Monoclonal antibody generated against Nile tilapia (Oreochromis niloticus) IgT heavy chain using a peptide-based strategy.

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

Synergic effect of anticancer peptide CIGB-552 and Cisplatin in lung cancer models.

BACKGROUND: The antitumor peptide CIGB-552 is a new targeted anticancer therapy which molecular mechanism is associated with the inhibition of the transcription factor NF-kB, mediated by COMMD1 protein stabilization. In this study, we examined the antiproliferative capacity of CIGB-552 in combination with chemotherapeutic agents in lung cancer models. METHODS AND RESULTS: We combined of CIGB-552 and the antineoplastic agent Cisplatin (CDDP) in concomitant and pre-treatment scenary in a dose matrix approach. This study was performed in the non-small cell lung cancer cell lines NCI-H460, A549 and in a mouse model of TC-1 lung cancer. Our results demonstrate a clear synergic effect between 37.5 µM of CIGB-552 and 5 µM of CDDP under concomitant scheme, on proliferation inhibition, cell cycle arrest, apoptosis induction and oxidative stress response. The effect of CIGB-552 (1 mg/kg) and CDDP (0.4 mg/kg) administrated as a combined therapy was demonstrated in vivo in a TC-1 mouse model where the combination achieved an effective antitumor response, without any deterioration signs or side effects. CONCLUSIONS: These findings demonstrate the efficacy of the concomitant combination of both drugs in preclinical studies and support the use of this therapy in clinical trials. This study is the first evidence of synergistic effect of the combination of  the antitumoral peptide CIGB-552 and CDDP.

Introducing an Asp-Pro linker in the synthesis of random one-bead-one-compound hexapeptide libraries compatible with ESI-MS analysis.

A random hexapeptide library (one-bead-one-compound), containing sixteen amino acids (16(6) different sequences) was synthesized on a Tentagel resin previously modified with a dipeptide linker (Asp-Pro). This peptide bond is highly susceptible to cleavage under mild acidic conditions in a salt-free solution prepared with H(2)(16)O/H(2)(18)O (60/40% v/v). In the hydrolysis, hexapeptides are released with an additional Asp residue partially labeled with (18)O at the C-terminus. These conditions are fully compatible with ESI-MS analysis and facilitate sequencing by MS, as N- and C-terminal ions can be easily differentiated in MS/MS spectra. The peptides were sequenced manually and also with de novo sequencing programs, and identifying them in a database containing all possible heptapeptide sequences or in a filtered database. The proposed strategy is also compatible with stepwise Edman degradation using either intact beads or the released free peptides.

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.

Immune response to synthetic peptides of dengue prM protein.

The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (P<0.05) of protection when challenged with DEN-2 virus. These results could constitute the basis for the establishment of the role of DEN virus pre and M antigens in the development of anti-flaviviral vaccines.