Yuhki Yanase

Detection of refractive index changes in individual living cells by means of surface plasmon resonance imaging

Real time imaging of living cell activation is an increasing demand in disciplines of life science and medicine. We previously reported that surface plasmon resonance (SPR) sensors detect large changes of refractive index with living cells, such as mast cells, keratinocyte, human basophils and B-cells activated by biological stimuli. However, conventional SPR sensors detect only an average change of refractive index with thousands of cells at detectable area on a sensor chip. In this study, we developed an SPR imaging (SPRI) sensor with a CMOS camera and an objective lens in order to analyze refractive index of individual living cells and their changes upon stimuli. The SPRI sensor could detect reactions of individual rat basophilic leukemia (RBL-2H3) cells, mouse keratinocyte (PAM212) cells, and human epidermal carcinoma (A431) cells in response to either specific or non-specific stimuli, such as antigen, phorbol ester or epidermal growth factor, with or without their inhibitors, resembling signals obtained by a conventional SPR sensor. Moreover, we distinguished reactions of different type cells, co-cultured on a sensor chip, and revealed that the increase of refractive index around nuclei is rapid and potent as compared to that in peripheries in the reaction of RBL-2H3 cells against antigen. This system may be a useful tool to investigate the mechanism of refractive index-changes evoked in near-membrane fields of living cells, and to develop a system of high-throughput screening for clinical diagnosis.

Development of an optical fiber SPR sensor for living cell activation

Surface plasmon resonance (SPR) sensors provide a useful means to study the interactions of biological molecules and the reaction of living cells on a sensor chip. However, conventional SPR sensors are bulky, expensive and complicated to use as common diagnostic equipment. In this study, we developed a relatively small and simple SPR system, using optical fibers of 250 microm diameter to detect the activation of living cells attached to the fiber tip. For this system, the core of 200 microm diameter with 1cm length of an optical fiber was coated by gold film with 50 nm thickness to cause plasmon resonance. The light provided by a white LED and attenuated due to a SPR phenomenon in the sensor part was detected and analyzed using a spectrum detector. The difference in solvents with various refractive indexes and protein-bindings to the sensor tip was detected with sufficient sensitivity. Moreover, it detected a sustained increase of AR in a real-time manner, when RBL-2H3 mast cells were fixed onto the fiber tip and stimulated by an antigen. This small fiber SPR system might serve as a useful tool for various clinical examinations either within or outside the body.

Fungal protein MGL_1304 in sweat is an allergen for atopic dermatitis patients

BACKGROUND Sweat is a major aggravating factor of atopic dermatitis (AD) and approximately 80% of patients with AD show type I hypersensitivity against sweat. OBJECTIVE To identify and characterize an antigen in sweat that induces histamine release from basophils of patients with AD. METHODS Basophil histamine-releasing activity in sweat was purified by a combination of chromatographies, and proteins were analyzed with mass spectrometry. Recombinant proteins of the sweat antigen were generated, and their biological characteristics were studied by immunoblots, histamine release tests, and neutralization assays. RESULTS We identified a fungal protein, MGL_1304, derived from Malassezia globosa (M globosa) in the purified sweat antigen. Recombinant MGL_1304 induced histamine release from basophils of most of the patients with AD, in accordance with the semi-purified sweat antigen. Moreover, recombinant MGL_1304 abolished the binding of serum IgE of patients with AD to the semi-purified sweat antigen, or vice versa in immunoblot analysis, and attenuated the sensitization of RBL-48 mast cells expressing human FcɛRI by serum IgE. Studies of truncated mutants of MGL_1304 indicated that IgE of patients with AD recognized the conformational structure of MGL_1304 rather than short peptide sequences. Western blot analysis of the whole lysate, the culture supernatant of M globosa, and the semi-purified sweat antigen showed that MGL_1304 was produced as a minor immunological antigen of M globosa with posttranslational modification, cleaved, and secreted as a 17-kDa major histamine-releasing sweat antigen. CONCLUSION MGL_1304 is a major allergen in human sweat and could cause type I allergy in patients with AD.

Protein kinase C‐α mediates TNF release process in RBL‐2H3 mast cells

1 To clarify the mechanism of mast cell TNF secretion, especially its release process after being produced, we utilized an antiallergic drug, azelastine (4‐(p‐chlorobenzyl)‐2‐(hexahydro‐1‐methyl‐1H‐azepin‐4‐yl)‐1‐(2H)‐ phthalazinone), which has been reported to inhibit TNF release without affecting its production in ionomycin‐stimulated RBL‐2H3 cells. 2 Such inhibition was associated with the suppression of an ionomycin‐induced increase in membrane‐associated PKC activity rather than the suppression of Ca2+ influx, suggesting that PKC might be involved in TNF release process. 3 To see whether conventional PKC family (cPKCs) are involved, we investigated the effects of a selective cPKC inhibitor (Gö6976) and an activator (thymeleatoxin) on TNF release by adding them 1 h after cell stimulation. By this time, TNF mRNA expression had reached its maximum. Gö6976 markedly inhibited TNF release, whereas thymeleatoxin enhanced it, showing a key role of cPKC in TNF post‐transcriptional process, possibly its releasing step. 4 To determine which subtype of cPKCs could be affected by azelastine, Western blotting and live imaging by confocal microscopy were conducted to detect the translocation of endogenous cPKC (α, βI and βII) and transfected GFP‐tagged cPKC, respectively. Both methods clearly demonstrated that 1 μM azelastine selectively inhibits ionomycin‐triggered translocation of αPKC without acting on βI or βIIPKC. 5 In antigen‐stimulated cells, such a low concentration of azelastine did not affect either αPKC translocation or TNF release, suggesting a functional link between αPKC and the TNF‐releasing step. 6 These results suggest that αPKC mediates the TNF release process and azelastine inhibits TNF release by selectively interfering with the recruitment of αPKC in the pathway activated by ionomycin in RBL‐2H3 cells.

Germline mutation in ATR in autosomal- dominant oropharyngeal cancer syndrome

ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a ∼16.8 cM interval in chromosomal region 3q22-24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain-which can activate p53-of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans.

Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-gamma in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.

Surface plasmon resonance biosensor detects the downstream events of active PKCβ in antigen-stimulated mast cells

Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors.

Surface plasmon resonance-biosensor detects the diversity of responses against epidermal growth factor in various carcinoma cell lines

Surface plasmon resonance (SPR) biosensor detects intracellular signaling events as a change of the angle of resonance (AR). We previously reported that the activation of epidermal growth factor receptor (EGFR) on keratinocytes causes a unique triphasic change of AR, whereas the activation of other receptors, such as IgE receptor and adenosine A3 receptor on mast cells, causes a transient monophasic increase of AR. To study the mechanism of AR changes induced by EGFR activation, we introduced wild and mutated EGFR cDNAs into Chinese hamster ovary (CHO) cells and analyzed changes of AR in response to EGF. CHO cells expressing wild-type EGFR showed a triphasic change of AR, whereas cells expressing kinase-dead EGFR (K721M) showed minimum change of AR. A phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated the third phase of AR change in CHO cells expressing wild-type EGFR. The pattern of AR change was independent on the concentration of EGF. We also analyzed changes of AR with a nontumorigenic keratinocyte cell line, HaCaT, and several cell lines of carcinoma to explore the feasibility of SPR biosensor as a tool for clinical diagnosis. The activation of HaCaT cells and one out of six carcinoma cell lines showed a full triphasic change of AR. In contrast, five out of the six cell lines showed mono- or bi-phasic change of AR. These results suggest that EGF induces the SPR signals via the phosphorylation of EGFR, and provide a possibility that the SPR biosensor could be applied to the real-time detection and diagnosis of malignant tumors.

Hydrolyzed Konjac Glucomannan Suppresses IgE Production in Mice B Cells

Background: Oral administration of pulverized Konjac glucomannan (KGM) reduces increased plasma IgE and the amount of Ε-germline transcript (ΕGT) in the spleen, as well as preventing the development of dermatitis in mice. To elucidate the mechanism of action of pulverized KGM, we solubilized KGM and studied its effect on IgE in vitro and in vivo. Methods: Solubilized KGM was prepared by acid hydrolysis, and we analyzed the effective molecular size for the suppression of IgE production and ΕGT in vitro and the level of plasma IgE induced by immunization with ovalbumin in BALB/c mice. Results: The production of IgE and ΕGT in splenic cells, but not purified B cells, was inhibited by hydrolyzed KGM (KGM hydrolyzed with 0.25 N HCl; H-KGM) at the optimal size of between 10 and 500 kDa. However, no effect was observed when H-KGM was substituted with unhydrolyzed KGM in vitro. IgE production from purified B cells cocultured with purified monocytes, but not with purified T cells, was inhibited by H-KGM. The release of IFNγ in cultures of monocytes but in purified B cells with or without T cells was enhanced in the presence of H-KGM. Injection of mice with H-KGM also suppressed the production of plasma IgE and IgG1 but not IgG2a in vivo. Conclusion: KGM at an optimal size prevents germline class-switching and IgE production both in vitro and in vivo. H-KGM may be useful as a tool to study the mechanism of action of KGM and as a dietary supplement to prevent atopic diseases.

Evaluation of peripheral blood basophil activation by means of surface plasmon resonance imaging

Basophil activation in response to antigen may represent specificities of type I allergy of individuals and their reactions in the body. We previously demonstrated that surface plasmon resonance (SPR) sensor could detect the activation of human basophils in response to antigens. In this study, we further developed a technique based on SPR imaging (SPRI) system to detect reactions of individual basophils isolated from human blood, and investigated the potential of this sensor as a tool for diagnosis of type I allergy. To detect the change of refractive index (RI) in individual basophils, human basophils were isolated by negative selection with antibodies conjugated with magnetic beads, fixed on a gold film with anti-basophil antibody and stimulated with various antigens under the measurement of SPRI. The sensor could detect the reactions of individual basophils in response to specific antigens as well as non-specific activators. Moreover, the sensor well allocated two spots of basophils on a sensor chip and detected individual reactions to antigen. Thus, the technique developed in this study can visualize the effect of various stimuli or inhibitors on basophils as change of intracellular RI distribution at the single cell level. In combination with a device to rapidly isolate basophils from peripheral blood, this technique may be a useful tool as a high throughput screening system in clinical diagnosis for type I allergy.