Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Sensitization to acid-hydrolyzed wheat protein by transdermal administration to BALB/c mice, and comparison with gluten.

BACKGROUND: An increasing number of studies have shown that hydrolyzed wheat protein (HWP) can induce IgE-mediated hypersensitivity by skin contact and/or food ingestion. However, there has been no study of the sensitizing potential of HWP. In this study, the possibility of transdermal pathway for sensitization to acid-HWP (HWP1) was investigated using BALB/c mice, and compared with that of gluten. METHODS: HWP1 or gluten (500 µg/mouse) was transdermally administered using patches. After three or four cycles of sensitization for 3 days/week, active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of the antigen, and rectal temperatures, scores of anaphylactic responses, and plasma histamine levels were determined. Because HWP1 was included in facial soap in Japan, the effect of detergent on the sensitizing potential was also investigated. RESULTS: Transdermal administration of HWP1 induced dose-dependent production of IgE and IgG1. After sensitization for 3 or 4 weeks, intraperitoneal injection of HWP1 caused ASA, leading to decreased rectal temperatures, increased anaphylaxis scores, and increased plasma histamine levels. In addition, splenocytes harvested after ASA produced IL-4, IL-5, and IL-10 by re-stimulation with HWP1. Transdermal exposure to gluten also induced IgE and IgG1 production, and intraperitoneal injection of gluten also induced ASA only in mice sensitized in the presence of sodium dodecyl sulfate. CONCLUSIONS: Transdermal exposure to HWP1 is sufficient to activate key immune pathways necessary for sensitizing mice for immediate hypersensitivity reactions. This study shows that HWP has a sensitizing potential as well as gluten, whereas its allergenicity may be different from that of gluten.

Incidence and risk factors of falling in ambulatory patients with rheumatoid arthritis: a prospective 1-year study.

UNLABELLED: A prospective 1-year study showed that fall incidence was 50% in women with rheumatoid arthritis. Multivariate analysis identified swollen joint count, use of antihypertensives or diuretics, one-leg standing time, and sway area measured by stabilometer as significant parameters associated with falls. INTRODUCTION: Patients with rheumatoid arthritis (RA) may be at increased risk of falling because they frequently experience muscle weakness and stiff or painful joints. The aim of this study was to use a prospective design to determine the incidence of falls and their risk factors in women with RA. METHODS: Eighty-four women aged 50 and over who had RA were enrolled. The mean age was 64.1 years. We evaluated postural stability, physical performance related to falls, disease activity, muscle volume, and bone density. The occurrence of falls was assessed every month for 1 year. Among 84 patients, 80 completed a 1-year observation. RESULTS: Forty patients (50.0%) reported one or more falls, and two of them (5.0%) had fractures during the follow-up period. The fall group had more swollen joints and took more antihypertensives and/or diuretics. The fall group also had lower postural stability and tended to have reduced physical performance. The one-leg standing time was shorter, and the step-up-and-down test score was lower in the fall group. The sway area was larger in the fall group. DISCUSSION: Multiple logistic regression analysis identified that number of swollen joints, use of antihypertensives or diuretics, shorter time standing on one foot, and the sway area were the most significant parameters associated with falls. CONCLUSION: We concluded that fall rates in RA patients were higher than in the general population and that balance impairment or side effects of drugs may play a role in increasing the risk of falls.

A convenient and sensitive allergy test: IgE crosslinking-induced luciferase expression in cultured mast cells.

BACKGROUND: For the detection of allergen-specific IgE in sera, solid-phase IgE-binding assays like the CAP test are commonly used. Although such immunochemical methods are very sensitive, they frequently produce false positives. Degranulation of the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) lines seems to be a possible indicator for human IgE, but spontaneous mediator release from these cells in the presence of human sera is not negligible. METHODS: The nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene was stably transfected into human FcεRI-expressing RBL-SX38 cells. One established clone (RS-ATL8) was sensitized with 1 : 100 dilution of sera from patients with egg white allergy and then stimulated with purified or a crude extract of egg white allergen. RESULTS: Sensitization with 15 pg/ml IgE was sufficient to detect IgE crosslinking-induced luciferase expression (EXiLE) by anti-IgE stimulation. Allergen-specific EXiLE was elicited by as little as 1 fg/ml of egg white protein without cytotoxicity. There was a good correlation between results with EXiLE and oral food challenge tests on patients with egg allergy (P = 0.001687, Fisher's exact test). The measured values of EXiLE and the CAP test also correlated well (R = 0.9127, Spearman's test). CONCLUSION: The EXiLE test using RS-ATL8 cells is a promising in vitro IgE test to evaluate the biological activity of the binding between IgE and allergens.

Measurement of human trabecular bone by novel ultrasonic bone densitometry based on fast and slow waves.

SUMMARY: Two longitudinal transmitted waves, fast and slow waves, were observed by employing a new quantitative ultrasound (QUS) method. The trabecular bone measurements generated by this method reflect three-dimensional structural information, and the new QUS parameters were able to identify vertebral fractures. INTRODUCTION: The aims were to identify new quantitative ultrasound (QUS) parameters that based on new QUS method reflecting not only bone volume but also the microstructures of trabecular bone ex vivo and to observe how much they predict fracture risk in vivo. METHODS: Ex vivo measurement: Three human femoral heads were used for the experiment. Attenuation of the slow wave, attenuation of the fast wave, speed of the slow wave, speed of the fast wave (SOFW), bone mass density of trabecular bone, and elastic modulus of the trabecular bone (EMTb) of each specimen were obtained using a new QUS method and compared with three-dimensional structural parameters measured by micro-computed tomography. In vivo measurement: Eighty-nine volunteers were enrolled, and the bone status in the distal radius was measured using a new QUS method. These parameters were compared with data evaluated by peripheral quantitative computed tomography and dual X-ray absorptiometry. RESULTS: Ex vivo measurement: SOFW and EMTb showed correlations with the parameter of trabecular anisotropy. In vivo measurement: The new QUS parameters were able to identify vertebral fractures. CONCLUSION: The newly developed QUS technique reflects the three-dimensional structure and is a promising method to evaluate fracture risk.

Sequential change in quality of life for patients with incident clinical fractures: a prospective study.

UNLABELLED: Health-related quality of life in elderly women with sustained incident fractures was assessed prospectively for 1 year, using the EuroQol standard. Loss of QOL was more severe in patients after hip or vertebral fractures than those with wrist fracture. QOL was not completely restored in patients suffering from hip fracture. INTRODUCTION: Osteoporosis-related fractures decrease mobility, social interaction, and emotional well-being. All of these characteristics determine health-related quality of life (HR-QOL). In this study, we assessed HR-QOL in elderly women following incident clinical fractures. METHODS: Thirty-seven patients with hip fractures (mean age 76.1 years), 35 with vertebral fractures (mean age 72.6 years), and 50 with wrist fractures (mean age 68.6 years) were enrolled. HR-QOL was prospectively measured using EuroQol (EQ-5D) before the fracture, 2 weeks, 3 months, 6 months, and 1 year after the fracture. RESULTS: During the observation period, reduction of EQ-5D values was greatest in the hip fracture group. In the wrist fracture group, EQ-5D values at 6 months after the fracture showed recovery; however, in the hip and vertebral fracture groups, recovery was significantly lower than before the fracture. One year after the fracture, EQ-5D values were not significantly different from prefracture values in the vertebral and wrist fracture groups, but remained significantly lower in the hip fracture group. CONCLUSIONS: Loss of QOL was more severe in patients after hip or vertebral fractures than in patients with wrist fracture. HR-QOL was not completely restored in patients suffering from hip fracture.

Recent trends in the incidence and lifetime risk of hip fracture in Tottori, Japan.

SUMMARY: Hip fracture incidence from 2004 to 2006 in the Tottori prefecture of Japan was investigated and compared with previously reported rates. The age- and gender-specific incidence of hip fracture in the Tottori prefecture has not plateaued, as has been reported for populations in Northern Europe or North America. INTRODUCTION: Recent data from Northern Europe and North America indicate that the incidence of hip fracture has plateaued, whereas most reports from Asia indicate that the incidence is increasing. The aims of this study were to investigate the recent incidence of hip fracture in the Tottori prefecture, Japan, and to compare it with previous reports. METHODS: All hip fractures in patients aged 35 years and older occurring between 2004 and 2006 were surveyed in all of the hospitals from the Tottori prefecture. The age- and gender-specific incidence rates were then calculated. Using these and previously reported data, the estimated number of hip fracture patients was determined using the age- and gender-specific incidence rates in each year from 1986 to 2006. RESULTS: The survey identified 851, 906, and 1,059 patients aged 35 years and older, in 2004, 2005, and 2006 respectively. The residual lifetime risk of hip fracture for individuals at 50 years of age was estimated to be 5.6% for men and 20.0% for women. The estimated number of patients from 1986 to 2006 showed a significant increase over time for both genders. CONCLUSIONS: The age- and gender-specific incidence of hip fracture in the Tottori prefecture, Japan has not plateaued for either gender.

Effects of herbimycin A and ST638 on Fc epsilon receptor-mediated histamine release and Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells.

We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.

Effect of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the cortical bone response to mechanical loading.

The influence of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the bone response to mechanical loading was evaluated. Six-month-old female Wistar rats were used and assigned to three groups (n = 12/group): Vehicle administration (EP4-V), low-dose ONO-4819 administration (EP4-L, 3 microg/kg BW), and high-dose ONO-4819 administration (EP4-H, 30 microg/kg BW). ONO-4819 was subcutaneously injected in the back twice a day for 3 weeks. Loads on the right tibia at 39.4 N for 36 cycles at 2 Hz were applied in vivo by 4-point bending every other day for 3 weeks. Whole-body bone mineral content showed a significant difference between EP4-V and EP4-H (P < 0.05). Bone mineral density (BMD) of the total and regional tibia (the region with maximal bending at the central diaphysis) was higher in EP4-H than EP4-V, showing a significant effect of loading (P < 0.001) and ONO-4819 (P < 0.05). BMD of the total femur was higher in EP4-H than EP4-V (P < 0.01) and that of the distal femur was higher in EP4-H than EP4-V (P < 0.001). Histomorphometry of the cortical bone showed that loading increased formation surface (FS/BS), mineral appositional rate (MAR), and bone formation rate (BFR/BS) significantly at the lateral periosteal surface (P < 0.001); however, the effect of ONO-4819 was not significant. At the medial periosteal surface, loading increased the three parameters (P < 0.001) and ONO-4819 increased FS/BS (P < 0.001) and MAR (P < 0.05) significantly. At the endocortical surface, the effects of both loading and ONO-4819 were significant on all three parameters (for loading; FS/BS P < 0.01, MAR P < 0.05, BFR/BS P < 0.03, for ONO-4819 all P < 0.001). It was concluded that ONO-4819 increased cortical bone formation in rats and there was an additive effect on the bone response to external loading by 4-point bending.

Possible involvement of phosphorylation of a 36,000-dalton protein of rat basophilic leukemia (RBL-2H3) cell membranes in serotonin release.

Phosphorylation of a 36,000-dalton (36k-Da) protein of rat basophilic leukemia (RBL-2H3) cell membranes was investigated. This phosphoprotein has been suggested to be the beta-subunit protein of the immunogloblin E (IgE) receptor of RBL-2H3 cells [Teshima et al., Biochem. biophys. Res. Commun. 125, 867-874 (1984)]. Phospholipids such as phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine, which are known to be activators of protein kinase C, enhanced the phosphorylation of the 36K-Da protein. In contrast, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) which has been identified as a potent inhibitor of protein kinase C in vitro decreased incorporation of radioactive phosphate from [gamma-32P]ATP into this protein. These results indicate that the phosphorylation of the 36K-Da protein of RBL-2H3 cell membranes is catalyzed by protein kinase C. H-7 also inhibited the release of serotonin from RBL-2H3 cells stimulated with an antigen or calcium ionophore A23187 and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Treatment of the antigen-stimulated cells with TPA caused a synergistic effect on the serotonin release. A similar effect was obtained by treatment of A23187-stimulated cells with TPA or 1-oleoyl-2-acetyl glycerol.

Enhancement of the phosphorylation of membrane bound myosin light chain by antigen stimulation in rat basophilic leukemia cells.

We have found that phosphorylation of the 18,000 mol. wt protein in rat basophilic leukemia cells (RBL-2H3 cells) is enhanced by stimulation by an antigen. This phenomenon was also observed when cells were treated with phorbol myristate (TPA) and a calcium ionophor, A23187. The phosphorylated 18,000 mol. wt protein was mainly located in the membrane fraction. It was identified as one of the myosin light chains as follows: (1) the mol. wt of one of the major myosin light chains of RBL-2H3 cells was 18,000; (2) more than half of the phosphorylated 18,000 mol. wt protein was recovered in an actomyosin fraction; (3) this phosphorylated 18,000 mol. wt protein was immunoprecipitated with anti-myosin antibody. Since the presence of Ca2+ in the cell culture medium was essential for the phosphorylation of the 18,000 mol. wt protein and, since trifluoperazine (a potent inhibitor of calmodulin as well as of the degranulation process of RBL-2H3 cells) inhibited the reaction, the phosphorylation may be catalyzed by a Ca2+-calmodulin-dependent process, most likely by myosin light chain kinase. These results, together with our previous observation [Teshima et al. Molec Immun. 23, 279-284 (1986)], suggest that simultaneous phosphorylation of the 18,000 mol. wt myosin light chain and a 36,000 mol. wt membranous protein is a prerequisite for the degranulation of RBL-2H3 cells.

Radial bone mineral changes in pre- and postmenopausal healthy Japanese women: cross-sectional and longitudinal studies.

Healthy premenopausal and postmenopausal Japanese women were evaluated by longitudinal and cross-sectional studies to assess changes in radial bone mineral density. A total of 300 healthy Japanese women were enrolled in the cross-sectional study, and 126 of them were chosen for the longitudinal study. In the cross-sectional study significantly lower bone mineral density was found in the women over 50 years old. In the longitudinal study, premenopausal women aged 45-54 years showed rate of change of -0.61%/year at the distal radius and 0.49%/year at the radial shaft. The perimenopausal changes were -1.95 and -0.93%/year and the postmenopausal changes were -1.88 and -1.37%/year at the distal radius and at the radial shaft, respectively, in the women aged 45-54 years. These rates of premenopausal and postmenopausal change were similar to those reported for whites in the United States. The strength of the relationship between the rate of change in radial bone mineral density and the anthropometric values, biochemical data, and menopausal status was evaluated by multiple linear regression analysis. Menopausal status had the greatest influence on the rate of change in radial bone mineral density. It is concluded that menopausal status was the most important variable related to bone loss in our longitudinal study and the rate of bone loss was greatest during the early postmenopausal period in healthy Japanese women.

A single cell observation of staurosporine effect on the Ca2+ signals in rat basophilic leukemia cells.

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen-dependent calcium signals in an individual rat basophilic leukemia cell (RBL-2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL-2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.

Effects of ovariectomy and estrogen replacement therapy on arthritis and bone mineral density in rats with collagen-induced arthritis.

We investigated the effects of ovariectomy (ovx) and estrogen replacement therapy (ERT) on bone mineral density (BMD) and arthritis severity in rats with collagen-induced arthritis (CIA). Seven-month-old female Sprague-Dawley rats were separated into a sham group (n = 8), CIA group (n = 14), ovx group (n = 10), CIA + ovx group (n = 11), and CIA + ovx + ERT group (n = 14). In these groups, ovx was performed at 7 days, and ERT (17beta-estradiol at 20 microg/kg three times per week) was initiated 8 days after sensitization. Every 2 weeks, until 8 weeks after sensitization, arthritis score and hind paw thickness were evaluated, and BMD of the trabecular and cortical bones in the metaphysis and diaphysis of the tibia were measured by peripheral quantitative computed tomography. The arthritis score was highest in the CIA + ovx group at all timepoints after sensitization. The hind paw thickness was significantly higher in the CIA + ovx group than in the CIA group at 8 weeks after sensitization (p < 0.05). Both the arthritis score and hind paw thickness were lower in the CIA + ovx + ERT group than in the CIA + ovx group. BMD in the metaphysis was significantly decreased in both the trabecular and cortical bones in the CIA + ovx group compared with those in the CIA group at 4, 6, and 8 weeks after sensitization. In the CIA + ovx group, trabecular BMD was changed by -34 +/- 11%, and cortical BMD changed by -14 +/- 7% in the metaphysis at 8 weeks compared with those at 0 week. In the CIA group, changes of BMD in the metaphysis were -7 +/- 11% in trabecular bone and 0 +/- 7% in cortical bone. These differences of trabecular and cortical bone loss in the metaphysis were significant (both p < 0.01). BMD reduction was significantly less in the CIA + ovx + ERT group than in the CIA + ovx group at 6 and 8 weeks after sensitization. Although BMD in the diaphysis was also reduced in the groups with CIA, the degree of reduction was smaller than in the metaphysis. We conclude that ovx in CIA rats could enhance the severity of arthritis and bone loss, and that ERT could suppress arthritis and bone loss.

Bone mass changes of tibial and vertebral bones in young and adult rats with collagen-induced arthritis.

To study the effect of arthritis on bone mass, bone mineral density (BMD) of cancellous and cortical bone in the tibial metaphysis and diaphysis in 2- and 7-month-old rats with collagen-induced arthritis (CIA) was serially measured using peripheral quantitative computed tomography (pQCT). BMD in the fourth lumbar vertebra in 7-month-old CIA rats was also measured by pQCT. The fourth lumbar vertebral body, distal femur, and proximal tibia in 7-month-old CIA rats were analyzed histomorphometrically. Changes in BMD differed between 2-month-old (young) and 7-month-old (adult) CIA rats. Although the BMD for the proximal tibia (2 mm and 5 mm distal from the growth cartilage) in young CIA rats decreased compared with that in control rats, the values exceeded the initial value during the arthritis course. On the other hand, bone loss in adult CIA rats occurred predominantly in the cancellous bone of the periarticular region of the tibia (2 mm distal from the growth cartilage), in which the enhancement of bone resorption and reduced bone formation were observed histomorphometrically. No remarkable changes were demonstrated in BMD or histomorphometrical analysis for the lumbar vertebra during the experimental course. These results suggest that bone loss in adult CIA rats resembles the osteoporosis that develops during the early stage of human rheumatoid arthritis. We conclude that adult CIA rats are more appropriate than young CIA rats as an experimental model of secondary osteoporosis due to rheumatoid arthritis.

Ca2+-ATPase inhibitor induces IL-4 and MCP-1 production in RBL-2H3 cells.

The effects of a Ca2(+)-ATPase inhibitor, cyclopiazonic acid (CPA), and two hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ) and 2,5-di-(tert-amyl)-1,4-hydroquinone (DTAHQ) on the release of IL-4 and MCP-1 from RBL-2H3 cells were investigated. CPA, DTBHQ and DTAHQ, all of which induce intracellular free Ca2+ concentration ([Ca2+]i) increase, induced IL-4 and MCP-1 release in a dose-dependent manner. The release of TNF-alpha required both a Ca2(+)-ATPase inhibitor and 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the Ca2(+)-ATPase inhibitors induced IL-4 and MCP-1 production without TPA. The release of IL-4 and MCP-1 reached a maximum at 9 and 6 h, respectively. IL-4 and MCP-I release was inhibited by treatment with the immunosuppressant FK-506 and actinomycin D. Therefore, in our system IL-4 and MCP-1 release involves Ca2(+)-dependent and FK-506-sensitive signaling pathways. This is the first report about Th-2 type cytokine and chemokine production in RBL-2H3 cells.

Correlation between cytosolic calcium concentration and degranulation in RBL-2H3 cells in the presence of various concentrations of antigen-specific IgEs.

We studied the dependence of beta-hexosaminidase release from RBL-2H3 cells on the antigen-specific IgE concentrations. The cells were sensitized with DNP-specific IgE (0.5-5000 ng/ml) or OVA-specific IgE (5-50 ng/ml) and stimulated with DNP(35)-HSA (10(-2)-100 ng/ml) or OVA (10(-1) ng/ml-10 microg/ml). It was found that the beta-hexosaminidase release increased in a dose-dependent manner with the concentration of the IgEs and antigens added to the mast-cell suspension. We also studied the correlation between the intracellular calcium concentration ([Ca(2+)]i) and degranulation in RBL-2H3 cells. The percentage of beta-hexosaminidase release from the cells was well correlated with [Ca(2+)](i) increase, and the correlation coefficient was 0.88 for DNP-specific IgE and 0.99 for OVA-specific IgE. The minimum [Ca(2+)](i) required to induce the beta-hexosaminidase release was 100 nM for DNP-specific IgE, and 70 nM for OVA-specific IgE. Therefore, the [Ca(2+)](i) monitoring system is a sensitive marker of degranulation from RBL-2H3 cells and can be used to measure even low amounts of antigen-specific IgE.

Induction of active systemic anaphylaxis by oral sensitization with ovalbumin in mast-cell-deficient mice.

Mast-cell-deficient W/W(v) mice were sensitized by oral administration of 0.1 and 1.0 mg ovalbumin (OVA) by gavage every day for 9 weeks, and active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of OVA. The production of OVA-specific IgE and IgG1 by oral immunization of the W/W(v) mice was high, and the production of IL-4 by splenocytes re-stimulated with OVA in vitro was increased. In contrast, production of OVA-specific IgG2a and IgG2b was low, and production of IFN-gamma by splenocytes after re-stimulation with OVA in vitro was rather decreased. These findings suggest that Th2-dominant helper T-cell activation had occurred. No increase in serum histamine level was observed following ASA induction. However, the plasma platelet-activating factor (PAF) levels of the mice sensitized with 0.1 and 1.0 mg OVA by gavage increased significantly. The increases in plasma PAF correlated well with the ASA-associated decreases in body temperature, suggesting that PAF plays an important role in ASA in W/W(v) mice. Taken together the above findings indicate that W/W(v) mice are a good model not only for studying induction of food allergy but also for examining the role of PAF in food-induced hypersensitivity.

Casein kinase II-like ectokinase activity on RBL-2H3 cells.

We studied the properties of the ectokinase activity on the outer cell surfaces of RBL-2H3 cells and examined the phosphorylation of exogenous substrates to clarify the substrate specificity of the ectokinases on RBL-2H3 cells. Among the several protein substrates tested, casein was the most strongly phosphorylated with [gamma-32P]ATP, and the net incorporation of 32P into casein was 0.65 pmol P/50 microg/10(6) cells. Casein kinase II peptide was also phosphorylated with [gamma-32P]ATP. The phosphorylation of casein and casein kinase II peptide was almost completely inhibited by the addition of 3 microg/ml of cell-impermeable K252b. Phosphorylation of casein and casein kinase II peptide was also observed by [gamma-32P]GTP. Western blot analysis using anti-casein kinase II antibody revealed a 44-kDa casein kinase band in the membrane fraction and Fc epsilonRI complexes. The immunofluorescence microscopic analysis using anti-casein kinase II antibody showed the existence of casein kinase II on the surface of the cells. This is the first report about the existence of ectokinase on mast cells.

Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice.

We studied the conditions needed to sensitize animals to the oral feeding of food allergens, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Brown Norway (BN) rats were sensitized by daily OVA (ovalbumin)-gavage or by drinking OVA containing water ad libitum and the ASA (active systemic anaphylaxis) response, as the immediate hypersensitivity response to antigen stimulation after oral sensitization, was examined. The oral administration of OVA by gavage produced a higher OVA-specific IgE response and an increase in serum histamine after antigen challenge, as compared to those produced by drinking water. Next, we examined the effect of murine age, the oral feeding technique and the oral feeding dose on sensitization using BALB/c, B10A and ASK mice. Twenty-week-old mice showed the strongest OVA-specific IgE and IgG1 responses and ASA-associated serum histamine contents increased with gavage in the three different age groups of BALB/c mice. Administering 0.1 mg of OVA by gavage daily for 9 weeks appeared to induce a higher response than administering 1 mg of OVA, in terms of OVA-specific IgE and IgG1 antibody responses and ASA responses. Among the three strains of mice, B10A mice exhibited the highest response in terms of OVA-specific IgE and IgG1 antibody and ASA responses. These findings suggested BN rats and B10A mice were suitable models for oral sensitization with antigen protein and that oral sensitization in mice requires low dose, intermittent antigen intakes.