Texts of Kolima dialect of Yukaghir

<p>Clinical chemistry data of monkeys fed on diets containing GM rice or non-GM rice.</p

CsBAFF, a Teleost B Cell Activating Factor, Promotes Pathogen-Induced Innate Immunity and Vaccine-Induced Adaptive Immunity

<div><p>B cell activating factor (BAFF) is a member of the tumor necrosis factor family that is known to play an important role in B cell activation, proliferation, and differentiation in mammals. However, studies of BAFF in teleosts are very limited and its function, in particular that under <i>in vivo</i> conditions, is essentially unknown. In this study, we conducted <i>in vivo</i> as well as <i>in vitro</i> functional analyses of a BAFF homologue (CsBAFF) from the teleost fish tongue sole (<i>Cynoglossus semilaevis</i>). CsBAFF is composed of 261 residues and shares moderate sequence identities with known BAFFs of other teleosts. <i>CsBAFF</i> expression was most abundant in immune organs and was upregulated during bacterial infection. Purified recombinant CsBAFF (rCsBAFF) bound to tongue sole lymphocytes and promoted cellular proliferation and survival. The results of an <i>in vivo</i> study showed that CsBAFF overexpression in tongue sole significantly enhanced macrophage activation and reduced bacterial infection in fish tissues, whereas knockdown of <i>CsBAFF</i> expression resulted in increased bacterial dissemination and colonization in fish tissues. Furthermore, vaccination studies showed that CsBAFF enhanced the immunoprotection of a DNA vaccine and augmented the production of specific serum antibodies. Taken together, these results provide the first <i>in vivo</i> evidence to indicate that teleost BAFF is an immunostimulator that significantly contributes to the innate antibacterial immune response and vaccine-induced adaptive immune response.</p></div

Malé levodistributivní kvazigrupy

<p><b>Effect of rCsBPI on bacterial membrane permeability (A) and cellular structure (B).</b> (A) <i>Pseudomonas</i> <i>fluorescens</i> was incubated with or without (control) rCsBPI or rTrx for different hours. After incubation, the cells were stained with PI solution, and PI-positive cells were determined. Values are shown as means ± SEM (N = 3). N, the number of times the experiment was performed. (B) P. fluorescens was treated with rCsBPI for 2 h (B3) and 4 h (B4) or treated with rTrx for 4 h (B2); the control cells (B1) were untreated. The cells were then subjected to microscopy. Bar, 500 nm. **<i>P</i> < 0.01.</p

Survival of vaccinated fish.

<p>Tongue sole vaccinated with or without (control) pCEsa1, pCEsa1 plus pCsBAFF, pCsBAFF, or pCN3 were challenged with <i>Edwardsiella tarda</i> and monitored daily for survival. The results are one representative vaccination trial. Significance between the survivals of the vaccinated fish and control fish was determined with logrank test. **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

Binding of rCsBAFF to lymphocytes.

<p>Tongue sole lymphocytes were incubated with rCsBAFF (A and B) or rGST (D and E), and the cells were treated with FITC-labeled antibody (A and D, for detecting His-tagged protein) or RBITC-labeled antibody (B and E, for detecting cell-surface IgM). The cells were observed under a fluorescence microscope. C, merge of A and B; F, merge of D and E. Magnification, 400 ×.</p

Effect of CsBAFF overexpression on bacterial infection.

<p>Tongue sole administered with pCsBAFF, pCN3, or PBS (control) were infected with <i>Edwardsiella tarda</i>, and the amount of bacteria in kidney (A), spleen (B), and blood (C) was determined at different time points. Data are expressed as means ± SEM (N = 3). N, the number of times the experiment was performed. **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

Alignment of the sequences of CsBAFF homologues.

<p>Dots denote gaps introduced for maximum matching. Numbers in brackets indicate overall sequence identities between CsBAFF and the compared sequences. The consensus residues are in black, and the residues that are ≥75% identical among the aligned sequences are in blue. The three conserved cysteine residues are indicated by asterisks, and the putative furin cleavage site is boxed in red. The GenBank accession numbers of the aligned sequences are as follows: <i>Epinephelus awoara</i>, AFN70720.1; <i>Miichthys miiu</i>, AHL44989.1; <i>Salmo salar</i>, ACI33633.1; <i>Takifugu obscurus</i>, AEB69781.1; <i>Oreochromis niloticus</i>, AHF20914.1; <i>Danio rerio</i>, NP_001107062.1; <i>Ctenopharyngodon idella</i>, AGG11791.1.</p

Primers used in this study.

<p>^aUnderlined nucleotides are restriction sites of the enzymes indicated in the brackets at the ends.</p><p>Primers used in this study.</p

Effect of CsBAFF overexpression on macrophage activation.

<p>Macrophages from tongue sole administered with pCsBAFF, pCN3, and PBS (control) were examined for respiratory burst activity (A) and acid phosphatase activity (B). The respiratory burst activity and phosphatase activity are shown as values relative to those of the control cells (<i>A</i>₆₃₀ of 0.46 and <i>A</i>₄₁₀ of 0.7 respectively). Data are expressed as means ± SEM (N = 3). N, the number of times the experiment was performed. *<i>P</i> < 0.05.</p

<i>CsBAFF</i> expression in fish tissues under normal physiological condition.

<p><i>CsBAFF</i> expression in the intestine, brain, gill, muscle, heart, blood, liver, kidney, and spleen was determined by quantitative real time RT-PCR. For the convenience of comparison, the expression level of <i>CsBAFF</i> in intestine (Ct value of 8.9) was set as 1. Vertical bars represent means ± SEM (N = 3). N, the number of times the experiment was performed.</p