Development and basic performance verification of a rapid homogeneous bioassay for agonistic antibodies against the thyroid-stimulating hormone receptor.

Graves' disease is a type of autoimmune hyperthyroidism caused by thyroid-stimulating antibodies (TSAb).1 The combination of a porcine thyroid cell bioassay and cyclic adenosine monophosphate (cAMP) immunoassay (TSAb-enzyme immunoassay; EIA) is a clinically approved TSAb measurement method. Due to the requirement of multiple procedures and a long assay time of 6 h in the TSAb-EIA, a simplified and rapid assay is desired. Herein, we developed a rapid homogeneous TSAb bioassay (rapid-TSAb assay) using the human embryonic kidney cell line (HEK293), engineered to express the human thyroid-stimulating hormone receptor (TSHR), along with a cAMP-dependent luminescence biosensor. The measurement consists of three steps: thawing frozen cells, blood sample addition, and luminescence detection. The procedures can be conducted within 1 h. The World Health Organization International Standard TSAb (NIBSC 08/204) stimulated the cells co-expressing TSHR and cAMP biosensor. The intra- and inter-assay coefficients of variance were < 10%. Stimulation activity using wild-type TSHR and chimeric TSHR (Mc4) almost completely correlated with the tested Graves' disease and normal samples. In the rapid-TSAb assay, the evaluation of 39 samples, including TSHR antibody-positive sera, yielded a sensitivity of 100.0% and a specificity of 90.9%, compared to the TSAb-EIA control. The rapid-TSAb assay enables simple and rapid measurement of TSAb and is promising for improving the diagnosis of autoimmune thyroid diseases.

Molecular cloning and analysis of Suncus murinus group IIA secretary phospholipase A2 expression.

The intestinal epithelial monolayer forms a mucosal barrier between the gut microbes and the host tissue. The mucosal barrier is composed of mucins and antimicrobial peptides and proteins (AMPs). Several animal studies have reported that Paneth cells, which occupy the base of intestinal crypts, play an important role in the intestinal innate immunity by producing AMPs, such as lysozyme, Reg3 lectins, α-defensins, and group IIA secretory phospholipase A2 (GIIA sPLA2). The house musk shrew (Suncus murinus) has only a few intestinal commensal bacteria and is reported to lack Paneth cells in the intestine. Although the expression of lysozyme was reported in the suncus intestine, the expression of other AMPs has not yet been reported. Therefore, the current study was focused on GIIA sPLA2 expression in Suncus murinus. GIIA sPLA2 mRNA was found to be most abundant in the spleen and also highly expressed in the intestine. Cells expressing GIIA sPLA2 mRNA were distributed not only in the crypt, but also in the villi. In addition, intragastric injection of lipopolysaccharide increased GIIA sPLA2 expression in the small intestine of suncus. These results suggest that suncus may host unique AMP-secreting cells in the intestine.

Identification and characterization of an antimicrobial peptide, lysozyme, from Suncus murinus.

Lysozyme is one of the most prominent antimicrobial peptides and has been identified from many mammalian species. However, this enzyme has not been studied in the order Insectivora, which includes the most primitive placental mammals. Here, we done the lysozyme cDNA from Suncus murinus (referred to as suncus, its laboratory name) and compare the predicted amino acid sequence to those from other mammalian species. Quantitative PCR analysis revealed a relatively higher expression of this gene in the spleen and gastrointestinal tract of suncus. The lysozyme-immunopositive (ip) cells were found mainly in the red pulp of the spleen and in the mucosa of the whole small intestine, including the follicle-associated epithelium and subepithelial dome of Peyer's patches. The lysozyme-ip cells in the small intestine were mostly distributed in the intestinal crypt, although lysozyme-expressing cells were found not only in the crypt but also in the villi. On the other hand, only a few lysozyme-ip cells were found in the villi and some granules showing intense fluorescence were located toward the lumen. As reported for other mammals, Ki67-ip cells were localized in the crypt and did not co-localize with the lysozyme-ip cells. Moreover, fasting induced a decrease in the mRNA levels of lysozyme in the intestine of suncus. In conclusion, we firstly identified the lysozyme mRNA sequence, clarified expression profile of lysozyme transcripts in suncus and found a unique distribution of lysozyme-producing cells in the suncus intestine.

ß-Oxidation in ghrelin-producing cells is important for ghrelin acyl-modification.

Ghrelin is a unique fatty acid-modified peptide hormone produced in the stomach and has important roles in energy homeostasis and gastrointestinal motility. However, the medium-chain fatty acid source for ghrelin acyl-modification is not known. We found that a fat-free diet and the removal of intestinal microbiota did not decrease acyl-ghrelin production in the stomach or plasma acyl-ghrelin levels in mice. RT-PCR analysis showed that genes involving fatty acid synthesis, metabolism, and transport were expressed in pancreas-derived ghrelinoma (PG-1) cells. Treatment with an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) strongly decreased acylated ghrelin levels but did not affect ghrelin or ghrelin o-acyl transferase (GOAT) mRNA levels in PG-1 cells. Our results suggest that the medium-chain fatty acid used for the acyl-modification of ghrelin is produced in ghrelin-producing cells themselves by ß-oxidation of long-chain fatty acids provided from the circulation.