Presence of high-endothelial venules correlates with a favorable immune microenvironment in oral squamous cell carcinoma.

Oral squamous cell carcinomas are associated with a poor prognosis, which may be partly due to functional impairment of the immune response. Lymphocyte recruitment to the tumor site is facilitated by high-endothelial venules, whereas expression of programmed-death ligand 1 (PD-L1) can impair T-cell function. Thus, we hypothesize that these factors are important in shaping the immune response in oral squamous cell carcinoma. In the present study, we characterized the immune infiltrate in formalin-fixed, paraffin-embedded tumor samples from 75 oral squamous cell carcinoma patients. We used immunohistochemistry to determine the distribution of immune cell subsets, high-endothelial venules, and PD-L1, as well as quantitative real-time polymerase chain reaction to assess the expression of inflammatory cytokines and chemokines associated with lymphocyte trafficking. Finally, we calculated correlations between the presence of immune cell subsets, the gene expression patterns, high-endothelial venules, PD-L1, and the clinicopathological parameters, including patient survival. The presence of high-endothelial venules correlated with increased number of CD3+ T cells and CD20+ B cells, higher levels of the chemokines CXCL12 and CCL21, and lower levels of CCL20, irrespective of the tumors' T stage. In univariate analysis, high levels of CD20+ B cells and CD68+ macrophages, positive high-endothelial venule status, and low T and N stages predicted longer patient survival. However, only the presence of high-endothelial venules and a low T stage were independent positive prognosticators. This indicates that high-endothelial venules are important mediators and a convenient marker of an antitumor immune response in oral squamous cell carcinoma. Our findings suggest that these vessels are a potential immunomodulatory target in this type of cancer. PD-L1 staining in tumor cells correlated with lower T stage, increased infiltration of CD4+ cells, and higher expression of several inflammation-related cytokines. Thus, oral squamous cell carcinomas rich in CD4+ cells may preferentially respond to PD-1/PD-L1 blockade therapy.

Francisella noatunensis subsp. noatunensis invades, survives and replicates in Atlantic cod cells.

Systemic infection caused by the facultative intracellular bacterium Francisella noatunensis subsp. noatunensis remains a disease threat to Atlantic cod Gadus morhua L. Future prophylactics could benefit from better knowledge on how the bacterium invades, survives and establishes infection in its host cells. Here, facilitated by the use of a gentamicin protection assay, this was studied in primary monocyte/macrophage cultures and an epithelial-like cell line derived from Atlantic cod larvae (ACL cells). The results showed that F. noatunensis subsp. noatunensis is able to invade primary monocyte/macrophages, and that the actin-polymerisation inhibitor cytochalasin D blocked internalisation, demonstrating that the invasion is mediated through phagocytosis. Interferon gamma (IFNγ) treatment of cod macrophages prior to infection enhanced bacterial invasion, potentially by stimulating macrophage activation in an early step in host defence against F. noatunensis subsp. noatunensis infections. We measured a rapid drop of the initial high levels of internalised bacteria in macrophages, indicating the presence and action of a cellular immune defence mechanism before intracellular bacterial replication took place. Low levels of bacterial internalisation and replication were detected in the epithelial-like ACL cells. The capacity of F. noatunensis subsp. noatunensis to enter, survive and even replicate within an epithelial cell line may play an important role in its ability to infect live fish and transverse epithelial barriers to reach the bacterium's main target cells-the macrophage.

Ultrapure LPS induces inflammatory and antibacterial responses attenuated in vitro by exogenous sera in Atlantic cod and Atlantic salmon.

Phagocyte recognition of lipopolysaccharide (LPS) is an early key event for triggering the host innate immune response necessary for clearance of invading bacteria. The ability of fishes to recognise LPS has been questioned as contradictory results have been presented. We show here that monocyte/macrophage cultures from Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) respond with an increased expression of inflammatory and antibacterial genes to both crude and ultrapure Escherichia coli LPS. Crude LPS produces higher induction than the ultrapure LPS type in both species in vitro as well as in vivo in cod injected with LPS. Crude LPS gave, in contrast to ultrapure LPS, an additional weak up-regulation of antiviral genes in salmon macrophages, most likely because of contaminants in the LPS preparation. Increased levels of chicken (c)-type lysozyme transcripts and enzyme activity were measured in salmon macrophages following ultrapure LPS stimulation demonstrating not only increased transcription but also translation. Simultaneous use and even pre-treatment with bovine sera suppressed the LPS-induced expression thereby reflecting the presence of transcription inhibitory components in sera. Together, these findings show that both cod and salmon recognise LPS per se and that the observed induction is highly dependent on the absence of sera.

Response to vaccination of Atlantic cod (Gadus morhua L.) progenies from families with different estimated family breeding values for vibriosis resistance.

The purpose of the study was to elucidate whether responses to vibriosis vaccination and gene expressions in parts of the innate immune system were different in families of Atlantic cod (Gadus morhua). The fish were progenies of families with differences in estimated breeding values (EBV) for vibriosis resistance. Families of coastal cod (CC) and northeast Arctic cod (AC) responded well to vaccination with a relative percent survival of 72-95. No correlation between response to vaccination and vibriosis resistance were found (p = 0.146). The AC family with medium low (M) resistance had significant (p ≤ 0.019) lowest mortality among all the unvaccinated fish but the CC-M family. Further, when comparing the vaccinated fish the AC family with very high (VH) resistance had significant (p ≤ 0.004) higher mortality than all except the CC-VL and CC-H families. Parts of the innate immune response were studied by measuring the gene expression of innate immune genes 2 and 4 days post dip vaccination. Vaccinated fish from two families had a weak but significant higher innate immune response compared to control fish of the same family. In vaccinated fish, the gene expression of interleukin (IL) 1b, IL-10, IL-12p40 and hepcidin were significant up-regulated. While, no measureable activations of interferon gamma (IFNγ), IL-8, cathelicidin, LBP/BPI and G-type lysozyme were found.

Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod (Gadus morhua L.) juveniles.

The purpose of this study was to investigate the efficacy of three monovalent and a trivalent vibriosis dip vaccines in juvenile Atlantic cod (Gadus morhua L.), examine whether the responses were specific and study the expression of selected immune genes after dip vaccination. In addition, the study addressed whether the deviating isolates of Vibrio anguillarum serotype O2 belongs to another sero-subgroup than the previously established sero-subgroups O2a, O2b and O2c. Rabbit V. anguillarum serotype O2 antiserum adsorbed with V. anguillarum O2a O-antigen was shown, by both ELISA and immunoblotting, to still contain serotype O2 specific antibodies. Cod V. anguillarum serotype O2 antiserum reacted only with isolate of homologous serotype and not with heterologous sero-subgroups. This indicates that the deviating V. anguillarum O2 isolates represent a new sero-subgroup differing from sero-subgroup O2a. The monovalent vaccines included formalin inactivated cultures of V. anguillarum sero-subgroup O2a, O2b or serotype O2, while the trivalent vaccine contained all three sero-subgroups. Cod mounted high protection 7 weeks post dip vaccination with monovalent vaccines when challenged with homologous isolates and significantly lower when challenged with heterologous isolates, regardless of sero-subgroups. The trivalent vaccine resulted in efficient protection against all sero-subgroups tested. Dip vaccination of cod juveniles did not result in detectable antibody production or alteration in gene expression of the heavy chain of IgM and IgD. In the trivalent vaccine group expression of IFNγ and IL-12p40 were significantly up-regulated 3 days post vaccination. However, in groups vaccinated against V. anguillarum sero-subgroups O2b or O2, IL-12p40 and IFNγ gene expression were slightly increased 3 and 55 days post vaccination, respectively.

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages.

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1ß and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.

Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses.

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F. noatunensis compared to saline formulation. The antigen epitope was a 20-25 kDa component of F. noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1ß and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1ß expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1ß and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F. noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F. noatunensis in cod.

Validation of Selected Head and Neck Cancer Prognostic Markers from the Pathology Atlas in an Oral Tongue Cancer Cohort.

The Pathology Atlas is an open-access database that reports the prognostic value of protein-coding transcripts in 17 cancers, including head and neck cancer. However, cancers of the various head and neck anatomical sites are specific biological entities. Thus, the aim of the present study was to validate promising prognostic markers for head and neck cancer reported in the Pathology Atlas in oral tongue squamous cell carcinoma (OTSCC). We selected three promising markers from the Pathology Atlas (CALML5, CD59, LIMA1), and analyzed their prognostic value in a Norwegian OTSCC cohort comprising 121 patients. We correlated target protein and mRNA expression in formalin-fixed, paraffin-embedded cancer tissue to five-year disease-specific survival (DSS) in univariate and multivariate analyses. Protein expression of CALML5 and LIMA1 were significantly associated with five-year DSS in the OTSCC cohort in univariate analyses (p = 0.016 and p = 0.043, respectively). In multivariate analyses, lymph node metastases, tumor differentiation, and CALML5 were independent prognosticators. The prognostic role of the other selected markers for head and neck cancer patients identified through unbiased approaches could not be validated in our OTSCC cohort. This underlines the need for subsite-specific analyses for head and neck cancer.

Nephronectin promotes breast cancer brain metastatic colonization via its integrin-binding domains.

This study demonstrates a role for the extracellular matrix protein nephronectin (NPNT) in promoting experimental breast cancer brain metastasis, possibly through enhanced binding to- and migration through brain endothelial cells. With the introduction of more targeted breast cancer treatments, a prolonged survival has resulted during the last decade. Consequently, an increased number of patients develop metastasis in the brain, a challenging organ to treat. We recently reported that NPNT was highly expressed in primary breast cancer and associated with unfavourable prognosis. The current study addresses our hypothesis that NPNT promotes brain metastases through its integrin-binding motifs. SAGE-sequencing revealed that NPNT was significantly up-regulated in human breast cancer tissue compared to pair-matched normal breast tissue. Human brain metastatic breast cancers expressed both NPNT and its receptor, integrin α8ß1. Using an open access repository; BreastMark, we found a correlation between high NPNT mRNA levels and poor prognosis for patients with the luminal B subtype. The 66cl4 mouse cell line was used for expression of wild-type and mutant NPNT, which is unable to bind α8ß1. Using an in vivo model of brain metastatic colonization, 66cl4-NPNT cells showed an increased ability to form metastatic lesions compared to cells with mutant NPNT, possibly through reduced endothelial adhesion and transmigration.

Molecular characterisation and expression analysis of interferon gamma in Atlantic cod (Gadus morhua).

Interferon gamma (IFN-gamma) has important roles in both innate and adaptive immune responses. In this study, the cDNA and genomic sequences of Atlantic cod IFN-gamma were cloned and found to encode a putative protein containing 194 amino acids with a 24 amino acid signal peptide sequence. The gene is composed of four exons and three introns similar to IFN-gamma genes of other vertebrates. The cod IFN-gamma showed only 14-29% amino acid identity with other fish IFN-gamma and 9-17% identity with IFN-gamma from higher vertebrates. However, cod IFN-gamma possesses the typical IFN-gamma motifs in the C-terminal end of the protein and displays an alpha-helix structure similar to mammalian IFN-gamma. The promoter region contains a putative ISRE element indicating up-regulation by type I IFNs and dsRNA. Real time RT-PCR analysis confirmed that IFN-gamma gene expression was up-regulated in organs of cod injected with the dsRNA polyinosinic:polycytidylic acid (poly I:C), which is a strong inducer of type I IFNs. Injection of cod with formalin-killed Vibrio anguillarum also increased IFN-gamma expression in head kidney, but to a much lesser extent than poly I:C. The gene expression results thus indicate a role for IFN-gamma in innate immune response against both virus and bacteria in Atlantic cod.

Molecular characterisation of a goose-type lysozyme gene in Atlantic cod (Gadus morhua L.).

Lysozymes are antibacterial enzymes important in the innate immune defense of several animal phyla. An Atlantic cod goose-type (g-type) lysozyme EST was identified in a suppression subtractive hybridisation (SSH) cDNA library and the full-length cDNA (codg1) was obtained by RACE-PCR. The lysozyme gene is organised in five exons and four introns similar to g-type lysozyme genes in other fish species. Two different cod lysozyme transcripts, named codg1 and codg2, seem to be produced by the use of alternative transcription start sites (TSS) in the lysozyme gene. The alternative TSS cause a different exon I usage where exon Ia transcripts possess a putative signal peptide (codg1) while exon Ib transcripts (codg2) lack this feature. Lysozyme without the signal peptide was produced recombinantly in Escherichia coli and displayed muramidase activity against Micrococcus luteus cells at an unusually low pH. Gene expression analysis of codg1 and codg2 showed that both were expressed in several tissues with highest expression in the head kidney, peritoneum and spleen. Codg1 and codg2 were differentially expressed in some tissues. In the non-immunised control group, codg2 was expressed significantly higher in the head kidney compared to codg1, while an opposite expression profile was observed in the gills. Compared to non-immunised fish, a significant up-regulation of codg2 transcripts was observed in the peritoneum and gills after injection of formalin inactivated Listonella anguillarum indicating a role for g-type lysozyme in the innate defense system of cod.

Susceptibility of Atlantic cod Gadus morhua juveniles to different routes of experimental challenge with infectious pancreatic necrosis virus (IPNV).

Atlantic cod Gadus morhua L. juveniles weighing 40 g were challenged with infectious pancreatic necrosis virus (IPNV) by intraperitoneal (i.p.) or intramuscular (i.m.) injection or by bath. The amount of infectious virus was determined over 6 wk in head kidney, heart and pylorus tissues. No mortality or clinical signs were observed in either of the challenged groups. However, 6 wk after challenge virus was still present in the fish, which shows that IPNV can persist asymptomatically in cod. I.p. and i.m. injections were the most efficient routes of challenge giving the highest virus recovery. The prevalence of individuals with a viral titre > or = 500 infectious units g(-1) tissue was lower in the group of fish challenged by bath; thus bath was a less efficient route of challenge than injection. Our data also show that pylorus and head kidney are target organs for IPNV in cod, and levels of virus recovery were not considerably different between these 2 organs. Challenged by injection, the cod heart is also a target organ for IPNV. Compared to head kidney and pylorus, the heart seems to have a minor role in virus multiplication. Virus was also recovered from cohabiting fish, demonstrating that covertly infected cod may represent a reservoir of infectious IPNV for surrounding fish populations. Expression analysis of selected cod immune genes showed that i.p. injection of IPNV induced gene expression of ISG15 and LGP2, markers for the innate antiviral defence, while expression of markers for the inflammatory response (interleukins IL-1 beta, IL-8, IL-10) was not significantly increased.

Characterisation and expression analysis of the interleukin genes, IL-1beta, IL-8 and IL-10, in Atlantic cod (Gadus morhua L.).

The mammalian interleukins IL-1beta and IL-8 are important pro-inflammatory cytokines often used as markers of an activated inflammatory response, while IL-10 is regarded as an anti-inflammatory cytokine and plays a crucial role in the regulation of inflammation. Few cytokines from gadoid fish have been described, and herein the sequence and expression of these interleukin genes are studied in Atlantic cod (Gadus morhua L.). IL-1beta, IL-8 and IL-10 from cod show similarities in gene organisation and predicted protein sequences, compared to counterpart genes in other teleosts. Gene expression was studied using quantitative real time PCR in response to several treatments both in vitro and in vivo. In adherent head kidney cells, infectious pancreatic necrosis virus (IPNV) and lipopolysaccharide (LPS) significantly stimulated gene expression of IL-1beta. The expression of IL-1beta was not increased after treatment with a viral imitator (poly I:C), and neither IL-8 nor IL-10 responded to any of these agents in vitro. However, in vivo administrated poly I:C and formalin-killed Vibrio anguillarum (In-V.ang) induced interleukin expression, varying in intensity between different organs. IL-1beta and IL-10 gene expression profiles showed an opposite induction pattern in the in vivo experiments. Injection of In-V.ang highly induced IL-1beta expression, while a low induction was evident for IL-10, whereas the opposite was observed after injection of poly I:C. This pattern was particularly marked in spleen, where also IL-8 followed the expression pattern of IL-1beta. The opposite expression profiles indicate a suppressive role for IL-10 on the transcription of IL-1beta, and to a lesser extent on IL-8 transcription. Along with the identification of important promoter regulatory motives, these results provide tools for studying inflammatory responses in cod.

Whole transcriptome analysis of the Atlantic cod vaccine response reveals subtle changes in adaptive immunity.

Atlantic cod has lost the Major Histocompatibility complex class II pathway - central to pathogen presentation, humoral response and immunity. Here, we investigate the immunological response of Atlantic cod subsequent to dip vaccination with Vibrioanguillarum bacterin using transcriptome sequencing. The experiment was conducted on siblings from an Atlantic cod family found to be highly susceptible towards vibriosis where vaccination has demonstrated improved pathogen resistance. Gene expression analyses at 2, 4, 21 and 42 days post vaccination revealed GO-term enrichment for muscle, neuron and metabolism-related pathways. In-depth characterization of immune-related GO terms demonstrated down-regulation of MHCI antigen presentation, C-type lectin receptor signaling and granulocyte activation over time. Phagocytosis, interferon-gamma signaling and negative regulation of innate immunity were increasingly up-regulated over time. Individual differentially expressed immune genes implies weak initiation of acute phase proteins with little or no inflammation. Furthermore, gene expression indicates presence of T-cells, NK-like cells, B-cells and monocytes/macrophages. Three MHCI transcripts were up-regulated with B2M and SEC61. Overall, we find no clear immune-related transcriptomic response which could be attributed to Atlantic cod's alternative immune system. However, we cannot rule out that this response is related to vaccination protocol/sampling strategy. Earlier functional studies demonstrate significant memory in Atlantic cod post dip vaccination and combined with our results indicate the presence of other adaptive immunity mechanisms. In particular, we suggest that further investigations should look into CD8+ memory T-cells, γδ T-cells, T-cell independent memory or memory induced through NK-like/other lymphoid cells locally in the mucosal lining for this particular vaccination strategy.

Disentangling the immune response and host-pathogen interactions in Francisella noatunensis infected Atlantic cod.

The genetic repertoire underlying teleost immunity has been shown to be highly variable. A rare example is Atlantic cod and its relatives Gadiformes that lacks a hallmark of vertebrate immunity: Major Histocompatibility Complex class II. No immunological studies so far have fully unraveled the functionality of this particular immune system. Through global transcriptomic profiling, we investigate the immune response and host-pathogen interaction of Atlantic cod infected with the facultative intracellular bacterium Francisella noatunensis. We find that Atlantic cod displays an overall classic innate immune response with inflammation, acute-phase proteins and cell recruitment through up-regulation of e.g. IL1B, fibrinogen, cathelicidin, hepcidin and several chemotactic cytokines such as the neutrophil attractants CXCL1 and CXCL8. In terms of adaptive immunity, we observe up-regulation of interferon gamma followed by up-regulation of several MHCI transcripts and genes related to antigen transport and loading. Finally, we find up-regulation of immunoglobulins and down-regulation of T-cell and NK-like cell markers. Our analyses also uncover some contradictory transcriptional findings such as up-regulation of anti-inflammatory IL10 as well as down-regulation of the NADPH oxidase complex and myeloperoxidase. This we interpret as the result of host-pathogen interactions where F. noatunensis modulates the immune response. In summary, our results suggest that Atlantic cod mounts a classic innate immune response as well as a neutrophil-driven response. In terms of adaptive immunity, both endogenous and exogenous antigens are being presented on MHCI and antibody production is likely enabled through direct B-cell stimulation with possible neutrophil help. Collectively, we have obtained novel insight in the orchestration of the Atlantic cod immune system and determined likely targets of F. noatunensis host-pathogen interactions.

Concomitant Temperature Stress and Immune Activation may Increase Mortality Despite Efficient Clearance of an Intracellular Bacterial Infection in Atlantic Cod.

The environmental temperature has profound effects on biological systems of marine aquatic organisms and plays a critical role in species distribution and abundance. Particularly during the warmer seasons, variations in habitat temperature may introduce episodes of stressful temperatures which the organisms must adapt to and compensate for to maintain physiological homeostasis. The marine environment is changing and predicted raises in water temperatures will affect numerous marine species. Translocation of pathogens follow migration of species and alternations in physical environmental parameters may have influence upon the virulence of pathogens, as well as the hosts immune responses. While pathogenicity of many true pathogens is expected to increase following climate induced temperature stress, the impact from environmental stressors on the occurrence and severity of opportunistic infections is unknown. Here we describe how thermal stress in the cold-water species Atlantic cod influenced the fish immune responses against an opportunistic intracellular bacterium. Following experimental infection with Brucella pinnipedialis at normal water temperature (6°C) and sub-optimal temperature (15°C), cod cleared the intracellular bacteria more rapidly at the highest temperature. The overall immune response was faster and of higher amplitude at 15°C, however, a significant number of cod died at this temperature despite efficient clearance of infection. An increased growth rate not affected by infection was observed at 15°C, confirming multiple energy demanding processes taking place. Serum chemistry suggested that general homeostasis was influenced by both infection and increased water temperature, highlighting the cumulative stress responses (allostatic load) generated by simultaneous stressors. Our results suggest a trade-off between resistance and tolerance to survive infection at sub-optimal temperatures and raise questions concerning the impact of increased water temperatures on the energetic costs of immune system activation in aquatic ectotherms.

Sequence and expression analysis of an interferon stimulated gene (ISG15) from Atlantic cod (Gadus morhua L.).

Suppression subtractive hybridization was used to examine gene expression in Atlantic cod head kidney cells treated with polyinosinic polycytidylic acid (poly I:C). One of the most abundant genes was ISG15, showing 24-53% amino acid similarity to ISG15 from both mammals and teleosts. The promoter was cloned by genome walking and three potential interferon-stimulated response elements (ISREs) were identified. Analysis of the gene structure revealed a single intron in the 5' untranslated region (UTR) of cod ISG15, which also seems to be present in zebrafish and pufferfish ISG15. A quantitative real time PCR assay was established to monitor the gene expression of cod ISG15. Injection of cod with poly I:C strongly induced the expression of ISG15 in all organs investigated. Stimulation was most pronounced the first day with a gradual decline the following days. The expression of ISG15 in head kidney cells was also induced in vitro by treatment with poly I:C, but not significantly with LPS. However, injection of formalin killed Vibrio anguillarum-induced ISG15 expression in head kidney.

The gene structure and expression of the non-specific cytotoxic cell receptor protein (NCCRP-1) in Atlantic cod (Gadus morhua L.).

The non-specific cell receptor protein (NCCRP-1) serves an important function in target cell recognition and activation of non-specific cytotoxic cells in teleosts. Atlantic cod NCCRP-1 was identified in a suppression-subtractive cDNA library and NCCRP-1 from Atlantic salmon, rainbow trout, Japanese medaka and fathead minnow was found deposited in the GenBank as EST sequences. The predicted amino acid sequences of these receptors contain the characteristic functional domains representing NCCRP-1, and phylogenetic analyses support the identification of five NCCRP-1 orthologues. Cod NCCRP-1 is shorter and has a different intron/exon organization from the common carp and channel catfish counterparts, but shows high extent of conservation in NCCRP-1 signature motives. Quantitative real-time PCR analyses showed that the gene expression of cod NCCRP-1 was higher in the lymphoid organs, head kidney (90-fold) and spleen (30-fold), compared to the organ with lowest expression. NCCRP-1 gene expression was not induced by in vitro treatment of head kidney cells with polyinosinic polycytidylic acid (poly I:C) or lipopolysaccharide (LPS), or by in vivo injections with poly I:C or formalin killed Vibrio anguillarum. These results show that the cod NCCRP-1 gene is differentially expressed in organs, and that gene expression is not induced by the tested treatments.

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.).

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.

Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod.

Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.