Graduation date: 1996

The immune systems of various teleost fish have been studied in some detail for the past several decades. One aspect of fish immunity, that of endogenously produced modulating factors, has recently received a great deal of attention. Understanding the functions and roles of endogenous factors that regulate fish immunity is paramount to expanding the fields of fish immunology and vaccinology. It is know that several lymphoid cell derived factors are detectable in in vitro cell culture systems and exhibit immune modulating effects similar to well studied proteins in mammals. However in comparison, few genes or gene products involved in the modulation of the trout immune responses have been isolated, cloned and characterized. The studies described herein were designed to isolate specific genes from rainbow trout (0ncorhynchus mykiss) and characterize their involvement in the modulation and regulation of the trout immune system. Two distinct genes were isolated cloned and sequenced. The first, non-specific cytotoxic cell enhancement factor (NCEF) gene is closely related to a human gene termed "natural killer enhancement factor" (NKEF) which is important in the modulation of human natural killer cell activity. The second gene is closely related to a group of recently characterized mammalian genes involved in the signal transduction of cytokines termed "STATs". The role of these genes and their respective protein products will be examined and discussed. The antigenic structures of the fish proteins (NCEF and STAT5) were examined by western blot and immunohistochemistry. Monoclonal antibodies derived against the respective human proteins were found to cross react with the corresponding trout proteins, demonstrating antigenic relatedness. The monoclonal regents were also used to analyze the expression of these proteins in fish cells of lymphoid and non lymphoid origin. In vitro cell culture analysis was used to determine the effects and roles of NCEF and STAT5 gene products in the trout immune system. The cytolytic and apoptotic killing activities of spleen, head kidney and peripheral blood leukocytes were found to be enhanced by NCEF. Mitogenic stimulation of peripheral blood lymphoid cells resulted in the trout STAT5 protein binding to a know sequences contained with in the promoters of genes transcriptionally activated in response to cytokine exposure. / Graduation date: 1996

Graduation date: 1995

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.

The characteristics of an inhibitor of infectious pancreatic necrosis virus (IPNV) found in normal rainbow trout serum (RTS) were studied. The serum inhibitor had a molecular weight of approximately 150 kDa and was dependent on divalent cations, either Ca����� or Mg�����. It was stable at temperatures up to 50��C and at a pH range between 4-10. The inhibitor directly inactivated the virus and the inhibition level was dependent on cell densities and on the time at which virus was exposed to RTS. The level of virus inhibition by RTS was altered by the cell line in which virus was produced. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines. Most of the salmonid sera tested showed inhibition, while non-salmonid sera did not inhibit IPNV replication. Rainbow trout continuously showed a significant level of inhibition in their serum after 23 weeks post hatch. Three isolates of IPNV were passaged five times in RTG-2 cells with either MEM-10 or MEM-10 with 1% rainbow trout serum and virus from each passage were tested for RTS sensitivity in vitro and virulence in vivo. The mortality level in brook trout fry was highly variable during viral passages, ranging between 30-89%. The RTS sensitivity and virulence were changed during viral passages, and these changes were dependent on cell culture conditions and IPNV isolate used. It was found that an IPNV crayfish isolate passaged in RTG-2 cells with MEM-10 showed significantly increased RTS sensitivity. This was, however, not correlated with decreased virulence. All three isolates showed identical antigenicity patterns with a panel of 11 monoclonal antibodies, irrespective of viral passage conditions. Clones prepared from an IPNV-Jasper (Ja) population which had been twice passed through brook trout were heterogeneous with respect to RTS sensitivity, serotype, and cDNA sequences. Eight percent of clones (4/50) were very sensitive to RTS (Ja-S), as was the parent strain, and eighty four percent of clones (42/50) showed a mid-range of RTS sensitivity. The final eight percent of clones (4/50) were RTS resistant (Ja-R). Enzyme immunodot assay revealed that Ja-S clones and Ja-R clones differed by several epitopes. Ja-S and Ja-R had significant differences in their cDNA sequences for the capsid protein VP2. These two strains shared 80.7% and 86% identity in nucleic acid and in amino acid sequences, respectively. / Graduation date: 2001

Currently little is known about the mechanisms and locations of lymphocyte development in teleosts. In this study several aspects of the underlying factors which govern B lymphocyte development in trout were investigated which included: the isolation and characterization of immunoglobulin heavy chain (IgH) genes, the recombination activating genes 1 and 2 (RAG1 and RAG2) and the use of cellular markers to identify tissues harboring precursor B-cells. Immunoglobulin heavy chains are part of the structural components which make up antibody molecules produced by B-cells. We isolated various full-length IgH cDNA clones, some of which contained the secreted while others contained the membrane bound form of IgH. Upon characterization of the membrane bound forms, typical features common to all IgH cDNAs were found including a leader peptide, a variable region and constant domain containing transmembrane (TM) segments as well. Further sequence analysis of this region revealed that the TM domains were spliced directly to the CH3 domains which results in the loss of the entire CH4 region. Our results support previous observations of unusual splicing events in fish IgH genes. RAG1 and -2 in mammals have been shown to be essential for carrying out V (D) J recombination of lymphocyte receptors and are found to be expressed within primary lymphoid tissues and precursor lymphocytes. We isolated the RAG locus from a rainbow trout genomic library and characterized their conservation and expression. Overall the complete amino acid sequences of RAG1 and RAG2 displayed 78% and 75% similarity when compared to RAG genes from higher vertebrates thus demonstrating the highly conserved nature of these genes. Tissue specific expression of both genes was primarily associated with the thymus and pronephros in both juvenile and adult trout. Based upon these observation we conclude that the thymus and pronephros likely serve as the tissue sites for V (D) J recombination in trout and are thus primary lymphoid organs. Finally we addressed the question as to where B-cell lymphopoiesis occurs in trout. Our results using both immunofluorescence and confocal microscopy putatively demonstrate that the thymus harbors precursor B-cells and thus alludes to a dual function for both B and T-cell development in trout. / Graduation date: 1996

Indole-3-carbinol (I3C) inhibits chemically induced tumor formation in rodents and rainbow trout. This study examines the effect of I3C and its analog, indole-3-acetonitrile (I3N) on xenobiotic-metabolizing enzyme systems. The modulation of these enzyme systems have been shown to have significant effects on the interaction of chemical carcinogens and cellular constituents. Rainbow trout were fed 500, 1000 and 2000 ppm dietary levels of I3C and 50, 500 and 1000 ppm dietary levels of I3N for 8 days. β-napthoflavone (BNF), which is also an effective anticarcinogen in the trout, was fed at a 500 ppm dietary level and was used as a positive LM4b (a cytochrome P-450 isozyme) inducing control. Enzyme activities assayed were: ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase (GST), and uridine diphosphoglucuronosyl transferase (UDPGT). Total cytochrome P-450 content was determined spectrophotometrically by the CO reduced method. The specific P-450 isozymes, LM2 and LM4b, were detected quantitatively using the western blot method. The BNF diet induced EROD and ECOD activities by an average of 17 fold and 5.5 fold, respectively. Total P-450 content was increased 2-fold; the P-450 isozyme LM4b was induced more than 5-fold, but LM2 content remained unchanged. This diet increased UDPGT activity 1.5-2-fold, but GST activity was not induced by dietary BNF. Neither I3C nor I3N induced the activity levels of the enzymes assayed at any administered dietary levels, which have previously shown to inhibit tumor formation and reduce formation of carcinogen-DNA adducts. Thus, the anticarcinogenic mechanism of I3C may proceed in trout by mechanisms other than enzyme induction. Further experiments on the effect of I3C and I3C acid condensation products (RXN) on in vitro AFB1-DNA binding resulted in a 40% and 48% inhibition of AFB1-DNA binding by I3C and RXN, respectively. Additions of RXN at levels much lower than those estimated to exist in vivo in hepatic tissue resulted in a significant reduction in AFB1-DNA formation suggesting that even small levels of RXN offers protection against the genotoxic effect of AFB1. However, in vitro additions of neither I3C nor RXN had an effect on DNA binding using AFBI-CI₂, an aflatoxin analog that does not require enzymatic activation. These results suggest that the primary mechanism for I3C inhibition of AFB1 induced carcinogenesis may proceed by inhibiton of formation of the ultimate electrophile, i.e. by reversible inhibition of cytochrome P-450. / Graduation date: 1989

This thesis tests the hypothesis that innate immunity may be enhanced immediately following a stressful event. The experiments characterize the acute effects of the fight or flight response on some immunological and endocrine parameters in rainbow trout (Oncorhynchus mykiss). Plasma cortisol and catecholamines were elevated within seconds of the initiation of an acute handling stressor consisting of 30 seconds in the air and five, 10 or 20 minutes in a shallow bucket of water. Plasma lysozyme activity increased after stress, however, the increases were not statistically significant unless variation was reduced by serial bleeding of the same individual trout before and after stress. A more "resting" fish was achieved by use of the anesthetic 2-phenoxy-ethanol which was surreptitiously introduced into the tanks before the initial bleed. Individual fish were then revived in freshwater and stressed as before. Enhancement of lysozyme activity was evident although levels of plasma stress hormones in fish that were anesthetized, revived and stressed were less than when fish were similarly stressed without anesthetic. Levels of cortisol and catecholamines increased within seconds of capture and aerial exposure, returned to near pre-stress levels after the fish had been placed in a shallow bucket of water for 30 seconds, then increased again. Evaluation of the influence of acute stress on survival following challenge with the pathogen Vibrio anguillarum yielded equivocal data. Results presented here suggest that enhancement of innate defenses as part of the fight or flight response merits further evaluation. / Graduation date: 1997