BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells.

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Decrease in cholesterol in the cell membrane is essential for Nrf2 activation by quercetin.

Quercetin is a flavonoid with various cytoprotective effects. We previously reported that quercetin exerts anti-allergic, anti-oxidative, and anti-fibrotic activities via the induction of heme oxygenase (HO)-1. However, the mechanisms by which quercetin induces HO-1 to exhibit cytoprotective effects are poorly understood. We focused on its action on the cell membrane, which is the first part of the cell to interact with the extracellular environment. The cell membrane contains lipid rafts and caveolae, which play important roles in cellular signaling. A recent study showed that nuclear factor E2-related factor 2 (Nrf2), a transcription factor regulating anti-oxidative enzymes including HO-1, interacts with caveolin-1 (Cav-1), a component of caveolae, to regulate cellular anti-oxidative capacity. In this study, we investigated the changes in the cell membrane that leads to the induction of HO-1 by quercetin. Quercetin decreased the amount of cholesterol in the raft fractions, which in turn promoted the induction of HO-1. It also changed the composition of the lipid rafts and decreased and increased the expression of Cav-1 in the raft and non-raft fractions, respectively. Nrf2, which was localized in the cell membrane under resting conditions, was translocated along with Cav-1 to the nucleus after exposure to quercetin. These findings indicate for the first time that the HO-1-dependent cytoprotective effects of quercetin are mediated by the structural changes in lipid rafts brought about by decreasing the amount of cholesterol in the cell membrane, which thereby results in the translocation of the Cav-1-Nrf2 complex to the nucleus and induces the expression of HO-1.

Correlation of theophylline levels in rat exhaled breath and lung tissue after its intravenous injection.

It is important to know the drug level in the target tissue to determine its dose. Some methods rely on blood levels of a drug to estimate its concentration in the tissues, which can be inaccurate. We thought that drug levels in exhaled breath aerosol (EBA) to give a more accurate value of the level of a test drug in the lung. Rats were intravenously injected with the bronchodilator theophylline and exhaled breath was collected up to 10-20 min after administration. Immediately after breath collection, lung, liver, kidney, and blood were collected and the pharmacokinetics were examined using these samples. Awake free-moving rats were used to efficiently collect exhaled breath from rats with low tidal volume. The amount of exhaled breath of rats was estimated by the amount of exhaled water vapor, and the drug concentration in exhaled breath sample was expressed by the amount of water vapor as the denominator. By using the active sampling method in which the adsorbent is sucked by a pump, theophylline in rat exhaled breath could be measured accurately. When the correlation of theophylline concentration in each sample was examined, a high correlation (r2= 0.74) was found only in exhaled breath and lung tissue. EBA was considered better than blood in pharmacokinetic analysis of lung tissue.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line.

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called "oncogene-induced senescence (OIS)", prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called "HBEC3KT"), immortalized with hTERT ("T") and Cdk4 ("K"), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated ß-galactosidase staining (SA-ßG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Miniaturization of Respiratory Measurement System in Artificial Ventilator for Small Animal Experiments to Reduce Dead Space and Its Application to Lung Elasticity Evaluation.

A respiratory measurement system composed of pressure and airflow sensors was introduced to precisely control the respiratory condition during animal experiments. The flow sensor was a hot-wire thermal airflow meter with a directional detection and airflow temperature change compensation function based on MEMS technology, and the pressure sensor was a commercially available one also produced by MEMS. The artificial dead space in the system was minimized to the value of 0.11 mL by integrating the two sensors on the same plate (26.0 mm × 15.0 mm). A balloon made of a silicone resin with a hardness of A30 was utilized as the simulated lung system and applied to the elasticity evaluation of the respiratory system in a living rat. The inside of the respiratory system was normally pressurized without damage, and we confirmed that the developed system was able to evaluate the elasticity of the lung tissue in the rat by using the pressure value obtained at the quasi-static conditions in the case of the ventilation in the animal experiments.

Involvement of heme oxygenase-1 in suppression of T cell activation by quercetin.

AIM: Acute rejection is still a major problem in transplantation and one of the most important causes of late graft loss. Cyclosporine and tacrolimus are widely used for suppression of T cell function to avoid graft rejection, but long-term use of these compounds is associated with serious toxicities. Quercetin, a flavonoid found in fruits and vegetables, has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO) -1, an enzyme involved in heme catabolism. We hypothesized that quercetin induces HO-1 in T cells and suppresses T cell function via HO-1. In the present study, we showed that quercetin suppressed the A23187-mediated expression of interleukin (IL) -2 in T cells. METHODS: Mouse splenocytes, enriched T cells, and EL4 cells, a mouse T cell line, were treated with quercetin, and then stimulated with A23187, a calcium ionophore, concanavalin A, or anti-CD3ε and anti-CD28 antibodies. Cell proliferation, expression of IL-2, calcium mobilization, apoptosis, cell cycle, and phosphorylation of extracellular signal-regulated kinase (ERK) were investigated. RESULTS: Quercetin induced HO-1, and this induction of HO-1 was implicated in the suppression of IL-2 production. Furthermore, the induction of HO-1 by quercetin suppressed the influx of calcium ions, a known trigger of IL-2 production. Additionally, quercetin suppressed T cell proliferation through promotion of cell cycle arrest via HO-1 induction, but quercetin did not induce apoptosis. To investigate the role of the signal transduction pathway in quercetin's effect on cell proliferation, we evaluated the phosphorylation of ERK in T cells. Quercetin suppressed the A23187-mediated stimulation of ERK, an effect that was mediated through HO-1. These results suggested that HO-1 is involved in the suppressive effects of quercetin on T cell activation and proliferation. CONCLUSION: Our findings indicate that the quercetin may be a promising candidate for inducing HO-1 in T cells, thereby facilitating immunosuppressive effects.

Mouse NC/Jic strain provides novel insights into host genetic factors for malaria research.

Malaria is caused by Plasmodium parasites and is one of the most life-threatening infectious diseases in humans. Infection can result in severe complications such as cerebral malaria, acute lung injury/acute respiratory distress syndrome, and acute renal injury. These complications are mainly caused by P. falciparum infection and are major causes of death associated with malaria. There are a few species of rodent-infective malaria parasites, and mice infected with such parasites are now widely used for screening candidate drugs and vaccines and for studying host immune responses and pathogenesis associated with disease-related complications. We found that mice of the NC/Jic strain infected with rodent malarial parasites exhibit distinctive disease-related complications such as cerebral malaria and nephrotic syndrome, in addition to a rapid increase in parasitemia. Here, we focus on the analysis of host genetic factors that affect malarial pathogenesis and describe the characteristic features, utility, and future prospects for exploitation of the NC/Jic strain as a novel mouse model for malaria research.

Modulation of immunological activity on macrophages induced by diazinon.

Diazinon is an organophosphorus (OP) insecticide and is widely used not only in agriculture but also homes and garden in Japan. Diazinon has been reported to increase TNF-α production in rat serum and brain, suggesting that it can modify the proinflammatory response. In this study, we investigated the effects of diazinon on macrophage functions, such as cytokine production, reactive oxygen species (ROS) generation, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expressions, cell-surface molecule expressions, and phagocytosis in RAW264.7 cells. In RAW264.7 cells, diazinon induced the production of TNF-α and IL-6. Diazinon induced ROS generation and the expressions of COX-2, iNOS, and cell-surface molecules CD40, CD86, and MHC class II, but reduced phagocytic activity in RAW264.7 cells. ERK and p38, but not JNK and p65 were involved in diazinon-induced IL-6 expression in RAW264.7 cells. We also examined these proinflammatory responses in bone marrow-derived macrophages (BMDM) and bronchoalveolar lavage fluid (BALF) cells. These results suggested that diazinon can activate macrophages and enhance inflammatory responses.

The Suppressive Effect of Quercetin on Toll-Like Receptor 7-Mediated Activation in Alveolar Macrophages.

Respiratory viral infections that cause chronic airway and lung disease can result in the activation of the innate immune response. Alveolar macrophages (AMs), one of the first lines of defense in the lung, are abundantly located in alveoli and the respiratory tract. Flavonoids found in fruits and vegetables exhibit cytoprotective effects on various cell types. In this study, we investigated the effect of quercetin on activation of AMs that had been exposed to imiquimod, a ligand of Toll-like receptor (TLR) 7. In both a mouse AM cell line (AMJ2-C11 cells) and mouse bronchoalveolar fluid cells, we demonstrated that quercetin attenuated TLR7-induced the expression of TNF-α and IL-6. In AMJ2-C11 cells, quercetin also attenuated the TLR7-induced CD40 expression; attenuated the translocation of p65; induced translocation of Nrf2 from cytosol to nucleus; and induced heme oxygenase (HO)-1 expression. Notably, tin protoporphyrin IX (SnPP), an inhibitor of HO-1, also attenuated TLR7-induced transcription of the TNF-α and IL-6 genes, suggesting that the effect of quercetin is mediated by HO-1. These results suggest that dietary supplementation with quercetin may have efficacy in the treatment of respiratory viral infection.

Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin's suppressive effects. METHODS: Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells. RESULTS: Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. CONCLUSIONS: Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.

Detection of loci for allergic asthma using SMXA recombinant inbred strains of mice.

Asthma is regarded as a multifactorial inflammatory disorder arising as a result of inappropriate immune responses in genetically susceptible individuals to common environmental antigens. However, the precise molecular basis is unknown. To identify genes for susceptibility to three asthma-related traits, airway hyperresponsiveness (AHR), eosinophil infiltration, and allergen-specific serum IgE levels, we conducted a genetic analysis using SMXA recombinant inbred (RI) strains of mice. Quantitative trait locus analysis detected a significant locus for AHR on chromosome 17. For eosinophil infiltration, significant loci were detected on chromosomes 9 and 16. Although we could not detect any significant loci for allergen-specific serum IgE, analysis of consomic strains showed that chromosomes 17 and 19 carried genes that affected this trait. We detected genetic susceptibility loci that separately regulated the three asthma-related phenotypes. Our results suggested that different genetic mechanisms regulate these asthma-related phenotypes. Genetic analyses using murine RI and consomic strains enhance understanding of the molecular mechanisms of asthma in human.

Quercetin protects against pulmonary oxidant stress via heme oxygenase-1 induction in lung epithelial cells.

The lung is a primary target for oxygen toxicity because of its constant exposure to high oxygen levels and environmental oxidants. Quercetin is one of the most commonly found dietary flavonoids, and it provides cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways. In the present study, we showed that quercetin increased the levels of heme oxygenase (HO)-1 expression and protected against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in lung epithelial cell lines. Quercetin suppressed H(2)O(2)-induced apoptotic events, including hypodiploid cells, activation of caspase 3 enzyme activity and lactate dehydrogenase release. This cytoprotective effect was attenuated by the addition of the HO inhibitor, tin protoporphyrin IX. In addition, the end products of heme metabolites catalyzed by HO-1, carbon monoxide and bilirubin, protect against H(2)O(2)-induced cytotoxicity in LA-4 cells. Quercetin may well be one of the promising substances to attenuate oxidative epithelial cell injury in lung inflammation.

CD40/CD40 ligand interactions in immune responses and pulmonary immunity.

The CD40 ligand/CD40 pathway is widely recognized for its prominent role in immune regulation and homeostasis. CD40, a member of the tumor necrosis factor receptor family, is expressed by antigen-presenting cells, as well as non-immune cells and tumors. The engagement of the CD40 and CD40 ligands, which are transiently expressed on T cells and other non-immune cells under inflammatory conditions, regulates a wide spectrum of molecular and cellular processes, including the initiation and progression of cellular and humoral adaptive immunity. Based on recent research findings, the engagement of the CD40 with a deregulated amount of CD40 ligand has been implicated in a number of inflammatory diseases. We will discuss the involvement of the CD40 ligand/CD40 interaction in the pathophysiology of inflammatory diseases, including autoimmune diseases, atherothrombosis, cancer, and respiratory diseases.

Comparison of salivary cortisol, heart rate, and oxygen saturation between early skin-to-skin contact with different initiation and duration times in healthy, full-term infants.

BACKGROUND: There are few studies that compare the physiological and biological efficacies between different early skin-to-skin contacts (SSC) post birth. AIM: To investigate physiologically and biochemically how early SSC with different initiation and duration time influence the stress post birth for full-term infants. STUDY DESIGN: Non-experimental study. SUBJECTS: Study I; Thirty-two infants who began SSC 5 min or less [birth SSC, mean initiation time (standard deviation): 1.6 (1.1) min] after birth and 36 infants who did so more than 5 min [very early SSC, 26.3 (5.0) min] in heart rate (HR) and oxygen saturation (SpO(2)) analysis. Study II; Eighteen infants who underwent SSC for 60 min or less [mean initiation time: 7.5 (12.2) min] and 61 infants who did so for more than 60 min [15.3 (12.5) min] in salivary cortisol analysis. OUTCOME MEASURES: HR and SpO(2) measured for 30 min post birth. Salivary cortisol concentration measured at 1 min, 60 min, and 120 min post birth. RESULTS: Birth SSC group reached HR stability of 120-160 bpm significantly faster than very early SSC group by Kaplan-Meier analysis (P=0.001 by log-rank test). As for SpO(2) stability of 92% and 96%, no significantly between-group difference was found. Salivary cortisol levels were significantly lower between 60 and 120 min after birth in SSC group, continuing for more than 60 min compared with SSC group for 60 min or less after adjustment for salivary cortisol level at 1 min besides the infant stress factors (P=0.046). CONCLUSIONS: Earlier SSC beginning within 5 min post birth and longer SSC continuing for more than 60 min within 120 min post birth are beneficial for stability of cardiopulmonary dynamics and the reduction of infant stress during the early period post birth.

Attenuation of transforming growth factor-ß-stimulated collagen production in fibroblasts by quercetin-induced heme oxygenase-1.

Quercetin is a flavonoid with a wide variety of cytoprotective and modulatory functions. Heme oxygenase-1 (HO-1) is an inducible enzyme. Its reaction product, carbon monoxide (CO), confers cellular protection in a number of conditions and diseases associated with oxidative or inflammatory lung injury. Furthermore, quercetin was reported to be a potent inducer of HO-1 in several cell types. We hypothesized that quercetin suppresses the production of collagen in fibroblasts via the induction of HO-1. Here, we showed that quercetin suppresses transforming growth factor-ß (TGF-ß)-induced collagen production in NIH3T3 cells and in normal human lung fibroblasts. This suppressive effect of quercetin was mediated by quercetin-induced HO-1. The suppression of collagen production was conferred by the reaction product of HO-1, CO, but not by bilirubin. Furthermore, the translocation of the nuclear factor E2-related factor-2 (Nrf2), an important transcription factor that regulates the expression of HO-1 from the cytoplasm to the nuclei, was demonstrated in NIH3T3 cells by exposure to quercetin. Assessment of the signal transduction pathway involved in TGF-ß signaling showed that quercetin stimulated the Smad and mitogen-activated protein kinase pathway to varying degrees. Our results demonstrate that quercetin exerts suppressive effects on the expression of collagen by the induction of HO-1. Idiopathic pulmonary fibrosis is the most lethal diffuse fibrosing lung disease, and is characterized by the deposition of extracellular matrix. Given that HO-1 is one of the important molecules emerging as a central player in diseases, quercetin or its derivatives, which effectively induced HO-1, will lead to new therapeutic strategies for promoting antifibrotic therapy in respiratory diseases.

Ghrelin and obestatin promote the allergic action in rat peritoneal mast cells as basic secretagogues.

Ghrelin is an endogenous ligand of the type 1a growth hormone secretagogue receptor (GHSR1a) that regulates energy balance. Ghrelin and obestatin, derived from the post-translational processing of preproghrelin, are involved in a diverse range of biological activities, yet their effect on the immune system is not fully understood. In the present study, we investigated the roles of ghrelin and obestatin on mast cell degranulation and found that both ghrelin and obestatin induce the release of histamine from rat peritoneal mast cells. This induced histamine release was inhibited by the pertussis toxin, an inhibitor of Gα(i) protein, and extracellular Ca(2+). Rat peritoneal mast cells and rat basophilic leukemia (RBL-2H3) cells did not express the ghrelin receptor GHSR1a, suggesting that histamine release induced by ghrelin occurs via a receptor-independent mechanism. We report here that ghrelin and obestatin, belonging to the family of basic secretagogues, stimulate mast cells independent of a receptor, and this may play a crucial role at the site of allergy or inflammation.

Hemoglobin induces the expression of indoleamine 2,3-dioxygenase in dendritic cells through the activation of PI3K, PKC, and NF-kappaB and the generation of reactive oxygen species.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme-free globin (Apo Hb), induced IDO expression in bone marrow-derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of I kappaB alpha. Hb-induced IDO expression was inhibited by inhibitors of PI3-kinase (PI3K), PKC and nuclear factor (NF)-kappaB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb-induced IDO expression was inhibited by anti-oxidant N-acetyl-L-cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3-amino-1,2,4-triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O(2) (-), H(2)O(2), and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF-kappaB through the PI3K-PKC-ROS and PI3K-Akt pathways is required for the Hb-induced IDO expression in BMDCs.

Peptide array-based analysis of the specific IgE and IgG4 in cow's milk allergens and its use in allergy evaluation.

Cow's milk (CM) is one of the major causes of food allergies in children. We constructed a peptide array consisting of a linear 16-mer peptide library with an offset of 3-mer, which corresponds to the primary sequences of six major CM allergens. The immune reactivity to cow's milk proteins diminishes with age and clinical tolerance commonly occurs. Although the central role of IgE in allergy is well established, the role of other specific antibody classes in obtaining immunotolerance is not well known. The hypothesis that patients become tolerant when they develop immunological changes particularly with the IgG4 isotype has been proposed. In this study, the binding pattern of the CM protein-specific IgE and IgG4 epitopes was measured using the peptide array with sera of 12 patients with persistent CM allergy (CMA), sera of 5 children who outgrew CMA, and sera of 7 CM-sensitized children without allergy symptoms. In CMA patients the IgG4/IgE fluorescence intensity ratios varied greatly from peptide to peptide, and the scatter plots of IgE versus IgG4 signals using significant IgE-binding peptides showed different distribution patterns. When setting the boundary line based on the IgG4/IgE ratio (IgG4/IgE=2), patients with persistent CMA and CM-sensitized children can be distinguished by the plot pattern of peptides. Furthermore, the number of peptide plots in these regions was less in children who outgrew CMA. The approach employed in this study will allow for the distinction between CMA and CM-sensitization, and will enable the estimation of CMA outgrow by monitoring the time elapsed data.

Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.

OBJECTIVE AND DESIGN: We investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of quercetin against degranulation of rat basophilic leukemia (RBL-2H3) cells, rat peritoneal mast cells, and mouse bone marrow-derived mast cells. METHODS: The strength of allergic reaction was evaluated by the extent of degranulation in mast cells sensitized with various stimulants. The levels of HO-1, HO-2, and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions were determined by quantitative RT-PCR, western blotting, or immunocytochemistry. RESULTS: Heme oxygenase activity was upregulated after short exposure to quercetin, followed by the induction of HO-1 expression after long exposure to quercetin. The inhibition of degranulation by quercetin was reversed using tin protoporphyrin IX (SnPP), an HO-1 inhibitor. HO-1 metabolites, bilirubin and CO, led to inhibit degranulation, and quercetin translocated Nrf2 from cytoplasm into nucleus in RBL-2H3 cells. CONCLUSION: These results strongly suggest that quercetin exerted anti-allergic actions via activation of Nrf2-HO-1 pathway.

Development of peptide arrays for detection of IgE-binding epitopes in cow's milk allergens.

Peptide arrays have become versatile tools for high throughput screening assays in biomedical and pharmaceutical research. In this study, we constructed a peptide array that contained linear peptide fragments reported as IgE-binding epitopes for cow's milk allergy (CMA). Various peptides with different solubility in aqueous solutions were dissolved in the buffer solutions containing sodium dodecyl sulfate, and we achieved a consistent spotting of peptide solutions using a piezoelectric ceramic micropump. The IgE-binding patterns were successfully detected by observing the binding of Alexa 647-labeled anti-human IgE using sera from CMA patients. Our technique in this study will provide a potent capability for the development of a peptide array for mapping IgE-epitopes in milk proteins, and it will help researchers better understand the IgE-epitopes associated with the clinical outcome of CMA.