The total number of X-gal-stained myofibres was decided microscopically from six sections per fish at regular intervals across the tissue block between gills and dorsal fin

The total number of X-gal-stained myofibres was decided microscopically from six sections per fish at regular intervals across the tissue block between gills and dorsal fin.15 Open in a separate window Rabbit polyclonal to JAKMIP1 Figure 4 Proliferative T-cell responses of splenocytes from groups of mice immunized (on days 0 and Vilazodone D8 56) by the intramuscular route with +pcDNA3 (?) or with and plasmid DNA-expressing mouse GM-CSF (?), 70 days after priming by standard technique.17 Results were expressed as stimulation indices (SI) of the mean counts per minute (c.p.m.) from triplicate cultures in the presence of -gal protein divided by mean c.p.m. In addition, plasmid DNA expressing mouse granulocyteCmacrophage colony-stimulating factor (GM-CSF) had a marked effect on cytotoxic T-cell-like activity in fish by reducing the average number of myofibres that expressed -gal, 28 days after co-injection with plasmid DNA expressing -gal. Although the mechanism by which the mouse GM-CSF exerted its biological effects in Vilazodone D8 fish is unknown, this obtaining might have important implications for fish Vilazodone D8 vaccination, particularly when cytotoxic T cells may play a critical role. Plasmid DNAs encoding protective antigens of pathogens have shown promise as vaccines for the control of infectious diseases. Plasmid DNA, apart from encoding the antigen, has pronounced immunostimulatory properties (adjuvant activity) that have been attributed to specific single-stranded oligonucleotide sequences made up of unmethylated CpG motifs.1 These CpG motifs have been shown to exert a mitogenic effect on B cells,2 and directly activate monocytes, macrophages, and dendritic cells leading to secretion of cytokines that favour the development of murine T helper 1 (Th1) effector cells.3,4 DNA from bacteria, insects and nematodes is immunostimulatory, whereas DNA from mammals and lower vertebrates, e.g. fish is not.5 Thus, bacterial DNA, but not Vilazodone D8 eukaryotic DNA, prompts the vertebrate innate immune system to sense danger by employing pattern recognition receptors with broad reactivity,6 as opposed to the antigen-specific receptors used by the adaptive immune system. Fish seroconvert more readily than mice following intramuscular injection of plasmid DNA expressing -galactosidase (-gal).7 In this study we asked whether this increased immunogenicity of plasmid DNA in fish was because of their increased responsiveness to immunostimulatory CpG. To test this, a synthetic oligodeoxynucleotide (ODN) TCCATGAgene9 was compared with another pUC-based plasmid made up of the non-CpG-gene9 in fish. Immune responses to DNA vaccines can be modulated by coinjecting plasmid DNA expressing various cytokines that play a critical regulatory role in immunity. GM-CSF in particular has been found to exert an adjuvant effect for antibody and cellular responses to DNA vaccines.10 In addition, a cytokine akin to GM-CSF has been described in fish.11 Therefore, we tested the adjuvanticity of plasmid DNA, expressing mouse GM-CSF around the immune responses to co-administered plasmid DNA expressing -gal (L.) were injected intramuscularly under anaesthesia into the epaxial muscle by a standard method7 with the following formulations; 50 g +50 g pcDNA3, 50 g +50 g plasmid expressing mouse GM-CSF, 10 g r-gal+50 g gene (donated by Vical, San Diego, CA, and emptied by removal of its luciferase gene), and finally 10 g r-gal. Serum was collected 4 weeks after injection.7 Mouse serum anti–gal immunoglobulin G (IgG) antibody responses, measured by enzyme-linked immunosorbent assay (ELISA) as previously described,7 were significantly increased ( 005) in mice immunized with r–gal protein and CpG ODN 1826 when compared with mice immunized with the protein and the GpG ODN (Fig. 1). Similarly, plasmid DNA expressing mouse GM-CSF was shown to increase the number of responding mice to -gal expressed by the co-administered plasmid Vilazodone D8 (Fig. 1). Open in a separate window Physique 1 Serum IgG antibody responses in BALB/c mice 42 days after a single injection by the intramuscular route with these injections: (a) +pcDNA3 or with plasmid DNA expressing mouse GM-CSF; (b) r-gal+GpG ODN or r-gal with the 1826 CpG motif. Antibody titres to -gal were measured by ELISA as previously described.7 The titre was the last dilution to give a binding twice the conjugate control, i.e. 0228. Coinjection of murine CpG ODN 1826 with r-gal in goldfish caused a small upward twofold shift in the scatter of serum anti–gal antibody titres [measured by ELISA as previously described7] when compared with the control groups immunized with the protein alone, or the protein with the GpG ODN (Fig. 2). The observed increase was not significant ( 005). However, co-injection of r–gal protein with vacant pcDNA3 or gene, increased the anti–gal antibody response by fourfold ( 005), and 13-fold ( 00001), respectively (Fig. 2). The vacant VR1223 plasmid made up of the non-CpG-gene did not have any effect on the anti–gal antibody responses (Fig. 2). Open in a separate window Physique 2 Serum antibody titres to -gal.