Diagnosis and discrimination of autoimmune Graves' disease and Hashimoto's disease using thyroid-stimulating hormone receptor-containing recombinant proteoliposomes.

Graves' disease (GD) is an autoimmune disease of the thyroid gland caused by autoantibodies against thyroid-stimulating hormone receptor (TSHR). Currently, the diagnostic test for TSHR autoantibodies is based on an indirect competitive binding assay that measures the ability of TSHR autoantibodies to inhibit the binding of thyroid-stimulating hormone (TSH) to TSHR. Here, we have developed a specific and direct diagnostic method for autoantibodies in GD that incorporates immobilized TSHR-containing recombinant proteoliposomes into an enzyme-linked immunosorbent assay (ELISA). To reduce non-specific binding of autoantibodies to recombinant proteoliposomes, we investigated the effect of polyethylene glycol (PEG)-lipid on the binding of commercially available anti-TSHR antibodies (aTSHRAb). The incorporation of PEG-lipids into liposomes decreased non-specific binding, as compared to liposomes that did not contain PEG-lipids, and the addition of blocking reagents further decreased non-specific reactivity. aTSHRAb exhibited higher reactivity towards PEG-modified TSHR recombinant proteoliposomes than PEG-modified liposomes without TSHR (bare liposomes). Importantly, serum autoantibodies from patients with GD, which is associated with hyperthyroidism, exhibited remarkably specific binding to TSHR recombinant proteoliposomes. Serum autoantibodies from patients with Hashimoto's disease (HD), which is associated with hypothyroidism, also reacted specifically with proteoliposomal TSHR. These results suggest that immobilized TSHR recombinant proteoliposomes can serve as a direct diagnostic test for GD and HD. Furthermore, given that there is no competition test currently available for detecting autoantibodies in HD, the combination of TSHR recombinant proteoliposome ELISA and indirect competitive TSHR binding assay might be an effective way to discriminate between GD and HD.

Development of a novel preparation method of recombinant proteoliposomes using baculovirus gene expression systems.

We have developed a novel method for the preparation of 'recombinant proteoliposomes'. Membrane proteins were expressed on budded virus (BV) envelopes using baculovirus gene expression systems, and proteoliposomes were prepared by fusion of these viruses with liposomes. First, plasmid DNA containing the gene for the thyroid-stimulating hormone receptor (TSHR) or the acetylcholine receptor alpha-subunit (AChRalpha) was co-transfected with wild type virus [Autographa californica nuclear polyhedrosis virus (AcNPV)] genomes into insect cells [Spodoptera frugiperda (Sf9)] to obtain recombinant viruses via homologous recombination. The recombinant viruses were again infected into Sf9 cells, and the resulting BVs were shown to express TSHR and AChRalpha. Next, the fusion behaviour of AcNPV-derived BVs and liposomes was examined via a fluorescence assay, and BVs were shown to fuse with phosphatidylserine-containing liposomes below pH 5.0, the pH at which fusion glycoprotein gp64 on the virus envelope becomes active. TSHR- or AChRalpha-expressed BVs were also shown to fuse with liposomes. Finally, TSHR- and AChRalpha-recombinant proteoliposomes were immobilized on enzyme-linked immunosorbent assay plates, and their reactivities were examined via a general immunoassay, which showed that the recombinant proteoliposomes were fully active. These results successfully demonstrate the development of a method based on a baculovirus gene expression system for the preparation of recombinant and functional proteoliposomes.

Biological activity of barbados cherry (acerola fruits, fruit of Malpighia emarginata DC) extracts and fractions.

Fractionation of barbados cherry (acerola fruit, a fruit of Malpighia emarginata DC.) extracts were performed by organic solvent extractions and column chromatographies, using two extraction methods. Higher cytotoxic activity was concentrated in fractions A4 and A6 (acetone extract), and H3 and HE3 (hexane extract). These four fractions showed higher cytotoxic activity against tumor cell lines such as human oral squamous cell carcinoma (HSC-2) and human submandibular gland carcinoma (HSG), when compared with that against normal cells such as human periodontal ligament fibroblasts (HPLF) and human gingival fibroblasts (HGF). HE2 (hexane extract), AE2 (ethyl acetate extract), AE3, AE4, AE5, A8, A9 and A10 showed some relatively higher anti-bacterial activity on the Gram-positive Staphylococcus epidermidis ATCC 1228 but were ineffective on the representative Gram-negative species E. coli and Ps. aeruginosa. The fractions were inactive against Helicobacter pylori, two representative Candida species, and human immunodeficiency virus (HIV). H3, H4 and HE3, which displayed higher tumor-specific cytotoxicity also showed higher multidrug resistance (MDR) reversal activity, than (+/-)-verapamil as positive control. ESR spectroscopy shows that the radical-mediated oxidation is not involved in the induction of tumor-specific cytotoxic activity. The tumor specific cytotoxic activity and MDR reversal activity of barbados cherry may suggest its possible application for cancer therapy.

Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by Barbados cherry, a fruit of Malpighia emarginata DC.

The extract of Barbados cherry (acerola fruit), a fruit of Malpighia emarginata DC., has been reported to display diverse biological activities such as prevention of age-related diseases. We investigated here the possible effect of Barbados cherry extract on nitric oxide (NO) production by activated macrophages. Barbados cherry was roughly separated into 4 or 5 fractions by two different methods, using various organic solvents such as hexane, acetone, methanol (70% and 100%) and water, and assayed for its ability to inhibit NO production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like Raw 264.7 cells. Among these fractions, AcOEt extracts (AE0) in Method I and acetone extract (A0) in Method II showed the highest inhibitory activity of NO production (SI > 20 and SI = 31, respectively). When these fractions were subjected to silica gel column chromatography, higher inhibitory activity for NO production was concentrated in AcOEt (AE6) (SI = 64) and benzene-AcOEt (1:4) (A10) fractions (SI > 59). Western blot analysis demonstrated that AE6 and A10 fractions reduced the intracellular concentration of inducible NO synthase (iNOS) by approximately one-third. ESR spectroscopy showed that these fractions scavenged various radical species such as superoxide anion (O2-) and NO radicals. These data suggest that the inhibitory effect on NO production by Barbados cherry extracts is partly due to the inhibition of iNOS expression, and scavenging of O2- and NO radicals.