50 research outputs found
NF kappa B induces overexpression of bovine FcRn: a novel mechanism that further contributes to the enhanced immune response in genetically modified animals carrying extra copies of FcRn
Among the many functions of the neonatal Fc receptor (FcRn) for
IgG, it binds to IgG-opsonized antigen complexes and propagates
their traffic into lysosomes where antigen processing occurs. We
previously reported that transgenic (Tg) mice and rabbits that
carry multiple copies and overexpress FcRn have augmented
humoral immune responses. Nuclear factor-kappa B (NFκB) is a
critical molecule in the signaling cascade in the immune
response. NFκB induces human FcRn expression and our previous in
silico analysis suggested NFκB binding sites in the promoter
region of the bovine (b) FcRn α-chain gene (FCGRT). Here, we
report the identification of three NFκB transcription binding
sites in the promoter region of this gene using luciferase
reporter gene technology, electromobility shift assay and
supershift analysis. Stimulation of primary bovine endothelial
cells with the Toll like receptor-4 ligand lipopolysaccharide
(LPS), which mediates its effect via NFκB, resulted in rapid
upregulation of the bFcRn expression and a control gene, bovine
E-selectin. This rapid bFcRn gene induction was also observed in
the spleen of bFcRn Tg mice treated with intraperitoneally
injected LPS, analyzed by northern blot analysis. Finally, NFκB-
mediated bFcRn upregulation was confirmed at the protein level
in macrophages isolated from the bFcRn Tg mice using flow
cytometry with a newly developed FcRn specific monoclonal
antibody that does not cross-react with the mouse FcRn. We
conclude that NFκB regulates bFcRn expression and thus optimizes
its functions, e.g., in the professional antigen presenting
cells, and contributes to the much augmented humoral immune
response in the bFcRn Tg mice
The PHENIX Experiment at RHIC
The physics emphases of the PHENIX collaboration and the design and current
status of the PHENIX detector are discussed. The plan of the collaboration for
making the most effective use of the available luminosity in the first years of
RHIC operation is also presented.Comment: 5 pages, 1 figure. Further details of the PHENIX physics program
available at http://www.rhic.bnl.gov/phenix
Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn
The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies
Could protein tertiary structure influence mammary transgene expression more than tissue specific codon usage?
High level expression of tissue-nonspecific alkaline phosphatase in the milk of transgenic rabbits
Alkaline phosphatase is a promising therapeutic agent in the Gram-negative bacterial lipopolysaccharide (LPS) mediated acute and chronic diseases. Contrary to other alkaline phosphatase isozymes, purified tissue-nonspecific alkaline phosphatase (TNAP) is not available in large quantities from tissue sources, which would enable to analyse its efficacy in animal sepsis models. Two transgenic rabbit lines were created by pronuclear microinjection with the whey acidic protein promoter-humanTNAP minigene (WAP-hTNAP). Lactating females of both lines produced biologically active human TNAP. As indicated by fractionation of milk samples the recombinant alkaline phosphatase was associated with the membrane of milk fat globules. Alkaline phosphatase enzymatic activity was two orders of magnitude higher compared to normal human serum levels. The demonstration that this TNAP is physiologically active would provide the clue to use transgenic animals as bioreactor for bulk production of the human tissue-nonspecific alkaline phosphatase in milk. This may be a valuable and possibly viable option with important implication in attenuating LPS mediated inflammatory responses