Comparative Genomic Analysis of Virulent Vibrio (Listonella) anguillarum Serotypes Revealed Genetic Diversity and Genomic Signatures in the O-Antigen Biosynthesis Gene Cluster.

Vibrio anguillarum is the most frequent pathogen affecting fish worldwide. The only known virulent strains of V. anguillarum are serotypes O1, O2, and O3. Genetic differences between the serotypes that could shed insight on the evolution and serotype differences of this marine pathogen are unknown. Here, we fully sequenced and characterized a strain of V. anguillarum O1 (J382) isolated from winter steelhead trout (Oncorhynchus mykiss irideus) in British Columbia, Canada. Koch's postulates using the O1 strain were replicated in naïve lumpfish (Cyclopterus lumpus) and compared to O2. Phenotypic and genotypic comparisons were conducted for serotypes O1, O2, and O3, using biochemical tests and bioinformatic tools, respectively. The genome of V. anguillarum O1 (J382) contains two chromosomes (3.13 Mb and 1.03 Mb) and two typical pJM1-like plasmids (65,573 and 76,959 bp). Furthermore, V. anguillarum O1 (J382) displayed resistance to colistin sulphate, which differs from serotype O2 and could be attributed to the presence of the ugd gene. Comparative genomic analysis, among the serotypes, showed that intra-species evolution is driven by insertion sequences, bacteriophages, and a different repertoire of putative ncRNAs. Genetic heterogeneity in the O-antigen biosynthesis gene cluster is characterized by the absence or the presence of unique genes, which could result in differences in the immune evasion mechanisms employed by the respective serotypes. This study contributes to understanding the genetic differences among V. anguillarum serovars and their evolution.

Antigen presentation genes in gadoid species (haddock: Melanogrammus aeglefinus and Atlantic cod: Gadus morhua) raise questions about cross-presentation pathways and glycosylated beta-2-microglobulin.

The Atlantic cod immune system deviates from antigen presentation processes seen in other vertebrates in that it lacks the necessary genes for exogenous antigen presentation (i.e., MHC-II and li) and a key MHC-II interacting molecule necessary for T-helper cell function (i.e., CD4), while possessing an expanded repertoire of MHC-I genes that facilitate endogenous antigen presentation. These observations, combined with the identification of putative endosomal sorting signals in MHC-I cytoplasmic tails, have led to speculation that cod rely on cross-presentation of exogenous antigens to elicit cytotoxic T-lymphocyte responses against extracellular threats. In light of this suggestion, we investigated MHC-I transcriptional profiles and endosomal sorting signals in a closely related gadoid species, the haddock. Analysis of transcripts from one individual identified 13 unique MHC-I molecules, including two non-classical molecules as determined by the level of conservation at their peptide anchoring sites. This suggests that like the cod, the haddock has an expanded MHC-I repertoire. Analysis of haddock MHC-I cytoplasmic tail sequences revealed that the dileucine- and tyrosine-based endosomal signaling motifs, that are suggested to facilitate cross-presentation in cod, were absent. Closer examination of the cod signaling motifs, including their relative position in the cytoplasmic tail region, indicates that these motifs might be non-functional, further supporting the need for functional studies to assess cross-presentation. Finally, in silico analysis and in vitro N-type de-glycosylation experiments demonstrate that haddock and cod beta-2-microglobulin (ß2M) are glycosylated at the same NQT sequon. Interestingly, whole genome tBLASTn searches also revealed that putative ß2 M glycosylation sites appear frequently within the Gadiformes lineage, as the predictive NQT and other N-X-S/T sequons were located in ß2M orthologues from 19 of the 25 additional gadoid genomes analyzed. Though the exact significance of ß2M glycosylation has yet to be elucidated, phylogenetic comparisons predict that the same NQT glycosylation sequence occurs in 13 additional species comprising four different orders of Actinopterygii (Gymnotiformes, Esociformes, Beryciformes and Perciformes). This suggests either that this feature has arisen independently in multiple lineages or that it comes from a common ancestor and has been lost or modified in many species. Together, the analysis of gadoid MHC-I genes and ß2M molecules highlights the challenges in generalizing immune system paradigms across the most diverse vertebrate lineage (i.e., fish) and between fish and more well-studied mammals.

Characterization of cDNA clones encoding major histocompatibility class II receptors from walleye (Sander vitreus).

The teleost major histocompatibility (MH) class II receptor presents peptides from exogenous sources to CD4+ T cells, leading to the initiation of the adaptive immune response. The genes encoding MH class II have been identified in a number of teleost species, but not in walleye, an important recreational fish and commercial fishery in North America. In this study, we cloned and characterized the sequences encoding walleye MH class II α and ß chains. These sequences contained all of the domains typical for functional MH class II α and ß chain proteins, and aligned with other teleost sequences of MH class II. The walleye MH class II α amino acid sequence, along with other members of the Supraorder Percomorpharia, contains a high concentration of methionine residues in the beginning of the leader peptide. Southern blotting indicated that there is more than one gene copy for both MH class II α and ß, while northern blotting analysis of both genes showed that expression of these genes is greatest in lymphoid tissues and at potential entry points for pathogens. These results help to further the understanding of MH class II receptors in teleosts, and could prove useful in the study of disease issues in walleye such as dermal sarcoma virus.

Transcriptome profiling of the antiviral immune response in Atlantic cod macrophages.

A study was conducted to determine the transcriptome response of Atlantic cod (Gadus morhua) macrophages to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC), using a 20K Atlantic cod microarray platform and qPCR. We identified 285 significantly up-regulated and 161 significantly down-regulated probes in cod macrophages 24 h after pIC stimulation. A subset of 26 microarray-identified transcripts was subjected to qPCR validation using samples treated with pIC or phosphate-buffered saline (control) over time (3, 6, 12, 24, 48 h), and 77% of them showed a significant response to pIC. The microarray and qPCR analyses in this study showed that pIC induced the expression of cod macrophage transcripts involved in RLR- and TLR-dependent pathogen recognition (e.g. tlr3, tlr7, mda5 and lgp2), as well as signal transducers (e.g. stat1 and nfkbia) and transcription activators (e.g. irf7 and irf10) in the MyD88-independent and dependent signalling pathways. Several immune effectors (e.g. isg15s, viperin, herc4, mip2 and ccl13) were significantly up-regulated in pIC-stimulated cod macrophages. The expression of some transcripts (e.g. irf7, irf10, viperin) was significantly up-regulated by pIC as early as 12 h. All pIC-induced transcripts had peak expression at either 24 h (e.g. tlr7, irf7, mip2) or 48 h (e.g. tlr3, lgp2, stat1). This study suggests possible roles of both vertebrate-conserved (e.g. tlr3 as an up-regulated gene) and fish-specific (tlr22g as a down-regulated gene) receptors in dsRNA recognition, and the importance of conserved and potentially fish-specific interferon stimulated genes in cod macrophages.

Transcriptome profiling reveals that feeding wild zooplankton to larval Atlantic cod (Gadus morhua) influences suites of genes involved in oxidation-reduction, mitosis, and selenium homeostasis.

BACKGROUND: Larval nutrition and growth are key issues for wild and cultured cod. While it was shown previously that larval cod fed wild zooplankton grow faster than those fed only rotifers, the mechanisms involved in this enhanced growth are not completely understood. We used microarrays to identify larval cod transcripts that respond to feeding with small amounts of wild zooplankton (5-10 % of live prey items). The larval transcriptome was compared between 3 treatment groups [fed rotifers (RA), rotifers with protein hydrolysate (RA-PH), or rotifers with zooplankton (RA-Zoo)] at 9-10 mm length [26-30 days post-hatch (dph)] to identify a robust suite of zooplankton-responsive genes (i.e. differentially expressed between RA-Zoo and both other groups). RESULTS: The microarray experiment identified 147 significantly up-regulated and 156 significantly down-regulated features in RA-Zoo compared with both RA and RA-PH. Gene ontology terms overrepresented in the RA-Zoo responsive gene set included "response to selenium ion" and several related to cell division and oxidation-reduction. Ten selenoprotein-encoding genes, and 2 genes involved in thyroid hormone generation, were up-regulated in RA-Zoo compared with both other groups. Hierarchical clustering of RA-Zoo responsive genes involved in oxidation-reduction and selenium homeostasis demonstrated that only the zooplankton treatment had a considerable and consistent impact on the expression of these genes. Fourteen microarray-identified genes were selected for QPCR involving 9-13 mm larvae, and 13 of these were validated as differentially expressed between RA-Zoo and both other groups at ~9 mm. In contrast, in age-matched (34-35 dph; ~11 mm RA and RA-PH, ~13 mm RA-Zoo) and size-matched (~13 mm) older larvae, only 2 and 3 genes, respectively, showed the same direction of RA-Zoo-responsive change as in ~9 mm larvae. CONCLUSIONS: The modulation of genes involved in selenium binding, redox homeostasis, and thyroid hormone generation in ~9 mm RA-Zoo larvae in this study may be in response to the relatively high levels of selenium, iodine, and LC-PUFA (potentially causing oxidative stress) in zooplankton. Nonetheless, only a subset of zooplankton-responsive genes in ~9 mm larvae remained so in older larvae, suggesting that the observed transcriptome changes are largely involved in initiating the period of growth enhancement.

Atlantic salmon (Salmo salar) liver transcriptome response to diets containing Camelina sativa products.

Due to increasing demand for fish oil (FO) and fish meal (FM) in aquafeeds, more sustainable alternatives such as plant-derived oils and proteins are needed. Camelina sativa products are viable feed ingredients given the high oil and crude protein content in the seed. Atlantic salmon were fed diets with complete or partial replacement of FO and/or FM with camelina oil (CO) and/or camelina meal (CM) in a 16-week trial [Control diet: FO; Test diets: 100% CO replacement of FO (100CO), or 100CO with solvent-extracted FM (100COSEFM), 10% CM (100CO10CM), or SEFM+10% CM (100COSEFM10CM)]. Diet composition, growth, and fatty acid analyses for this feeding trial were published previously. A 44K microarray experiment identified liver transcripts that responded to 100COSEFM10CM (associated with reduced growth) compared to controls, yielding 67 differentially expressed features (FDR<5%). Ten microarray-identified genes [cpt1, pcb, bar, igfbp-5b (2 paralogues), btg1, dnph1, lect-2, clra, klf9, and fadsd6a], and three additional genes involved in lipid metabolism [elovl2, elovl5 (2 paralogues), and fadsd5], were subjected to QPCR with liver templates from all 5 dietary treatments. Of the microarray-identified genes, only bar was not QPCR validated. Both igfbp-5b paralogues were significantly down-regulated, and fadsd6a was significantly up-regulated, in all 4 camelina-containing diet groups compared with controls. Multivariate statistics were used to correlate hepatic desaturase and elongase gene expression data with tissue fatty acid profiles, indicating the involvement of these genes in LC-PUFA biosynthesis. This nutrigenomic study provides molecular biomarkers for use in developing novel aquafeeds using camelina products.

Genome-wide expression analysis of hereditary hyperplastic gingivitis in silver foxes (Vulpes vulpes) using canine microarrays.

Hereditary hyperplastic gingivitis (HHG) is an autosomal recessive condition found predominantly in farmed silver foxes, first documented in Europe in the 1940s. Hereditary gingival fibromatosis (HGF) is an analogous condition occurring in humans. HGF has a heterogeneous aetiology with emphasis placed on the autosomal dominant forms of inheritance for which there are three known loci: HGF1, HGF2, and HGF3. Among these, only one causative mutation has been determined, in the Son of sevenless homolog 1 (SOS1) gene. The goal of this study was to explore potential molecular or cellular mechanisms underlying HHG by analysis of global gene expression patterns from Affymetrix Canine 2.0 microarrays cross-referenced against candidate genes within the human loci. We conclude that the SOS1 gene involved in HGF1 is not significantly up-regulated in HHG. However, the structurally and functionally similar SOS2 gene is up-regulated in affected foxes, and we propose this as a candidate gene for HHG. At HGF2 we identify RASA1 (rat sarcoma viral p21 protein activator 1) as a candidate gene for HHG, as it is up-regulated in affected foxes and is involved in MAPK signalling. From comparison to the genes within the HGF3 locus, we find evidence for a role of androgens in HHG phenotype severity by differential up-regulation of SRD5A2 in HHG-affected foxes. We hypothesize that the putative mutation occurs upstream of RAS in the extracellular signal-regulated kinase component of MAPK signalling.

Toxicological effect of single contaminants and contaminant mixtures associated with plant ingredients in novel salmon feeds.

Increasing use of plant feed ingredients may introduce contaminants not previously associated with farming of salmonids, such as pesticides and PAHs from environmental sources or from thermal processing of oil seeds. To screen for interaction effects of contaminants newly introduced in salmon feeds, Atlantic salmon primary hepatocytes were used. The xCELLigence cytotoxicity system was used to select optimal dosages of the PAHs benzo(a)pyrene and phenanthrene, the pesticides chlorpyrifos and endosulfan, and combinations of these. NMR and MS metabolic profiling and microarray transcriptomic profiling was used to identify novel biomarkers. Lipidomic and transcriptomic profiling suggested perturbation of lipid metabolism, as well as endocrine disruption. The pesticides gave the strongest responses, despite having less effect on cell viability than the PAHs. Only weak molecular responses were detected in PAH-exposed hepatocytes. Chlorpyrifos suppressed the synthesis of unsaturated fatty acids. Endosulfan affected steroid hormone synthesis, while benzo(a)pyrene disturbed vitamin D3 metabolism. The primary mixture effect was additive, although at high concentrations the pesticides acted in a synergistic fashion to decrease cell viability and down-regulate CYP3A and FABP4 transcription. This work highlights the usefulness of 'omics techniques and multivariate data analysis to investigate interactions within mixtures of contaminants with different modes of action.

Variation in embryonic mortality and maternal transcript expression among Atlantic cod (Gadus morhua) broodstock: a functional genomics study.

Early life stage mortality is an important issue for Atlantic cod aquaculture, yet the impact of the cod maternal (egg) transcriptome on egg quality and mortality during embryonic development is poorly understood. In the present work, we studied embryonic mortality and maternal transcript expression using eggs from 15 females. Total mortality at 7days post-fertilization (7 dpf, segmentation stage) was used as an indice of egg quality. A 20,000 probe (20K) microarray experiment compared the 7hours post-fertilization (7 hpf, ~2-cell stage) egg transcriptome of the two lowest quality females (>90% mortality at 7 dpf) to that of the highest quality female (~16% mortality at 7 dpf). Forty-three microarray probes were consistently differentially expressed in both low versus high quality egg comparisons (25 higher expressed in low quality eggs, and 18 higher expressed in high quality eggs). The microarray experiment also identified many immune-relevant genes [e.g. interferon (IFN) pathway genes ifngr1 and ifrd1)] that were highly expressed in eggs of all 3 females regardless of quality. Twelve of the 43 candidate egg quality-associated genes, and ifngr1, ifrd1 and irf7, were included in a qPCR study with 7 hpf eggs from all 15 females. Then, the genes that were confirmed by qPCR to be greater than 2-fold differentially expressed between 7 hpf eggs from the lowest and highest quality females (dcbld1, ddc, and acy3 more highly expressed in the 2 lowest quality females; kpna7 and hacd1 more highly expressed in the highest quality female), and the 3 IFN pathway genes, were included in a second qPCR study with unfertilized eggs. While some maternal transcripts included in these qPCR studies were associated with extremes in egg quality, there was little correlation between egg quality and gene expression when all females were considered. Both dcbld1 and ddc showed greater than 100-fold differences in transcript expression between females and were potentially influenced by family. The Atlantic cod ddc (dopa decarboxylase) complete cDNA was characterized, and has a 1461bp open reading frame encoding a 486 amino acid protein that contains all eight residues of the conserved pyridoxal 5'-phosphate binding site including the catalytic lysine. This study provides valuable new information and resources related to the Atlantic cod egg transcriptome. Some of these microarray-identified, qPCR-confirmed, Atlantic cod egg transcripts (e.g. ddc, kpna7) play important roles during embryonic development of other vertebrate species, and may have similar functions in Atlantic cod.

Evaluation of the impact of camelina oil-containing diets on the expression of genes involved in the innate anti-viral immune response in Atlantic cod (Gadus morhua).

To improve sustainability of aquaculture, especially for carnivorous species like Atlantic cod, replacement of fish oil-based diets with vegetable oil-based diets has been studied. The use of vegetable oil in fish feeds can significantly change the fatty acid composition of fish tissues, and given the importance of fatty acids in inflammation and immunity, this change could potentially impact the immune response and health of the fish. The oilseed Camelina sativa is a promising source for this vegetable oil, because of the high oil content of its seeds (40%), a higher n-3 fatty acid content than most other oilseeds, and a high amount of γ-tocopherol. This study aims to investigate the effect of the replacement of dietary fish oil with oil from Camelina sativa on the immune response of Atlantic cod, as measured by the gene expression in spleen. Juvenile cod were fed on a fish oil-based diet (FO) or one of two diets in which camelina oil replaced 40% or 80% of fish oil (40CO and 80CO respectively) for 67 days, after which they were injected with either the viral mimic polyriboinosinic polyribocytidylic acid (pIC), or phosphate-buffered saline (PBS) as a control. Microarray analysis was used to determine the effect of the diet on the basal spleen transcriptome (pre-injection), and on the response to pIC (24 h post-injection). No marked differences in the spleen transcriptome were found between the three diets, either before or after injection with pIC. All fish, regardless of diet, showed a strong anti-viral response 24 h after pIC injection, with more than 500 genes having a significant difference of expression of 2-fold or higher compared to the PBS-injected fish for the FO, 40CO and 80CO diets. Gene Ontology annotation analysis of the three pIC-responsive gene lists indicated they were highly similar, and that the term 'immune system process' was significantly enriched in the pIC-responsive gene lists for all three diets. QPCR analysis for 5 genes with a known function in the anti-viral innate immune response (LGP2, STAT1, IRF1, ISG15 and viperin) showed modestly (smaller than 2-fold) up-regulated basal expression of LGP2, IRF1 and STAT1 in fish fed 40CO compared to the other diets. After pIC injection, all 5 genes were significantly and strongly up-regulated in pIC-injected fish compared to PBS-injected fish, but no significant differences were found between any of the diets. In conclusion, replacement of up to 80% of fish oil with camelina oil in Atlantic cod diets does not have a strong effect on basal spleen gene expression. Atlantic cod fed on camelina oil-containing diets are capable of mounting a strong anti-viral immune response, which is comparable to that in cod fed with a fish oil diet.

The impact of a moderate chronic temperature increase on spleen immune-relevant gene transcription depends on whether Atlantic cod (Gadus morhua) are stimulated with bacterial versus viral antigens.

Exposure to elevated temperature is an inherent feature of Atlantic cod (Gadus morhua) sea-cage culture in some regions (e.g., Newfoundland) and may also become an increasingly prevalent challenge for wild fish populations because of accelerated climate change. Therefore, understanding how elevated temperatures impacts the immune response of this commercially important species may help to reduce the potential negative impacts of such challenges. Previously, we investigated the impacts of moderately elevated temperature on the antiviral responses of Atlantic cod (Hori et al. 2012) and reported that elevated temperature modulated the spleen transcriptome response to polyriboinosinic polyribocytidylic acid (pIC, a viral mimic). Herein, we report a complementary microarray study that investigated the impact of the same elevated temperature regime on the Atlantic cod spleen transcriptome response to intraperitoneal (IP) injection of formalin-killed Aeromonas salmonicida (ASAL). Fish were held at two different temperatures (10 °C and 16 °C) prior to immune stimulation and sampled 6 and 24 h post-injection (HPI). In this experiment, we identified 711 and 666 nonredundant ASAL-responsive genes at 6HPI and 24HPI, respectively. These included several known antibacterial genes, including hepcidin, cathelicidin, ferritin heavy subunit, and interleukin 8. However, we only identified 15 differentially expressed genes at 6HPI and 2 at 24HPI (FDR 1%) when comparing ASAL-injected fish held at 10 °C versus 16 °C. In contrast, the same comparisons with pIC-injected fish yielded 290 and 339 differentially expressed genes (FDR 1%) at 6HPI and 24HPI, respectively. These results suggest that moderately elevated temperature has a lesser effect on the Atlantic cod spleen transcriptome response to ASAL (i.e., the antibacterial response) than to pIC (i.e., antiviral response). Thus, the impacts of high temperatures on the cod's immune response may be pathogen dependent.

A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection.

BACKGROUND: Atlantic cod (Gadus morhua) reared in sea-cages can experience large variations in temperature, and these have been shown to affect their immune function. We used the new 20K Atlantic cod microarray to investigate how a water temperature change which, simulates that seen in Newfoundland during the spring-summer (i.e. from 10°C to 16°C, 1°C increase every 5 days) impacted the cod spleen transcriptome response to the intraperitoneal injection of a viral mimic (polyriboinosinic polyribocytidylic acid, pIC). RESULTS: The temperature regime alone did not cause any significant increases in plasma cortisol levels and only minor changes in spleen gene transcription. However, it had a considerable impact on the fish spleen transcriptome response to pIC [290 and 339 significantly differentially expressed genes between 16°C and 10°C at 6 and 24 hours post-injection (HPI), respectively]. Seventeen microarray-identified transcripts were selected for QPCR validation based on immune-relevant functional annotations. Fifteen of these transcripts (i.e. 88%), including DHX58, STAT1, IRF7, ISG15, RSAD2 and IκBα, were shown by QPCR to be significantly induced by pIC. CONCLUSIONS: The temperature increase appeared to accelerate the spleen immune transcriptome response to pIC. We found 41 and 999 genes differentially expressed between fish injected with PBS vs. pIC at 10°C and sampled at 6HPI and 24HPI, respectively. In contrast, there were 656 and 246 genes differentially expressed between fish injected with PBS vs. pIC at 16°C and sampled at 6HPI and 24HPI, respectively. Our results indicate that the modulation of mRNA expression of genes belonging to the NF-κB and type I interferon signal transduction pathways may play a role in controlling temperature-induced changes in the spleen's transcript expression response to pIC. Moreover, interferon effector genes such as ISG15 and RSAD2 were differentially expressed between fish injected with pIC at 10°C vs. 16°C at 6HPI. These results substantially increase our understanding of the genes and molecular pathways involved in the negative impacts of elevated ambient temperature on fish health, and may also be valuable to our understanding of how accelerated global climate change could impact cold-water marine finfish species.

An integrated approach to gene discovery and marker development in Atlantic cod (Gadus morhua).

Atlantic cod is a species that has been overexploited by the capture fishery. Programs to domesticate this species are underway in several countries, including Canada, to provide an alternative route for production. Selective breeding programs have been successfully applied in the domestication of other species, with genomics-based approaches used to augment conventional methods of animal production in recent years. Genomics tools, such as gene sequences and sets of variable markers, also have the potential to enhance and accelerate selective breeding programs in aquaculture, and to provide better monitoring tools to ensure that wild cod populations are well managed. We describe the generation of significant genomics resources for Atlantic cod through an integrated genomics/selective breeding approach. These include 158,877 expressed sequence tags (ESTs), a set of annotated putative transcripts and several thousand single nucleotide polymorphism markers that were developed from, and have been shown to be highly variable in, fish enrolled in two selective breeding programs. Our EST collection was generated from various tissues and life cycle stages. In some cases, tissues from which libraries were generated were isolated from fish exposed to stressors, including elevated temperature, or antigen stimulation (bacterial and viral) to enrich for transcripts that are involved in these response pathways. The genomics resources described here support the developing aquaculture industry, enabling the application of molecular markers within selective breeding programs. Marker sets should also find widespread application in fisheries management.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Specific expression of miR-17-5p and miR-127 in testicular and central nervous system diffuse large B-cell lymphoma.

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P<0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas (P<0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.

From brain to testis: immune escape and clonal selection in a B cell lymphoma with selective outgrowth in two immune sanctuaries [correction of sanctuariesy].

We describe a patient with a primary diffuse large B-cell lymphoma of the central nervous system who developed a localized testicular relapse after 8 years. Both tumours lacked HLA-DR expression, the relapse additionally lost HLA class I expression. Immunoglobulin heavy chain gene rearrangements were identical in both lymphomas with extensive and ongoing somatic hypermutations resulting in extensive idiotype modulation. We hypothesize that these immune sanctuaries initially provided a safe haven for the tumour cells. When the environment becomes more permissive for an anti-tumour response, the continuous idiotype modulation and progressive loss of HLA expression on the tumour cells facilitates further immune escape.

Array comparative genomic hybridization reveals a very high frequency of deletions of the long arm of chromosome 6 in testicular lymphoma.

Despite the fact that numerous studies have been performed on diffuse large B-cell lymphoma (DLBCL), only few have concerned extranodal lymphomas occurring in the testis. We performed a cytogenetic and molecular study of 17 testicular non-Hodgkin lymphomas, of which 14 were proven primary DLBCL of the testis. Cytogenetic analysis revealed in 8 out of 11 evaluable cases a structural abnormality of the long arm of chromosome 6, with deletion or addition of material of unknown origin, and with breakpoints spanning the region 6q12-6q23. The cytogenetic findings were confirmed by fluorescent in situ hybridization (FISH) with a chromosome 6 painting probe. Using array based-comparative genomic hybridization on 16 evaluable cases, including 5 cases not tested by cytogenetics or FISH, 14 (88%) showed chromosome 6q deletions. We identified two regions of minimal deletion (RMD), at 104-113 Mb (6q16.3-q21) and 137.5-138.8 Mb (6q23.3), respectively. In one case, we observed a 2.7 Mb homozygous deletion ranging from 135.3 to 138.0 Mb that partly overlapped with the RMD at 6q23.3. Our study indicates that 6q deletions play a major pathogenetic role in DLBCL of the testis and that many of these deletions are part of unbalanced translocations.

Mechanisms and effects of loss of human leukocyte antigen class II expression in immune-privileged site-associated B-cell lymphoma.

PURPOSE AND EXPERIMENTAL DESIGN: Loss of human leukocyte antigen (HLA) expression on tumor cells is frequent in diffuse large B-cell lymphoma (DLBCL) arising in immune-privileged sites, such as the testis and central nervous system, and is associated with small homozygous deletions of HLA-DQ/HLA-DR and larger hemizygous deletions of the MHC region. To better understand the significance of down-regulation of HLA class II expression in relation to the homozygous and hemizygous deletions, we analyzed global gene expression patterns in a series of 26 testicular DLBCL after characterization of these deletions. RESULTS: Low levels of HLA-DR mRNA in whole testicular DLBCL samples were associated with a strong down-regulation of numerous immune-related genes specific for T cells, macrophages, antigen presentation and processing, lymphocyte activation, chemokines and chemokine receptors, and the complement system. The number of CD3+ tumor-infiltrating T cells was also significantly lower in low expressors of HLA-DR mRNA. Interestingly, hemizygous and homozygous deletions in the MHC region did not have any additional effect on global gene expression. CONCLUSION: In conclusion, we found that loss of HLA class II mRNA expression in testicular DLBCL is associated with a significant change in global gene expression patterns. This effect is independent of the mechanism causing the down-regulation of HLA class II genes in the lymphoma cells.

BCL6 alternative translocation breakpoint cluster region associated with follicular lymphoma grade 3B.

Translocations involving band 3q27, affecting the major breakpoint region (MBR) of BCL6, are common in diffuse large B-cell lymphomas (DLBCLs). Recent data suggest an alternative breakpoint cluster region (ABR) located between 245 and 285 kb 5' of BCL6, which might be associated with Follicular Lymphoma (FL). Ten DLBCLs and 9 FLs grade 3B with cytogenetic rearrangements at 3q27 were studied by fluorescence in situ hybridization (FISH) to discriminate between breakpoints at the ABR and MBR. Eight DLBCLs contained a breakpoint in the MBR, and 6 FL grade 3B (FL3B) cases contained a breakpoint in the ABR. No specific chromosomal partners could be identified in both groups. Previously published data have suggested that FL3B cases with 3q27 aberrations are closely related to the majority of DLBCLs of germinal center cell origin. However, our findings suggest that the mechanism of 3q27 rearrangement in FL3B cases is similar to the mechanism in follicular lymphomas grade 1,2, and 3A cases.