showed that they were intronless. The recombinant proteins of PmSERPINB3 (rPmSERPINB3) and PmSERPIN3 (rPmSERPIN6) were over-produced in Escherichia coli system and used for antibody production as well as inhibitory activity assay. Immunofluorescence revealed that PmSERPINB3 and PmSERPIN3 were produced in shrimp hemocyte. The purified rPmSERPINB3 and rPmSERPIN3 strongly inhibited subtilisin and partially inhibited chymotrypsin, trypsin and elastase. Moreover, they exhibited inhibitory activity on prophenoloxidase activation. They reduced prophenoloxidase activation when added to the LPS-induced hemocyte lysate supernatant. Moreover, injecting rPmSERPIN3 into shrimp reduced the bacterial clearance efficacy of shrimp. According to these evidences, these shrimp serpins might play roles in regulating the prophenoloxidase activation pathway in shrimp. * Corresponding author. E-mail address: [email protected] (K. Somboonwiwat)
O-401. Characterization of peptides loaded into MHC class I in IHNV infection of rainbow trout (Oncorhynchus mykiss) V. Soto Lampe 1, F. Takizawa 2, M. Keller 1, U. Fischer 1,*. 1 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald, Germany; 2 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, PA, USA
Abstract Cytotoxic T lymphocytes (CTL) have proven to execute efficient adaptive immune responses to virus infections of higher vertebrates. Also in fish, adaptive cell-mediated cytotoxicity against virus infected cells has been demonstrated by functional assays. Moreover, the existence of CTLs could be confirmed by gene expression profiling of the corresponding effectors cells, and more recently using antibodies against their population specific surface marker CD8. Virus infected target cells are recognized through binding of the CTLs T cell receptor (TCR) to short antigenic peptides derived from processed viral proteins that are loaded into major histocompatibility complex (MHC) class I molecules. The interaction between effector CTLs and virus infected target cells triggers clonal expansion of antigen-specific CTLs in an MHC class I restricted fashion, meaning that the TCRs of a certain individual do only bind to the host's body own but not to another MHC class I. In vitro, such binding can only be achieved if the donor of CTLs bears the same MHC class I allele as the virus infected target cell. The identification of MHC class I loaded peptides as potential CTL epitopes is crucial for the understanding of antiviral immune responses and for vaccine development. In order to predict potential viral peptides that are involved in MHC class I restricted presentation during infection of rainbow trout with infectious haematopoietic necrosis virus (IHNV) a computational prediction system has been used. For this, the full length protein sequence of the rainbow trout MHC class I allele UBA*150101, as well as of the glycoprotein of IHNV was uploaded resulting in the prediction of several nonapeptides of different binding intensities. In parallel, the extracellular heavy chain domain of UBA*150101 and of the rainbow trout b2-microglobulin (b2m) were expressed in E. coli. The recombinant proteins were then incubated in the presence of the predicted synthetic nonapeptides and tested for the formation of properly refolded peptideUBA*150101-b2m complexes by size exclusion chromatography followed by PAGE and Western blot analysis. Those peptides that have induced proper complexing will be employed for preparation of fluorescent labelled MHC class I tetramers necessary for the recognition, isolation and characterization of antigen-specific T cells induced by viral infection or vaccination. * Corresponding author. E-mail address: uwe.fischer@fli.bund.de (U. Fischer)
O-277. Distinct roles of interferon A, B, C and D in antiviral immunity of Atlantic salmon revealed by differences in antiviral activity and cell dependent expression T. Svingerud x, T. Solstad x, B. Sun, M.L.J. Nyrud, O. Kileng, L. GreinerTollersrud, B. Robertsen*. Norwegian College of Fishery Science, University of Tromsø, 9037 Tromsø, Norway Abstract Antiviral activities and expression properties of the 2-cysteine-containing type I IFNs IFNa and IFNd and the 4-cysteine-containing IFNb and IFNc in antiviral immunity of Atlantic salmon were compared in this work. While IFNa and IFNc showed similar antiviral activities and ability to induce antiviral genes, IFNb was less active and IFNd showed no activity. Expression of IFNs was compared by treatment of cells or fish with the dsRNA poly (I:C), which induces IFNs via the viral RNA receptors MDA5,