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The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells. Our results suggest that the extracellular Drgal1-L2 and Drgal3-L1 interact directly and in a carbohydrate-dependent manner with the IHNV glycosylated envelope and glycans on the epithelial cell surface, significantly reducing viral adhesion.

High-dose, continuous infusion interleukin-2 (IL-2) regimens generate greater Lymphokine Activated Killer cell (LAK) cytotoxicity in vitro and a higher rebound lymphocytosis in vivo than do bolus IL-2 regimens. Lymphocytes initially activated by continuous infusion IL-2 then subsequently pulsed with IL-2 have increased cytotoxicity against cancer cells. Famotidine may enhance the lysis of tumors by cytotoxic lymphocytes. Fourteen patients with melanoma were treated with famotidine 20 mg intravenously twice per day and continuous infusion IL-2 (18 MIU/sq m/24 hours) for 72 hours, followed by a 24-hour rest, then IL-2 18 MIU/sq m over 15-30 minutes for 1 dose (12 patients) or daily for 3 doses (2 patients). Most common toxicities were fever, nausea/emesis, hypophosphatemia, hypomagnesemia, and rigors. Nine partial responses (64% response rate; 95% Confidence Interval: 39%-84%) have been seen. Median survival has not been reached at greater than 10 months. Two patients responding to therapy showed an increase in detectable CD 56(+) cells in serial subcutaneous or lymph node biopsies, while 1 patient undergoing progression of disease had no such infiltrate. High-dose, 72-hour continuous infusion plus pulse interleukin-2 with famotidine has activity in melanoma. CD 56(+) cells may play a role in responding patients.

γδ TCR that have structural differences from conventional γδ TCR in mammals have been observed in non-eutherian vertebrates. Three different types of Vδ regions are present in Xenopus tropicalis, similar to Vδ, Vα, and VHδ (derived from the V regions of Ig H chains). Semi-quantitative PCR analyses in adult and larval Xenopus laevis show that expression levels of TCRδC and TCRγC, as well as of TCRδV and TCRγV repertoires, vary between tissues. Comparative analyses of adult and larval CDR3 sequences suggest ontogenic-, tissue- and perhaps V family-specific expression. We have detected changes at the VDJ junctions including N-nucleotide addition, nucleotide trimming, and homologous recombination events. γδ T cell populations in mammals differentially express either IL-17 or IFNγ. In mammals, six IL-17 forms have been described (A-E), the least well characterized of which is IL-17D. Most non-mammalian IL-17 homologs, including those in chicken, fish, lamprey, nematode, oyster, sea urchin, and tunicate, and especially, Xenopus, are the IL-17D form. We have cloned two types of IL-17A/F, IL-17B, and IL-17D from X. laevis. Semi-quantitative PCR analyses in adult and larval Xenopus show that the expression of these IL-17 family members vary during ontogeny and between tissues. Study of γδ TCR and IL-17 family expression in Xenopus will illuminate evolutionary and developmental changes in γδ T cell antigen recognition and effector strategies.

γδ T cell receptors (TCR) that have structural differences from conventional γδ T cells in mammals have been observed in non-eutherian vertebrates. Three different types of V regions are present in the frog Xenopus tropicalis genomic TCRδ locus (Eur. J. Immunol. 40: 2319, 2010), most similar to Vδ, Vα, and VHδ (evolutionarily derived from the V regions of Ig H chains). We have begun a survey of the tissue distribution of TCRδ expression in X. laevis tadpoles and adults by 5’ RACE PCR. The deduced amino acid sequences predicted from X. tropicalis genome sequences were aligned with those from X. laevis TCRδ V RACE clones, revealing similar Vδ, Vα, and VHδ families in X. laevis. Analyses of the TCRδ RACE nucleotide sequences have identified the putative D and J gene segments in X. laevis as well. Although we find representatives of all TCRδ V sub-families in all tested adult tissues, the distribution of the data suggests that the expressed repertoire is skewed in some tissues, especially in the skin, where a majority of recovered sequences were of the Vα type. Interestingly, analyses of the Vδ type indicate a lack of N-nucleotide addition in the CDR3 in adults, suggestive of a population of T cells that is generated during larval life that persists through metamorphosis. Continued study of these unique TCR in Xenopus will illuminate apparent plasticity in TCR evolution, and shed light on how γδ TCR recognize antigen in all vertebrates.

The galectin family of β-galactoside-binding proteins is evolutionarily conserved. Galectins are known to interact with and affect the infectivity and/or pathogenicity of several viruses. We hypothesize that galectin interactions with viruses influence anti-viral immune responses. We investigate the interactions of zebrafish galectins with the glycosylated (G) protein of the rhabdovirus IHNV. We (a) characterized the carbohydrates present on IHNV proteins, and (b) identified direct interactions between zebrafish galectins (Drgal) and IHNV. We assessed the effects of immune challenge on (c) galectin and (d) cytokine expression in zebrafish. Several IHNV proteins exhibit complex glycosylation patterns. One or multiple IHNV proteins exhibit terminally linked mannose, sialic acid terminally linked to galactose, galactose-β(1-3)-GalNAc, sialic acid terminally linked to galactose or GalNAc, and galactose-β(1-4)-GlcNAc. The proto type Drgal1-L2, the chimera type Drgal3-L1 and the tandem-repeat type Drgal9-L2 all appear to bind to one or more IHNV proteins. All tested Drgal appear to bind to the IHNV G protein. ZFL cells endogenously express mRNA encoding Drgal1-L2, -L3, and -L4, Drgal3-L1 and -L2, and Drgal9-L1 mRNA. Drgal mRNA expression in ZFL cells was not induced by treatment with polyI:C. ZFL cells were found to endogenously express mRNA encoding Mx, TNFα, and IFNαβ. Treatment of these cells with polyI:C induced the expression of mRNA encoding IL-10 and IFNγ2.

The galectin family of β-galactoside-binding proteins is evolutionarily conserved. In homeotherm vertebrates, galectin-virus interactions can affect viral infectivity and/or pathogenicity. We hypothesize that in teleost fish, galectin interactions with viral proteins modulate viral infectivity. We examined fish cell lines for expression of galectin-like molecules, and assessed changes in expression of identified galectins as well as of cytokines in response to viral infection. Minnow (EPC), trout (RTG-2), bluegill (BF-2), salmon (CHSE-214, ASK), zebrafish (ZFL), and catfish (COO, 42TA, G14D, 28S.3, 1G8, and 3B11) cells were tested. A proto-type galectin-like protein is expressed in EPC, RTG-2, and ZFL cell lines, and a chimera-type in EPC and RTG-2 cell lines. All tested cell lines express mRNA that exhibits high conservation with the zebrafish Drgal1-L2 proto-type galectin. Ten of the 12 cell lines express Drgal1-L3-like mRNA, 7 express Drgal1-L1-like mRNA, and 6 express Drgal1-L4-like mRNA. With the exception of 3B11, all tested cell lines express Drgal3-L1-like mRNA. Eight of the 12 express Drgal3-L2-like mRNA. Two of the 12 cell lines expressed Drgal9-like mRNA. Following infection of EPC cells with IHNV or IPNV, we observe modulations in expression of proto-type and tandem-repeat-type galectin-like molecules. We see modulations in expression of various cytokines, including type I and II interferons and TNF family members.