Characterization of the native form of anthrax lethal factor for use in the toxin neutralization assay.

The cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

Chorionic gonadotropin regulates prostaglandin E synthase via a phosphatidylinositol 3-kinase-extracellular regulatory kinase pathway in a human endometrial epithelial cell line: implications for endometrial responses for embryo implantation.

Successful implantation necessitates modulation of the uterine environment by the embryo for a specific period of time during the menstrual cycle. Infusion of chorionic gonadotropin (CG) into the oviducts of baboons to mimic embryo transit induces a myriad of morphological, biochemical, and molecular changes in the endometrium. Endometrial epithelial cells from both baboons and humans when stimulated by CG in vitro, activates a cAMP-independent MAPK pathway leading to prostaglandin E(2) (PGE(2)) synthesis. This study shows that in the human endometrial cell line, HES, CG, acting via its G-protein coupled receptor, phosphorylates protein kinase B, c-Raf, and ERK1/2 in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Furthermore, ERK1/2 phosphorylation is independent of the signaling paradigms of Galpha(s), Galpha(I), and epidermal growth factor receptor (EGFR) transactivation, typical of gonadal cells, indicating an alternative signaling pattern in the endometrium. After phosphorylation by CG, ERK1/2 translocates to the nucleus in a time-dependent manner. Downstream of ERK1/2, CG activates the nuclear transcription factor, Elk1, also in a PI3K-MAPK-dependent manner. Lastly, we show that in HES cells, this pathway regulates the expression of the microsomal enzyme PGE(2) synthase (mPTGES), a terminal prostanoid synthase responsible for PGE(2) synthesis. CG regulates the mPTGES promoter and also induces mPTGES synthesis in HES cells via the PI3K-ERK1/2 pathway. We suggest that this alternative PI3K-ERK-Elk pathway activated by CG regulates prostaglandin production by the endometrial epithelium and serves as an early trigger to prepare the endometrium for implantation.