Dinitrophenylated Bovine Serum Albumin Research Articles

Inhibition of Histamine Release from RBL-2H3 Cells by Zoledronate Did Not Affect Rab27a/Doc2a Interaction.

Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.

Anti-allergic and anti-inflammatory effects of hydrosol extracted from Zanthoxylum schinifolium branch

Zanthoxylum schinifolium Sieb. et Zucc. (syn. Fagara schinifolia Engler) was studied for its potential anti-inflammatory properties. The hydrosol extract prepared from the Z. schinifolium branch was analyzed by gas chromatography/mass spectrometry. Here, five main chemical components were identified in the hydrosol of the branches of this shrub. The main chemical compounds in the branch inhibited both an Immunoglobulin E (IgE)-antigen complex and a dinitrophenyl-bovine serum albumin (DNP-BSA)-induced β-hexosaminidase release in a dose-dependent manner in RBL-2H3 mast cells, and at the tested concentrations did not show cytotoxicity to RBL-2H3 cells. Moreover, hydrosol obtained from the branch substantially inhibited a lipopolysaccharide (LPS) induced overproduction of intracellular active oxygen (ROS) and nitric oxide (NO). Consistently, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins of SNAP23, syntaxin4, VAMP7, and VAMP8 were remarkably decreased through hydrosol treatment. Hydrosol suppressed the activation of SNARE proteins in DNP-BSA-stimulated RBL-2H3 cells and inhibited ROS and NO in LPS-stimulated RAW264.7 cells. Camphor and estragole are the main chemical components of hydrosol and downregulate the LPS-induced phosphorylation of the SNARE proteins. The hydrosol obtained from the branch of Z. schinifolium has therapeutic benefits for allergic inflammatory diseases.

Identification of key pathways and gene expression in the activation of mast cells via calcium flux using bioinformatics analysis

Mast cells are the main effector cells in IgE-associated allergic disorders, and we have reported that mucosal mast cells (MMCs) play a more important role in the development of food allergy (FA). IgE with antigen or calcium ionophore stimulation can lead to the activation of MMCs via a calcium-dependent pathway. The purpose of the present study was to identify gene signatures with IgE/antigen (dinitrophenyl-bovine serum albumin, DNP-BSA) or calcium ionophore (A23187) on the activation of MMCs. Differentially expressed genes between the two types of samples were identified with microarray analysis. Gene ontology functional and pathway enrichment analyses of differentially expressed genes were performed using the database for annotation, visualization, and integrated discovery software. The results showed that IgE/antigen and A23187 could induce degranulation, increase vacuoles, and elevate the cytosolic calcium concentration in MMCs. Furthermore, GeneChip analysis showed that the same 134 mRNAs were altered with IgE/DNP-BSA and A23187, suggesting that DNP-BSA/IgE and A23187 affect the same signal pathway partly in degranulation. KEGG analysis showed that the data were enriched in NF-κB, TNF, MAPK, transcription factor activity, DNA binding, and nucleic acid binding, suggesting that activation of MMCs is a complex process. The results provide new insights on MMCs activation.

Antiallergic Effects of Phenolic Compounds Isolated From Stellera chamaejasme on RBL-2H3 Cells

The current study was undertaken to isolate and identify anti-inflammatory and antiallergic active compounds in the ethyl acetate and butanol fractions of an ethanolic extract of the aerial parts of Stellera chamaejasme (SCAE). A new maltol glucoside, maltol 3-(6″-(2-( E)-butenoyl)-glucoside) and benzoic acid, daphnoretin (a coumarin), 6 flavonoids (apigenin, genkwanin, taxifolin, apigenin 7- O-glucoside, luteolin 7- O-sambubioside, genkwanin 5- O-glucoside), and 2 lignans (matairesinoside and lariciresinol) were isolated from SCAE by chromatographic separation. Their chemical structures were elucidated by analyzing spectroscopic data, including 1-dimensional and 2-dimensional nuclear magnetic resonance (Fig1). Maltol 3-(6″-(2-( E)-butenoyl)-glucoside) significantly inhibited β-hexosaminidase release by 85.5% in immunoglobulin E and dinitrophenyl/bovine serum albumin (DNP/BSA)-induced RBL-2H3 cells. Benzoic acid from S. chamaejasme inhibited β-hexosaminidase release (by 80.2%) in IgE and DNP/BSA-induced RBL-2H3 cells and interleukin-4 messenger ribonucleic acid expression (by 21.9% inhibition) in propidium iodide-induced RBL-2H3 cells.

Clinacanthus nutans aqueous extract suppresses the release of histamine and β-Hexosaminidase inin-vitro model of IgE-mediated mast cell degranulation

Introduction: Clinacanthus nutans (Burm. f.) Lindau, a shrub found in South East Asia, particularly in Malaysia, Thailand, and Indonesia has many potential medicinal uses. It is used as a traditional herbal medicine for treating many diseases including skin rashes. Skin rash often appear in an allergic reaction. Recently, we have shown that C. nutans aqueous extract has the ability to alleviate ovalbumin-induced active systemic anaphylaxis rats from anaphylaxis – the acute form of allergy. This present study is aimed at comparing the ability of ethanolic and aqueous extracts of C. nutans to suppress IgE-mast cell degranulation. Materials and Methods: IgE-presensitized rat basophilic leukemic (RBL-2H3) cells pretreated with C. nutans ethanolic (100% ethanolic, 70% and 50% aqueous ethanolic) or 100% aqueous extracts were challenged with dinitrophenyl-bovine serum albumin to analyze the release of early and late-phase pro-inflammatory mediators. Results: We found that at concentrations of 5 mg/ml and above, C. nutans aqueous extract was able to suppress the levels of β-hexosaminidase and histamine while suppression was not seen in ethanolic extracts pretreated RBL-2H3 cells.Conclusion: Hence, we proposed that C. nutans aqueous extract is more active compared to the ethanolic extracts in suppressing the mediators of IgE-mast cell degranulation.

Clinacanthus nutans Aqueous Extract But Not Ethanolic Extract Inhibits the Release of Preformed Mediators in In Vitro Model of IgE-Mediated Mast Cell Degranulation

C. nutans (Burm. f.) Lindau is a shrub that is commonly found in South East Asia, particularly in Malaysia, Thailand and Indonesia. This plant has diverse and potential medicinal uses in traditional herbal medicine for treating many diseases which include skin rashes. Skin rash is one of the symptoms that primarily occur in people with allergies. Till date, there has been no scientific evidence to support this traditional use of C. nutans as well as its potential of possessing anti-allergic properties. IgE and mast cells play an important role in the pathogenesis of allergy. Upon cross-linking of allergen to IgE antibodies bound to FcεRI on mast cells, pro-inflammatory mediators will be released as a result of degranulation. The release of pro-inflammatory mediators by mast cells activated by allergen in the skin causes large, itchy, red swellings of the skin. This study aimed to identify the most active C. nutans extract in inhibiting in vitro IgE-mast cell degranulation. Rat basophilic leukaemic cells (RBL-2H3) were pre-sensitised with IgE, pre-treated with C. nutans ethanolic (100%, 70% and 50%) or aqueous extracts and challenged with dinitrophenyl-bovine serum albumin (DNP-BSA) accordingly to analyse the release of β-hexosaminidase, histamine, interleukin (IL)-4 and tumour necrosis factor (TNF)-α. It was found that C. nutans aqueous extract was the most active extract compared to the ethanolic extracts as it was able to inhibit the release of β-hexosaminidase and histamine at concentrations of 5 mg/ml and above.

Inhibitory effect of Zizania latifolia chloroform fraction on allergy-related mediator production in RBL-2H3 cells.

Zizania latifolia exhibits anti-inflammatory and anti-allergic effects; however, the mechanisms behind these effects are unknown. Here the ethanol extract of Z. latifolia was partitioned using hexane, chloroform, ethyl acetate, butanol, and water. Subsequently, the anti-allergic effects of these fractions were evaluated in vitro. The results showed that the chloroform fraction of Z. latifolia inhibited the release of β-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells stimulated with dinitrophenyl-bovine serum albumin (DNP-BSA). In addition, this fraction suppressed the expression of cyclooxygenase-2 (COX-2) and inhibited the activation of mitogen-activated protein kinases (MAPKs). The results obtained suggest that the chloroform fraction of Z. latifolia inhibited mast cell-mediated allergic inflammatory responses.

A novel mast cell co-culture microfluidic chip for the electrochemical evaluation of food allergen

In this study a novel cell-to-cell electrochemical microfluidic chip was developed for qualitative and quantitative analysis of food allergen. Microfluidic cell culture, food allergen-induced cell morphological changes, and cell metabolism measurements were performed simultaneously using the aforementioned device. RBL-2H3 mast cells and ANA-1 macrophages have been used within a cell co-culture model to observe their allergic response when they are introduced to the antigen stimulus. Two cell cultivation microfluidic channels are located in the microfluidic chip, which is fabricated with four groups of gold electrodes, with an additional “capillary”. In order to detect the allergic response, the cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) without anti-DNP IgE incubation. When exocytosis occurs, the cell-secreted inflammatory cytokines were measured by enzyme-linked immuno sorbent assay (ELISA) and cell impedance changes were detected using cell-based electrochemical assay. Results indicate that the real-time cell allergic response are accurately monitored by this electrochemical microfluidic chip, which provides a general example of rapidly prototyped low-cost biosensor technology for applications in both food allergen detection and investigation.

Dietary Compound Kaempferol Inhibits Airway Thickening Induced by Allergic Reaction in a Bovine Serum Albumin-Induced Model of Asthma.

Asthma is characterized by aberrant airways including epithelial thickening, goblet cell hyperplasia, and smooth muscle hypertrophy within the airway wall. The current study examined whether kaempferol inhibited mast cell degranulation and prostaglandin (PG) release leading to the development of aberrant airways, using an in vitro model of dinitrophenylated bovine serum albumin (DNP-BSA)-sensitized rat basophilic leukemia (RBL-2H3) mast cells and an in vivo model of BSA-challenged asthmatic mice. Nontoxic kaempferol at 10–20 μM suppressed β-hexosaminidase release and cyclooxygenase 2 (COX2)-mediated production of prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α) in sensitized mast cells. Oral administration of ≤20 mg/kg kaempferol blocked bovine serum albumin (BSA) inhalation-induced epithelial cell excrescence and smooth muscle hypertrophy by attenuating the induction of COX2 and the formation of PGD2 and PGF2α, together with reducing the anti-α-smooth muscle actin (α-SMA) expression in mouse airways. Kaempferol deterred the antigen-induced mast cell activation of cytosolic phospholipase A2 (cPLA2) responsive to protein kinase Cμ (PKCμ) and extracellular signal-regulated kinase (ERK). Furthermore, the antigen-challenged activation of Syk-phospholipase Cγ (PLCγ) pathway was dampened in kaempferol-supplemented mast cells. These results demonstrated that kaempferol inhibited airway wall thickening through disturbing Syk-PLCγ signaling and PKCμ-ERK-cPLA2-COX2 signaling in antigen-exposed mast cells. Thus, kaempferol may be a potent anti-allergic compound targeting allergic asthma typical of airway hyperplasia and hypertrophy.

Gallic acid-grafted chitooligosaccharides suppress antigen-induced allergic reactions in RBL-2H3 mast cells

In this study, a bioactive derivative of chitooligosaccharides (3–5kDa) was synthesized via grafting of gallic acid onto chitooligosaccharides (G-COS) to enhance anti-allergic activity. Hence, G-COS was evaluated for its capabilities against allergic reactions in RBL-2H3 mast cells sensitized with dinitrophenyl-specific immunoglobulin E antibody and stimulated by antigen dinitrophenyl-bovine serum albumin. It was revealed that G-COS exhibited significant inhibition on histamine release and production as well as intracellular Ca2+ elevation at the concentration of 200μg/ml. Likewise, the suppressive effects of G-COS on expression and production of interleukin (IL)-4 and tumor necrosis factor (TNF)-α were evidenced. Moreover, G-COS treatment caused a remarkable blockade on degradation of inhibitory κB-α (IκB-α) protein, translocation of nuclear factor (NF)-κB, and phosphorylation of mitogen-activated protein kinases (MAPKs). Notably, the inhibitory activities of G-COS on allergic reactions were found as a consequence of suppression of FcεRI expression in antigen-stimulated cells. Accordingly, G-COS was suggested to be a promising candidate of novel inhibitors against allergic reactions.

Inhibitory Effects of Wasabi Isothiocyanates on Chemical Mediator Release in RBL-2H3 Rat Basophilic Leukemia Cells

Wasabi is a plant of Japanese origin. It belongs to the family Brassicaceae and produces various isothiocyanates (ITCs). To clarify the type I allergies inhibited by wasabi ITCs, we investigated the inhibitory effect on chemical mediator release from dinitrophenylated bovine serum albumin (DNP-BSA)-stimulated RBL-2H3 rat basophilic leukemia cells. Allyl ITC (AITC), sec-butyl ITC (s-BuITC), and 3-butenyl ITC (3-BuITC), which have 3 or 4 carbon chains, inhibited histamine release but did not inhibit the release of leukotriene B4 (LTB4) or cysteinyl LTs (CysLTs). 4-Pentenyl ITC (4-PeITC) and 5-hexenyl ITC (5-HeITC), which have 5 or 6 carbon chains and an unsaturated bond at the end, inhibited LTB4 release but did not inhibit the release of histamine or CysLTs. 6-Methylthiohexyl ITC (6-MTITC), 6-methylsulfinylhexyl ITC (6-MSITC), and 6-methylsulfonylhexyl ITC (6-MSFITC), which have a sulfur atom inserted at the end of a 6-carbon chain, inhibited the release of histamine, LTB4, and CysLTs and the elevation in intracellular Ca(2+). These results suggest that wasabi ITCs inhibited type I allergies by inhibiting chemical mediator release and that the inhibitory effects on each chemical mediator were due to differences in the side chain structure of the wasabi ITCs.

Protective effect of chitosan oligosaccharides against FcɛRI-mediated RBL-2H3 mast cell activation

The activation of mast cells by immunoglobulin E-mediated stimuli is considered as a central event in allergic responses. In this regard, chitosan oligosaccharides (COS) of two different molecular weight ranges (1–3 kDa and 3–5 kDa) were investigated for their capabilities against the activation of RBL-2H3 mast cell sensitized with dinitrophenyl-specific immunoglobulin E antibody and stimulated by antigen dinitrophenyl-bovine serum albumin. It was found that COS significantly inhibited RBL-2H3 cell degranulation via attenuating the releases of histamine and β-hexosaminidase. Moreover, the inhibitory activity of COS was accompanied by a reduction in intracellular Ca 2+ elevation. Notably, the expression of immunoglobulin Fc epsilon receptor I (FcɛRI) in RBL-2H3 cells was down-regulated by COS treatment in a dose-dependent manner. The suppressive effect of COS on RBL-2H3 cell activation suggested that COS may be potential candidates of novel inhibitors against allergic reactions.

Antiasthmatic Compounds Isolated from Antiasthmatic “JAMU” Ingredient Legundi Leaves (Vitex trifolia L.)

“JAMU” Indonesian traditional medicine preparations have been utilized extensively by Indonesian people. Due to lack of scientific and clinical data, JAMU has not been recommended for formal medication although it has been practiced long before recorded history. Therefore, this study aimed to screen those plant materials (0.25 and 0.50 mg/ml, n=3) for tracheospasmolytic activity in vitro (spasms induced by histamine 10-7-10-3 M) in order to obtain the promising active compounds to be studied for their mechanisms of actions. The leaves of Vitex trifolia were among the most promising material and further studied. By bioassay guided isolation, 3 compounds were isolated and identified as viteosin-A (1), vitexicarpin (2) and vitetrifolin-E (3) based on their spectroscopic and literature data. Compound 2 was the most active as compared to 1 and 3 in a tracheospasmolytic test using sensitized (ovalbumin 5, 50, 500, 5000 ng/ml, n=3) guinea pig trachea. These compounds were not harmful to the organ as it was proved by inducing the organ after the test with carbachol, followed by saturated KCl. Their mechanism seems to be non-competitive antagonistic to histamine, and the compounds are possibly able to stabilize the mast cells membrane function. Upon testing of those compounds (1 x 10-4 M) on RBL-2H3 (Rat Basophilic Leukemia cells represent mucosal mast cells) induced by DNA-BSA (Dinitrophenylated Bovine Serum Albumin), compound 2 still showed the strongest effect to inhibit the histamine release as compared to that of 1 and 2.

The Role of Haptic Macrophages in Regulation of Idiosyncratic Drug Reactions

Idiosyncratic drug reactions (IDR) account for approximately 6%-10% of all adverse drug reactions. The unpredictable and serious nature of these reactions makes them a significant economic burden and safety concern to the health care community and the pharmaceutical industry. Clinical and laboratory evidence suggests that adverse immune responses against drug-protein adducts play a role in the pathogenesis of IDR. However, it remains unclear why only a small percentage of patients are susceptible to developing these reactions. We hypothesized that most patients develop immunological tolerance against drug-protein adducts as a default mechanism, and that IDRs can only occur when this tolerance is deficient or abrogated in susceptible individuals. Using a murine model of 2,4-dinitrochlorobenzene (DNCB)-induced delayed type hypersensitivity (DTH) reaction, our previously published data demonstrated that intravenous pretreatment of mice with dinitrophenyl-bovine serum albumin (DNP-BSA) induced immunological tolerance to subsequent DNCB sensitization, and that hepatic macrophages (Kupffer cells, KC) played an important role in mediating such tolerance. Further mechanistic investigation revealed that KC, acting as incompetent antigen-presenting cells, cannot elicit strong T cell reactions, and that they actively suppress T cell activation through production of prostaglandins. These findings suggest that KCs may play a critical role in regulating immune reactions within the liver and contributing to liver-mediated systemic immune tolerance.

Sustained Activation of the Tyrosine Kinase Syk by Antigen in Mast Cells Requires Local Ca2+ Influx through Ca2+ Release-activated Ca2+ Channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.

Enhancement of the release of inflammatory mediators by substance P in rat basophilic leukemia RBL-2H3 cells

Substance P (SP), a neurotransmitter, may play an important role in neurogenic inflammation. Ginseng has been used extensively in traditional medicine; however, few studies were focused on their anti-allergic effect. Therefore, the effect and mechanism of ginsenoside Rb1 on the SP enhancement of allergic mediators were explored. In this study, SP and dinitrophenyl-bovine serum albumin (DNP-BSA) were used to activate rat basophilic leukemia (RBL)-2H3 cells. The cultured supernatants were assayed for histamine, leukotriene C(4)(LTC(4)) and interleulin-4 (IL-4) production. The mitogen-activated protein kinases (MAPKs) signaling pathway was determined by Western blotting analysis. We found that IgE/DNP-BSA, SP, ginsenoside Rb1, or MAPK specific inhibitors had no effect on cell viability and cytotoxicity. SP (30 microM) alone, did not induce histamine and LTC(4) release, but it enhanced allergen-induced histamine and LTC(4) release. In addition, SP significantly induced and enhanced allergen-activated IL-4. Ginsenoside Rb1 dose-dependently inhibited these effects. SP enhanced the allergen-activated ERK pathway in RBL-2H3 cells, and Rb1 effectively inhibited the ERK pathway activation. Although MAPK specific inhibitors suppressed LTC(4) and IL-4, only U0126 inhibited the SP enhanced histamine release. These results demonstrate that Rb1 dose-dependently inhibited SP enhanced allergen-induced mediator release and its mechanism was through the inhibition of the ERK pathway.

Application of on-chip cell cultures for the detection of allergic response

In this report, the development of a microfluidic cell chip for monitoring allergic response is described. A rat basophilic leukemia cell line (RBL-2H3), a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus. The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods. The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods. In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine. The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE. When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope.

Enhancement of rat tear IgA antibody responses following intranasal immunization with antigen and CpG ODN

Purpose. To determine the effect of unmethylated oligodeoxynucleotides containing bacterial CpG motifs (CpG ODN) on the induction of rat tear IgA antibody responses. Methods. Rats were immunized intranasally with either soluble dinitrophenylated bovine serum albumin (DNP-BSA) or poly(lactide-co-glycolide) (PLG) encapsulated DNP-BSA in combination with CpG ODN. The animals received two immunizations 21 days apart. Following the second immunization, tear, saliva and serum samples were collected for 28 days and analyzed for antigen specific antibodies. Tear IgA, saliva IgA and serum IgG antibody concentrations were determined by ELISA. Results. Co-administration of CpG ODN with either soluble or encapsulated antigen resulted in significantly elevated levels of both tear and salivary IgA antibodies as well as levels of serum IgG antibodies. Microencapsulated DNP-BSA plus CpG ODN elicited higher levels of IgA antibodies in tears than did soluble antigen plus CpG ODN. Conclusions. CpG ODN is an effective mucosal immune modulator for enhancing rat tear IgA antibody responses to both soluble and microencapsulated antigens.

Differential measurement with a microfluidic device for the highly selective continuous measurement of histamine released from rat basophilic leukemia cells (RBL-2H3).

We fabricated a micro-fluidic device for the highly selective detection of the histamine released from rat basophilic leukemia (RBL) 2H3 cells. The device has two thin layer flow channels, each with one working electrode. One electrode was modified with Os-polyvinylpyridine based mediator containing horseradish peroxidase (Os-gel-HRP) and histamine oxidase (HAOx), the other was modified with Os-gel-HRP without any HAOx. We employed the device for differential measurement by using the HAOx modified electrode for detection and the unmodified electrode as a reference. The detection limit was greatly improved from 190 to 25 nM since the baseline noise level was suppressed. We used differential measurement to observe the histamine released from RBL-2H3 cells when stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) as an antigen. We injected 5 microM of histamine solution into our device and it remained stable for more than 8 h.

Continuous measurement of histamine from rat basophilic leukemia cells (RBL-2H3) with an on-line sensor using histamine oxidase

Histamine release from rat basophilic leukemia (RBL-2H3) cells was continuously measured using a histamine oxidase (HO)-immobilized on-line electrochemical sensor. The sensor has a carbon-based electrode modified with a bilayer of osmium-polyvinylpyrridine-based gel containing horseradish peroxidase (HRP) and BSA containing HO. The high specificity of the enzyme made the sensor highly selective with regards to diamines such as putrescine and cadaverine. We obtained a low detection limit of 23 nM owing to the low operation potential of the sensor (below 0 V vs. Ag/AgCl) and the high activity of the enzyme after immobilization. Moreover, the sensor had a high conversion efficiency. The sensor response was unaffected by 400 min of continuous histamine measurement and by more than 2 weeks of storage in a refrigerator. We combined the histamine sensor with a microdialysis (MD) sampling probe and used it to measure the histamine concentration of RBL-2H3 cells stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with IgE. We also fabricated a small volume histamine sensor by the microfabrication technique and were able to measure the histamine release from a small mast cell colony in real time with the sensor by drawing the extracellular solution continuously through a sampling capillary located near the cells.