Pilot study on novel skin care method by augmentation with Staphylococcus epidermidis, an autologous skin microbe--A blinded randomized clinical trial.

BACKGROUND AND OBJECTIVE: Staphylococcus epidermidis is an autologous bacterium that is beneficial to skin health. Our goal was to develop a novel, personalized basic cosmetic that exploits this characteristic. METHODS: We conducted a double-blinded, randomized clinical trial on augmentation with S. epidermidis as a pilot study, in which S. epidermidis was collected from the subject, cultured for proliferation, and then continuously applied to the subject's own face before sleep twice per week for four weeks in order to increase colonization levels. RESULTS: The results showed that this treatment increased the lipid content of the skin and suppressed water evaporation, thereby markedly improving skin moisture retention. Moreover, augmentation with S. epidermidis maintained a low acidic condition on the skin surface. The low risk of undesirable effects induced by augmentation with S. epidermidis was also confirmed by measuring erythema and melanin levels. CONCLUSIONS: These results may serve as a driving force to accelerate the development of novel, personalized basic cosmetics.

Preparation and immunological characterization of ß-lactoglobulin-amylose conjugate.

ß-Lactoglobulin (BLG), a major allergen of cow's milk, was conjugated with the N-hydroxysuccinimide ester of the amylose-glycylglycine adduct (AG-ONSu) to reduce its immunogenicity, and the biochemical and immunological properties of the resulting conjugate (AG-BLG) were studied. The conjugate was prepared by modifying BLG with AG-ONSu, and was purified in a Sephadex G-100 column. The analytical data for AG-BLG indicated that 10.5 moles of AG-ONSu, with a mean molecular weight of 2,800, was covalently attached to the amino groups of the BLG molecule. Conjugation with AG-ONSu greatly decreased the reactivity of BLG with anti-BLG polyclonal antibodies owing to its shielding action for epitopes on the protein's surface. These findings suggest that AG-ONSu can be used advantageously to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.

Reduction of the immunogenicity of beta-lactoglobulin from cow's milk by conjugation with a dextran derivative.

Beta-lactoglobulin (BLG) was conjugated with the N-hydroxysuccinimide ester of the dextran-glycylglycine adduct (DG-ONSu) to reduce the immunogenicity of BLG, a major allergen of cow's milk, and some immunological properties of the conjugate (DG-BLG) were studied. The conjugate was prepared by modifying BLG with DG-ONSu and purified in a Sephadex G-100 column. The analytical data for DG-BLG indicated that 5.2 moles of DG-ONSu with a mean molecular weight of 9,300 were covalently attached to the amino groups of the BLG molecule. Conjugation with DG-ONSu greatly decreased the reactivity of BLG with anti-BLG antibodies and suppressed their production in vivo due to its shielding action for epitope(s) on the protein's molecular surface. It was also found that DG-BLG was resistant to proteolytic enzymes. These findings allow us to suggest that DG-ONSu could be advantageously used to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.

Identification and characterization of the hypoxia-responsive element of the human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K, identical to PFKFB3) is expressed in a variety of cells and tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, primary blood mononuclear cells and cancer cells. We observed previously that the enhancer region of the HP2K gene, which has been identified in the 5'-flanking region between -1265 and -1329, could respond to serum stimulation following the transfection of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. The HP2K enhancer region also contains two copies of the hypoxia-inducible factor-1 (HIF-1) binding motif (5'-ACGTG-3'). In this study we performed characterization of the HP2K gene expression in response to hypoxic conditions. Both electrophoretic mobility shift and co-transfection assays of the HP2K promoter-luciferase reporter with HIF-1 expression vectors indicated that HIF-1 binds to the hypoxia-responsive element (HRE) of HP2K, thereby upregulating its gene expression. In addition, we demonstrated using site-directed mutagenesis that a complete tandem repeat of the HIF-1 binding motif with a 4-bp interruption is required for full induction of HP2K expression (up to 22-fold) under hypoxic conditions, and that this response is much stronger than that of the erythropoietin (EPO) gene. These results suggest that the sequence 5'-ACGTGNNNNACGTG-3' in the HP2K enhancer is the authentic HRE consensus motif that mediates increased transcription, under hypoxic conditions, via HIF-1.

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin.

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.