CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coil, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. (C) 2010 Elsevier Ltd. All rights reserved.

The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system. (c) 2006 Elsevier Ltd. All rights reserved.

A single amino acid defines cross-species reactivity of tree shrew (Tupaia belangeri) CD1d to human invariant natural killer T (iNKT) cells

P>The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evoluti

Detection of serum and mucosal antibody production and antibody secreting cells (ASCs) in large yellow croaker (Pseudosciaena crocea) following vaccination with Vibrio harveyi via different routes

The kinetics of mucosal and serum antibody response is well as antibody secreting cells (ASCs) production were studied in large yellow croaker following vaccination with inactivated Vibrio harveyi by different routes: oral administration. intraperitoneal (IP) injection and immersion. Indirect ELISA was used to measure the antibody level in serum and cutaneous mucus, and ELISPOT was used to monitor the ASCs derived from gill, blood and head kidney. The data demonstrated that IP injection resulted in the highest antibody levels in the systemic circulation, whereas immersion induced significant antibody levels in mucous. As for the ASCs response, IP injection induced high numbers of ASCs in the head kidney and blood; oral intubation only induced a slight ASCs response in the head kidney: immersion induced a much stronger ASCs response in the gill. These results indicate that mucosal antibodies following immersion immunization are independent of a systemic response and more sensitive, since it could be triggered earlier than serum antibodies. The mucosal antibodies following IP injection immunization may depend oil a systemic immune response. The protective effects of the three vaccination methods were compared by challenging with live V. harveyi. Survival of the three groups of vaccinated fish varied front 40 to 60%. while 100% mortality was found in control fish. Compared with IP and oral vaccination, immersion stimulated higher specific antibody titers in the mucosal system and achieved similar protection, so it is in effective and efficient method for immunizing a large number of fish against V harveyi (C) 2008 Elsevier B.V. All rights reserved.

Detection of cutaneous antibodies in excised skin explants from grass carp, Ctenopharyngodon idella (Valenciennes), immune to Scophthalmus maximus rhabdovirus

This study determined whether cutaneous antibodies were present in excised skin explants of grass carp, Ctenopharyngodon idella, immune to Scophthalmus maximus rhabdovirus (SMRV). Culture fluid from immune skin explants were assayed by indirect enzyme-linked immunosorbent assay (iELISA), Western blot, indirect immunofluorescent assay (IFA) and flow cytometry (FCM). iELISA showed that cutaneous antibody titres were much lower (1:12) than antiserum titres (1:1458) from intraperitoneally immunized grass carp. The phosphoprotein and matrix protein antigens of purified SMRV proteins were recognized by cutaneous antibodies from skin culture fluid using Western blot. The skin culture fluid produced staining signals in viral assembly sites and cytoplasm of SMRV-infected epithelioma papulosum cyprini (EPC) cells by IFA. FCM showed that 4.39% SMRV-infected EPC cells were detected, while non-specific reaction was seen in 2% of control cells. This is the first description of cutaneous antibodies against SMRV in grass carp.

The first non-mammalian CXCR3 in a teleost fish: Gene and expression in blood cells and central nervous system in the grass carp (Ctenopharyngodon idella)

A novel fish chemokine receptor gene, chemokine (C-X-C motif) receptor 3 (CXCR3)-like was isolated from the grass carp Ctenopharyngodon idella , with its full-length genomic sequence. The cDNA of grass carp CXCR3-like (gcCXCR3-like) consists of 1261 bp with a 49bp 5'-UTR and a 189 bp 3'-UTR. An open reading frame of 1023 bp encodes a 341-amino acid peptide, with seven transmembrane helices. The deduced amino acid sequence showed the same sequence identities (37.8%) with its counterparts in goat and human. The gcCXCR3-like gene consists of two exons, with one intervening intron, spaced over approximately 2 kb of genomic sequence. Phylogenetic analyses clearly demonstrated that the gcCXCR3-like resembles the CXCR3s of other vertebrates. Real-time PCR analysis showed that gcCXCR3-like was expressed in all tested organs except heart and the expression level of gcCXCR3-like was highest in brain. Flow cytometric analyses showed the positive rate of labelled leukocytes from the healthy grass carp was 17.3%, and the labelled leukocytes were divided into three types by cell sorting. Immunohistochemical localization revealed that gcCXCR3-like expressed in whole brain regions including cerebel, diencephalon, medulla oblongata, optic lobe, and rhinencephalon, and that the labelled leukocytes are actually populations of monocyte and/or phagocyte, lymphocyte and the granulocyte. It is considered that fish CXCR expression and their function may need to be investigated in both nervous and immune systems. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zalpha). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs. (C) 2004 Elsevier Ltd. All rights reserved.

Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (crucian carp (Carassius auratus L.) blastulae) cells, and thus defend host cells against the virus invasion. The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes. In this study, suppressive subtractive hybridization is applied to constructing a subtracted cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells. 272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library. Sequencing analysis reveals 69 genes, including 46 known gene homologues, and 23 unknown putative genes. The known genes include the genes involved in interferon signaling pathways, such as Stat1 and Jak1, the antiviral genes, such as Mx and Viperin, and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknown putative genes contain AU-rich element in their sequences. Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR. The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells, but also leads to the expression of a series of antiviral-relevant genes or immune-relevant genes, and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.

Alterations of immune functions induced by 12C6+ion irradiation in mice

Purpose: To estimate the biological risks to the immune system of the type of space radiation, 12C6+, encountered by cosmonauts during long-term travel in space. Materials and methods: The Kun-Ming strain mice were whole-body irradiated by 12C6+ ion with 0, 0.01, 0.05, 0.075, 0.2, 0.3, 0.5, 0.75, 1 or 2 Gy, at a dose rate of 1 Gy/min. At 35 days after irradiation, the thymus and spleen weights were measured, the natural killer (NK) cells activity of spleen was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), and the interferon-gamma (IFN-gamma) levels in serum and thymus were detected with enzyme-linked immunosorbent assays (ELISA). Results: The results showed that the thymus weight, IFN-gamma levels in serum and the activity of splenic NK-cells had significantly increased at a dose of 0.05 Gy. With further dose increase, the weight of spleen continued to increase but the weight of thymus, IFN-gamma level and NK-cells activity declined. Conclusions: These results suggest that the dose of 0.05 Gy irradiation has a stimulatory effect on mouse immunity; this effect declined with increasing dose.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 degrees C, 17 degrees C and 25 degrees C respectively. Thereafter, a recovery period of 24 h at 17 degrees C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immumomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 degrees C and 24 h of air exposure at 17 degrees C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 degrees C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 degrees C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 degrees C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops. (c) 2007 Elsevier B.V. All rights reserved.