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

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

The role of immunoediting in lymphomas of immune-privileged sites

Diffuse large B cell lymphomas (DLBCL) can be found at several different locations. DLBCL in the central nervous system or the testis are 'Immune-privileged site-associated' diffuse large B-cell lymphomas (IP-DLBCL). They differ from other DLBCL in several clinical and molecular aspects. The most striking characteristic of IP-DLBCL is the loss of expression of human leukocyte antigen (HLA), often associated with deletions of the HLA region on chromosome 6p21.3. The research in this thesis aims to gain more insight into the molecular background of the biological behavior of IP-DLBCL. Loss of HLA expression in IP-DLBCL was associated with loss of expression of numerous genes involved in the immune response and with a lower number of immune cells. The common chromosomal aberrations also deregulated genes that are involved in the immune response or cell death. These results indicate the importance of the regulation of the anti-tumor immune response in IP-DLBCL, loss of HLA expression probably being an immune escape mechanism. Apart from the loss of HLA expression, additional characteristics of IP-DLBCL that separate them from other DLBCL were investigated. IP-DLBCL have an 'activated B cell-like' expression profile. IP-DLBCL also share some characteristic genomic aberrations. These findings support the classification of IP-DLBCL as a separate subgroup within DLBCL in general. To complete the thesis a model is proposed for IP-DLBCL lymphomagenesis based on the principle of immunoediting, in which the interaction between the lymphoma and its environment shapes the final characteristics of the IP-DLBCL.

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 20 K Atlantic cod microarray to investigate how a water temperature change which simulates that seen in Newfoundland during the spring-summer (i.e. from 10[DEGREE SIGN]C to 16[DEGREE SIGN]C, 1[DEGREE SIGN]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[DEGREE SIGN]C and 10[DEGREE SIGN]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 IkappaBalpha, 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[DEGREE SIGN]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[DEGREE SIGN]C and sampled at 6HPI and 24HPI, respectively. Our results indicate that the modulation of mRNA expression of genes belonging to the NF-kappaB 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[DEGREE SIGN]C vs. 16[DEGREE SIGN]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

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Not AvailableExposure 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’simmuneresponsemaybe pathogen dependent.Not Availabl

Additional file 3: Table S3. of 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

Complete list of overrepresented and underrepresented GO terms in test set of genes (303 genes differentially expressed between RA-Zoo and both RA and RA-PH) compared with reference set of genes (20K cod microarray). (PDF 90kb

Additional file 2: Table S2. of 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

Microarray-identified genes significantly down-regulated in RA-Zoo compared with both RA and RA-PH. (PDF 310kb

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, II cases of primary testicular and II 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