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Background: Inhaled corticosteroids (ICSs) are widely used in combination with other classes of drugs for treatment of childhood asthma. The efficacy and the safety of montelukast added to low-dose ICS therapy were compared with those of sustained-release theophylline added to low-dose ICS therapy in asthmatic children in the present study., Methods: Following the 2-week run-in period, 6-to 14-year old patients receiving treatment with ICSs were randomized to treatment for 4 weeks with either montelukast 5 mg once daily or sustained release theophylline 5-8 mg/kg (dry syrup) or 100-200 mg (tablet) twice daily. Patients also received a fixed dose of ICS throughout the run-in and treatment periods. The primary efficacy endpoint was the change from baseline in peak expiratory flow (PEF) at Week 2., Results: A significant increase in morning PEF was observed in the add-on montelukast group as compared with the add-on theophylline group at Week 2 (change from baseline of 22.8 L/minvs. 8.7 L/min; p = 0.041 for between-group difference) and at Week 4 (31.0 L/minvs. 9.8 L/min; p = 0.012). A significant increase in evening PEF was observed in the add-on montelukast group as compared with the add-on theophylline group at Week 4 (24.7 L/minvs. 8.7 L/min; p = 0.027). There were no significant differences between the treatment groups in incidences of clinical and laboratory adverse experiences., Conclusions: The results indicate that montelukast added to low-dose ICS is an effective and safe option for the treatment of asthma in children.

To identify novel genes related to the clinical signs of atopic dermatitis (AD), differentially expressed genes were sought in peripheral blood eosinophils from both AD patients and healthy volunteers. RNA was prepared from eosinophils, expression of various genes was monitored using the Affymetrix GeneChip, and expression was quantified by real-time RT-PCR. Two genes, Nur77 and NOR1, members of NR4A orphan nuclear receptor family, were expressed at a significantly higher level in AD patients than in healthy volunteers. Expression of another gene in the NR4A receptor family, Nurr1, was also higher in AD patients than in healthy volunteers. When peripheral blood leukocytes from healthy volunteers were fractionated, NOR1 expression was highest in eosinophils, but expression of Nur77 and Nurr1 genes was not eosinophil-specific. Extremely intense apoptosis was induced in both eosinophils and an eosinophil cell line, AML14.3D10, by treatment with antibody (Ab) to both CD30 and Fas. Rapid expression of the genes for the NR4A receptor family was observed with anti-CD30 Ab treatment but not with anti-Fas Ab. The NR4A orphan nuclear receptor family gene expression and the subsequent eosinophil apoptosis were downregulated by the MAPK inhibitor, U0126. These results suggest that the expression of the NR4A receptor family genes through CD30 signaling may regulate eosinophil apoptosis in allergic conditions such as AD., (Copyright 2005 S. Karger AG, Basel.)

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritic chronic eczema. The immunopathogenesis of this condition is still not well understood. We assessed the transcription and production of IFN-gamma, the Th1 cytokine, and the Th2 cytokine IL-5 in peripheral blood mononuclear cells (PBMCs) from patients with severe AD., Methods: The subjects included 17 severe (serum IgE: 5,000-92,000 U/ml, median: 20,000 U/ml), 4 mild AD (IgE: 2-520 U/ml) and 8 nonatopic controls (IgE: <100 U/ml). The severe AD patients were classified into two groups according to the response to standard treatment with topical glucocorticoids. Individuals were classified as poorly responsive (AD-P) if the clinical score decreased less than one third after 2 weeks of hospital treatment and as responsive (AD-R) if the score decreased more than one third. PBMCs isolated from the subjects were stimulated with PHA and PMA., Results: The expression of IFN-gamma in PBMCs in the AD-P group was much lower than that observed in the other groups at both mRNA and protein levels. There were no significant differences in the levels of IL-5 both in mRNA and protein levels between the groups. There were no significant differences in STAT4 DNA-binding activity following PHA/IL-2/IL-12 stimulation between AD-P and controls., Conclusions: These results suggest that the decreased INF-gamma production may account for the abnormal immunopathogenesis of severe, intractable AD., (Copyright 2004 S. Karger AG, Basel)

To identify new genes related to atopic dermatitis (AD), we screened for differentially expressed genes in peripheral blood eosinophils derived from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils obtained from both AD patients and healthy volunteers, and the expression of various genes was monitored using fluorescent differential display and real-time RT-PCR. One of the expressed sequence tags (ESTs) was expressed at a significantly higher level in AD patients than in healthy volunteers. A full-length cDNA was identified that encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1, also named hASB-8. The expression of HSOCP-1 was increased in cultured peripheral blood eosinophils after IL-4 stimulation, and overexpression of HSOCP-1 caused cell death in an eosinophil cell line, AML14.3D10. p34(SEI-1) was identified as a HSOCP-1-interacting protein by a yeast two-hybrid system. It is a protein that also interacts with the cyclin-dependent kinase inhibitor p16(INK4), suggesting that HSOCP-1 is involved in cell cycle control and apoptosis., (Copyright 2004 S. Karger AG, Basel)

Background: Little is known about airway inflammation in childhood asthma. The aims of this study were to analyze the expression of a wide range of mediators of airway inflammation in childhood asthma., Methods: Eight asthmatic children with acute exacerbations were recruited for the study. Peripheral blood was drawn from the patients at the time of exacerbation and after improvement. Total RNA was extracted from the isolated T lymphocytes. The differential display (DD) RT-PCR method was used to detect expressed genes, and the quantification of candidate gene expression was performed using real-time quantitative RT-PCR., Results: The only gene for which significant expression differences were detected in both DD analysis and quantitative RT-PCR was lipocortin II (annexin II) (exacerbation > remission, p < 0.05). In quantitative RT-PCR of interleukin-4 (IL-4), IL-5, interferon-gamma, IL-12 receptor-beta and integrin alpha6, a significant difference was found only in the expression of IL-4 mRNA (exacerbation > remission, p < 0.05). The IL-4 plasma concentration tended to be higher in exacerbation than in remission., Conclusions: Our findings suggest activation of T cells and IL-4 production may be involved not only in the basic pathogenesis of childhood asthma but also in its acute exacerbation, and that lipocortin II may be a marker or contribute to asthma exacerbation., (Copyright 2004 S. Karger AG, Basel)

Background: Monocytes, macrophages, and antigen-presenting cells (APCs) are key effectors of both innate and acquired immune responses. Such cells have been implicated in the pathogenesis of some inflammatory diseases. Differential gene expression in CD14-positive cells from patients with atomic dermatitis (AD) was studied using real-time quantitative RT-PCR to measure transcription levels of selected genes., Methods: PBMCs were prepared by Ficoll gradient separation from 30 AD patients (the anti-mite-specific IgE RAST score: 0.75 to >100 UA/ml) and 10 healthy adult individuals (the RAST score: <0.34-0.37 UA/ml) and CD14-positive cells were selected. A total of 64 genes was selected for study from groups of genes with different molecular function., Results: Genes involved in MHC class I antigen presentation, such as beta(2)-microglobulin, subunits of an immunoproteasome and ATP-binding cassette transporter TAP2 were expressed at higher levels in the AD patients than in the healthy controls. The genes for Toll-like receptors, CD36 and IFNgamma receptor were also upregulated in the AD patients. These genes are involved in MHC class I antigen presentation, recognition of bacterial pathogens and apoptotic cells., Conclusions: The upregulation of genes suggests that circulating monocytes in AD patients may be primed to differentiate into effector cells by conditions associated with AD. The upregulation of genes may prove to be a useful marker for AD., (Copyright 2003 S. Karger AG, Basel)

To identify the genes related to atopic dermatitis (AD), we compared gene expression in eosinophils from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils. Gene expression was monitored by fluorescent differential display (DD) and real-time RT-PCR. Eighteen new sequences, including expressed sequence tags (ESTs), were expressed at higher levels in eosinophils from AD patients than in those from healthy volunteers. The functions of most of these genes are unknown. We found no correlation between the expression of a particular gene and clinical markers such as the number of eosinophils and the amount of IgE. Multivariate analysis of the gene expression data in each sample showed a very high coefficient of correlation among the copy numbers of each gene. The genes under investigation were also expressed in cultured blood eosinophils after IL-4, IL-5 and IFN-gamma stimulation. We were able to predict the function of some of the sequences by scanning for homologies within either the human or mouse genome databases. The mouse counterpart of one of these genes, intersectin 2, was expressed dramatically, as measured by ear edema, in 1-fluoro-2,4-dinitrobenzene-induced mouse contact dermatitis and in NC/Nga mouse dermatitis., (Copyright 2003 S. Karger AG, Basel)

Investigation of differentially expressed genes in eosinophils of patients with allergic diseases such as atopic dermatitis (AD) will provide important information for elucidating possible mechanisms of pathology. To identify novel genes that are expressed in AD, we compared gene expression in samples of peripheral blood eosinophils from AD patients and healthy volunteers. RNA was extracted from peripheral blood eosinophils. The expression of various genes, such as those for cytokine receptors, eosinophil activation marker, platelet activating factor (PAF) receptor, eosinophil-specific granular proteins and apoptosis-related genes, was confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR). Peripheral blood eosinophils of healthy volunteers were also isolated and stimulated for introduction of various cytokines. RNA was extracted and gene expression was monitored. Several genes, such as those for cytokine receptors (granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha and beta chain and interleukin (IL)-3 receptor alpha chain), CD44 and PAF receptor were expressed at significantly higher levels in AD patients than in healthy volunteers. In addition, the anti-apoptotic genes, bcl-2 and bcl-xL, were expressed at increased levels in AD patients. No single gene expression correlated with clinical markers, such as eosinophil count or IgE levels. Expression of GM-CSF receptor beta chain and IL-3 receptor alpha chain in isolated blood eosinophils of healthy volunteers was stimulated by IL-5, IL-4, interferon (IFN)-gamma and GM-CSF. Expression of bcl-2 and bcl-xL was also increased after stimulation with IL-5, IL-4 or IFN-gamma. The in vitro enhancement of cytokine-stimulated gene expression correlated well with the enhancement observed in clinical samples of eosinophils, suggesting that cytokines may affect gene expression in vivo in eosinophils of patients with AD.

Background: Analysis of genes that are differentially expressed in patients with atopic dermatitis (AD) and normal individuals will provide important information on the underlying molecular pathogenetic mechanisms of AD., Methods: Transcript of freshly isolated peripheral blood T cells from 59 individuals were analyzed with a fluorescent differential display (FDD) method. Ninety-two differentially expressed genes were identified in this manner. Additionally, real-time quantitative RT-PCR was employed to investigate the expression of the FDD-selected genes and also genes related to T cell function., Results: A number of genes, including CC chemokine receptor 4, T cell-specific tyrosine kinase (Emt/Itk), integrin beta1, integrin alpha6, IQGAP1 and MAR/SAR DNA-binding protein (SATB1), were shown to be more highly expressed in patients with moderate and/or severe AD than in controls or patients with mild AD. Because the products of these upregulated genes influence chemotaxis, adhesion, migration and Th2 polarization, it is suggested that in more severe AD, circulating T cells may function differently in this regard. Several other genes, the role of which in T cell function is currently unknown, were also found to be differentially expressed in AD. These included the heat shock protein 40 and vasopressin-activated calcium-mobilizing receptor 1., Conclusion: The upregulated genes identified in this work may serve as useful markers for moderate to severe AD as opposed to normal or mild AD and also as markers indicating progression to more severe AD. Further functional characterization will provide a better understanding of the pathophysiology of circulating T cells in AD., (Copyright 2002 S. Karger AG, Basel)

Background: There are few laboratory tests for evaluating atopic dermatitis (AD) with the exception of IgE levels or the eosinophil count. We attempted to identify new diagnostic markers by screening the genome-wide expression of transcripts in peripheral blood mononuclear cells (PBMC)., Methods: For this study, we enrolled 7 nonatopic healthy volunteers, 5 AD patients who responded well to treatment and 6 who responded poorly. We compared genome-wide transcript levels in PBMC derived from patients with severe AD and healthy volunteers using high-density oligonucleotide arrays (GeneChip, Affymetrix). After the first screening with GeneChip, we employed real-time quantitative PCR to confirm differential expression levels., Results: Screening with GeneChip showed that the levels of a total of 92 transcripts increased at least 3-fold in one population compared to another. After further evaluation of these genes with real-time quantitative PCR, the levels of 4 transcripts were confirmed to be significantly different in PBMC from AD patients compared to controls, namely IFN-gamma, TRAIL (TNF-related apoptosis-inducing ligand), ISGF-3 (STAT1) and defensin-1. With the exception of IFN-gamma, none of these genes has previously been implicated in AD pathology., Conclusion: These 4 transcripts in PBMC are expected to be useful markers for evaluating AD., (Copyright 2002 S. Karger AG, Basel)

As a complication of atopic dermatitis (AD), the incidence of hypoproteinemia is increasing among infants with severe AD in Japan. It can be a life-threatening condition owing to hypovolemic shock as a result of hypoproteinemia and vascular infarction as a result of thrombocythemia. However, the pathophysiology of this condition remains unclear. The objectives of the present study were two-fold. The first objective was to determine the main route of protein loss, i.e. through the damaged skin or the gastrointestinal tract, or as a result of insufficient food intake. The second objective was to identify whether allergy or infection was the cause of severe skin inflammation. Fifteen patients with AD were enrolled who had serum protein levels of 3.2-5.8 g/dl. Specific immunoglobulin E (IgE) and skin test to allergens, stool eosinophils, alpha1-antitrypsin clearance, skin Staphylococcus aureus colonization and superantigens (SAgs) produced by the organism, serum SAg-specific IgE antibodies, serum interleukin (IL)-5, IL-6, IL-12, and interferon-gamma (IFN-gamma) were evaluated. Prominent serous skin discharge was seen in all of the patients and was found to have almost the same protein concentration as serum. Marked thrombocytosis, with a maximum of 1,060 x 103/ml, was seen. Skin culture revealed S. aureus colonization in all patients. SAg-producing S. aureus were found in 84.6% of the patients. The concentration of serum IL-5 was significantly increased and correlated well with the blood eosinophil count. Hence, the main route of protein loss was believed to be through damaged skin. The cause of severe inflammation was thought to be a combination of allergic inflammation and skin colonization by SAg-producing S. aureus. Serum cytokines showed a T helper 2 (Th2) T-cell-mediated pattern. To prevent hypovolemic shock, vascular occlusion, and growth retardation, it is of vital importance to diagnose hypoproteinemia at an early stage and start appropriate therapy.

Background: It remains unclear whether the number of circulating mast cell progenitors is increased in patients with atopic diseases. Distinct genotypes are reported to affect mast cell/basophil activation., Objective: We compared the number and function of mast cell progenitors present in the peripheral blood from donors with normal IgE (IgE < 400 U/mL) and those with atopic dermatitis accompanied by high serum IgE (IgE > 5000 U/mL)., Methods: Purified peripheral blood cells were cultured in serum-free methylcellulose containing stem cell factor (SCF), IL-6 plus IL-3. Fresh methylcellulose containing the cytokines was layered over every 2 weeks. The cultured mast cells were retrieved from the methylcellulose and were functionally analysed., Results: Mast cell colonies were distinguished at 6 weeks of culture as other colony types had been degenerated. The number of mast cell colony-forming cells varied depending on donors and was not significantly increased in peripheral blood from the hyper-IgE atopic patients. A significant inversed correlation was found between the number of mast cells per one colony and the ages of donors. The cultured mast cells derived from atopic patients and those from normal IgE donors equally expressed Fc epsilon RI and released histamine through Fc epsilon RI, although IL-4 priming in vitro markedly enhanced the function of mast cells regardless of donors., Conclusions: These results indicate that the number of circulating mast cell progenitors may be regulated by unknown individual factors unrelated to IgE levels. Mast cell function may be regulated largely by environmental factors, such as IL-4, but not determined by their progenitors' genotypes.

Mast cells (MCs) and eosinophils are thought to play important roles in evoking allergic inflammation. Cell-type--specific gene expression was screened among 12,000 genes in human MCs and eosinophils with the use of high-density oligonucleotide probe arrays. In comparison with other leukocytes, MCs expressed 140 cell-type--specific transcripts, whereas eosinophils expressed only 34. Among the transcripts for expected MC-specific proteins such as tryptase, major basic protein (MBP), which had been thought to be eosinophil specific, was ranked fourth in terms of amounts of increased MC-specific messenger RNA. Mature eosinophils were almost lacking this transcript. MCs obtained from 4 different sources (ie, lung, skin, adult peripheral blood progenitor--derived and cord blood progenitor--derived MCs, and eosinophils) were found to have high protein levels of MBP in their granules with the use of flow cytometric and confocal laser scanning microscopic analyses. The present finding that MCs can produce abundant MBP is crucial because many reports regarding allergic pathogenesis have been based on earlier findings that MBP was almost unique to eosinophils and not produced by MCs. (Blood. 2001;98:1127-1134)

Background: beta(2)-Adrenergic agonists are the most widely used bronchodilators for the treatment of asthma. On the other hand, there is concern that excessive use of beta(2)-agonists may contribute to the exacerbation of asthma. However, the mechanism of such adverse effects of beta(2)-agonists is not completely clear., Objective: The aim of this study was to assess the direct influence of beta(2)-agonists on airways by analyzing the effect of a beta(2)-agonist, fenoterol, on airway sensitivity in an animal model and on tachykinin neurokinin-2 receptor expression in bovine tracheal smooth muscle., Methods: We performed an acetylcholine challenge test on ovalbumin sensitized guinea pigs that were exposed to daily inhalation of ovalbumin and fenoterol. We also investigated the effects of fenoterol on neurokinin-2 receptor messenger RNA and density with Northern blot analysis and receptor binding assay., Result: The increase of airway responsiveness and the decrease of beta(2)-adrenergic receptors were found in guinea pigs that were treated with fenoterol. There were time- and dose-dependent increases of neurokinin-2 receptor mRNA and of density in tracheal smooth muscle that was treated with fenoterol., Conclusion: This increased airway responsiveness, increased neurokinin-2 receptor expression, and decreased beta(2)-adrenergic receptor density may be relevant to asthma exacerbation.

Background: Adrenocortical suppression is a potential complication of the use of topical corticosteroids in patients with atopic dermatitis (AD)., Objectives: To determine whether or not the adrenocortical suppression observed in patients with severe AD is a sole result of the application of topical steroids., Methods: A total of 45 patients with severe AD that required hospitalization for treatment were enrolled. These patients were divided into two groups according to the treatment received before hospitalization: group 1 had not used topical corticosteroids for at least three months (n = 17), while group 2 had used topical corticosteroids daily (n = 28). Otherwise, these two groups were matched to clinical characteristics. A rapid ACTH test was performed upon hospital admission. Topical corticosteroids were then applied to both groups. The second ACTH test was performed just before discharge, an average of 23 days after the first test., Results: The basal serum cortisol levels as well as the response to ACTH stimulation in the first examination were significantly lower in the AD patients than in the controls (P < .001), although there were no significant differences in the results between groups 1 and 2. The followup study of adrenocortical function at hospital discharge showed that morning basal serum cortisol levels were significantly increased in group 1 (P < .01), despite their topical corticosteroid treatment, while no significant increase or decrease was seen in group 2., Conclusion: Our findings suggest that the adrenocortical suppression seen in patients with AD may be caused by the percutaneous absorption of topical corticosteroids as well as by other factors related to the disease.

Histamine, released from activated mast cells, causes bronchoconstriction mediated by H(1) receptors, whereas beta(2)-agonists are widely used for the relief of bronchoconstriction. In this study, we examined the effects of the beta(2)-adrenoceptor agonist, fenoterol, on the expression of H(1) receptors at the mRNA and protein levels, and functional responses. Incubation of bovine tracheal smooth muscle with fenoterol (10(-7) M) for 2 h increased H(1) receptor mRNA (maximum approximately 190%). The number of H(1) receptors was increased after 12 and 18 h without any change in binding affinity. In the contraction experiments, the concentration-response curves for histamine-induced contraction were shifted significantly to the left after 18-h exposure to fenoterol, consistent with the increase in receptor number. The fenoterol-induced increase in H(1) receptor mRNA was concentration-dependent and was abolished by propranolol and ICI 118551, but not by CGP 20712A, indicating that fenoterol acts via beta(2)-adrenoceptors. These effects were mimicked by other cAMP-elevating agents forskolin and prostaglandin E(2), and by the stable cAMP analog 8-bromo-cAMP. Cycloheximide alone produced superinduction of H(1) receptor mRNA and augmented the fenoterol-induced increase in H(1) receptor mRNA. Fenoterol increased both the stability and the transcription rate of H(1) receptor mRNA. Pretreatment with dexamethasone did not prevent fenoterol-induced up-regulation of H(1) receptor mRNA. Thus, fenoterol increases the expression of airway smooth muscle H(1) receptors via activation of the cAMP system through increased gene transcription and mRNA stability. This mechanism may be involved in the adverse responses encountered with the clinical use of short-acting beta(2)-agonists.

Neurokinin A (NKA) induces bronchoconstriction mediated by tachykinin NK(2) receptors in animals and humans, and may be increased in asthma. Because beta(2)-adrenoceptor agonists are the most widely used bronchodilators in asthma, we investigated the effects of the beta(2)-adrenoceptor agonist fenoterol on NK(2) receptor messenger RNA (mRNA) and receptor density as well as the functional responses of bovine tracheal smooth muscle to the NK(2) receptor agonist [beta-Ala(8)]-NKA(4-10) in vitro, using Northern blot analysis, receptor binding, and organ bath studies. Incubation with fenoterol induced a time- and concentration-dependent upregulation of NK(2) receptor mRNA (71% increase after 12 h at 10(-7) M fenoterol), which was abolished by propranolol (a nonselective beta-adrenoceptor agonist) and ICI118551 (a selective beta(2)-adrenoceptor antagonist), but not by CGP20712A (a selective beta(1)-adrenoceptor antagonist), indicating that fenoterol acts via beta(2)-adrenoceptors. These effects were mimicked by forskolin and prostaglandin E(2) (PGE(2)), both agents that increase cyclic adenosine monophosphate (cAMP), and by the cAMP analogue 8-bromo-cAMP. The upregulation was blocked by cycloheximide, indicating that it requires new protein synthesis, and was accompanied by an increase in both the stability of NK(2) receptor mRNA and the rate of NK(2) receptor gene transcription. Radioligand binding assay using the selective NK(2) receptor antagonist [(3)H]SR48968 showed a significant increase in the number of receptor binding sites after 12 h and 18 h, which was accompanied by an increased contractile responsiveness to the NK(2) receptor agonist [beta-Ala(8)]-NKA(4-10). Dexamethasone completely prevented the fenoterol-induced increase in NK(2) receptor mRNA and in the contractile response. We conclude that beta(2)-adrenoceptor agonists induce upregulation of functional NK(2) receptors in airway smooth muscle by increasing cAMP, and that this can be prevented by a corticosteroid. The increased responsiveness could be relevant to asthma control and mortality.

Background: Superantigenic exotoxins produced by Staphylococcus aureus and their specific IgE antibodies are thought to be important precipitating factors of atopic dermatitis (AD), but there are few reports evaluating these 2 factors at the same time., Objective: We examined whether the presence of the exotoxins sampled from the skin of patients with AD and the levels of anti-exotoxin IgE antibodies in their sera correlated with their severity of AD., Methods: Patients with mild-to-severe AD, 1 to 22 years of age, were evaluated by using Leicester's scoring system. Specific IgE antibodies against these exotoxins were determined by using ELISA. S aureus was isolated from 3 different areas of the skin. We examined whether the exotoxin (staphylococcal enterotoxin [SE]A, SEB, SEC, SED, and toxic shock syndrome toxin-1) could be detected., Results: The levels of SEB-specific IgE were correlated with the severity of AD. Five of 6 patients having very high SEB-specific IgE antibody titers were under 6 years of age, and SEB was most frequently isolated (41%). There was no difference in severity between patients with or without exotoxin-producing S aureus. The severity of 9 patients who had both exotoxin-producing S aureus on the skin and specific IgE antibody against the same exotoxin in sera was significantly higher than that of the other patients., Conclusions: Anti-SEB IgE titers correlate well with the severity of AD. The presence of exotoxin-producing S aureus may precipitate AD through its specific IgE antibody.

Neurokinin A is not only a potent bronchoconstrictor, but also has immuno-modulatory effects in animals and man, mediated via tachykinin NK2 receptors. We have examined the effect of the glucocorticoid, dexamethasone, on tachykinin NK2 receptor mRNA and the number of tachykinin NK2 receptors in bovine tracheal smooth muscle in vitro by Northern blot analysis using a human tachykinin NK2 receptor cDNA probe and receptor binding assay using [3H]SR48968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2(3,4-dichlorophenyl) butyl]benzamide]. Tachykinin NK2 receptor mRNA showed a time-dependent suppression (62% reduction after 6 h at 10(-7) M of dexamethasone), as well as a concentration-dependent suppression after the incubation with dexamethasone (IC50 = 1.3 x 10(-8) M). This suppression was abolished by the glucocorticoid receptor antagonist, mifepristone (RU38486), indicating that dexamethasone acts via the glucocorticoid receptor. It was also abolished by the protein synthesis inhibitor, cycloheximide (10 microg/ml), indicating that new protein synthesis is required on this suppression. Using the RNA polymerase inhibitor actinomycin D (5 microg/ml), we showed that the stability of tachykinin NK2 receptor mRNA was not affected by dexamethasone (t1/2 = 5 h). Nuclear run-on assays revealed a 51% reduction in the rate of tachykinin NK2 receptor gene transcription after treatment with dexamethasone for 6 h. Radioligand binding assay using an selective tachykinin NK2 receptor antagonist, [3H]SR48968 showed a significant decrease in the number of receptor binding sites after 16 h (Bmax = 262 +/- 23 versus 213 +/- 13 fmol/mg protein for vehicle and dexamethasone treatment respectively, P < 0.05), with no significant change at the earlier time points. These results suggest that glucocorticoids act on glucocorticoid receptors to decrease tachykinin NK2 receptor expression by decreasing the rate of tachykinin NK2 receptor gene transcription.

Serum glutamic-oxaloacetic transaminase (GOT) levels were determined in 214 infants (133 males and 81 females) with atopic dermatitis during their first visit to the Department of Allergy, National Children's Hospital, Tokyo, Japan. Compared with the normal hospital range, their levels were found to be significantly higher, a tendency which was more conspicuous in lower age groups. We carried out a 13C-methacetin breath test (MBT), administering the stable-isotope-labeled compound to 11 children with higher serum GOT values and 5 within the normal range to investigate hepatic metabolism of methacetin in infants with atopic dermatitis. 13C-methacetin was given orally, and the 13CO2 level in the breath was determined immediately before and after administration, by mass spectrometry. Compared to the normal controls, the atopic infants demonstrated significantly lower 13CO2 peak excretion and delayed peak time. The clearance rate of 13CO2 was also decreased. These results suggest some relationship between atopic dermatitis and liver function in infants.

The adrenal function of children with near fatal asthma was evaluated by a modified rapid ACTH test. The rapid ACTH tests, which were performed within six months of each life-threatening asthmatic episode, showed extremely low responsiveness. The response in the subjects was significantly lower than that of patients who had received high dose of corticosteroid therapy. It was suggested that the adrenocortical function of children with near fatal asthma had been already suppressed and that adrenal suppression could easily occur in such patients. We advocate the following measures: (1) environmental control, education of patients and their families, physical training accurate medication should be supplied to reduce the use of corticosteroids, (2) sufficient doses of corticosteroids should, however be given to patients with acute exacerbation, (3) not only lung function tests or allergic examinations, but also adrenocortical function tests should be performed on severe asthmatic patients.

Sinusitis is common in children with allergic diseases, and the relationship between sinusitis and reactive airway diseases involving asthma has been reported. Most pediatricians and physicians base their diagnosis of sinusitis on findings from plain radiographs of the sinuses, especially Waters projection radiographs. We compared the diagnoses made by 11 pediatric allergists using 56 Waters projection radiographs with transverse CT findings. The ratio for the two diagnosis being consistent (normal plain radiographic findings and normal CT findings, or abnormal plain radiographic findings and abnormal CT findings) was approximately 60%. Sinusitis in children is often misdiagnosed on the basis of findings from Waters projection radiographs alone. Therefore, the use of CT findings for the diagnosis of sinusitis together with Waters projection radiographs is recommended.

We investigated the effect of 300 micrograms of inhaled beclomethasone dipropionate (BDP) daily on bronchial responsiveness to methacholine and pulmonary function of 22 subjects with asthma in a single-blind, crossover study. The severity of bronchial hyperresponsiveness lessened significantly during treatment with BDP (P < .01). No significant changes occurred in FEV1 or in the control value of airway conductance. We conclude that a 300-micrograms total daily dose of BDP is an efficacious treatment for patients with asthma. Because suppression of adrenal function and systemic adverse effects can occur in asthmatic patients treated with inhaled corticosteroids, especially children, long-term treatment with inhaled steroids should employ minimal daily doses necessary.

The function of the hypothalamic-pituitary-adrenal (HPA) axis was investigated in nine asthmatic children treated by inhalation of beclomethasone dipropionate (300 micrograms/day) for 12 weeks. The trial was designed as a prospective study. The 24-hour urinary free cortisol, serum cortisol, response to ACTH, and nocturnal serum cortisol followed by 20-minute sampling were measured before and after the study period. No significant changes were found, suggesting that inhaled beclomethasone, in a daily dose of 300 micrograms, does not cause suppression of the HPA axis.

We present a case of wheat-dependent exercise-induced anaphylaxis, in which pretreatment with sodium bicarbonate inhibited reappearance of anaphylactic symptoms following wheat and exercise provocation. Decrease in blood pH relative to elevation in plasma histamine levels was also inhibited. These results suggest possible efficacy of pretreatment with sodium bicarbonate as a preventive measure in patients with exercise-induced anaphylaxis.

We studied the protective effect of sustained-release theophylline granules on early or late response in exercise-induced asthma (EIA) in 16 patients ranging in age from 7 to 18 years. A standardized exercise test was examined 2 hours after administration of theophylline granules on one day and after no medication on another day. On both days, the lung functions and the serum theophylline and cortisol concentrations of the patients were examined before exercise and over a period of 8 hours after exercise. On days without medication, early response was seen in 16 patients and late response in 6. On days with medication, the theophylline granules inhibited early response in 13 of the 16 patients and late response in 5 of the 6 patients. This suggests that theophylline preparation is a useful drug for inhibiting the dual responses of EIA.

When cord blood mononuclear cells were cultured in the presence of rhIL-3 for 5 weeks or more, 40-90% of cultured cells became morphologically mature basophils. We analyzed the kinetics of histamine release, changes in filament actin (F-actin), and movement of intracellular Ca2+ ([Ca2+]i) induced by IgE-dependent (anti-IgE) and -independent (fMLP) stimuli in these cultured basophils. Anti-IgE and fMLP released 24.5 +/- 5.4% and 14.5 +/- 4.5% histamine from the cells, respectively. Anti-IgE caused actin polymerization with a peak response at 15 min, which began much later than the elevation of [Ca2+]i. In contrast to anti-IgE stimulation, fMLP induced rapid actin polymerization with a peak response at 30 s in correlation with kinetics of histamine release. Our results indicate that cord blood-derived cultured basophils show similar cell functions to mature basophils, and are useful models with which to investigate the mechanisms of degranulation, specifically when a large amount of highly purified cells are required.

A 64-year-old man was referred to our hospital because of little improvement of hemoptysis lasting three days after drug therapy. A chest roentgenogram and fiberoptic bronchoscopic examination performed on the second hospital day when the patient experienced a massive hemoptysis of about 2,000 ml revealed arterial bleeding from the left upper lobe. Even after extensive embolizations of the left upper bronchial, the first, second, third and 4th intercostal arteries, the patient's hemoptysis did not improve. On the 73rd hospital day the patient underwent left upper lobectomy. Macroscopic and microscopic examinations in the resected specimen revealed lung injury due to a torn segment of pleural calcification after tuberculous pleuritis, resulting in the massive hemoptysis. Although physicians encounter many patients complaining of hemoptysis and/or hemosputum, this case is considered to be very rare.

We present a case of exercise-induced anaphylaxis with improvement following the removal of dental amalgam. Although her symptoms were unresponsive to various kinds of therapy until removal of the amalgam, her symptoms related to exercise improved remarkably after the removal. The increase in plasma histamine levels for exercise provocation test also improved. This suggests that sensitivity to metals might cause exercise-induced asthma in some patients.

The relationship between chymotrypsin-inhibitory and immunoenhancing activity of alpha-1-antichymotrypsin was studied. alpha-1-Antichymotrypsin was treated at 50 degrees C, 55 degrees C or 60 degrees C for 15 min. It was found that antichymotryptic activity was reduced by half when alpha-1-antichymotrypsin was heated at 55 degrees C and was not detected at all when heating was carried out at 60 degrees C. alpha-1-Antichymotrypsin which was heated at 60 degrees C did not form a complex with chymotrypsin, but became a substrate for chymotrypsin. The effect of native and heated alpha-1-antichymotrypsin on antibody response was studied in mice. alpha-1-Antichymotrypsin increased the number of anti-sheep erythrocytes antibody producing cells even when it was heated at 60 degrees C. Circular dichroism and single radial immunodiffusion were used to detect conformational changes. Circular dichroism in the region of side chain absorption showed that the intensities of the spectra at 296, 284, and 265 nm decreased with a rise in temperature from 50 to 60 degrees C. In single radial immunodiffusion analysis, alpha-1-antichymotrypsin did not form a halo after being heated at 60 degrees C. In conclusion, when alpha-1-antichymotrypsin was heated at 60 degrees C, the immunoenhancing activity remained intact while the antichymotryptic activity was lost with the conformational change.

alpha 1-Antichymotrypsin (ACT), which is known as an efficient serum protease inhibitor and is detected in tumor cell nuclei, was found to inhibit the activity of DNA polymerase alpha purified from human stomach adenocarcinoma. The concentration of ACT required for 50% inhibition was 1.0 mg/ml and the manner of its inhibition showed the partially competitive relationship between ACT and DNA in the assay system. Furthermore the removal of ACT by anti-ACT antibody lost its antichymotryptic and anti-DNA polymerase activities in parallel. On the other hand, it did not inhibit the activity of human DNA polymerase beta. Other human serum proteins including serum albumin, alpha 1-acid glycoprotein, alpha 1-antitrypsin, and immunoglobulin G as well as other protease inhibitors such as leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and chymostatin did not affect the activity of DNA polymerase alpha. Furthermore ACT heated at 60 degrees C did not inhibit DNA polymerase alpha, although it could still bind to DNA as well as native ACT. It was therefore concluded that the inhibitory action of ACT on DNA polymerase alpha was a direct phenomenon unrelated to its protease inhibitory or DNA binding activities.

The serum levels of alpha-1-antichymotrypsin (ACT) were studied in 168 patients with various liver diseases and cancers in conjunction with other liver function tests, serum sialic acid, AFP and CEA. The ACT levels in acute viral hepatitis and chronic hepatitis were not significantly altered compared with the normal level (220 +/- 40 microgram/ml), although the level was slightly increased or decreased temporarily during the acute phase of the former. In liver cirrhosis, the mean level was significantly lower than the normal in spite of the absence of signs of hepatic decompensation (168 +/- 51 microgram/ml, p less than 0.001). In contrast to cirrhosis, the levels were increased to various extents in 65% of cases with hepatoma, in spite of the association of liver cirrhosis in the majority of them. Much higher levels were observed in all cases of metastatic liver cancers and cancers of the pancreas and the biliary tract. The elevations were observed even in cases without the increase of AFP or CEA. Both in cirrhosis and cancers, ACT levels were not correlated with any of serum bilirubin and serum enzyme activities, but were positively correlated with the levels of plasma fibrinogen and serum sialic acid. The measurement of serum ACT level can be taken advantage of for the diagnosis and monitoring of liver cirrhosis and liver cancers, particularly of hepatoma without AFP elevation.

The activities and kinetic properties of argininosuccinate synthetase in cultured skin fibroblasts of three citrullinemic patients with qualitative or quantitative abnormalities of hepatic argininosuccinate synthetase were examined. The cultured skin fibroblasts of a citrullinemic patient with a low hepatic argininosuccinate synthetase activity showed low activity and abnormal kinetics similar to those of the hepatic enzyme. This suggests a similar genetic origin of the fibroblast argininosuccinate synthetase as of the hepatic enzyme. However, we found normal argininosuccinate synthetase activity in cultured fibroblasts of two citrullinemic patients who had a low hepatic enzyme activity resulting from decrease in the amount of enzyme protein but with normal kinetic properties. This suggests that part of the quantitative abnormality of hepatic argininosuccinate synthetase of citrullinemic patients may be due to an abnormal organ-specific gene expression in the liver.

Our preliminary data suggest that HPL was present in the largest quantity in chorionic villi of 16-18 weeks of pregnancy but the concentration of HPL in mother blood was maximum at 30-40 weeks of pregnancy. To clarify the discrepancy of term of those maximum values, the following experiments were done. The immuno-precipitate caused by the reaction with anti-HPL serum in the crude extract from chorionic villi incubated with MEM medium containing [3H]-leucine was subjected to SDS gel electrophoresis. Three radioactive bands were observed. Their molecular weights were 22000, 29000 and 48000, native HPL is 22000 molecular weights and other heavier molecular weights protein are surmised pre- or pro-HPL. Native HPL was few in 30 min. incubated cases but increased in over night incubated cases. Other heavier molecular weights protein changed into native HPL with the lapse of time. The concentration of HPL in mother blood is surmised to reflect the production of two pro-HPL is syncytium cells, change into free HPL and furthermore their secretion.

Sialidase activity of peripheral mononuclear cells, which are mostly lymphocytes, was found to be increased by lectin stimulations in in vitro experiments, but this induction was suppressed in the presence of 50 mM ethanol. This increasing change and its suppressive effect by lectins and ethanol were parallel to blastogenic change of the cells determined by 3H-thymidine uptake. In in vivo, sialidase activity of peripheral MNC prepared from patients with alcoholic liver disease was found to be decreased or not increased in 50% of the cases, in contrast to the marked increase of the activity in non-alcoholic liver diseases observed in our previous study.

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.

Intraperitoneal (IP) injection of a fecal anorexigenic substance (FS-T) induced significant suppression of feeding and this suppression recovered on the second day. At 2 hr after IP injection, at the time of maximum feeding suppression, plasma glucose, insulin and free fatty acid (FFA) levels did not change but amino acid level decreased. Intra-third cerebral ventricle (ICV) infusion of FS-T induced parallel but more potent feeding suppression. Analysis of meal patterns demonstrated that suppression of feeding after ICV treatment continued into the second day. FS-T was applied electrophoretically to glucose-sensitive and non glucose-sensitive neurons in the lateral hypothalamic area (LHA) and to glucoreceptor and non glucoreceptor neurons in the ventromedial hypothalamic nucleus (VMH). It significantly inhibited glucose-sensitive neurons but not non glucose-sensitive neurons, and excited both neuron types in the VMH. FS-T might thus work directly through the hypothalamic feeding control centers to suppress feeding. Even after pronase treatment of FS-T, a non-dialysable fraction of large molecular weight, consisting of protein and carbohydrate, maintained the original anorexigenic activity.

A protease degrading type IV collagen was purified more than 8000-fold from human stomach carcinoma tissue. This protease degraded type IV collagen, while type I, II, III and V collagen, laminin, fibronectin, casein, albumin and hemoglobin were not affected. This enzyme had a pH optimum of pH 7.0-8.0 and was inhibited completely by EDTA and o-phenanthroline, but not by seryl, thiol and carboxyl protease inhibitors. Furthermore, the molecular mass of this enzyme was estimated to be 1 MDa by Sepharose 6B column and HPLC-gel filtration. The molecular mass and substrate specificity of this metalloprotease from human carcinoma tissue indicate it to be a new protease.

A method for separation of serum (pseudo) cholinesterase isozymes was studied in order to take advantage of it for clinical research. By employing modifications on a previously reported method, analytical time was shortened to a half (1.5 days) of the original method without abolishing qualitative and quantitative accuracy. Thus, the present method facilitated its clinical application for the study of this isozyme. A normal pattern of the isozyme in Japanese in this method was determined by the analysis of sixteen normal subjects, which appeared to be very consistent in each individual under the present conditions. The distribution of relative activities of respective isozymes measured on a densitogram coincided well with that measured calorimetrically. Abnormalities of the zymogram were newly found in patients with liver cirrhosis and metastatic liver cancer, which seemed to be characteristic to the respective diseases. Isozyme patterns in the liver and ascites were also measured and compared with those in serum.

Enzymological and immunochemical analyses of the liver were preformed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlony's double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The Km values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher Km values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.

Human stomach adenocarcinomas containing alpha-1-antichymotrypsin (ACT) in their cell nuclei were transplanted into nude mice. The presence of ACT was monitored using an immunohistochemical technique with horseradish peroxidase-labeled rabbit anti-ACT Fab' as well as single radial immunodiffusion. Two weeks after transplantation, ACT could be found neither in transplanted carcinoma cells nor in the sera of carcinoma-bearing nude mice. However, if human ACT was injected i.v., it could be detected in the transplanted carcinoma cell nuclei 2 h after injection. The ACT was detected immunohistochemically and was confirmed by biochemical fractionation using 125I-labeled ACT. On the other hand, the amount of ACT production was not sufficient to indicate biosynthesis. These results demonstrated that ACT detected in stomach carcinoma cell nuclei was not synthesized in carcinoma cells but was incorporated from the blood circulation.

The levels of alpha 1-antichymotrypsin (ACT) was monitored in patients who underwent bone marrow transplantation. Seven received HLA-identical sibling bone marrow grafts, two received transplants from twins and one was given HLA-nonidentical marrow from his father. A dramatic increase of ACT was observed in all patients who developed graft-versus-host disease (GvHD). ACT did not rise at all in the case of patients who received marrow from twins, even in a patient who was given three transplants from the same donor. The patient transplanted from his father died from GvHD and the increase of ACT was the greatest fluctuation measured.