Catch of the Day: New Serum Amyloid A (SAA) Antibody Is a Valuable Tool to Study Fish Health in Salmonids.

Serum amyloid A (SAA) proteins belong to a family of acute-phase reactants, playing an integral role in defending the organism from pathological damage. Despite a wealth of data on the regulation of SAA transcripts in teleosts, there is only limited information on these proteins' abundance in fish. The aim of this study is to characterise SAA protein levels in salmonids using a newly developed antibody specific to salmonid SAA. The salmonid SAA antibody detected SAA and accurately discriminated between stimulated and control specimens from rainbow trout macrophage cell line (RTS-11) in vitro, as well as rainbow trout challenged with Aeromonas salmonicida- or flagellin-stimulated Atlantic salmon in vivo. The presence of SAA protein was analysed in RTS-11 cell line supernatants, liver, and spleen samples using ELISA, immunoblotting, and immunohistochemistry. This study is the first to characterise SAA protein levels in salmonids in vivo and in vitro. The newly developed salmonid SAA antibody was able to discriminate between stimulated and unstimulated specimens, showing that it can be used to study the acute-phase response in salmonids with the potential to be further developed into assays to monitor and evaluate health in wild and farmed fish.

Identification of a prognostic signature in colorectal cancer using combinatorial algorithm-driven analysis.

Colorectal carcinoma is one of the most common types of malignancy and a leading cause of cancer-related death. Although clinicopathological parameters provide invaluable prognostic information, the accuracy of prognosis can be improved by using molecular biomarker signatures. Using a large dataset of immunohistochemistry-based biomarkers (n = 66), this study has developed an effective methodology for identifying optimal biomarker combinations as a prognostic tool. Biomarkers were screened and assigned to related subsets before being analysed using an iterative algorithm customised for evaluating combinatorial interactions between biomarkers based on their combined statistical power. A signature consisting of six biomarkers was identified as the best combination in terms of prognostic power. The combination of biomarkers (STAT1, UCP1, p-cofilin, LIMK2, FOXP3, and ICOS) was significantly associated with overall survival when computed as a linear variable (χ2  = 53.183, p < 0.001) and as a cluster variable (χ2  = 67.625, p < 0.001). This signature was also significantly independent of age, extramural vascular invasion, tumour stage, and lymph node metastasis (Wald = 32.898, p < 0.001). Assessment of the results in an external cohort showed that the signature was significantly associated with prognosis (χ2  = 14.217, p = 0.007). This study developed and optimised an innovative discovery approach which could be adapted for the discovery of biomarkers and molecular interactions in a range of biological and clinical studies. Furthermore, this study identified a protein signature that can be utilised as an independent prognostic method and for potential therapeutic interventions.

Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss.

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3-5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Induction of IL-22 protein and IL-22-producing cells in rainbow trout Oncorhynchus mykiss.

IL-22 is a critical cytokine which is involved in modulating tissue responses during inflammation, and is produced mainly by T cells and innate leucocytes. In mammals, IL-22 is a key component in mucosal defences, tissue repair, epithelial cell survival and proliferation. In teleosts, IL-22 has been cloned and studied in several species, and the transcript is highly expressed in mucosal tissues and induced by pathogen associated molecular patterns (PAMPs), suggesting IL-22 also functions as an important component of the innate immune response in fish. To investigate these immune responses further, we have validated and characterised two monoclonal antibodies (mAbs) which were raised against two different peptide immunogens of salmonid IL-22. Our results show that both mAbs specifically react to their own peptide immunogens and recombinant IL-22, and are able to detect the induction of native protein expression after stimulation. In flow cytometry, an increase in IL-22 positive cells was detected after stimulation in vitro with cytokines and PAMPs and in vivo after bacterial challenge. The immunohistochemistry results showed that IL-22 is highly upregulated in the gills after challenge, both in cells within the gill filaments and in the interbranchial lymphoid tissue. Such results suggest IL-22 may have a role in triggering local antimicrobial defences in fish that may facilitate efficient microbial clearance. Hence monitoring IL-22 producing cells/protein secretion may provide an alternative mean to assess the effectiveness of mucosal vaccines.

The expression of brown fat-associated proteins in colorectal cancer and the relationship of uncoupling protein 1 with prognosis.

Colorectal carcinoma is one of the most common types of malignancy and a leading cause of cancer related death. The aberrant expression of a brown fat-like phenotype in cancer cells has been previously implicated in tumour growth. Therefore, the expression of brown fat-associated proteins in colorectal cancer could be associated with tumour prognosis. Monoclonal antibodies to brown fat-associated proteins CIDEA, ELOVL3, ELOVL5, and UCP1 were developed. The antibodies were used to profile the expression of protein targets by immunohistochemistry in a discovery cohort comprising 50 normal colonic mucosa samples and 274 primary colorectal cancers and a validation cohort comprising 549 colorectal cancers. Immunostaining for UCP1 was observed in the majority of colorectal tumours while no immunostaining was observed in normal colonic mucosa (p < 0.001). The expression of UCP1 was significantly associated with better overall survival in both the discovery cohort (HR = 0.615, 95%CI = 0.416-0.909, χ2 = 6.119, p = 0.013) and the validation cohort (HR = 0.629, 95%CI = 0.480-0.825, χ2 = 11.558, p = 0.001). Furthermore, UCP1 was independently prognostic in multivariate analysis (p = 0.004). This study has identified the brown fat-like phenotype as a novel pathway associated with survival in colorectal cancer. The expression of UCP1 was identified as a significant prognostic biomarker for colorectal cancer.

Immunohistochemical examination of immune cells in adipose tissue of rainbow trout (Oncorhynchus mykiss) following intraperitoneal vaccination.

Mammalian perivisceral adipose has been shown to play an important role in the regulation of the peritoneal immune responses. Recently it has been demonstrated that peritoneal antigens are collected by leukocytes within the visceral adipose mass, and a broad range of immunomodulatory genes are differentially expressed in adipose tissue after intraperitoneal vaccination in rainbow trout. To assess the immune cell component in adipose, immunohistochemical analysis was used to examine B-cell, T-cell and antigen presenting cell (APC) numbers and distribution in rainbow trout adipose tissue 24 and 72 h post vaccination in comparison to control fish. The results of this study support previous work on mammals with omental milky spots in naïve fish found to contain APCs and T-cells which then increased in size, number and complexity following vaccination. It suggests that following peritoneal stimulation the visceral adipose mass in fish likely plays an important role in vaccine antigen uptake and presentation by APCs, as well as subsequent T-cell activation and differentiation.

Analysis of adipose tissue immune gene expression after vaccination of rainbow trout with adjuvanted bacterins reveals an association with side effects.

Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. Whilst very effective they are frequently associated with inflammatory responses that can result in clinically significant side-effects often involving the adipose tissue that is in direct contact with the vaccine. To explore the potential of immune gene expression changes in the adipose tissue of fish to be markers of vaccination efficacy or development of side-effects we have studied the response to a bacterial (Aeromonas salmonicida) vaccine administered with two different adjuvants. The first adjuvant was Montanide™ ISA 763A VG, thought to induce a mostly humoral response, and the second was Montanide™ ISA 761 VG that gives a more balanced humoral and cell mediated response. Following vaccination tissue samples were collected at days 3, 14 and 28 for RTqPCR analysis. Fifty immune genes were studied with a focus on a) pro-inflammatory associated molecules and b) adaptive immune response related molecules linked with possible Th1, Th2, Th17 and T-regulatory pathways, with the expression data analysed for associations with Speilberg post-vaccination side effect scores. The results showed that the adipose tissue is a particularly sensitive and discriminatory tissue for studying adjuvant effects. A clear upregulation of many immune genes occurred in response to both vaccine groups, which persisted over time and overlapped with the appearance of visible adhesions. Our analysis revealed a relationship between adipose tissue immune function and the development of vaccine-induced adhesions giving the potential to use immune gene expression profiling in this tissue to predict the side-effects seen.

The differential expression of omega-3 and omega-6 fatty acid metabolising enzymes in colorectal cancer and its prognostic significance.

BACKGROUND: Colorectal cancer is a common malignancy and one of the leading causes of cancer-related deaths. The metabolism of omega fatty acids has been implicated in tumour growth and metastasis. METHODS: This study has characterised the expression of omega fatty acid metabolising enzymes CYP4A11, CYP4F11, CYP4V2 and CYP4Z1 using monoclonal antibodies we have developed. Immunohistochemistry was performed on a tissue microarray containing 650 primary colorectal cancers, 285 lymph node metastasis and 50 normal colonic mucosa. RESULTS: The differential expression of CYP4A11 and CYP4F11 showed a strong association with survival in both the whole patient cohort (hazard ratio (HR)=1.203, 95% CI=1.092-1.324, χ2=14.968, P=0.001) and in mismatch repair-proficient tumours (HR=1.276, 95% CI=1.095-1.488, χ2=9.988, P=0.007). Multivariate analysis revealed that the differential expression of CYP4A11 and CYP4F11 was independently prognostic in both the whole patient cohort (P=0.019) and in mismatch repair proficient tumours (P=0.046). CONCLUSIONS: A significant and independent association has been identified between overall survival and the differential expression of CYP4A11 and CYP4F11 in the whole patient cohort and in mismatch repair-proficient tumours.

Correction: First Demonstration of Antigen Induced Cytokine Expression by CD4-1+ Lymphocytes in a Poikilotherm: Studies in Zebrafish (Danio rerio).

[This corrects the article DOI: 10.1371/journal.pone.0126378.].

Analysis of interferon gamma protein expression in zebrafish (Danio rerio).

IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.

Characterisation of the oxysterol metabolising enzyme pathway in mismatch repair proficient and deficient colorectal cancer.

Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes and tumour-derived oxysterols have been implicated in tumour growth and survival. The aim of this study was to profile the expression of oxysterol metabolising enzymes in primary colorectal cancer and assess the association between expression and prognosis.Immunohistochemistry was performed on a colorectal cancer tissue microarray containing 650 primary colorectal cancers using monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1, which we have developed. Unsupervised hierarchical cluster analysis was used to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis.Cluster analysis of the whole patient cohort identified a good prognosis group (mean survival=146 months 95% CI 127-165 months) that had a significantly better survival (χ2=12.984, p<0.001, HR=1.983, 95% CI 1.341-2.799) than the poor prognosis group (mean survival=107 months, 95% CI 98-123 months). For the mismatch repair proficient cohort, the good prognosis group had a significantly better survival (χ2=8.985, p=0.003, HR=1.845, 95% CI 1.227-2.774) than the poor prognosis group. Multi-variate analysis showed that cluster group was independently prognostically significant in both the whole patient cohort (p=0.02, HR=1.554, 95% CI 1.072-2.252) and the mismatch repair proficient group (p=0.04, HR=1.530, 95% CI 1.014-2.310).Individual oxysterol metabolising enzymes are overexpressed in colorectal cancer and an oxysterol metabolising enzyme expression profile associated with prognosis has been identified in the whole patient cohort and in mismatch repair proficient colorectal cancers.

The expression and prognostic significance of bcl-2-associated transcription factor 1 in rectal cancer following neoadjuvant therapy.

AIMS: bcl-2-associated transcription factor 1 (BCLAF1) is a nuclear protein that binds to bcl-related proteins and can induce apoptosis and autophagy. The aim of this study was to investigate the expression of BCLAF1 in a series of rectal cancers following neoadjuvant therapy. METHODS AND RESULTS: Immunohistochemistry was performed on a post-neoadjuvant therapy rectal cancer tissue microarray. It contained rectal cancers (n = 248), lymph node metastases (n = 76), and non-neoplastic rectal mucosal samples (n = 73). A monoclonal antibody against BCLAF1 that we have developed was used. Non-neoplastic rectal epithelium showed nuclear localization of BCLAF1 in both crypt and surface epithelial cells, whereas rectal cancers showed both nuclear and cytoplasmic BCLAF1 expression. Most rectal cancers showed moderate or strong nuclear immunoreactivity, but showed weak cytoplasmic immunoreactivity. Cytoplasmic BCLAF1 expression was increased in primary rectal cancers as compared with non-neoplastic rectal mucosa (P = 0.008). Negative and weak nuclear BCLAF1 expression was associated with a poor prognosis [hazard ratio (HR) 0.502, 95% confidence interval (CI) 0.269-0.939, χ(2) = 4.876, P = 0.027]. Nuclear BCLAF1 expression was independently prognostic in a multivariate model (HR 0.431, 95% CI 0.221-0.840, P = 0.013). CONCLUSIONS: This study has shown that both cytoplasmic BCLAF1 expression and nuclear BCLAF1 expression are increased in post-neoadjuvant therapy rectal cancer, and that negative and weak nuclear BCLAF1 expression are independently associated with a poor prognosis.

First Demonstration of Antigen Induced Cytokine Expression by CD4-1+ Lymphocytes in a Poikilotherm: Studies in Zebrafish (Danio rerio).

Adaptive immunity in homeotherms depends greatly on CD4+ Th cells which release cytokines in response to specific antigen stimulation. Whilst bony fish and poikilothermic tetrapods possess cells that express TcR and CD4-related genes (that exist in two forms in teleost fish; termed CD4-1 and CD4-2), to date there is no unequivocal demonstration that cells equivalent to Th exist. Thus, in this study we determined whether CD4-1+ lymphocytes can express cytokines typical of Th cells following antigen specific stimulation, using the zebrafish (Danio rerio). Initially, we analyzed the CD4 locus in zebrafish and found three CD4 homologues, a CD4-1 molecule and two CD4-2 molecules. The zfCD4-1 and zfCD4-2 transcripts were detected in immune organs and were most highly expressed in lymphocytes. A polyclonal antibody to zfCD4-1 was developed and used with an antibody to ZAP70 and revealed double positive cells by immunohistochemistry, and in the Mycobacterium marinum disease model CD4-1+ cells were apparent surrounding the granulomas typical of the infection. Next a prime-boost experiment, using human gamma globulin as antigen, was performed and revealed for the first time in fish that zfCD4-1+ lymphocytes increase the expression of cytokines and master transcription factors relevant to Th1/Th2-type responses as a consequence of boosting with specific antigen.

The expression and prognostic significance of retinoic acid metabolising enzymes in colorectal cancer.

Colorectal cancer is one of the most common types of cancer with over fifty percent of patients presenting at an advanced stage. Retinoic acid is a metabolite of vitamin A and is essential for normal cell growth and aberrant retinoic acid metabolism is implicated in tumourigenesis. This study has profiled the expression of retinoic acid metabolising enzymes using a well characterised colorectal cancer tissue microarray containing 650 primary colorectal cancers, 285 lymph node metastasis and 50 normal colonic mucosal samples. Immunohistochemistry was performed on the tissue microarray using monoclonal antibodies which we have developed to the retinoic acid metabolising enzymes CYP26A1, CYP26B1, CYP26C1 and lecithin retinol acyl transferase (LRAT) using a semi-quantitative scoring scheme to assess expression. Moderate or strong expression of CYP26A1was observed in 32.5% of cancers compared to 10% of normal colonic epithelium samples (p<0.001). CYP26B1 was moderately or strongly expressed in 25.2% of tumours and was significantly less expressed in normal colonic epithelium (p<0.001). CYP26C1 was not expressed in any sample. LRAT also showed significantly increased expression in primary colorectal cancers compared with normal colonic epithelium (p<0.001). Strong CYP26B1 expression was significantly associated with poor prognosis (HR = 1.239, 95%CI = 1.104-1.390, χ(2) = 15.063, p = 0.002). Strong LRAT was also associated with poorer outcome (HR = 1.321, 95%CI = 1.034-1.688, χ(2) = 5.039, p = 0.025). In mismatch repair proficient tumours strong CYP26B1 (HR = 1.330, 95%CI = 1.173-1.509, χ(2)= 21.493, p<0.001) and strong LRAT (HR = 1.464, 95%CI = 1.110-1.930, χ(2) = 7.425, p = 0.006) were also associated with poorer prognosis. This study has shown that the retinoic acid metabolising enzymes CYP26A1, CYP26B1 and LRAT are significantly overexpressed in colorectal cancer and that CYP26B1 and LRAT are significantly associated with prognosis both in the total cohort and in those tumours which are mismatch repair proficient. CYP26B1 was independently prognostic in a multivariate model both in the whole patient cohort (HR = 1.177, 95%CI = 1.020-1.216, p = 0.026) and in mismatch repair proficient tumours (HR = 1.255, 95%CI = 1.073-1.467, p = 0.004).

Cellular apoptosis susceptibility (chromosome segregation 1-like, CSE1L) gene is a key regulator of apoptosis, migration and invasion in colorectal cancer.

Cellular apoptosis susceptibility (chromosome segregation 1-like, CSE1L) gene maps to chromosomal region 20q13.13, a region frequently amplified in solid tumours. In this study, we investigated the roles played by CSE1L in colorectal cancer by examining CSE1L expression and clinico-pathological parameters in colorectal cancer and investigating the effect of CSE1L on the viability, adhesion and migration of colorectal cancer cells. RT-PCR showed that CSE1L mRNA was over-expressed in colorectal cancer. CSE1L depletion by knock-down with CSE1L-specific siRNA significantly reduced viability in HCT116 cells (p = 0.004) and SW480 cells (p = 0.003) whilst significantly increasing the proportion of apoptotic HCT116 cells (p < 0.001) and SW480 cells (p < 0.001). Furthermore, CSE1L depletion significantly reduced the adhesive capacity of HCT116 (p = 0.003) and SW480 cells (p = 0.004). Analysis by qRT-PCR following CSE1L siRNA treatment of HCT116 and SW480 cells showed significant modulation of key apoptotic (p53, p73 and BAK) and adhesive (E-cadherin, Ep-CAM and ICAM-1) molecules. Immunohistochemistry of a colorectal cancer tissue microarray showed that CSE1L had a significantly increased level in colorectal cancer compared to normal colorectal epithelium (p < 0.001). There were significant decreases in both nuclear (p = 0.006) and cytoplasmic (p = 0.003) staining of CSE1L in tumours with lymph node metastasis (stage 3 tumours) compared with lymph node-negative tumours (stage 1 and 2 tumours). In lymph node-negative patients, poor survival was associated with increased CSE1L cytoplasmic expression (p = 0.042). These results indicate that CSE1L is associated with viability and apoptosis, cellular adhesion and invasion, thus implicating CSE1L in the progression of colorectal cancer.

Discovery of zebrafish (Danio rerio) interleukin-23 alpha (IL-23α) chain, a subunit important for the formation of IL-23, a cytokine involved in the development of Th17 cells and inflammation.

This paper reports the cloning and sequencing of interleukin (IL)-23 p19 subunit for the first time within a non-mammalian species, the zebrafish (Danio rerio), which was discovered using a synteny approach. In addition, amino acid sequences were for IL-23 p19 subunits were also predicted from the stickleback, Fugu and Tetraodon genomes and included in this investigation. The zebrafish IL-23 p19 cDNA consisted of a 66bp 5' UTR, a 249bp 3' UTR and a single open reading frame of 567bp giving a predicted 188 aa IL-23 p19 molecule. Multiple alignment of zebrafish IL-23 p19, with other known IL-23 p19 and IL-12 p35 amino acid sequences revealed areas of amino acid conservation, such as the presence of four predicted α-helixes, cysteines important for disulphide bond formation and the conservation of a tryptophan known to interact with the receptor. Amino acid homologies and phylogenetic analysis confirmed the relationship of the fish IL-23 p19 subunits with their mammalian homologues. All the teleost fish IL-23 p19 subunits were found to have 4 exons and 3 introns similar to that of human and mouse IL-23 p19 and a limited degree of synteny was found between the organisms for the regions containing the IL-23 p19 genes with only PAB-dependent poly(A)-specific ribonuclease subunit 2 (PAN2) and IL-23 p19 found in the same order on human chromosome 12 and all the fish genomes looked at. Lastly using real-time PCR, constitutive expression of IL-12 p40 and IL-23 p19 was observed in the kidney, liver, gut and muscle with IL-12 p40 expression higher than IL-23 p19. As soon as an hour after stimulation with LPS, there was an increase of IL-23 p19 in zebrafish leukocytes and an increase of IL-1ß, IL-12 p40 and IL-23 p19 expression was found after infection of zebrafish for 1 or 6 days with Mycobaterium marinum strain E11.

Molecular cloning and characterization of interferon regulatory factors 4 and 8 (IRF-4 and IRF-8) in rainbow trout, Oncorhynchus mykiss.

Mammalian interferon regulatory factor (IRF)4 (PIP, LSIRF, and ICSAT) and IRF8 (ICSBP) are known to be critical in regulating a spectrum of functional and developmental processes in lymphomyeloid cell lineages either through direct binding to IRF-E motifs in target gene promoters or indirectly by binding to composite motifs recognized by Ets family members, PU.I and Sp.I. Here we report, for the first time in fish, the sequencing and characterization of full-length cDNA homologues of rainbow trout (rt) IRF4 and rtIRF8. The rtIRF4 molecule consists of 1848 bp with a 45 bp 5' UTR and a predicted 378 bp 3' UTR translating into a 474 aa protein. RtIRF8 consists of 1951 bp with a 52 bp 5' UTR and a 564 bp 3' UTR translating into a 444 aa protein. Each gene possesses a putative DNA binding domain (DBD) containing the tryptophan pentad-repeat domain found in all IRF family members. Both molecules also possess a well conserved IRF association domain (IAD). The presence of these domains along with phylogenetic analysis places the two genes in the IRF4 subfamily. Both genes were detected in a range of trout tissues where IRF8 was the overall predominant transcript. Consistent with mammalian studies, the highest expression levels of IRF4 and IRF8 were observed in the lymphomyeloid-rich fish tissues, spleen, head kidney and gills. IRF8 expression in stimulated trout splenocytes was significantly up-regulated by polyinosinic:polycytidylic acid (poly I:C), trout recombinant (r)IL-15, phorbol 12-myristate 13-acetate (PMA), and phytohaemagglutinin (PHA) treatment whilst remaining refractory towards lipopolysaccharide (LPS) treatment. IRF4 was significantly down-regulated by LPS stimulation and remained refractory towards poly I:C, trout rIL15, and PHA. PMA stimulation elicited a significant upregulation of IRF4 expression. Overall, these data support the premise that these IRFs are likely to play important roles in the functional and developmental processes occurring in fish lymphomyeloid tissues.

Identification and characterization of the transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio.

The discovery of cytokines expressed by T-helper 1 (Th1), Th2, Th17 and T-regulatory (T(reg)) cells has prompted speculation that these types of responses may exist in fish, arising early in vertebrate evolution. In this investigation, we cloned three zebrafish transcription factors, T-box expressed in T cells (t-bet), signal transducer and activator of transcription 6 (stat6) and fork-head box p3 (foxp3), in which two transcripts are present, that are important in the development of a number of these cell types. They were found within the zebrafish genome, using a synteny approach in the case of t-bet and foxp3. Multiple alignments of zebrafish t-bet, stat6 and foxp3 amino acids with known vertebrate homologues revealed regions of high conservation, subsequently identified to be protein domains important in the functioning of these transcription factors. The gene organizations of zebrafish t-bet and foxp3 were identical to those of the human genes, with the second foxp3 transcript lacking exons 5, 6, 7 and 8. Zebrafish stat6 (21 exons and 20 introns) was slightly different from the human gene, which contained 22 exons and 21 introns. Immunostimulation of zebrafish head kidney and spleen cells with phytohaemagglutinin, lipopolysaccharide or Poly I:C, showed a correlation between the expression of t-bet, stat6 and foxp3 with other genes involved in Th and T(reg) responses using quantitative PCR. These transcription factors, together with many of the cytokines that are expressed by different T-cell subtypes, will aid future investigations into the Th and T(reg) cell types that exist in teleosts.

The roles of heterogeneous nuclear ribonucleoproteins in tumour development and progression.

The heterogeneous nuclear ribonucleoproteins (hnRNP) are a family of proteins which share common structural domains, and extensive research has shown that they have central roles in DNA repair, telomere biogenesis, cell signaling and in regulating gene expression at both transcriptional and translational levels. Through these key cellular functions, individual hnRNPs have a variety of potential roles in tumour development and progression including the inhibition of apoptosis, angiogenesis and cell invasion. The aims of this review are to provide an overview of the multi functional roles of the hnRNPs, and how such roles implicate this family as regulators of tumour development. The different stages of tumour development that are potentially regulated by the hnRNPs along with their aberrant expression profiles in tumour tissues will also be discussed.