Rabbit Anti-Human B2M (a.a. 22-119) Polyclonal antibody

Background. Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect animals at high trophic levels, such as cetaceans and humans, via the food chain. Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in the modulation of immune system and can be classified into two types: Th1 and Th2. Th1 /Th2 balance can be evaluated by the ratios of their polarizing cytokines (i.e., interferon [IFN]-gamma/Interleuldn [IL]-4), and animals with imbalanced Thl/Th2 response may become more susceptible to certain kinds of infection. Therefore, the present study evaluated the in vitro cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP20) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Methods. Blood samples were collected from six captive common bottlenose dolphins (Tursiops truncatus). The cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses. The cytokines evaluated included IL-2, IL-4, IL-10, IL-12, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. The geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta 2-microglobulin (B2M), of each sample were determined and used to normalize the mRNA expression levels of target genes. Results. The ratio of late apoptotic/necrotic cells of cPBMCs significantly increased with or without concanavalin A (ConA) stimulation after 24 h of 10 mu g/ml C-AgNP20 treatment. At 4 h of culture, the mRNA expression level of IL-10 was significantly decreased with mu g/ml C-AgNP20 treatment. At 24 h of culture with 1 mu g/ml C-AgNP20, the mRNA expression levels of all cytokines were significantly decreased, with the exceptions of IL-4 and IL-10. The IFN-gamma/IL-4 ratio was significantly decreased at 24 h of culture with 1 mu g/ml C-AgNP20 treatment, and the IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 mu g/ml C-AgNP20 treatment, respectively. Furthermore, the mRNA expression level of TNF-alpha was significantly decreased by 1 mu g/ml C-AgNP20 after 24 h of culture. Discussion. The present study demonstrated that the sublethal dose of C-AgNP20 (<= 1 mu g/ml) had an inhibitory effect on the cytokine mRNA expression levels of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased the mRNA expression level of TNF-alpha. Th2 cytokine bias is associated with enhanced immunity against parasites but decreased immunity to intracellular microorganisms. TNF-alpha is a contributing factor for the inflammatory response against the infection of intracellular pathogens. In summary, our data indicate that C-AgNP20 suppresses the cellular immune response and thereby increases the susceptibility of cetaceans to infection by intracellular microorganisms.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis is a popular method for the measurement of mRNA expression level and is a critical tool for basic research. The identification of suitable reference genes that are stable and not affected by experimental conditions is a critical step in the accurate normalization of RT-PCR. On the other hand, the levels of numerous transcripts exhibit circadian oscillation in various peripheral tissues and it is thought to be regulated by feeding rhythms in addition to the molecular circadian clock. Here, we investigated the effects of feeding schedule on the temporal expression profiles of 13 common housekeeping genes in metabolic tissues of mice fed during either the sleep or the active phase. The expression of most of these genes fluctuated dependently on feeding rhythms in the liver and WAT, but not in skeletal muscle. Two-way analyses of variance (ANOVA) identified 18S ribosomal RNA (Rn18s) as the only gene that was stably expressed throughout the day independently of feeding schedules in the liver and WAT, although RefFinder software showed that peptidylprolyl isomerase A (Ppia) was the most stably expressed housekeeping gene. Both ANOVA and RefFinder software determined that Actb was the preferred reference gene for skeletal muscle. Furthermore, NormFinder proposed that the optimal pairs of reference genes were beta-2 microglobulin (B2m)-Ppia in the liver, Ppia-TATA box binding protein (Tbp) in WAT, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz)-glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in skeletal muscle, and that their stability value was better than that of a single stable gene. The appropriate reference gene pairs for normalizing genes of interest in mouse circadian studies are B2m-Ppia in the liver, Ppia-Tbp in WAT, and Ywhaz-Gapdh in skeletal muscle. (C) 2017 Elsevier Inc. All rights reserved.