Development of a recombinant murine tumour model using hepatoma cells expressing hepatitis C virus nonstructural antigens.

Hepatitis C virus (HCV) chronically infects 2%-3% of the world's population, causing liver disease and cancer with prolonged infection. The narrow host range of the virus, being restricted largely to human hepatocytes, has made the development of relevant models to evaluate the efficacy of vaccines a challenge. We have developed a novel approach to accomplish this by generating a murine hepatoma cell line stably expressing nonstructural HCV antigens which can be used in vitro or in vivo to test HCV vaccine efficacies. These HCV-recombinant hepatoma cells formed large solid-mass tumours when implanted into syngeneic mice, allowing us to test candidate HCV vaccines to demonstrate the development of an HCV-specific immune response that limited tumour growth. Using this model, we tested the therapeutic potential of recombinant anti-HCV-specific vaccines based on two fundamentally different attenuated pathogen vaccine systems-attenuated Salmonella and recombinant adenoviral vector based vaccine. While attenuated Salmonella that secreted HCV antigens limited growth of the HCV-recombinant tumours when used in a therapeutic vaccination trial, replication-competent but noninfectious adenovirus expressing nonstructural HCV antigens showed overall greater survival and reduced weight loss compared to non-replicating nondisseminating adenovirus. Our results demonstrate a model with anti-tumour responses to HCV nonstructural (NS) protein antigens and suggest that recombinant vaccine vectors should be explored as a therapeutic strategy for controlling HCV and HCV-associated cancers.

Gonadotropin-releasing hormone neuronal system of the freshwater snails Helisoma trivolvis and Lymnaea stagnalis: possible involvement in reproduction.

Peptides of the gonadotropin-releasing hormone (GnRH) family are present in neural and nonneural tissues throughout the chordate phylum. Although GnRH peptides have been implicated in nonreproductive functions, their primary function is to control reproduction by regulating sexual behaviors and inducing gonadotropin hormone release from the pituitary. Evidence suggesting the presence of a similar peptide in the central nervous system (CNS) of the gastropod mollusc Helisoma trivolvis has recently been provided. In the present study, we examined the tissue distribution of the peptide and found that it is likely restricted to the nervous system. The neuronal system containing the endogenous GnRH-like peptide is described further and is shown, in part, to innervate the male reproductive tract. Immunostaining in the closely related snail, Lymnaea stagnalis, showed a conservation in the locations of some immunoreactive neurons. Notably, staining occurred in and adjacent to the lateral lobes of both snails. Because these lobes contain neurons involved in the stimulation of egg laying and GnRH staining occurred in additional areas in the Helisoma CNS that are involved in reproduction, we suggest that the endogenous GnRH-like peptide plays a role in regulating reproduction in freshwater snails.

Involvement of protein kinase C in 5-HT-stimulated ciliary activity in Helisoma trivolvis embryos.

1. During development, embryos of the pulmonate gastropod, Helisoma trivolvis, undergo a rotation behaviour due to the co-ordinated beating of three bands of ciliated epithelial cells. This behaviour is in part mediated by the neurotransmitter serotonin (5-HT) released from a pair of identified embryonic neurons. Using time-lapse videomicroscopy to measure ciliary beat frequency (CBF) in response to pharmacological manipulations, we determined whether protein kinase C (PKC) is involved in mediating 5-HT-stimulated ciliary beating. 2. Diacylglycerol (DAG) analogues sn-1,2-dioctanoyl glycerol (DiC8; 100 microM) and 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 microM), partially mimicked the 5-HT-induced increase in CBF. In contrast, application of OAG in the absence of extracellular Ca2+ did not result in an increase in CBF. 3. 5-HT-stimulated CBF was effectively blocked by PKC inhibitors bisindolylmaleimide (10 and 100 nM) and calphostin C (10 nM). In addition, bisindolylmaleimide (100 nM) inhibited DiC8-induced increases in CBF. At a higher concentration (200 nM), bisindolylmaleimide did not significantly reduce 5-HT-stimulated cilio-excitation. 4. Two different phorbol esters, phorbol 12-myristate 13-acetate (TPA; 0.1, 10 or 1000 nM) and phorbol 12beta, 13alpha-dibenzoate (PDBn; 10 microM) did not alter basal CBF. TPA (1 microM) did not alter 5-HT-stimulated CBF. Likewise, the synthetic form of phosphatidylserine, N-(6-phenylhexyl)-5-chloro-1-naphthalenesulphonamide (SC-9; 10 microM), did not increase CBF, whereas a strong increase in CBF was observed upon exposure to 5-HT. 5. The results suggest that a DAG-dependent, phorbol ester-insensitive isoform of PKC mediates 5-HT-stimulated CBF in ciliated epithelial cells from embryos of Helisoma trivolvis.