Tomoki Yamadori

Flavonoids such as luteolin, fisetin and apigenin are inhibitors of interleukin-4 and interleukin-13 production by activated human basophils.

Background: We have previously shown that fisetin, a flavonol, inhibits IL-4 and IL-13 synthesis by allergen- or anti-IgE-antibody-stimulated basophils. This time, we investigated the inhibition of IL-4 and IL-13 production by basophils by other flavonoids and attempted to determine the fundamental structure of flavonoids related to inhibition. We additionally investigated whether flavonoids suppress leukotriene C4 synthesis by basophils and IL-4 synthesis by T cells in response to anti-CD3 antibody. Methods: Highly purified peripheral basophils were stimulated for 12 h with anti-IgE antibody alone or anti-IgE antibody plus IL-3 in the presence of various concentrations of 18 different kinds of flavones and flavonols. IL-4 and IL-13 concentrations in the supernatants were then measured. Leukotriene C4 synthesis was also measured after basophils were stimulated for 1 h in the presence of flavonoids. Regarding the inhibitory activity of flavonoids on IL-4 synthesis by T cells, peripheral blood mononuclear cells were cultured with flavonoids in anti-CD3-antibody-bound plates for 2 days. Results: Luteolin, fisetin and apigenin were found to be the strongest inhibitors of both IL-4 and IL-13 production by basophils but did not affect leukotriene C4 synthesis. At higher concentrations, these flavonoids suppressed IL-4 production by T cells. Based on a hierarchy of inhibitory activity, the basic structure for IL-4 inhibition by basophils was determined. Conclusions: Due to the inhibitory activity of flavonoids on IL-4 and IL-13 synthesis, it can be expected that the intake of flavonoids, depending on the quantity and quality, may ameliorate allergic symptoms or prevent the onset of allergic diseases.

Proteomics-based identification of α-enolase as a tumor antigen in non-small lung cancer

Autoantibodies against tumor antigens represent one type of biomarker that may be assayed in serum for detection of cancer and monitoring of disease progression. In the present study, we used a proteomics‐based approach to identify novel tumor antigens in non‐small cell lung cancer (NSCLC). By combining two‐dimensional electrophoresis, western blotting, mass spectrometry and enzyme‐linked immunosorbent assay technology, we detected autoantibodies against α‐enolase in a subset of NSCLC patients’ sera. When ‘Mean ODhealthy control sera + 3 SDhealthy control sera’ was used as the cut‐off point, the prevalence of this autoantibody was 27.7% in patients with NSCLC (26 of 94), 1.7% in healthy control subjects (1 of 60), and not detectable in sera from 15 patients with small cell lung cancer, 18 patients with gastrointestinal cancer and nine patients with Mycobacterium avium complex infection of lung. Immunohistochemical staining showed that expression of α‐enolase was increased in cancer tissues of NSCLC patients, and flow cytometric analysis confirmed the expression of α‐enolase at the surface of cancer cells. The combined detection of autoantibodies against α‐enolase, carcinoembryonic antigen and cytokeratin 19 fragment (CYFRA21‐1) enhanced sensitivity for the diagnosis of NSCLC. Therefore, autoantibodies against α‐enolase may constitute a promising biomarker for NSCLC. (Cancer Sci 2007; 98: 1234–1240)

Luteolin, a Flavonoid, Inhibits CD40 Ligand Expression by Activated Human Basophils

Background: We have previously shown that flavonoids such as luteolin, apigenin and fisetin inhibit interleukin 4 and interleukin 13 production. In this study, we investigated whether luteolin can suppress CD40 ligand expression by basophils. Methods: A human basophilic cell line, KU812, was stimulated with A23187 and phorbol myristate acetate (PMA) with or without various concentrations of luteolin or other flavonoids for 12 h, and CD40 ligand expression was analyzed by FACS. The effect of luteolin on CD40 ligand mRNA expression was studied by semiquantitative reverse transcription PCR analysis. In addition, CD40 ligand expression was also measured in purified basophils that had been stimulated for 12 h with A23187 plus PMA with or without various concentrations of luteolin. Results: CD40 ligand expression by KU812 cells was enhanced noticeably in response to A23187 and even more strikingly augmented by A23187 plus PMA. The expression was significantly suppressed by 10 or 30 µM of luteolin, whereas myricetin failed to inhibit. Reverse transcription PCR analyses demonstrated that luteolin inhibited CD40 ligand mRNA expression by stimulated KU812 cells. Of the six flavonoids examined, luteolin, apigenin, fisetin and quercetin at 30 µM showed a significant inhibitory effect on CD40 ligand expression. The incubation of purified basophils with A23187 plus PMA significantly enhanced CD40 ligand expression, and the presence of luteolin again had an inhibitory effect. Conclusions: Luteolin inhibits CD40 ligand expression by activated basophils.

Proteomic analysis of autoantigens associated with systemic lupus erythematosus: Anti-aldolase A antibody as a potential marker of lupus nephritis.

To screen for autoantibodies associated with systemic lupus erythematosus (SLE), we used proteomic approaches combining 2‐D PAGE and Western blot analysis, followed by protein identification by LC‐MS/MS analysis, resulting in the identification of aldolase A as a novel autoantigen in SLE. ELISA showed the prevalence of anti‐aldolase A antibodies to be 29.3% in SLE, 8.2% in rheumatoid arthritis, 18.1% in polymyositis and absent in healthy controls. Furthermore, 43.4% of SLE patients suffering from nephritis showed anti‐aldolase A autoantibodies, which was significantly higher than the prevalence for those without nephritis (11.1%). In lupus nephritis, there are few reliable diagnostic methods, other than urinalysis. Therefore, these results indicate that autoantibodies against aldolase A may serve as an alternative clinical biomarker of SLE associated with nephritis.

Assignment of SH3BP5/Sh3bp5 encoding sab, an SH3 domain-binding protein which preferentially associates with Bruton's tyrosine kinase, to human chromosome 1q43 and mouse chromosome 14B by in situ hybridization.

Btk (Bruton’s tyrosine kinase), whose gene is located on the X chromosome, is a cytoplasmic tyrosine kinase crucial for B lymphocyte development and the loss of its function leads to human X-linked agammaglobulinemia (XLA) (Tsukada et al., 1993) and murine X-linked immunodeficiency (XID) (Rawlings et al., 1993). It has been shown that Btk is involved in several cytoplasmic signaling pathways in hematopoietic cells. We recently cloned a novel protein Sab (the SH3 domain-binding protein which preferentially associates with Btk) (Matsushita et al., 1998) and demonstrated that Sab binds to the SH3 domain of Btk and negatively regulates its kinase activity (Yamadori et al., 1999). In this study, we determined the chromosome locations of human and mouse Sab genes (SH3BP5/ Sh3bp5) by using fluorescence in situ hybridization and identified a novel conserved homology in human chromosome 1 and mouse chromosome 14, which has not previously been reported. Materials and methods