Evidence for antimicrobial and anticancer activity of pituitary adenylate cyclase-activating polypeptide (PACAP) from North African catfish (Clarias gariepinus): Its potential use as novel therapeutic agent in fish and humans.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide that belongs to the secretin/glucagon superfamily, of which some members have shown antimicrobial activities. Contrasting to mammals, published studies on the action of PACAP in non-mammalian vertebrate immune system remain scarce. Some of our recent studies added this peptide to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in teleost fish. Regulation of PACAP and expression of its receptor genes has been demonstrated during an immune response mounted against acute bacterial infection in fish, though the direct effect of PACAP against fish pathogenic bacteria has never been addressed. Current work provides evidence of antimicrobial activity of Clarias gariepinus PACAP against a wide spectrum of Gram-negative and Gram-positive bacteria and fungi of interest for human medicine and aquaculture, in which computational prediction studies supported the putative PACAP therapeutic activity. Results also indicated that catfish PACAP not only exhibits inhibitory effects on pathogen growth, but also affects the proliferation of human non-small cell lung cancer cell line H460 in a dose-dependent manner. The observed cytotoxic activity of catfish PACAP against human tumor cells and pathogenic microorganisms, but not healthy fish and mammalian erythrocytes support a potential physiological role of this neuropeptide in selective microbial and cancer cell killing. All together, our findings extend the mechanisms by which PACAP could contribute to immune responses, and open up new avenues for future therapeutic application of this bioactive neuropeptide.

Humoral and cellular immune response in mice induced by the classical swine fever virus E2 protein fused to the porcine CD154 antigen.

The development of subunit vaccines against classical swine fever is a desirable goal, because it allows discrimination between vaccinated and infected animals. In this study, humoral and cellular immune response elicited in inbred BALB/c mice by immunization with a recombinant classical swine fever virus (CSFV) E2 protein fused to porcine CD154 antigen (E2CD154) was assessed. This model was used as a predictor of immune response in swine. Mice were immunized with E2CD154 emulsified in Montanide ISA50V2 or dissolved in saline on days 1 and 21. Another group received E2His antigen, without CD154, in the same adjuvant. Montanide ISA50V2 or saline served as negative controls for each experimental group. Animals immunized with 12.5 and 2.5 µg/dose of E2CD154 developed the highest titers (>1:2000) of CSFV neutralizing antibodies. Moreover, CSFV specific splenocyte gamma-interferon production, measured after seven and twenty-eight days of immunization, was significantly higher in mice immunized with 12.5 µg of E2CD154. As a conclusion, E2CD154 emulsified in Montanide ISA50 V2 was able to induce a potent humoral and an early cellular immune response in inbred BALB/c mice. Therefore, this immunogen might be an appropriate candidate to elicit immune response in swine, control CSF disease and to eliminate CSFV in swine.

Pituitary adenylate cyclase-activating polypeptide (PACAP): a regulator of the innate and acquired immune functions in juvenile fish.

To date, published in-vivo studies on the action of the PACAP in fish are few and these are concerned with reproduction, brain development and feeding behavior. Recently, we demonstrated for the first time that PACAP, apart from its neuroendocrine role, influences immune functions in fish larvae. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on important immune parameters in juvenile fish. We observed that a single injection of the recombinant peptide (0.1 microg per g of body weight) was able to increase the nitric oxide synthase-derived metabolites (NOS) and total immunoglobulin M (IgM) concentration in serum of juvenile catfish C. gariepinus and tilapia Orechromis niloticus respectively, after 24 h of its administration. In addition, our results showed that recombinant PACAP increases IgM, NOS and lysozyme in serum correlated with its ability to enhance growth performance in juvenile fish. Finally, the PACAP mRNA expression and PACAP immunoreactivity detected in peripheral blood leucocytes from juvenile catfish suggest a direct autocrine or/and paracrine mechanism of regulation of this peptide to mediate immune functions in fish.

The biological role of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth and feeding behavior in juvenile fish.

To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase-activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth-promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture.

Competitive RT-PCR Strategy for Quantitative Evaluation of the Expression of Tilapia (Oreochromis niloticus) Growth Hormone Receptor Type I.

UNLABELLED: Quantization of gene expression requires that an accurate measurement of a specific transcript is made. In this paper, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) by competition for tilapia growth hormone receptor type I is designed and validated. This experimental procedure was used to determine the abundance of growth hormone receptor type I transcript in different tilapia tissues. The results obtained with this developed competitive RT-PCR were similar to real-time PCR results reported recently. This protocol provides a reliable alternative, but less expensive than real-time PCR to quantify specific genes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (DOI:10.1007/s12575-009-9002-3) contains supplementary material, which is available to authorized users.

A novel GH secretagogue, A233, exhibits enhanced growth activity and innate immune system stimulation in teleosts fish.

In teleosts fish, secretion of GH is regulated by several hypothalamic factors that are influenced by the physiological state of the animal. There is an interaction between immune and endocrine systems through hormones and cytokines. GH in fish is involved in many physiological processes that are not overtly growth related, such as saltwater osmoregulation, antifreeze synthesis, and the regulation of sexual maturation and immune functions. This study was conducted to characterize a decapeptide compound A233 (GKFDLSPEHQ) designed by molecular modeling to evaluate its function as a GH secretagogue (GHS). In pituitary cell culture, the peptide A233 induces GH secretion and it is also able to increase superoxide production in tilapia head-kidney leukocyte cultures. This effect is blocked by preincubation with the GHS receptor antagonist [d-Lys(3)]-GHRP6. Immunoneutralization of GH by addition of anti-tilapia GH monoclonal antibody blocked the stimulatory effect of A233 on superoxide production. These experiments propose a GH-mediated mechanism for the action of A233. The in vivo biological action of the decapeptide was also demonstrated for growth stimulation in goldfish and tilapia larvae (P<0.001). Superoxide dismutase levels, antiprotease activity, and lectin titer were enhanced in tilapia larvae treated with this novel molecule. The decapeptide A233 designed by molecular modeling is able to function as a GHS in teleosts and enhance parameters of the innate immune system in the fish larvae.