Immunological strategy for detecting the pro-inflammatory cytokine TNF-alpha in salmonids

Background: Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which exerts a variety of immunological functions in vertebrates. TNF-alpha has been identified and cloned in a number of teleost fish species; nevertheless, the functions displayed by this cytokine in fishes remain poorly understood, given that the low sequence identity compared to their mammalian counterpart, limit fish TNF-alpha detection using mammalian antibodies. Then, for fish immune response characterization is fundamental the production of specific fish anti-TNF-alpha antibody. Results: We have developed a monoespecific antibody against the pro-inflammatory molecule TNF-alpha of salmonid fish. TNF-alpha epitope region was identified and characterized using bioinformatic tools. The epitope sequence was chemically synthesized using Fmoc strategy, analyzed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was used to immunize mice and antibodies from ascitic fluid were purified. The resulting antibody was used for molecular and histochemical detection in gut samples from salmonid fishes treated with different food. By ELISA, we detected a differential expression of TNF-alpha, the western blot analysis shows recognition of the whole TNF molecule and by immunohistochemistry TNF-alpha positive cells were observed. Conclusions: We provide an immunological tool, validated through classical immunological assays, which can be a useful tool for characterizing fish TNF-alpha function.

Effects of TNF alpha on expression of SREBP-1c mRNA and triglyceride contents in cultured steatosis hepatocytes / 第三军医大学学报

Objective To explore the effects of TNF alpha on the expression of sterol-regulatory element binding protein-1c, SREBP-1c mRNA and the content of triglyceride (TG) in the models of cultured steatosis hepatocytes. Methods Steatosis models of hepatocytes were established by adding oleic acid to the growing L-02 cells, and then cultured in present with TNF alpha or its antibody. The expressions of SREBP-1c and fatty acid synthetase (FAS) mRNA were measured with RT-PCR, lipid droplets in the hepatocytes were observed with oil red staining and the TG contents in hepatocytes were measured with analyzed kit. Results SREBP-1c mRNA was upregulated in the TNF alpha treatment group in comparison with the normal control group(P