Intrauterine sensitization of ovalbumin in the third trimester increases the risk of food allergy in progeny

Intrauterine sensitization caused by food allergens plays an important role in the food allergy development in progeny. The aim of our study was to determine the critical period of intrauterine sensitization during pregnancy. Female mice were exposed to ovalbumin (OVA) during different trimesters of pregnancy. Lymphocytes from their offspring were isolated and cultured, and proliferation was evaluated by CCK-8 assay. The levels of IFN-γ and IL-4 in serum were measured using ELISA. In addition, the expressions of IFN-γ and IL-4 mRNAs and proteins were detected by real-time PCR and western blot. The mice were divided into the first trimester pregnancy (FTP1 and FTP2) group, the second trimester pregnancy (STP1 and STP2) group, and the third trimester pregnancy (TTP1 and TTP2) group based on the stages of pregnancy in which their mothers were exposed to OVA and their ages. The OVA-specific lymphocyte proliferation of the TTP1 group was statistically significantly greater that in the FTP1 and STP1 groups. The serum level of IFN-γ in the TTP1 group was significantly decreased, and the serum level of IL-4 in the TTP1 group was significantly increased compared with the levels in the FTP1 and STP1 groups. The mRNA and protein expression levels of IFN-γ in the TTP1 group were significantly decreased and the mRNA and protein expression levels of IL-4 in this group were significantly increased compared with the levels in the FTP1 and STP1 groups. Our results suggest that OVA-induced intrauterine sensitization in the third trimester may increase the risk of food allergy after birth.     

Serine protease inhibitor attenuates ovalbumin induced inflammation in mouse model of allergic airway disease

Background

Serine proteases promote inflammation and tissue remodeling by activating proteinase-activated receptors, urokinase, metalloproteinases and angiotensin. In the present study, 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) a serine protease inhibitor was evaluated for prophylactic and therapeutic treatment in mouse model of airway allergy.

Methods

BALB/c mice were sensitized by i.p route and challenged with ovalbumin. They were treated i.n. with 2, 10 and 50 µg of AEBSF, one hour before or after challenge and euthanized to collect BALF (bronchoalveolar lavage fluid), blood and lungs. Proteolytic activity, total cell/eosinophil/neutrophil count eosinophil peroxidase activity (EPO), IL-4, IL-5, IL-10, IL-13, cysteinyl leukotrienes and 8-isoprostane were determined in BALF and immunoglobulins were measured in serum. H&E and PAS stained lung sections were examined for cellular infiltration and airway inflammation.

Results

Mice exposed to ovalbumin and treated with PBS showed increased cellular infiltration in lungs and higher serum IgE, IgG1 and IgG2a levels as compared to sham mice. Treatment with AEBSF reduced total cells/eosinophil/neutrophil infiltration. Both prophylactic and therapeutic AEBSF treatment of 10 or 50 µg reduced serum IgE and IgG1 significantly (p<0.05) than control. AEBSF treatment reduced the proteolytic activity in BALF. IL-4 IL-5 and IL-13 levels decreased significantly (p<0.05) after AEBSF treatment while IL-10 levels increased significantly (p<0.05) in BALF. Airway inflammation and goblet cell hyperplasia reduced as demonstrated by lung histopathology, EPO activity and cysteinyl leukotrienes in BALF after treatment. AEBSF treatment also suppressed oxidative stress in terms of 8-isoprostane in BALF. Among the treatment doses, 10 or 50 µg of AEBSF were most effective in reducing the inflammatory parameters.

Conclusions

Prophylactic and therapeutic treatment with serine protease inhibitor attenuates the airway inflammation in mouse model of airway allergy and have potential for adjunct therapy.     

Comparison of allergic lung disease in three mouse strains after systemic or mucosal sensitization with ovalbumin antigen

Murine models of allergic lung disease have many similar traits to asthma in humans and can be used to investigate mechanisms of allergic sensitization and susceptibility factors associated with disease severity. The purpose of this study was to determine strain differences in allergic airway inflammation, immunoglobulin production, and changes in respiratory responses between systemic and mucosal sensitization routes in BALB/cJ, FVB/NJ, and C57BL/6J, and to provide correlations between immune and pathophysiological endpoints. After a single intranasal ovalbumin (OVA) challenge, all three strains of mice systemically sensitized with OVA and adjuvant exhibited higher airflow limitation than non-sensitized mice. No changes were seen in mice that were pre-sensitized via the nose with OVA. Systemic sensitization resulted in an elevated response to methacholine (MCH) in BALB/cJ and FVB/NJ mice and elevated total and OVA-specific IgE levels and pulmonary eosinophils in all three strains. The mucosal sensitization and challenge produced weaker responses in the same general pattern with the C57BL/6J strain producing less serum IgE, IL5, IL13, and eosinophils in lung fluid than the other two strains. The converse was found for IL6 where the C57BL/6J mice had more than twice the amount of this cytokine. The results show that the FVB/NJ and BALB/cJ mice are higher Th2-responders than the C57BL/6J mice and that the levels of pulmonary eosinophilia and cytokines did not fully track with MCH responsiveness. These differences illustrate the need to assess multiple endpoints to provide clearer associations between immune responses and type and severity of allergic lung disease.     

Oral probiotic bacterial administration suppressed allergic responses in an ovalbumin-induced allergy mouse model

This study investigated whether orally administered probiotic bacteria (Bifidobacterium bifidum and Lactobacillus casei) and a gram-negative bacterium (Escherichia coli) function as allergic immune modulators to prevent food allergy, according to the hygiene hypothesis. C3H/HeJ mice were sensitized with ovalbumin (OVA) and cholera toxin for 5 weeks. After sensitization, the OVA-induced mice that were not treated with bacteria had significantly increased levels of OVA-specific IgE, total IgE, and IgG1 in sera, as well as scab-covered tails. In comparison, groups treated with B. bifidum BGN4 (BGN4), L. casei 911 (L. casei), or Escherichia coli MC4100 (E. coli) had decreased levels of OVA-specific IgE, total IgE, and IgG1, and decreased levels of mast cell degranulation and tail scabs. OVA-specific IgA levels were decreased in BGN4- and L. casei-treated groups. In conclusion, administration of E. coli, BGN4, or L. casei decreased the OVA-induced allergy response. However, a normal increase in body weight was inhibited in the E. coli-treated mice and in the montreated mice groups during allergy sensitization. Thus, BGN4 and L. casei appear to be useful probiotic bacteria for the prevention of allergy.     

Novel T-cell epitopes of ovalbumin in BALB/c mouse: Potential for peptide-immunotherapy

The identification of food allergen T-cell epitopes provides a platform for the development of novel immunotherapies. Despite extensive knowledge of the physicochemical properties of hen ovalbumin (OVA), a major egg allergen, the complete T-cell epitope map of OVA has surprisingly not been defined in the commonly used BALB/c mouse model. In this study, spleen cells obtained from OVA-sensitized mice were incubated in the presence of 12-mer overlapping synthetic peptides, constructed using the SPOTS^® synthesis method. Proliferative activity was assessed by 72-h in vitro assays with use of the tetrazolium salt WST-1 and led to identification of four mitogenic sequences, i.e., A39R50, S147R158, K263E274, and A329E340. ELISA analyses of interferon (IFN)-γ and interleukin (IL)-4 productions in cell culture supernatants upon stimulation with increasing concentrations of peptides confirmed their immunogenicity.Knowledge of the complete T-cell epitope map of OVA opens the way to a number of experimental investigations, including the exploration of peptide-based immunotherapy.     

Lactobacillus helveticus SBT2171 alleviates allergic symptoms in a murine model for pollen allergy

ABSTRACT

Lactic acid bacteria are known to have various health-promoting effects and are highly expected to find applications in anti-allergic food materials. In this study, we focused on Lactobacillus helveticus SBT2171 (LH2171), which reportedly modifies some unique immune responses and ameliorated symptoms of patients allergic to mites and house dust in the previous studies. We examined the effect of LH2171 on cytokine production by antigen-stimulated murine naïve splenocytes in vitro and demonstrated that it inhibited IL-4 and IL-13 production while enhancing IFN-γ and IL-10 production. Then, we examined the anti-allergic effect of LH2171 in vivo using a murine model of pollen allergy and found that LH2171 reduced the sneezing frequency when orally administered to mice. We successfully confirmed the immune modulatory activity of LH2171 and its anti-allergic activity against inhaled antigens. These evidences would contribute to identifying the anti-allergic mechanism of LH2171.

Abbreviations: ALDH: aldehyde dehydrogenase; EGCG: epigallocatechin gallate; LAB: lactic acid bacteria; LH2171: Lactobacillus helveticus SBT2171; NALT: nasal-associated lymphoid tissue; OVA: ovalbumin     

Decrease in Rice Allergenic Proteins of Polished Rice Grains by Incubating with a Miso Solution

The effect of miso on allergenic proteins in rice seeds was investigated. When polished rice grains were incubated at 37°C for 30-120 min with a 10% miso solution, but not with heat-treated miso or 1% NaCl, the amount of soluble proteins extracted from the rice grains with 1 M NaCl markedly decreased. SDS-PAGE, immunoblotting and densitometric analyses of these soluble proteins and insoluble proteins indicate that 26 kDa globulin and 14-16 kDa allergens in the grains were decreased to 15-60% during incubation with the miso solution, especially soybean-koji miso, without any large change in the content of major insoluble proteins.     

CGS21680 attenuates symptoms of Huntington's disease in a transgenic mouse model

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A(2A) adenosine receptor (A(2A)-R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-microMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. (1)H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5'AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.     

Treatment of malaria in a mouse model by intranasal drug administration

The goal of this work was to investigate intranasal dihydroartemisinin (DHA) delivery as a non-invasive method for treatment of malaria. ICR female mice were infected with Plasmodium berghei ANKA, a model for severe malaria with similarities to the human disease. DHA, at a dose of 2 × 5 mg/kg/day, was administered to mice either intranasally or i.p. Two dosage regimens were tested: prophylaxis and treatment. Parasitemia was monitored every other day, from the time of infection, by thin smears prepared from tail blood. The survival rates in prophylaxis and treatment regimens were 93% and 75%, respectively, for intranasal DHA and this route was at least as effective as the i.p. route used for comparison. All mice in the untreated control and placebo groups succumbed due to the parasitemia. The results show that DHA nasal administration to mice was highly efficient in the treatment of Plasmodium infection in infected rodents. This novel mode of drug administration may be considered as an alternative to conventional treatment.     

Downregulation in aquaporin 4 and aquaporin 8 expression of the colon associated with the induction of allergic diarrhea in a mouse model of food allergy

Food allergies have become increasingly prevalent during the past few decades. Diarrhea is one of the most frequent intestinal symptoms caused by food allergens and is characterized by imbalanced ion exchange and water transfer; however, the underlying mechanism of allergic diarrhea remains unclear. Water transfer across the intestinal epithelial membrane seems to occur via aquaporins (AQPs). However, the molecular mechanism of water transfer and the pathophysiological roles of aquaporins in the intestine have not been fully established. The present studies have focused on the alterations of AQPs in a mouse model of allergic diarrhea in which BALB/c mice developed diarrhea following repeated challenges of orally administered ovalbumin. Quantitative real-time PCR analysis and immunohistochemical technique were used for expression of mRNA and protein of AQPs, respectively. AQP4 and AQP8 mRNA levels were significantly decreased in the proximal colon of allergic mice compared to controls; likewise, expression of AQP4 and AQP8 proteins was reduced in the proximal colon of the allergic mice. These results suggest that allergic diarrhea is associated with a downregulation in AQP4 and AQP8 expression.     

Polysaccharides L900/2 and L900/3 isolated from Lactobacillus rhamnosus LOCK 0900 modulate allergic sensitization to ovalbumin in a mouse model

Here, we compared the abilities of polysaccharides L900/2 and L900/3, which were previously isolated from Lactobacillus rhamnosus LOCK 0900, to modulate the immune response to bystander antigens in a mouse model of ovalbumin (OVA) sensitization. In vivo, both polysaccharides reduced the levels of OVA‐specific IgE, IgE‐dependent basophil degranulation and IgG2a antibodies, but had no effect on the levels of OVA‐specific IgA or IgG1. Interestingly, both polysaccharides triggered recall cellular responses with distinct properties. L900/3 significantly suppressed the OVA‐induced upregulations of IL‐4, IL‐5, IL‐10 and IL‐13 in re‐stimulated spleen cells and mesenteric lymph nodes. Our findings support and expand on our previous in vitro studies by demonstrating that polymer L900/3 might modulate the Th1/Th2 balance and could be a promising candidate molecule for preventing allergic sensitization.     

Intranasal administration of a combination of choline chloride,vitamin C,and selenium attenuates the allergic effect in a mouse model of airway disease

Respiratory allergic disease is an inflammatory condition accompanied by oxidative stress. Supplementation of an anti-inflammatory agent with antioxidants may have a therapeutic effect. In this study, the effects of choline chloride in combination with antioxidants were evaluated via the intranasal route in a mouse model of allergic airway disease. Balb/c mice were sensitized on days 0, 7, and 14 and challenged on days 25–30 with cockroach extract (CE) and with a booster challenge on day 38. They were treated with choline chloride (ChCl; 1 mg/kg), vitamin C (Vit C; 308.33 mg/kg), and selenium (Se; 1 mg/kg) alone or in combination via the intranasal route on days 31, 33, 35, 37, and 39. The mice were sacrificed on day 40 to collect blood, bronchoalveolar lavage fluid, lungs, and spleen. Mice immunized with CE showed a significant increase in airway hyperresponsiveness (AHR), lung inflammation, Th2 cytokines, and the oxidative stress markers intracellular reactive oxygen species and 8-isoprostanes compared to the phosphate-buffered saline control group. A significant decrease was observed in these parameters with all the treatments (p<0.01). The highest decrease was noticed in the ChCl+Vit C+Se-treated group, with AHR decreased to the normal level. This group also showed the highest decrease in airway inflammation (p<0.001), IL-4 and IL-5 (p<0.001), IgE and IgG1 (p<0.001), NF-κB (p<0.001), and 8-isoprostane levels (p<0.001). Glutathione peroxidase activity, which was decreased significantly in CE-immunized mice, was restored to normal levels in this group (p<0.001). IL-10 level was decreased in CE-immunized mice and was restored to normal by combination treatment. The combination treatment induced FOXP3⁺ cells in splenocyte culture, responsible for the upregulation of IL-10. In conclusion, the combination of choline chloride, vitamin C, and selenium via the intranasal route reduces AHR, inflammation, and oxidative stress, probably by causing IL-10 production by FOXP3⁺ cells, and possesses therapeutic potential against allergic airway disease.     

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28–58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.     

Downregulation of leukotriene biosynthesis by thymoquinone attenuates airway inflammation in a mouse model of allergic asthma

Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.     

Intracellular Localization of Several Enzymes in Candida tropicalis Grown on Different Carbon Sources

Intracellular localization of several enzymes related to tricarboxylic acid cycle was investigated during the aerobic growth of Candida tropicalis on acetate, n-alkane and glucose. NADP-linked isocitrate dehydrogenase in acetate-grown cells was mostly found in S₂ fraction (20,000 × g supernatant fraction of protoplast lysate), whereas more than half of this activity in n-alkane-grown cells was recovered in P₂ fraction (20,000 × g pellet fraction). Large parts of NAD-linked isocitrate dehydrogenase and malate dehydrogenase were present in P₂ fraction, while NADP- and NAD-linked glutamate dehydrogenases were found preferentially in S₂ fraction, irrespective of the growth substrates used. Isocitrate lyase was detected in both fractions. Citrate synthase and aconitase in acetate-grown cells were almost particulate. Catalase activity recovered in P₂ fraction was far higher in alkane-grown cells than in acetate- or glucose-grown cells.     

Absorption of cadmium after a long-term oral administration of cadmium to dogs

A long-term experiment using beagle dogs to investigate the absorption of cadmium was conducted. The dogs in the experimental groups were given a commercial diet and pelleted food containing 1, 3, 10, 50, and 100 mg of cadmium per day. The cadmium concentration in the blood increased continuously, gradually reaching a steady state following the administration of cadmium. The cadmium excreted daily in urine increased continuously. The cumulative excreted amount of cadmium in urine was calculated by using the trapezoidal rule based on the data of excretion of cadmium in urine. Then the absorbed fraction of administered cadmium was estimated on the basis of the relationship between the cumulative excreted amount of cadmium in urine and the cumulative administered dose of cadmium after the cadmium concentration in blood reached a steady state. The absorbed fraction of cadmium decreased with an increase in the administered dose of cadmium. A dose-dependent increase between the absorbed amount and the administered dose was observed.     

The expression of MC4Rs in D1R neurons regulates food intake and locomotor sensitization to cocaine

While it is known that mice lacking melanocortin 4 receptor (MC4R) expression develop hyperphagia resulting in early‐onset obesity, the specific neural circuits that mediate this process remain unclear. Here, we report that selective restoration of MC4R expression within dopamine‐1 receptor‐expressing neurons [MC4R/dopamine 1 receptor (D1R) mice] partially blunts the severe obesity seen in MC4R‐null mice by decreasing meal size, but not meal frequency, in the dark cycle. We also report that both acute cocaine‐induced anorexia and the development of locomotor sensitization to repeated administration of cocaine are blunted in MC4R‐null mice and normalized in MC4R/D1R mice. Neuronal retrograde tracing identifies the lateral hypothalamic area as the primary target of MC4R‐expressing neurons in the nucleus accumbens. Biochemical studies in the ventral striatum show that phosphorylation of DARPP‐32^Thr‐34 and GluR1^Ser‐845 is diminished in MC4R‐null mice after chronic cocaine administration but rescued in MC4R/D1R mice. These findings highlight a physiological role of MC4R‐mediated signaling within D1R neurons in the long‐term regulation of energy balance and behavioral responses to cocaine.     

Lack of association between acupoint sensitization and microcirculatory structural changes in a mouse model of knee osteoarthritis: A pilot study

As a stimulating point in acupuncture, acupoint has unique microcirculatory features, and its dynamics vary greatly depending on health status. Acupoint sensitization is defined as the transformation of an acupoint from a “silenced status” (healthy) to an “activated status” (disease). Our previous study demonstrated that acupoint sensitization is associated with an increase in the level of local blood perfusion. However, the structural changes in microcirculation during acupoint sensitization have yet to be elucidated because the high‐resolution microcirculation imaging of acupoints has been difficult to obtain. In this study, the structural changes in microcirculation at the Zusanli (ST36), Yanglingquan (GB34) and nonacupoint sites on days 0, 7 and 21 were dynamically observed during acupoint sensitization in an experimental knee osteoarthritis mouse model by using optical‐resolution photoacoustic microscopy. The results showed that no significant differences in microvessel density, the distribution of vessel diameters or vascular tortuosity were observed at the GB34, ST36 or nonacupoint sites among days 0, 7 and 21. We proposed that acupoint sensitization may not be associated with the structural changes in microcirculation and that the microcirculatory changes during acupoint sensitization are more likely to be functional. The functional characteristics of the sensitized acupoints warrant further investigation.      

Nitration of the egg-allergen ovalbumin enhances protein allergenicity but reduces the risk for oral sensitization in a murine model of food allergy

Background

Nitration of proteins on tyrosine residues, which can occur due to polluted air under “summer smog” conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route.

Methodology/Principal Findings

BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y₁₀₇) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization.

Conclusions/Significance

These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes.