[Show abstract][Hide abstract] ABSTRACT: Noninvasive real-time evaluation of living cell conditions and functions is increasingly sought after in life science. Surface plasmon resonance (SPR) sensors can sensitively detect refractive index changes on the surface of a sensor chip without any labeling in a real-time manner. In this chapter, we first review the principle of SPR sensors and the applications of SPR sensors for detection of living cell reactions. We then introduce the technique to isolate and fix basophils on an SPR sensor chip. Finally, we describe the method to visualize individual basophil activation by means of SPR imaging (SPRI) sensor and the potential of basophil activation test by SPRI for clinical diagnosis of type I allergy.
Methods in Pharmacology and Toxicology 01/2015; 53:373-385. DOI:10.1007/978-1-4939-2617-6_21
[Show abstract][Hide abstract] ABSTRACT: The identification of antigen that induces activation of mast cells and basophils by crosslinking IgE bound to the cell surface is crucial to avoid symptoms of allergic diseases. Surface plasmon resonance imaging (SPRI) possesses a great potential for clinical diagnosis of allergy, in that it reveals living cell activation following the binding of antigens to IgE, on real-time and single cell basis without artificial labeling. However, present technique of SPRI requires freshly isolated basophils of patients and cannot analyze multiple samples in parallel. To overcome such problems, we developed devices for SPRI to make a broad observation area and a multi-well SPRI sensor chip with a hydrophobic membrane. The employment of human IgE receptor-expressing mast cell lines (RBL-48 cells) sensitized with serum, collected and stored from less than a microliter of patient’s blood, allowed us to detect specific reactions of RBL-48 cells in response to antigens. This technique may be a useful tool as a high throughput screening system of type I allergy not only for freshly prepared basophils but also for sera stored in clinical practices.
Sensing and Bio-Sensing Research 11/2014; 2. DOI:10.1016/j.sbsr.2014.10.014
[Show abstract][Hide abstract] ABSTRACT: Hereditary angioedema (HAE) presents as severe angioedema, which is mostly due to the C1 inhibitor (C1-INH) gene mutations. Environmental factors, minor trauma and oral contraceptives have been reported to induce angioedema attack, but the trigger may often be uncertain. Activated factor XII controlled by C1-INH facilitates bradykinin generation and also regulates coagulation cascade, but the relationship between edema formation and coagulation is still unclear. We have described a 35-year-old female patient with HAE, presenting with frequent angioedema attacks in the absence of an apparent triggering factor. She showed higher levels of FDP and D-dimer during angioedema than those in remission. In addition, tissue factor (TF), an initiator of the extrinsic coagulation cascade, was expressed on the surface of monocytes. It was significantly higher than that of monocytes from healthy controls and tends to further increase during attacks. The expression of TF on monocytes may play a role in the induction of angioedema attacks in HAE by activating the coagulation pathway in association with reduced functions of C1-INH.
The Journal of Dermatology 09/2014; 41(10). DOI:10.1111/1346-8138.12610 · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immortal mast cell lines, such as RBL-2H3 and HMC-1 cells, are commonly utilized to investigate the function of mast cells. However, they are tumor cells carrying a gain-of-function mutation of Kit. We established an immortal mast cell line without Kit mutation, NCL-2, derived from NC mouse bone marrow. NCL-2 cells could be maintained without additional growth factors and thus could respond to exogenous growth signals. Moreover, NCL-2 cells expressed FcεRI and KIT, and release histamine and LTB4 in response to antigen stimulation. This cell line could be a useful tool to analyze proliferation, differentiation, and function of normal mast cells.
FEBS Open Bio 04/2014; 4. DOI:10.1016/j.fob.2014.03.011 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-invasive real-time observations and the evaluation of living cell conditions and functions are increasingly demanded in life sciences. Surface plasmon resonance (SPR) sensors detect the refractive index (RI) changes on the surface of sensor chips in label-free and on a real-time basis. Using SPR sensors, we and other groups have developed techniques to evaluate living cells' reactions in response to stimuli without any labeling in a real-time manner. The SPR imaging (SPRI) system for living cells may visualize single cell reactions and has the potential to expand application of SPR cell sensing for clinical diagnosis, such as multi-array cell diagnostic systems and detection of malignant cells among normal cells in combination with rapid cell isolation techniques.
[Show abstract][Hide abstract] ABSTRACT: Background:
MGL_1304 secreted by Malassezia globosa is contained in human sweat and induces histamine release from basophils in patients with atopic dermatitis (AD) at a high positive rate. The aims of this study were to establish the enzyme-linked immunosorbent assay (ELISA) measuring specific immunoglobulins against MGL_1304 and to investigate the levels of these immunoglobulins in sera of patients with various allergic diseases.
Purified MGL_1304 from human sweat (QRX) and recombinant MGL_1304 (rMGL_1304) were prepared for ELISA. To quantify the amount of MGL_1304-specific immunoglobulins, the standard serum was created by pooling sera of 20 patients with AD whose basophils released histamine in response to QRX. A monoclonal antibody which exhibited the highest neutralizing ability against QRX was established as Smith-2, and used as a capture antibody for the assay of QRX-specific IgE. A total of 156 subjects [normal controls (n = 23), AD (n = 63), cholinergic urticaria (CU) (n = 24), bronchial asthma (n = 32), and allergic rhinitis (n = 14)] were enrolled in this study.
ELISA methods to quantify the specific IgE, IgG and IgG4 against MGL_1304 in sera were successfully established. Levels of QRX-specific IgE in sera of patients with AD and CU were significantly higher than those of normal controls. Moreover, the levels of QRX-specific IgE and rMGL_1304-specific IgE in patients with AD were significantly correlated with their disease severities.
These ELISA methods to quantify the specific immunoglobulins against MGL_1304 are easy and useful means to assess allergy to MGL_1304. MGL_1304 contained in sweat is an important antigen for patients with AD and CU.
Allergology International 01/2014; 63(1). DOI:10.2332/allergolint.13-OA-0611 · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Non-steroidal anti-inflammatory drugs (NSAIDs), especially aspirin, and food additives (FAs) may exacerbate allergic symptoms in patients with chronic idiopathic urticaria and food-dependent exercise-induced anaphylaxis (FDEIA). Augmentation of histamine release from human mast cells and basophils by those substances is speculated to be the cause of exacerbated allergic symptoms. We sought to investigate the mechanism of action of aspirin on IgE-mediated histamine release.
The effects of NSAIDs, FAs or cyclooxygenase (COX) inhibitors on histamine release from human basophils concentrated by gravity separation were evaluated.
Benzoate and tartrazine, which have no COX inhibitory activity, augmented histamine release from basophils similar to aspirin. In contrast, ibuprofen, meloxicam, FR122047 and NS-398, which have COX inhibitory activity, did not affect histamine release. These results indicate that the augmentation of histamine release by aspirin is not due to COX inhibition. It was observed that aspirin augmented histamine release from human basophils only when specifically activated by anti-IgE antibodies, but not by A23187 or formyl-methionyl-leucyl-phenylalanine. When the IgE receptor signaling pathway was activated, aspirin increased the phosphorylation of Syk. Moreover, patients with chronic urticaria and FDEIA tended to be more sensitive to aspirin as regards the augmentation of histamine release, compared with healthy controls.
Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.
Allergology International 10/2013; 62(4). DOI:10.2332/allergolint.13-OA-0536 · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 FcεRI 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.
The Journal of allergy and clinical immunology 05/2013; 132(3). DOI:10.1016/j.jaci.2013.03.047 · 11.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A technique to visualize living cell activation in a real time manner without any labeling is required in the fields of life sciences and medicine. We have reported that surface plasmon resonance (SPR) sensors detect large changes of refractive index (RI) with living cells, such as mast cells, human basophils and lymphocytes. However conventional SPR sensors detect only an average change of RI with thousands of cells at detectable area on a sensor chip. Therefore, we developed an SPR imaging (SPRI) sensor with a CMOS camera and an objective lens in order to visualize RI of individual living cells and their changes upon stimuli. The SPRI sensor we developed could detect reactions of individual rat basophilic leukemia (RBL-2H3) cells and mouse keratinocyte cells in response to specific or nonspecific stimuli. Moreover, the sensor could detect the reactions of individual human basophils isolated from patients in response to antigens (allergens). Thus the technique can visualize the effect of various stimuli, inhibitors and/or conditions on cell reactions as change of intracellular RI distribution at single cell levels. Establishment of the technique to rapidly isolate cells from patient blood should enable us to utilize SPRI system as a high throughput screening system in clinical diagnosis, such as type I hypersensitivity and drug hypersensitivity, and as a tool to reveal novel phenomena in evanescent fields around plasma membranes.
Allergology International 02/2013; 62(2). DOI:10.2332/allergolint.12-RA-0505 · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunoglobulin (Ig) E plays an important role in the pathogenesis of allergic diseases such as atopic dermatitis and allergic asthma.We previously reported that a sulfate polysaccharide, fucoidan, suppressed IgE production by murine B cells in vitro. However, the mechanism by which fucoidan suppresses IgE production remains unclear.
We incorporated sulfate groups into cellulose and studied their biological characteristics in vitro to explore the possibility of converting biologically neutral polysaccharides to active reagents with antiallergic functions.
Cellulose was chemically processed using N,N-dimethylformamide (DMF) and DMF-sulfurtrioxide and recovered as cellulose sulfate with a molecular weight of around 10 kDa. We then studied the effect of cellulose sulfate on IgE production from B cells, IgE class-switching, and populations of IgE-secreting B cells prepared from murine spleen. We also investigated the effects of sulfated cellulose on the production of interleukin (IL) 4 and interferon (IFN) gamma and the expression of T-bet mRNA by splenic T cells. The cytotoxicity of cellulose sulfate was also examined.
Cellulose sulfate suppressed IgE production in B cells stimulated with IL-4 and anti-CD40 antibody by inhibiting IgE class-switch recombination and decreasing the number of IgE-secreting B cells in vitro. Moreover, both cellulose sulfate and fucoidan suppressed IL-4 production and enhanced IFN-gamma production by murine T cells stimulated with anti-CD3 and anti-CD28 antibodies, despite the decrease in T-bet mRNA expression.
Cellulose gains an antiallergic effect on B cells and T cells with the addition of sulfate groups.
Journal of investigational allergology & clinical immunology: official organ of the International Association of Asthmology (INTERASMA) and Sociedad Latinoamericana de Alergia e Inmunología 06/2012; 22(3):180-7. · 2.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously reported that about 80 % of patients with atopic dermatitis and 60 % with cholinergic urticaria revealed type I allergy against sweat, by means of skin test against autologous sweat and/or histamine-release test for peripheral blood basophils with semi-purified sweat antigen. In this study, we developed an assay for sera to neutralize histamine-releasing activity of semi-purified sweat antigen. The semi-purified sweat antigen was pre-incubated with serially diluted sera for 30 min at 37 °C and was subjected to histamine-release activity. Histamine release-neutralization (HRN) activities were calculated by measuring the amount of histamine release from basophils in the presence or absence of semi-purified sweat antigen. Of 62 subjects, 39 showed positive histamine release (≥5 %) from their basophils in response to semi-purified sweat antigen, and sera of 34 out of 39 subjects (87.2 %) were also positive in HRN activity (≥10 %). The specificity of the HRN assay was 0.522. Moreover, HRN activities in sera were largely correlated with degrees of histamine release from peripheral blood basophils of the same donors in response to sweat antigen. To identify the substance that neutralizes histamine-release activity, we removed IgE and IgG from the sera of HRN (+) subjects by column chromatography. The HRN activities in 30 out of 42 sera were largely reduced by the removal of IgG. On the other hand, sera of four subjects lost HRN activity by the removal of IgE, suggesting that the majority of HRN (+) subjects have serum IgG against the sweat antigen as well as IgE bound to peripheral basophils. Thus, the HRN assay maybe useful for the screening of type I allergy against sweat antigen.
Archives for Dermatological Research 04/2012; 304(8):647-54. DOI:10.1007/s00403-012-1236-2 · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The American Journal of Human Genetics 02/2012; 90(3):511-7. DOI:10.1016/j.ajhg.2012.01.007 · 10.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production in B cells from mice spleen in vitro and ovalbumin-sensitized mice in vivo. In this study, we examined the effect of fucoidan on IgE production in human peripheral blood mononuclear cells (PBMC) in vitro. PBMC, obtained from healthy donors or patients with atopic dermatitis (AD) with high levels of serum IgE, were cultured with IL-4 and anti-CD40 antibody in the presence or absence of fucoidan. Fucoidan significantly reduced IgE production in PBMC without affecting cell proliferation and IFN-γ production. Fucoidan also inhibited immunoglobulin germline transcripts of B cells in PBMC, and decreased the number of IgE-secreting cells. The inhibitory effects of fucoidan were similarly observed for both PBMC from patients with AD and those with healthy donors. Our findings indicate that fucoidan suppresses IgE induction by inhibiting immunoglobulin class-switching to IgE in human B cells, even after the onset of AD.
Archives for Dermatological Research 12/2010; 303(6):425-31. DOI:10.1007/s00403-010-1115-7 · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.