[A CASE OF VOCAL CORD DYSFUNCTION, WHO USED ADRENALIN AUTOINJECTOR (EPIPEN®) FREQUENTLY AFTER BEING DIAGNOSED AS ANAPHYLAXIS].

We experienced a case of vocal cord dysfunction (VCD) in a child to whom an adrenaline autoinjector (Epipen®) had been prescribed and frequently used following a diagnosis of exercise-induced anaphylaxis. An exercise test was performed to investigate her frequent episodes of anaphylaxis-like symptoms. A few minutes after starting the test, signs of dyspnea, such as throat tightness and stridor, appeared, although hypoxia was not present and her respiratory sounds were normal. Medications were not effective for treating her respiratory symptoms. Laryngoscopy performed at the test revealed bizarre vocal cord movement, which was diagnosed as VCD. The symptoms gradually diminished after the initiation of biofeedback therapy, including pursed lips breathing and abdominal breathing. Thereafter, she did not use an adrenaline autoinjector when symptoms appeared; instead, she would perform biofeedback therapy before using the adrenaline autoinjector. Thus, VCD should be included in the differential diagnosis of patients who show anaphylactic symptoms that are resistant to preventive therapy.

[A PEDIATRIC CASE OF DIFFUSE PANBRONCHIOLITIS WHO PREDOMINANTLY SHOWED RESTRICTIVE PULMONARY DYSFUNCTION AND DRAMATICALLY RESPONDED TO MACROLIDE LOW-DOSE LONG-TERM THERAPY.]

A 12-year-old boy visited our hospital with complaints of chronic cough and dyspnea. Chest X-ray and CT revealed diffuse granular shadow in the bilateral lung fields and "Tree-in-bud appearance" in the peripheral airways, respectively. Sinusitis was present, and restrictive disorder was predominantly found in pulmonary function. The patient was diagnosed with DPB, and long-term therapy was started with low-dose clarithromycin (CAM), The patient showed a dramatic response to CAM, with improvements of both the clinical symptoms and pulmonary function within 1-2 months. According to the relevant literature, in adult patients with this disease, pulmonary dysfunction starts from an obstructive pattern; however, this is not the case in pediatric patients. It was therefore suggested that the mechanisms underlying the development of pulmonary dysfunction in cases of childhood onset differs from those with an adult onset.

Airway reversibility and inflammation in stable pre- to late-adolescent asthmatics without long-term control medications.

Objective: Pulmonary function and airway inflammation were investigated in stable pre- to late-adolescent asthmatics without long-term control medications and compared with those in currently medicated asthmatics.Methods: Subjects comprised 34 well-controlled asthmatic children (aged 8.1-18.0 years; group without medication). Flow volume curves before and after inhaling a ß2 agonist, a bronchodilator (BD), were compared and fractional exhaled nitric oxide (FENO) concentrations were measured. All patients were attack-free for at least 12 months prior to testing without the use of asthma medications for at least three months. Fifty-one age-matched stable asthmatics with medications at the time of the present study (group with current medication) underwent the same examinations.Results: The rate of children whose respiratory function after BD improved by 20% or more in both the central and peripheral airways (High responder at total airways subtype: HTA) was significantly higher in the group without medication than in that with current medication (17.6 and 2.0%, respectively; p < 0.01). Furthermore, FEV1.0% pred after BD was significantly lower for HTA than for the low responder subtype in the same group (94.8 ± 3.5 and 104.1 ± 1.5% respectively, p < 0.05). FENO concentrations in the group without medication were high, but not significantly different from those in the group with current medication.Conclusions: Stable asthmatic children without medication include a certain percentage of those with irreversible airflow limitation possibly due to airway remodeling. The control of daily asthma symptoms with long-term control medications may effectively prevent airway remodeling.

[Successful treatment with chloramphenicol in four pediatric cases of intractable bacterial meningitis].

Chloramphenicol (CP) is recently one of the rarely-used antibiotics. In this study, we present four patients with intractable bacterial meningitis, who were successfully treated with CP and discuss the therapeutic indications of CP in these pediatric cases. The patients were diagnosed as bacterial meningitis at the ages ranging from 2 months to 1 year and 4 months. The causative organisms found in three of the patients were H. influenzae and in the fourth patient, S. pneumoniae. According to the microbial sensitivity tests, these organisms were highly sensitive to antibiotics including ceftriaxone, meropenem and/or panipenem/betamipron. Treatment with these antibiotics was initially effective; however, recurrences of meningitis appeared in all patients. Administration of CP (100 mg/kg/day) started between the 11th and the 58th days, and was continued for 9 days up to 19 days. Their fever had disappeared within four days after the administration of CP, and it was confirmed that all patients completely recovered from meningitis. Two of the patients developed a mild degree of anemia, but soon recovered after the discontinuation of CP. None of them had neurological sequela. We recommend CP as one of the choices for the treatment of intractable bacterial meningitis.

Usefulness of modified Pulmonary Index Score (mPIS) as a quantitative tool for the evaluation of severe acute exacerbation in asthmatic children.

BACKGROUND: Acute exacerbation of asthma is divided qualitatively into mild, moderate, and severe attacks and respiratory failure. This system is, however, not suitable for estimating small changes in respiratory condition with time and for determining the efficacy of treatments, because it has a qualitative, but not quantitative nature. METHODS: To evaluate the usefulness of quantitative estimation of asthma exacerbation, modified Pulmonary Index Score (mPIS) values were measured in 87 asthmatic children (mean age, 5.0 ± 0.4 years) during hospitalization. mPIS was calculated by adding the sum of scores for 6 items (scores of 0-3 were given for each item). These consisted of heart rate, respiratory rate, accessory muscle use, inspiratory-to-expiratory flow ratio, degree of wheezing, and oxygen saturation in room air. Measurements were made at visits and at hospitalization and were then made twice a day until discharge. RESULTS: mPIS values were highly correlated among raters. mPIS values at visits were 9.1 ± 0.1 and 12.6 ± 0.4 in subjects with moderate and severe attacks, respectively (p < 0.001). mPIS values of subjects requiring continuous inhalation therapy (CIT) with isoproterenol in addition to systemic steroids were significantly higher than the values of those without CIT (12.0 ± 0.5 and 9.3 ± 0.2, respectively, p < 0.001). A score of 10 was suggested to be the optimal cutoff for distinguishing between subjects requiring and not requiring CIT, from the perspectives of both sensitivity and specificity. mPIS at hospitalization correlated well with the period until discharge, suggesting that this score was a useful predictor for the clinical course after hospitalization. CONCLUSIONS: mPIS could be a useful tool for several aspects during acute asthma attacks, including the determination of a treatment plan, and prediction of the period of hospitalization in admitted patients, although prospective studies would be required to establish our hypothesis.

Surveillance of pollen-food allergy syndrome in elementary and junior high school children in Saitama, Japan.

BACKGROUND: Because few studies have epidemiologically evaluated pollen-food allergy syndrome (PFAS), relevant information about this disease is limited in children. OBJECTIVE: We wanted to clarify the epidemiological details of PFAS by creating a questionnaire which enables to distinguish class 2 food allergy from that of class 1. METHODS: We conducted a questionnaire survey for schoolchildren attending to public elementary and junior high schools. In this questionnaire, we asked about both the allergy to fruits and/or vegetables and allergic rhinitis (AR). PFAS was, then, defined as allergy for fruits and/or vegetable which occurred after the symptoms of AR appeared. RESULTS: A total of 2,346 children (median age, 10.6±2.5 years; 1,157 boys) were evaluated. The prevalence of PFAS was 6.9% among subjects. The mean ages in the onset of AR and PFAS were 4.59±2.76 and 7.38±3.17 years old, respectively. Various kinds of foods were shown to be causative, among which kiwifruits were the commonest. As high as approximately 30% of children with PFAS experienced systemic symptoms including cutaneous (21.8%) and respiratory symptoms (9.6%). Anaphylaxis was diagnosed in 5.8% children. CONCLUSION: Our results indicated that the prevalence of PFAS was getting higher and the mean age of onset was getting lower. These may be attributed to the increasing number of patients with AR and also to the lower age of onset of AR. We have to be careful to not only local but also systemic symptoms when examining children with PFAS.

TNF-α is a key regulator of MUC1, an anti-inflammatory molecule, during airway Pseudomonas aeruginosa infection.

Muc1 is a heterodimeric mucin that is expressed on the apical surface of airway epithelial cells as well as hematopoietic cells. Both in vivo and in vitro studies revealed that Muc1 suppresses inflammatory responses induced by Pseudomonas aeruginosa (PA). In this study, we sought to determine, using intact animals (C57BL/6 mice), whether the expression of Muc1 is important during airway PA infection, and how Muc1 levels are controlled during inflammation. Our results showed that: (1) Muc1 levels in the wild-type (WT) mice were initially low, but gradually increased after PA inhalation, reaching a peak on Day 2, remaining elevated until Day 4, and then gradually decreasing to basal levels on Day 7; (2) TNF receptor 1(-/-) mice failed to increase Muc1 levels after PA infection; (3) after PA inhalation, more inflammatory cells were present in the bronchoalveolar lavage fluid from either Muc1(-/-) or TNF receptor(-/-) mice compared with their WT control animals; (4) more apoptotic neutrophils were present in bronchoalveolar lavage fluid from WT mice compared with Muc1(-/-) mice. We conclude that Muc1(-/-) mice are more inflammatory than WT mice during airway PA infection as a result of both an increase in neutrophil influx and a decrease in neutrophil apoptosis. These results suggest that the up-regulation of Muc1 during airway PA infection might be crucial for suppressing excessive and prolonged inflammatory responses, and is induced mainly by TNF-α, the key proinflammatory mediator.

The aromatic ring of phenylalanine 334 is essential for oligomerization of Vibrio vulnificus hemolysin.

Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.

MUC1 mucin is a negative regulator of toll-like receptor signaling.

MUC1 (MUC1 in humans and Muc1 in nonhuman species) is a transmembrane mucin-like glycoprotein expressed in epithelial cells lining various mucosal surfaces as well as hematopoietic cells. Recently, we showed that Muc1(-/-) mice exhibited greater inflammatory responses to Pseudomonas aeruginosa or its flagellin compared with their wild-type littermates, and our studies with cultured cells revealed that MUC1/Muc1 suppressed the Toll-like receptor (TLR) 5 signaling pathway, suggesting its anti-inflammatory role. Here we demonstrate that other TLR signaling (TLR2, 3, 4, 7, and 9) is also suppressed by MUC1/Muc1. The results from this study suggest that MUC1/Muc1 may play a crucial role during airway infection and inflammation by various pathogenic bacteria and viruses.

The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure.

SIRT1 (silent information regulator 2 homolog 1) is a crucial cellular survival protein especially in oxidative stress environments, and has been thought to locate within the nuclei, but also known to shuttle between cytoplasm and nuclei in some cell types. Here, we show for the first time the dynamics of SIRT1 in the presence of single or concurrent cigarette smoke extract (CSE) exposure in human bronchial epithelial cells (HBEC). In BEAS-2B HBEC or primary HBEC, SIRT1 was localized predominantly in cytoplasm, and the CSE (3%) induced nuclear translocation of SIRT1 from cytoplasm in the presence of L-buthionine sulfoximine (an irreversible inhibitor of γ-glutamylcystein synthetase), mainly through the activation of phosphatidylinositol 3-kinase (PI3K) α subunit. This SIRT1 nuclear shuttling was associated with FOXO3a nuclear translocation and the strong induction of several anti-oxidant genes including superoxide dismutase (SOD) 2 and 3; therefore seemed to be an adaptive response. When BEAS-2B cells were pretreated with repeated exposure to a lower concentration of CSE (0.3%), the CSE-induced SIRT1 shuttling and resultant SOD2/3 mRNA induction were significantly impaired. Thus, this result offers a useful cell model to mimic the impaired anti-oxidant capacity in cigarette smoking-associated lung disease such as chronic obstructive pulmonary disease.

The signaling pathway involved in neutrophil elastase stimulated MUC1 transcription.

We previously reported that neutrophil elastase (NE) stimulated MUC1 gene expression in A549 lung epithelial cells through binding of Sp1 to the MUC1 promoter element. The current study was undertaken to elucidate the complete signaling pathway leading to Sp1 activation. Using a combination of pharmacologic inhibitors, dominant-negative mutant, RNA interference, and soluble receptor blocking techniques, we identified a protein kinase Cdelta (PKCdelta) --> dual oxidase 1 (Duox1) --> reactive oxygen species (ROS) --> TNF-alpha-converting enzyme (TACE) --> TNF-alpha --> TNF receptor (TNFR)1 --> extracellular signal-regulated kinase (ERK)1/2 --> Sp1 pathway as responsible for NE-activated MUC1 transcription. This cascade was identical up to the point of TACE with the signaling pathway previously reported for NE-stimulated MUC5AC production. However, unlike the MUC5AC pathway, TNF-alpha, TNFR1, ERK1/2, and Sp1 were unique components of the MUC1 pathway. Given the anti-inflammatory role of MUC1 during airway bacterial infection, up-regulation of MUC1 by inflammatory mediators such as NE and TNF-alpha suggests a crucial role for MUC1 in the control of excessive inflammation during airway bacterial infection.

Exocytotic release of alanine from cultured cerebellar neurons.

Many neurons release a variety of amino acids in response to depolarizing stimuli. Although some of these amino acids, namely, glutamate, aspartate, and gamma-aminobutyric acid (GABA), have been qualified as neurotransmitters, functional roles of the other amino acids including alanine remain obscure. We investigated the mechanism and the origin of alanine release from cultured rat cerebellar cells. High-K(+)-induced depolarization produced a considerable amount (139+/-8 pmol/2 min/dish) of alanine release, comparable to that of glutamate (103+/-7 pmol/2 min/dish). Other depolarizing agents including veratridine or 4-aminopyridine also induced alanine release, suggesting that the major source is excitable neurons, rather than non-excitable glial cells. Depolarization-evoked alanine release was suppressed in the absence of extracellular Ca(2+), and was almost abolished by treating the cells with botulinum type B neurotoxin (BoNT/B), indicating that alanine is released by Ca(2+)-dependent exocytosis of vesicle-associated membrane protein-2 (VAMP-2)-containing vesicles. The properties of alanine release were different from those of glutamate and GABA in several aspects: (a) Depolarization-dependent alanine release appeared as early as 7 days in vitro, much earlier than that of GABA. (b) Fifty microM kainate, which causes selective cell death of GABAergic neurons in the culture, only partially reduced alanine release, whereas it had no effect on glutamate release. (c) Alanine release was not affected by phorbol ester, which enhanced glutamate and GABA release in a kinase-dependent manner. We therefore conclude that alanine release occurs via exocytosis of a pool of synaptic vesicles distinct from those containing glutamate or GABA.

Quantal size of catecholamine release from rat chromaffin cells is regulated by tonic activity of protein kinase A.

To address the roles of tonic activity of protein kinase A (PKA) in the regulation of exocytosis, we examined the effects of selective PKA inhibitors on catecholamine (CA) release from rat chromaffin cells in the absence of PKA activators. Myristoylated protein kinase inhibitor (myr-PKI) reduced the total amount of CA released from a single cell, whereas H-89 did not affect the total amount and frequency of CA release. Amperometric recording revealed that myr-PKI had only a small effect on the number of amperometric spikes per cell, but preferentially inhibited exocytotic events of large quantal size. The change in quantal size distribution was not associated with a reduction of cellular CA content, quantified by using HPLC. These results suggest that the tonic activity of PKA near the cell membrane regulates neurotransmitter release by modulating quantal size.

Nicotine primarily suppresses lung Th2 but not goblet cell and muscle cell responses to allergens.

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C(4); and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.

TNF-alpha induces MUC1 gene transcription in lung epithelial cells: its signaling pathway and biological implication.

The current study was conducted to elucidate the mechanism through which TNF-alpha stimulates expression of MUC1, a membrane-tethered mucin. A549 human lung alveolar cells treated with TNF-alpha exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Increased MUC1 protein levels were correlated with significantly higher levels of MUC1 mRNA in TNF-alpha-treated cells compared with controls. However, TNF-alpha did not alter MUC1 transcript stability, implying increased de novo transcription induced by the cytokine. TNF-alpha increased MUC1 gene promoter activity in A549 cells transfected with a promoter-luciferase reporter plasmid. Both U0126, an inhibitor of MEK1/2, and dominant negative ERK1 prevented TNF-alpha-induced MUC1 promoter activation, and anti-TNFR1 antibody blocked TNF-alpha-stimulated ERK1/2 activation. MUC1 promoter activation by TNF-alpha also was blocked by mithramycin A, an inhibitor of Sp1, as well as either deletion or mutation of a putative Sp1 binding site in the MUC1 promoter located between nucleotides -99 and -90. TNF-alpha-stimulated binding of Sp1 to the MUC1 promoter in intact cells was demonstrated by chromatin immunoprecipitation assay. We conclude that TNF-alpha induces MUC1 gene transcription through a TNFR1 --> MEK1/2 --> ERK1 --> Sp1 pathway.

Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice.

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1(-/-) mice with Muc1(+/+) littermates following intranasal instillation of PA or flagellin. Compared with Muc1(+/+) mice, Muc1(-/-) mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-alpha and KC in bronchoalveolar lavage fluid, higher levels of TNF-alpha in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.