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Cytological studies on haematopoietic stem cells of redclaw crayfish, Cherax quadricarinatus
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
involved in immune responses and inflammation was also observed in resveratrol-treated leucocytes after 3 and 6 h: the suppressor of cytokine signalling 3, LPS-induced tumour necrosis alpha, hepcidin, metallothionein, TLR8 and the calcium dependent lectin A. Resveratrol also decreased the expression of CCL20, IL-8, apolipoprotein E and glutathione S-transferase, after incubation of leucocytes for 6 and 24 h, and of TNF-a, after incubation for 3 and 24 h. Several genes were also regulated separately after 3, 6 and 24 h. We conclude that resveratrol modulates (by downregulation) the expression of many genes involved in inflammation and immune responses, which confirms the anti-inflammatory activity of this polyphenol. Resveratrol affects the expression of many genes at the same time, apparently by targeting several cell signalling pathways, although the molecular basis of this process is still unknown. * Corresponding author. E-mail address: [email protected] (J. Lamas) xThese authors have contributed equally to this work.
P-226. Cytological studies on haematopoietic stem cells of redclaw crayfish, Cherax quadricarinatus H. Duan 1, 2, J. Xiang 1, *, Y. Zhang 1, S. Jin 1. 1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; 2 University of Chinese Academy of Sciences, Beijing, China
Abstract Circulating haemocytes play important roles in the immunity of crustacean. Some cellular immune process, such as phagocytosis, encapsulation and lysis of foreign cells were carried out in haemocytes to eliminate the invaded pathogens. Haematopoietic tissue (Hpt) in crustacean was regarded as the source of new haemocytes to complement the lost haemocytes in haemolymph. However, knowledge about the Hpt of crustacean is still very limited. In the present study, cytological studies were undertaken to describe the cell characteristics in Hpt of redclaw crayfish, Cherax quardricarinatus and their function in phagocytosis was investigated. Cells in Hpt were divided into 5 different types based on their morphological characteristics through light microscope and transmission electron microscope (TEM) observation. In order to learn the relations between Hpt cells and haemocytes, Hpt cells and haemocytes were incubated with fluorescent (FITC) labeled beads (0.5 mm) overnight, then they were observe with laser scan confocal microscope (LSCM) and scanning electron microscope (SEM). Fluorescent labeled beads were observed in some Hpt cells which suggested that they might have phagocytosis function. Fluorescent labeled beads were observed in the hyaline and small granular haemocytes, but not in large granular haemocytes, which indicated that hyaline and small granular haemocytes had phagocytosis function. These data might help us to understand the relationship between Hpt cells and haemocytes cells in crustacean. * Corresponding author. E-mail address: [email protected] (J. Xiang)
P-206. European sea bass leucocytes do not show cell-mediated cytotoxic activity against nodavirus-infected cells in vitro M.A. Esteban, J. Meseguer, A. Cuesta*. Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, Campus Regional de Excelencia Internacional “Campus Mare Nostrum”, University of Murcia, 30100 Murcia, Spain Abstract Nodavirus (family Nodaviridae, genus Betanodavirus) is a bi-segmented, single-stranded, positive-sense RNA virus that causes the nervous necrosis viral (NNV) or viral encephalopathy and retinopathy (VER) disease in fish.
1705
While European sea bass (Dicenctrarchus labrax) is among the most susceptible species the gilthead seabream (Sparus aurata) is an asymptomatic reservoir. Therefore, while most available information focuses on the mechanisms involved in pathogen susceptible fish further knowledge is also important in pathogen-reservoir fish systems in order to avoid or control the spread of pathogens. Among the major immune mechanisms to fight viral infections in fish, the interferon (IFN) pathway and the cell-mediated cytotoxic (CMC) activity are the most important, but most efforts have only focused on the IFN pathway. In a previous work, we have infected seabream and sea bass specimens with nodavirus and found that the innate CMC activity of isolated head-kidney leucocytes (HKLs) against xenogeneic tumor cells was much higher in VNNV-infected than in uninfected fish, especially in sea bass. Afterwards, we have also evaluated the innate CMC activity in vitro. Thus, isolated seabream and sea bass HKLs were incubated for 4 h with nodavirus-free or -infected cell lines (SSN-1 and SAF-1) and the innate cytotoxic activity determined by the lactate dehydrogenase (LDH) viability test. Surprisingly, the activity of seabream HKLs against the cell lines was greatly increased upon virus infection whilst in the case of sea bass the CMC was unaltered. Moreover, we have evaluated the gene expression pattern of CMC-related genes (natural killer enhancing factor, non-specific cytotoxic cell receptor-1, granzymes and perforin). Overall, data revealed that seabream gene expression was significantly upregulated after incubation with nodavirus-infected cells compared to uninfected-cells. Strikingly, gene expression and cytotoxic activity followed a very similar pattern. This data indicate that the ability of sea bass HKLs to kill nodavirus-infected cells is not properly activated and would suggest the cause to the great susceptibility of sea bass to nodavirus infections. Further studies are in progress to assess this possibility. Acknowledgements This work was co-funded by Ministerio de Economía y Competitividad (AGL2010-20801-C02-02) and FEDER. Nodavirus strain and SSN-1 cells were kindly donated by Pilar Fernández Somalo (Laboratorio Central de Veterinaria de Algete, Ministerio de Agricultura, Alimentación y Medio Ambiente). * Corresponding author. E-mail address: [email protected] (A. Cuesta)
P-371. Identification of common carp IFNs 4 and analysis of their antiviral activity against SVCV A. Falco*, M. Forlenza, G.F. Wiegertjes. Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands Abstract Type-I Interferons (IFNs) are cytokines with diverse roles in regulating the immune system and inducing an anti-viral state. IFNs signal through the conserved JAK/STAT pathway, upregulating the expression of numerous IFN-stimulated genes (ISGs). In mammals, three major classes of gene families are identified: type I (including IFN-a and b), type II (IFN-g) and type III (IFN-l). Whereas type II IFN expression is restricted to immune cells, type I and type III IFNs are usually expressed by a variety of cell types. Fish IFNs cannot easily be classified as either type I or type III based on the features commonly distinguishing IFNs in mammals, hence they are often grouped in a separate family called IFN4. The IFN4 family contains at least four subtypes that may be classified into two major groups based on the presence of two or four cysteines, forming one or two disulfide bonds, respectively. IFN41 and IFN44 contain two cysteines (group I – 2C IFNs), whereas IFN42 and IFN43 contain four cysteines (group II – 4C IFNs). In Salmonids, a similar IFN repertoire is present but classified differently: IFNa corresponds to IFNf1 in cyprinids, IFNc to IFNf2-3 and IFNd to IFNf4, thus no IFNb homologs have been found in cyprinids yet. In Cyprinids, all four IFN4s are capable of inducing an anti-viral state but signal through different receptor chains: IFN41 and IFN44 bind to a CRFB1/CRFB5 complex whereas IFN42 and IFN43 bind to CRFB2/CRFB5 receptors.
involved in immune responses and inflammation was also observed in resveratrol-treated leucocytes after 3 and 6 h: the suppressor of cytokine signalling 3, LPS-induced tumour necrosis alpha, hepcidin, metallothionein, TLR8 and the calcium dependent lectin A. Resveratrol also decreased the expression of CCL20, IL-8, apolipoprotein E and glutathione S-transferase, after incubation of leucocytes for 6 and 24 h, and of TNF-a, after incubation for 3 and 24 h. Several genes were also regulated separately after 3, 6 and 24 h. We conclude that resveratrol modulates (by downregulation) the expression of many genes involved in inflammation and immune responses, which confirms the anti-inflammatory activity of this polyphenol. Resveratrol affects the expression of many genes at the same time, apparently by targeting several cell signalling pathways, although the molecular basis of this process is still unknown. * Corresponding author. E-mail address: [email protected] (J. Lamas) xThese authors have contributed equally to this work.
P-226. Cytological studies on haematopoietic stem cells of redclaw crayfish, Cherax quadricarinatus H. Duan 1, 2, J. Xiang 1, *, Y. Zhang 1, S. Jin 1. 1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; 2 University of Chinese Academy of Sciences, Beijing, China
Abstract Circulating haemocytes play important roles in the immunity of crustacean. Some cellular immune process, such as phagocytosis, encapsulation and lysis of foreign cells were carried out in haemocytes to eliminate the invaded pathogens. Haematopoietic tissue (Hpt) in crustacean was regarded as the source of new haemocytes to complement the lost haemocytes in haemolymph. However, knowledge about the Hpt of crustacean is still very limited. In the present study, cytological studies were undertaken to describe the cell characteristics in Hpt of redclaw crayfish, Cherax quardricarinatus and their function in phagocytosis was investigated. Cells in Hpt were divided into 5 different types based on their morphological characteristics through light microscope and transmission electron microscope (TEM) observation. In order to learn the relations between Hpt cells and haemocytes, Hpt cells and haemocytes were incubated with fluorescent (FITC) labeled beads (0.5 mm) overnight, then they were observe with laser scan confocal microscope (LSCM) and scanning electron microscope (SEM). Fluorescent labeled beads were observed in some Hpt cells which suggested that they might have phagocytosis function. Fluorescent labeled beads were observed in the hyaline and small granular haemocytes, but not in large granular haemocytes, which indicated that hyaline and small granular haemocytes had phagocytosis function. These data might help us to understand the relationship between Hpt cells and haemocytes cells in crustacean. * Corresponding author. E-mail address: [email protected] (J. Xiang)
P-206. European sea bass leucocytes do not show cell-mediated cytotoxic activity against nodavirus-infected cells in vitro M.A. Esteban, J. Meseguer, A. Cuesta*. Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, Campus Regional de Excelencia Internacional “Campus Mare Nostrum”, University of Murcia, 30100 Murcia, Spain Abstract Nodavirus (family Nodaviridae, genus Betanodavirus) is a bi-segmented, single-stranded, positive-sense RNA virus that causes the nervous necrosis viral (NNV) or viral encephalopathy and retinopathy (VER) disease in fish.
1705
While European sea bass (Dicenctrarchus labrax) is among the most susceptible species the gilthead seabream (Sparus aurata) is an asymptomatic reservoir. Therefore, while most available information focuses on the mechanisms involved in pathogen susceptible fish further knowledge is also important in pathogen-reservoir fish systems in order to avoid or control the spread of pathogens. Among the major immune mechanisms to fight viral infections in fish, the interferon (IFN) pathway and the cell-mediated cytotoxic (CMC) activity are the most important, but most efforts have only focused on the IFN pathway. In a previous work, we have infected seabream and sea bass specimens with nodavirus and found that the innate CMC activity of isolated head-kidney leucocytes (HKLs) against xenogeneic tumor cells was much higher in VNNV-infected than in uninfected fish, especially in sea bass. Afterwards, we have also evaluated the innate CMC activity in vitro. Thus, isolated seabream and sea bass HKLs were incubated for 4 h with nodavirus-free or -infected cell lines (SSN-1 and SAF-1) and the innate cytotoxic activity determined by the lactate dehydrogenase (LDH) viability test. Surprisingly, the activity of seabream HKLs against the cell lines was greatly increased upon virus infection whilst in the case of sea bass the CMC was unaltered. Moreover, we have evaluated the gene expression pattern of CMC-related genes (natural killer enhancing factor, non-specific cytotoxic cell receptor-1, granzymes and perforin). Overall, data revealed that seabream gene expression was significantly upregulated after incubation with nodavirus-infected cells compared to uninfected-cells. Strikingly, gene expression and cytotoxic activity followed a very similar pattern. This data indicate that the ability of sea bass HKLs to kill nodavirus-infected cells is not properly activated and would suggest the cause to the great susceptibility of sea bass to nodavirus infections. Further studies are in progress to assess this possibility. Acknowledgements This work was co-funded by Ministerio de Economía y Competitividad (AGL2010-20801-C02-02) and FEDER. Nodavirus strain and SSN-1 cells were kindly donated by Pilar Fernández Somalo (Laboratorio Central de Veterinaria de Algete, Ministerio de Agricultura, Alimentación y Medio Ambiente). * Corresponding author. E-mail address: [email protected] (A. Cuesta)
P-371. Identification of common carp IFNs 4 and analysis of their antiviral activity against SVCV A. Falco*, M. Forlenza, G.F. Wiegertjes. Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands Abstract Type-I Interferons (IFNs) are cytokines with diverse roles in regulating the immune system and inducing an anti-viral state. IFNs signal through the conserved JAK/STAT pathway, upregulating the expression of numerous IFN-stimulated genes (ISGs). In mammals, three major classes of gene families are identified: type I (including IFN-a and b), type II (IFN-g) and type III (IFN-l). Whereas type II IFN expression is restricted to immune cells, type I and type III IFNs are usually expressed by a variety of cell types. Fish IFNs cannot easily be classified as either type I or type III based on the features commonly distinguishing IFNs in mammals, hence they are often grouped in a separate family called IFN4. The IFN4 family contains at least four subtypes that may be classified into two major groups based on the presence of two or four cysteines, forming one or two disulfide bonds, respectively. IFN41 and IFN44 contain two cysteines (group I – 2C IFNs), whereas IFN42 and IFN43 contain four cysteines (group II – 4C IFNs). In Salmonids, a similar IFN repertoire is present but classified differently: IFNa corresponds to IFNf1 in cyprinids, IFNc to IFNf2-3 and IFNd to IFNf4, thus no IFNb homologs have been found in cyprinids yet. In Cyprinids, all four IFN4s are capable of inducing an anti-viral state but signal through different receptor chains: IFN41 and IFN44 bind to a CRFB1/CRFB5 complex whereas IFN42 and IFN43 bind to CRFB2/CRFB5 receptors.