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1. 14 C-Labelled E/Z isomers of a synthetic pyrethroid metofluthrin ((E/Z)-(1 R,3 R)-2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylate, abbreviated as RTE/RTZ, respectively) were used for rat metabolism studies. 14 C-RTE or RTZ labelled at the carbonyl-carbon [acid-14C] or the methoxymethylbenzyl-α-carbon [alcohol-14 C] was administered orally to rats at 1 and 20 mg/kg. 2. Dosed compounds were mostly absorbed, metabolised, and rapidly excreted. Dose-related increase in blood AUC suggested no saturation of absorption at the high dose. Blood 14 C was maximal at 3–8 h and decreased with a half-life of 52–163 h. Radioactivity in tissues, blood and plasma decreased basically at the same rate and the sum fell below 0.2% of the dose at 168 h. 3. Although the major metabolic pathways of the isomers, that is, ester cleavage, O-demethylation and ω-oxidation, were similar, there was a notable difference. The RTZ double bond commonly undergoes epoxidation while RTE double bond mainly undergoes glutathione conjugation, which causes faster elimination from plasma and greater excretion into faeces on RTE. Faster urinary excretion and elimination from blood were observed for the alcohol moiety than the acid moiety. 4. In conclusion, this study described the overall metabolic profiles of metofluthrin and identified the differences in metabolic breakdown between the isomers. No marked sex-/dose-related differences were observed.

The agricultural fungicide procymidone can cause external genitalia abnormalities in rats but not monkeys or rabbits. To investigate the relevance of developmental findings in rats to humans, we conducted in vitro plasma protein binding studies, in vitro metabolism (biotransformation) studies using liver S9 fractions and hepatocytes, and in vivo metabolism and excretion studies using chimeric mice with humanized hepatocytes. On the basis of these results, we concluded that the metabolic and excretion profiles of procymidone in humans are similar to those in monkeys and rabbits but differ from those in rats. From the findings of this and previous studies, we judge the developmental toxicity potential of procymidone to be very low in humans.

The pathomechanism of inflammatory bowel disease (IBD) has not yet been fully demonstrated. However, it is well known that mast cells are present in the gastrointestinal tract, suggesting that mast cells may take part in it. So, we investigated the number of mast cells in IBD, such as ulcerative colitis (UC) and eosinophilic colitis, and showed that the number of mast cells was increased in the inflammatory lesions. We also presented a case of UC which was treated successfully with an antiallergic drug, tranilast. Furthermore, possible new approaches to treating the disease with immunomodulators including suplatast are introduced. However, our investigations were performed with a limited number of patients with IBD, and additional further studies are required to confirm the findings.

Background Aspirin-exacerbated respiratory disease (AERD) is nonatopic asthma, and the role of heat shock protein (HSP) 70 in AERD remains unknown. We analyzed HSP70 gene polymorphisms in Japanese patients with AERD. Methods The single-nucleotide polymorphisms in HSPA1B-179C>T and 1267A>G gene were examined in patients with AERD and those with aspirin-tolerant asthma (ATA). All patients were in a stable condition. Results There were significant differences in total serum IgE levels, peripheral blood eosinophil count, and prevalence of atopy between AERD and ATA. The patients with AERD showed higher frequencies of the CT/TT genotype of the HSPA1B-179C>T than that of the CC genotype compared to ATA ( P < 0.001). They showed higher frequencies of the GG genotype of the HSPA1B1267A>G than that of the GA/AA genotype compared to ATA ( P < 0.001). These differences were irrespective of the sex for the genotypes analyzed. The frequency of HSPA1B-179C/1267A haplotype was significantly higher in AERD compared to ATA ( P < 0.001; odds ratio, 3.154; 95% confidence interval, 1.916-5.193). Among the clinical and hematological characteristics investigated, AERD showed a significant variance in peripheral blood eosinophil count according to the association of the 2 HSP70 gene polymorphisms ( P = 0.033), but not in ATA. Conclusions Our findings first suggest that the association between HSPA1B-179C>T and 1267A>G gene sequence variations might be implicated in the development of AERD.

The role of interleukin (IL) 13 and IL-17A in aspirin-exacerbated respiratory disease (AERD) remains unknown.To analyze the IL-13 and IL-17A gene polymorphisms in Japanese patients with AERD.The single-nucleotide polymorphisms in each gene were examined in patients with AERD, patients with aspirin-tolerant asthma (ATA), and healthy controls.Frequencies of the TT/CT genotype of the IL-13 -1111CT gene were higher than frequencies of the CC genotype in AERD patients compared with ATA patients (P.001). In female patients with AERD, frequencies of the TT/CT genotype were higher than those of the CC genotype compared with female patients with ATA (P.001). However, genotype frequencies of IL-13 Arg110Gln did not differ between AERD and ATA patients. Frequencies of the CC genotype of the IL-17A -737CT gene were higher than those of the TT/CT genotype in AERD patients compared with ATA patients (P = .02). In female patients with AERD, frequencies of the CC genotype were higher than those of the TT/CT genotype compared with female patients with ATA (P = .03). Forced expiratory volume in 1 second (percentage predicted) in AERD patients with the CC genotype of the IL-13 -1111CT gene was lower than that in the patients with the TT/CT genotype. AERD patients with the TT/CT genotype of the IL-17A -737CT gene had a higher peripheral total eosinophil count compared with the patients with the CC genotype. The comparison of the clinical characteristics according to the IL-13 Arg110Gln gene polymorphism showed no difference.These findings suggest that the IL-13 -1111CT and IL-17A -737CT gene sequence variations might have a role in the development of AERD.

Background: There has been no report that investigated β2-adrenergic receptor (ADRB2) gene polymorphism in patients with aspirin-exacerbated respiratory disease (AERD). Methods: DNA in the specimens in three groups of study subjects classified patients with AERD, patients with aspirin-tolerant asthma (ATA) and normal controls was extracted, and the target DNA sequence of the ADRB2 was amplified using a set of primers to generate an amplicon of 219 bp in length. Allelic discrimination assay for single nucleotide polymorphisms relating to the ADRB2 gene expression was carried out by using a previously described single nucleotide polymorphism detective system, sequence-specific thermal-elution chromatography. Results: The frequency of the Gly variant allele in patients with AERD was significantly lower than that in patients with ATA (p = 0.007), and the odds ratio (OR) of AERD to ATA associated with wild-type ArgArg homozygote was 3.300. Frequencies of wild-type ArgArg homozygote are significantly higher than those of variant-type ArgGly/GlyGly genotype in patients with AERD compared with those with ATA (p < 0.001, OR = 3.153). In patients with AERD, frequencies of wild-type ArgArg homozygote in both female and male patients are significantly higher than those of variant-type ArgGly/GlyGly genotype in male patients compared with those with ATA (p < 0.001, OR = 5.128 and p = 0.007, OR = 4.367, respectively). Also, in patients with AERD, frequencies of wild-type ArgArg homozygote in female patients are significantly higher than those of variant-type ArgGly/GlyGly genotype in female patients compared with those with ATA (p = 0.002, OR = 2.825). Conclusions: We were the first to analyze Arg16Gly ADRB2 gene polymorphism in Japanese patients with AERD, and showed that Arg16Gly ADRB2 gene polymorphism in Japanese patients with AERD is different from that in the patients with ATA.

L-Propargylglycine was efficiently labeled with 14C at the 1- and 2- positions. The synthesis was achieved according to the scheme illustrated in Fig.1. Ni-complex of Schiff's base, Gly-Ni-BPB(4) was derived from [U-14C]glycine(2) and (S)-BPB(3). Diastereoselective alkylation of 4 with propargyl bromide followed by acid hydrolysis afforded L-propargyl[1, 2-14C]glycine(1). The optical purity of 1was proved to be 97.0% and the overall yield of 1 from 2 was 28.2%.

Background Comparison of the number of mast cells in the active stage and that in remission in the same patients with ulcerative colitis with immunohistochemical staining remains to be elucidated, and analysis of the number of mast cells in benign and malignant colonic lesions is insufficient. Methods Using immunohistochemical methods, morphological examinations of mast cells were undertaken in colonic tissues from 8 patients with ulcerative colitis and 10 patients with colonic primary cancer, which were formalin-fixed and paraffin-embedded. Changes in the number of mast cells in the active stage and in remission in the same patients with ulcerative colitis were investigated. Then, the number of mast cells in malignant tissues and adjacent healthy tissues obtained from the same patients with colonic primary cancer were compared, and finally the number of mast cells was compared among the samples from benign and malignant colonic lesions. Results Accumulation of mast cells was found to be significant in the active stage of ulcerative colitis compared with remission in the same patients. The number of mast cells in colonic primary cancer was significantly increased compared with that in adjacent healthy tissues. The number of mast cells in ulcerative colitis was significantly greater than that in adjacent healthy tissues from patients with colonic primary cancer, irrespective of the stages of ulcerative colitis. Conclusions We were the first to analyse mast cells in the active stage and in remission in the same patients with ulcerative colitis using immunohistochemical methods, and compared the number of mast cells between benign and malignant colonic lesions.

Background: It has been reported that measurements of eosinophil-derived neurotoxin (EDN) may be useful for identifying eosinophil activities in allergic diseases including atopic dermatitis. Methods: EDN concentrations in the urine were measured by enzyme-linked immunosorbent assay, and the number of eosinophils in the peripheral blood was counted in 30 patients with atopic dermatitis. The severity of atopic dermatitis was graded on the criteria proposed by Rajka and Langeland. The disease activity was assessed by each patient on a visual analogue scale (VAS). Results: Urinary concentrations of EDN in patients with atopic dermatitis showed a significant positive correlation with disease severity. Urine EDN concentrations also correlated with VAS scores for itching, skin condition, overall skin symptoms and total VAS score, but not with the VAS score for skin dryness. Urinary EDN concentrations did not correlate with the number of eosinophils in the peripheral blood. Conclusions: The urinary EDN concentration in patients with atopic dermatitis is a useful clinical marker for monitoring disease activity.

When [benzyl-α-(14)C]-labeled (Z)-(1R,3R)-profluthrin (2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2-dimethyl-3-(prop-1-enyl) cyclopropanecarboxylate, a newly developed pyrethroid) was administered orally to rats at 1 mg/kg, around 70% was absorbed, metabolized, and mainly excreted into urine within 48 h. Radioactivity in plasma reached Cmax at 6-8 h, and decreased (half-life; 37-52 h). A similar tendency was observed also in tissues. Absorption rate was slightly lower at high dose, while kinetics and distribution did not change. Eight metabolites were detected in urine and one in feces. Most of the (14)C in feces was unabsorbed (Z)-(1R,3R)-profluthrin. The main metabolic reactions were ester cleavage, hydroxylation of the methyl group on the C4-position of the benzene ring, and its glucuronidation or oxidation to carboxylic acid. Oxidation of the geminal dimethyl on the cyclopropane-C2 to carboxylic acid, oxidation followed by hydration of the propenyl double bond, and ω-oxidation to carboxylic acid and mercapturic acid conjugation of the benzyl alcohol were observed as minor reactions.

Background: Betaine/gamma-aminobutyric acid (GABA) signaling pathway in the airway epithelium has been revealed to play a critical role in bronchial asthma. To elucidate any genetic influence of the GABAergic in aspirinexacerbated respiratory disease (AERD), we investigated the association of solute carrier family 6 (neurotransmitter transporter, betaine/GABA) member 12 (SLC6A12) gene in Japanese patients with AERD. Methods: DNA specimens were obtained from 103 AERD patients, 300 patients with aspirin-tolerant asthma (ATA) and 100 normal controls. Allelic discrimination assay for two single nucleotides polymorphisms in SLC6A12 gene (rs499368 and rs557881) was carried out. Results: The minor allele frequencies in SLC6A12 intron 2 A>T genotype (rs49936) were higher in AERD patients than in normal controls, and that in SLC6A12 exon 4 T>C genotype (rs557881) were higher in AERD patients than in ATA patients and normal controls. The frequencies of the combined TC/CC genotype group of SLC6A12 exon 4 T>C were higher than those of the TT genotype in AERD patients compared with those in ATA patients (P=0.021; odds ratio, 1.724; 95% confidence interval, 1.087-2.732). In male patients with AERD, frequencies of the TC/CC genotype group were higher than those of the TT genotype compared with male patients with ATA (P = 0.010; odds ratio, 3.177; 95% confidence interval, 1.311-7.699). Forced expiratory volume in one second (percentage predicted) in AERD patients with the TC/CC genotype group of the SLC6A12 exon 4 T>C gene was lower than that in the patients with the TT genotype (P=0.039). Conclusion: This is the first Japanese study to investigate the SLC6A12 intron 2 A>T and SLC6A12 exon 4 T>C genotype polymorphisms in patients with AERD. Our findings suggest that the association between SLC6A12 intron 2 A>T and exon 4 T>C gene sequence variations might be implicated in the development of AERD in a Japanese population.

Background: Chronic obstructive pulmonary disease (COPD) and bronchial asthma might have common genetic factors. Interleukin 13 (IL-13) gene polymorphism has been suggested to be one of the candidates; however inconsistent results have been reported. Studies of the gene polymorphisms in IL-17A gene and mast cell chymase gene (CMA1) in COPD and bronchial asthma have not been reported. Methods: The single nucleotide polymorphisms in IL-13 -1111C>T, IL-13 Arg130Gln, IL-17A -737C>T, and CAM1 -1903G>A genes were examined in 100 COPD patients, 250 asthmatics and 100 normal control. All patients were Japanese who were in a stable condition. Results: The frequency of TT/CT genotype of the IL-13 -1111C>T was higher than that of CC genotype in COPD patients compared with asthmatics. Subgroup analyses with gender showed that in female COPD patients the frequency of TT/CT genotype of the IL-13 -1111C>T was higher than that of CC genotype compared with female asthmatics. The frequency of TT/CT genotype of the IL-17A -737C>T was lower than that of CC genotype in COPD patients compared with asthmatics. Subgroup analyses with gender showed that in male COPD patients the frequency of TT/CT genotype of the IL-17A -737C>T was lower than that of CC genotype compared with male asthmatics. The frequency of AA/GA and GG genotypes of the CMA1 -1903G>A in COPD patients did not differ from that of asthmatics. Asthmatics with CC genotype of the IL-13 Arg130Gln showed higher levels of total serum IgE than that of the patients with TT/CT genotype. Conclusion: This study suggested the IL-13 -1111C>T and IL-17A -737C>T gene sequence variations might have a role in COPD and asthma in a Japanese population.

Background: Safety concerns have been raised regarding the regular use of long-acting s2-agonists (LABAs) with inhaled corticosteroids (ICSs), and some studies suggested patients with asthma who are homozygous for arginine (ArgArg) at the 16th amino acid position of the s2-adrenergic receptor (ADRB2) gene benefit less from treatment with ICS/LABA than do those homozygous for glycine (GlyGly). Phase III trial of fluticasone/formoterol, which consisted of 8-week, multicenter randomized single-blinded parallel-group study and 52-week, multicenter open-label study, have been conducted in Japan. In the trials, we performed a pilot study on effect of ADRB2 gene polymorphisms on lung function and asthma control to regular use of the new ICS/LABA inhaler. Methods: As number of patients was limited to each institution, nine patients were allowed to participate in this pilot study. DNA in the specimens in patients with persistent asthma was extracted, and the target DNA sequence of the gene was amplified. Allelic discrimination assay for single nucleotide polymorphisms relating to the ADRB2 was carried out. Results: In the 8-week study, patients with either ArgArg or ArgGly genotype showed good changes of peak expiratory flow (PEF) values from baseline with fluticasone/formoterol treatment. In the 52-week study, the improvement of lung function tests (forced expiratory volume in one second and PEF) from baseline was observed with the treatment, and all of the daily diary-based measures of asthma control were improved from baseline in each patient over the treatment period irrespective of their genotypes of ADRB2. Conclusion: This is the first prospective pilot study, which might suggest inheritance of ADRB2 gene polymorphisms may not appear to effect clinical outcomes with fluticasone/formoterol inhaler.

Background: Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine, implicated in the development and progression of allergic diseases. Several studies indicated polymorphisms in TSLP gene were associated asthma, and two single nucleotides polymorphisms (SNPs) in TSLP (rs1837253 and rs2289276) were shown to be associated with asthma in a sex-specific fashion. However, there has been no report that investigated TSLP gene polymorphisms in patients with aspirin-exacerbated respiratory disease (AERD). Methods: DNA specimens were obtained from the following three groups: 105 patients with AERD, 270 patients with aspirin-tolerant asthma (ATA) and 90 normal controls. The target DNA sequence of the TSLP gene was amplified using a set of primers. Allelic discrimination assay for the two SNPs in TSLP gene (rs1837253 and rs2289276) was carried out. All patients were Japanese, and they were in a stable condition. Results: The frequency of the T minor allele of TSLP -5717C>T in patients with AERD and those with ATA was significantly higher than that in normal controls. There were no significant difference of the T minor allele frequency of TSLP -82C>T among the three groups. Analysis of genotype frequencies of the CT/TT genotype group and CC genotype both in TSLP -5717C>T and in TSLP -82C>T showed no differences between AERD and ATA patients. In addition, subgroup analysis of the genotype frequencies with gender did not differ between AERD and ATA patients. Conclusion: This is the first pilot study to investigate TSLP gene polymorphisms in AERD, which didn’t find an association between the TSLP gene polymorphisms and AERD susceptibility in a Japanese population, suggesting polymorphisms in TSLP gene may contribute to asthma, but not to aspirin hypersensitivity.

Aspirin-exacerbated respiratory disease (AERD) is characterized by severe asthmatic attack after taking aspirin and/or nonsteroidal anti-inflammatory drugs (NSAIDs). The typical patient with AERD is an adult who develops refractory chronic rhinitis in the third or fourth decade of life. Natural history and clinical features of AERD indicates that during the evaluation of chronic rhinitis persistent bronchial asthma develops, and finally after exposure to NSAID acute respiratory reactions begin to occur. The inhibitory action of aspirin and/or NSAIDs on cyclooxygenase activity may cause diversion to the 5-lipoxygenase pathway, which leads to the overproduction of cysteinyl leukotrienes (LTs). Thus, a general consensus exists that increased levels of cysteinyl LTs are key inflammatory mediators in AERD. As aspirin intolerance is found in a specific population, genetic predisposition has been considered as a crucial determinant. Investigations on candidate genes have been concentrated especially on cysteinyl LTs-related genes, however conflicting results have been reported. So, future areas of investigations need to focus on comprehensive approaches towards other genetic biomarkers. We’ve recently reported possible presence of other gene polymorphisms in Japanese patients with AERD. The natural history and clinical characteristics of AERD indicate that the respiratory mucosal inflammatory process in AERD begins and continues in the absence of ongoing or even intermittent exposure to NSAIDs. So, in this review, we propose a hypothetical progress of AERD over time based on mainly the results of our investigations, and present a schematically sketching the course of NSAIDs-triggered hypersensitivity with genes, such as asthma-associated genes, that may initiate susceptibility to AERD, and that may accelerate pathogenesis and induce onset of AERD. The findings of our studies were based on small-sized samples from a Japanese population, and future validation studies in independent populations are required to provide reassurance about our hypothesis.

Uptake and transformation of 14C-labeled metabolites from several pesticides, 3-methyl-4-nitrophenol (1), 3,5-dichloroaniline (2), 3-phenoxybenzoic acid (3), (R,S)-2-(4-chlorophenyl)-3-methylbutanoic acid (4), and (1RS)-trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (5), were examined by using duckweed (Lemna gibba) in Hoagland's medium. More uptake into duckweed from the exposure water at pH 7.0 was observed for non-ionized 1 and 2 than for 3-5 in an ionized form, and their hydrophobicity accounted for these differences. While carboxylic acids 4 and 5 were scarcely transformed in duckweed, 1-3 mainly underwent phase II conjugation with glucose for 1 and 2, malic acid for 3, glutamic acid for 2, and malonylglucose for 3, the chemical identities of which were confirmed by various spectrometric analyses (LC-MS, LC-MS/MS, and NMR) and/or HPLC cochromatography with reference synthetic standards.

Background It has been reported that measurements of eosinophil-derived neurotoxin (EDN) may be useful to determine eosinophil activities in allergic diseases. Methods Serum, plasma and urine concentrations of EDN in patients with allergic diseases were measured by enzyme-linked immunosorbent assay. Samples of blood and urine were obtained from the same patients when their symptoms were active and when patients were well. The number of eosinophils in peripheral blood was also counted. Results The median concentrations of EDN in blood and urine and the number of eosinophils in peripheral blood from patients with active symptoms were all significantly higher compared with values obtained when patients were well. Compared with healthy control subjects, EDN concentrations in the serum, plasma and urine from asymptomatic asthmatic patients, as well as EDN concentrations in the serum and plasma from patients with mild atopic dermatitis and asymptomatic patients with seasonal allergic rhinitis, were significantly higher. Blood and urine concentrations of EDN in patients with active symptoms, but not urine concentrations of EDN from symptomatic patients with seasonal allergic rhinitis, were significantly increased compared with values obtained for healthy control subjects. The concentrations of EDN in the serum and plasma from bronchial asthmatic patients, serum from patients with atopic dermatitis and plasma from patients with mild atopic dermatitis correlated with the number of eosinophils in peripheral blood; however, the concentration of EDN in the serum correlated with the number of eosinophils from asymptomatic patients with seasonal allergic rhinitis. Conclusions Determination of blood and urinary concentrations of EDN is useful to monitor eosinophil activity in allergic diseases.

Background: It has been reported that the number of mast cells was significantly greater in malignant breast carcinomas than in benign breast lesions. This was due to tryptase-containing mast cells while tryptase, chymase-containing mast cells had no effect. However, analysis of mast cells in breast carcinomas and benign breast lesions based on their histological findings remains to be elucidated. Methods: Using immunohistochemical methods morphological examinations of mast cells were undertaken in benign and malignant breast tissues from 51 patients (30 benign, 21 malignant), which were formalin-fixed and paraffin-embedded. In the study with malignant breast tissues, samples of malignant tissues and adjacent healthy tissues were obtained from a single patient, and the number of mast cells was compared. Results: Among benign breast tissues, the number of mast cells in intracanalicular fibroadenoma was significantly lower than that in pericanalicular fibroadenoma as well as that in mastopathy. The number of mast cells was significantly greater in malignant lesions than that in benign lesions. The number of mast cells in scirrhous carcinoma and that in solid-tubular carcinoma were significantly increased compared with that in adjacent healthy tissues. In addition, the number of mast cells in scirrhous carcinoma was highest among breast carcinomas, and significantly greater than that in papillotubular carcinoma. Conclusion: We were the first to find the significant lower number of mast cells in intracanalicular breast fibroadenoma when compared with that in pericanalicular fibroadenoma as well as that in mastopathy. Moreover, the number of mast cells in scirrhous carcinoma was significantly greater than that in papillotubular carcinoma.

We report here a 47-year-old female who had episode of acute pancreatitis three times after banana ingestion. After each admission clinical symptoms such as epigastric pain, nausea, vomiting and diarrhea were disappeared within a few days with a light diet and intravenous fluids in parallel with normalizing serum amylase levels. Serum total IgE level was elevated and serum specific IgE level to banana was 2.18 UA/ml. No remarkable abnormalities were present in sonography, computed tomography (CT), magnetic resonance image (MRI) on the abdomen, and magnetic resonance cholangiopancreatography (MRCP). Endoscopic examination of the upper digestive tract, showed that the ampulla of Vater was swollen and edematous. Toluidine blue staining and immunohistochemical staining against human mast cell tryptase with biopsy specimens showed mast cells accumulation in mucosa and submucosa of the stomach. Even though the provocation test by banana could not be performed, the patient was diagnosed as having pancreatitis possibly caused by allergy to banana because she has not suffered any attacks of pancreatitis since the third attack by avoiding ingestion of banana.

Background: An increase in mast cell number at sites of inflamed tissues has been observed. However, the expression of CXC chemokine receptors on human mast cells is poorly understood. Methods: Cultured human mast cells were raised from human umbilical cord blood cells in the presence of stem cell factor and interleukin-6 (IL-6). The expression of surface chemokine receptors on the mast cells was analyzed by flow cytometry and that of mRNA was examined by the method of reverse transcriptase-polymerase chain reaction (RT-PCR). As functional assays for the receptors, mast cell migration was examined by a microchemotaxis assay and changes in the cytosolic free intracellular calcium concentrations ([Ca2+]i) was measured using fura-2-loaded mast cells, respectively. Results: Expression of IL-8 receptors CXCR1 and CXCR2 was demonstrated by flow cytometry and of both mRNA by RT-PCR; however, CC chemokine receptors including CCR3 were not expressed on cord-blood-derived cultured human mast cells. IL-8 and its homologues showed chemotactic activity toward them in a dose-dependent manner, and IL-8 induced a dose-dependent rapid and transient increase in [Ca2+]i in the mast cells. Conclusions: Our results suggest the surface expression of functional CXCR1 and CXCR2 on cord-blood-derived cultured human mast cells.

Background Accumulating data indicates that pholcodine (PHO)-consuming countries have higher sero-prevalences of immunoglobulin E (IgE)-antibodies to PHO and suxamethonium (SUX) and increased frequencies of IgE-mediated anaphylaxis to neuromuscular blocking agents (NMBAs) than nonconsuming. Withdrawing PHO-containing cough syrups resulted in a significant decrease of cases with anaphylaxis in Scandinavia. Nevertheless, the European Medicines Agency in 2011 advised to continue the unrestricted use throughout the European Union. Objective To extend studies on PHO consumption and prevalence of IgE-sensitization to morphine (MOR), PHO, and SUX to countries representing high (Australia), and low (Korea and Japan), consumers, respectively. Methods IgE-antibodies to SUX, MOR, and PHO in atopic subjects were determined by immunoassay and compared with official figures for PHO consumption and reported anaphylaxis to NMBA. Results The prevalences of IgE-antibodies to PHO, MOR, and SUX were 10%, 8.6%, and 4.3%, respectively, in Australia. The corresponding figures for Japan were 0.8%, 0.8%, and 1.5%, and for Korea 1.0% to PHO and 0.5% to MOR and SUX. Of the SUX-positive sera, 100% were positive to PHO or MOR in Australia and 0% in Japan and Korea. Conclusion The study supports previous findings; exposure to PHO may induce IgE-antibodies to the substituted ammonium ion epitope of NMBAs, thus increasing risk of NMBA-induced anaphylaxis considerably. However, other, still unknown factors occasionally might induce IgE-antibodies to SUX.

Aspirin-exacerbated respiratory disease (AERD) is a complex clinical syndrome characterised by severe asthmatic attack upon treatment with aspirin and/or non-steroidal anti-inflammatory drugs (NSAIDs). Genetic predisposition has been considered as a crucial determinant and candidate genes have concentrated especially on cysteinyl leukotrienes (LTs)-related genes as the inhibitory action of aspirin and NSAIDs on cyclooxygenase activity may cause overproduction of cysteinyl LTs. However, conflicting results have been reported, in parallel with replication studies in different ethnic groups. Thus, future areas of investigations need to focus on comprehensive approaches towards the discovery of other genetic biomarkers. Unfortunately, few papers have been reported about gene polymorphisms in Japanese patients with AERD. Here, we described on our recent genetic investigations on B2ADR, IL-13, IL-17A, CYP2C19, TBXA2R, CRTH2 and HSP70. This review indicates potential genetic biomarkers contributing to the early diagnosis of AERD, which may include CYP2C19 and HSP70 gene polymorphisms, and future validation studies in independent population are required to provide reassurance about our findings.

Both endotoxic and antagonistic [ 3 H]-labeled 2-(phosphonooxy)ethyl (PE) analogs of lipid A were synthesized with high purity and high specific radioactivity. Lipid A-binding proteins were detected by using the endotoxic analog of hexaacyl Escherichia coli -type designated [ 3 H] PE-506. The plasma membrane fractions from peritoneal macrophages derived from LPS-responder C3H/HeN mice and LPS-hyporesponder C3H/HeJ mice were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then incubated with the [ 3 H] PE-506. Several [ 3 H] PE506 binding proteins were detected in both C3H/HeN and C3H/HeJ macrophages. Unlabeled hexaacyl lipid A inhibited the interaction between [ 3 H] PE-506 and these proteins. The result suggests that there exist multiple binding sites for lipid A on macrophages. LPS-induced change in the profile of the cell surface lipid A binding proteins was observed in C3H/HeN macrophages, but not in C3H/HeJ macrophages, by preincubation of macrophages with LPS.

Background: Mast cells are a potential source of cytokines, but their contribution to nonallergic inflammatory conditions, such as fibrosis, remains unclear. Objective: We investigated whether cord blood–derived cultured human mast cells could produce fibrogenic cytokines by IgE-mediated activation. Methods: Mast cells were obtained from human cord blood mononuclear cells by culture with stem cell factor and IL-6. The mast cells were incubated with human myeloma IgE and were activated with anti-IgE. The expression of messenger RNA for fibrogenic cytokines was examined by the reverse-transcription polymerase chain reaction, and cytokine protein was assayed by enzyme-linked immunosorbent assay, or bioassay. Results: Cultured human mast cells constitutively expressed mRNA for transforming growth factor-β 1 , and its expression was not increased by anti-IgE stimulation. The cells released this factor into the culture medium spontaneously, which showed bioactivity after heat treatment. The mast cells also expressed mRNA for platelet-derived growth factor A, which was enhanced with a peak at 3 hours by stimulation with anti-IgE. Conditioned medium from nonactivated mast cells did not contain basic fibroblast growth factor, but this cytokine was released into the medium in a time-dependent manner after stimulation with anti-IgE. Conclusion: Human mast cells activated by IgE-mediated stimulation show production of fibrogenic cytokines that varies depending on the cytokine, which suggests possible involvement of mast cell cytokines in the development of fibrosis. (J Allergy Clin Immunol 2000;106:S85-90.)

Extracellular matrix-destructive enzymes, like matrix metalloproteinases (MMP), have been recognized in the process of inflammation and tissue remodeling and repair. The affected tissues often contain markedly increased numbers of mast cells. Although mast cells are capable of activating latent collagenase and proMMP, it has so far been unknown whether human mast cells themselves produce and secrete MMP9. In this study, MMP9 production by cord blood-derived cultured human mast cells and HMC-1 human mast cells was examined by reverse-transcriptase PCR, gelatin zymography and Western blot analysis using an antibody against MMP9. Cultured mast cells and HMC-1 cells treated with phorbol 12-myristate 13-acetate were shown to express MMP9 mRNA, and the cultured conditioned media from these cells showed gelatinolytic activity, identical with MMP9. Immunohistochemical examination was performed to detect MMP9 in tissue mast cells; mast cells localized in the skin, lung and synovial tissue showed strongly positive reactions for MMP9. Thus, these findings indicate that human mast cells can produce MMP9, which might contribute to extracellular matrix degradation and absorption in the process of allergic and nonallergic responses.

Background Mast cells frequently accumulate at the site of fibrosis and their contribution has been suspected in the pathogenesis of fibrotic conditions. However, it still remains unknown whether human mast cells synthesize transforming growth factor β1 (TGF-β1). Objective We have investigated whether cord blood-derived human cultured mast cells express messenger RNA (mRNA) for TGF-β and produce bioactive TGF-β1. Methods Mast cells were obtained by culturing mononuclear cells from cord blood in the presence of stem cell factor and interleukin-6. Expression of mRNA for TGF-β1 was examined by the method of reverse transcriptase-polymerase chain reaction (RT-PCR). Immunocytochemical staining for TGF-β and growth-inhibitory assay using Mv1Lu cells were also performed. Results The cultured human mast cells constitutively expressed mRNA for TGF-β1. With calcium ionophore A23187, the intensity of the PCR-amplified band for TGF-β1 was not increased. Immunocytochemical staining showed that the cultured mast cells were positive for both latency-associated peptide and activated forms of TGF-β. Bioassay with Mv1Lu cells and R 4-2 mutant cells showed that mast-cell conditioned medium had a bioactivity of TGF-β1. Conclusion Cord blood-derived human cultured mast cells constitutively express mRNA for TGF-β1 and produce functional TGF-β1. Because TGF-β1 has been shown to be highly fibrogenic, these results may highlight a novel role for human mast cells in tissue fibrosis.

Background Mast cell tryptase has been shown to be mitogenic for fibroblasts, however, it still remains unknown whether mast cell tryptase stimulates collagen production by human derrmal fibroblasts. Objective We have investigated the effect of mast cell tryptase on type I collagen production by human dermal fibroblasts as well as the proliferation of the fibroblasts. Methods Tryptase isolated from human lung tissue was added to the culture of fibroblasts from normal dermis, and the fibroblast proliferation and the activity of type I collagen synthesis in the supernatants were assayed, respectively. Results Fibroblast proliferation was increased with tryptase in a concentration-dependent manner, and a significant increase was observed in the presence of tryptase at concentrations from 0.01 to 10 microg/mL. The increase of fibroblast proliferation with 3 microg/mL tryptase was significantly reduced by 15 microg/mL antitryptase IgG antibody, which was demonstrated to inhibit fibrinogenolysis of tryptase. On the other hand, the production of type I collagen by the fibroblasts was significantly increased with tryptase at a concentration of 10 microg/mL. The collagen production in the presence of 10 microg/mL tryptase was significantly inhibited by 50 microg/mL antitryptase IgG antibody. Conclusion Tryptase increases not only the proliferation of human dermal fibroblasts but also type I collagen production.

Background Isolating human mast cells is a laborious procedure. Recently, cultured human mast cells raised from umbilical cord blood cells have become available. It is necessary to investigate whether IgE-mediated activation of these cells is mediated by exocytosis. Objective To verify IgE-mediated activation of these cultured human mast cells morphologically. Methods The mast cells were raised from human umbilical cord blood cells in the presence of stem cell factor and interleukin-6. IgE-sensitized cultured human mast cells were activated by anti-IgE, and morphological changes of the cells were examined under phase-contrast microscopy using cinematographic techniques and scanning electron microscopy. Histamine release from the cells was measured with high-performance liquid chromatography. Results Under the condition in which a significant histamine release was observed from the mast cells, phase-contrast microscopy showed that the cultured human mast cells became swollen and extruded granules. Scanning electron microscopy disclosed the extrusion of smooth and round bodies from pores formed on the activated mast cell surface. Conclusion IgE-mediated histamine release from cultured human mast cells is accompanied by exocytosis morphologically, indicating that cultured human mast cells will help in studying the functional properties of human mast cells.

Background: Mastocytosis is a disorder of mast cell proliferation that occurs in both cutaneous and systemic forms. The most frequent site is the skin. Objective: The mast cell subtype of two patients with mastocytosis was investigated. Methods: Immunohistochemical studies were performed on the skin or gastric mucosa or both of the two patients. Blood and urine levels of various mediators were measured for one patient. Results: Mast cells containing tryptase and chymase were the only type seen in the skin lesions of an 11-month-old boy with urticaria pigmentosa. Mast cells containing tryptase were predominant in lesions of the skin and gastric mucosa of a 41-year-old man with indolent systemic mastocytosis. However, mast cells containing tryptase and chymase were predominant in the nonlesional and the normal skin of this patient. Tryptase-positive cells were more numerous in lesional skin than nonlesional skin and normal skin. Elevated blood and urine levels of various mediators were decreased by means of combination therapy with ketotifen and ranitidine. Conclusion: In indolent systemic mastocytosis, mast cell dynamics involve only cells containing tryptase. Release of mediators from mast cells may be inhibited by means of combination therapy with histamine H1 and H2 receptor antagonists. (J Allergy Clin Immunol 1997;100:S25-32.)

Summary Background Ultraviolet light irradiation has been shown to suppress immunological reactions of irradiated individuals, however, less attention has been paid to the effects on neurogenic inflammation. Objective We have investigated the effect of ultraviolet light B (UVB) irradiation on substance P (SP)-induced histamine release from rat mast cells. Methods Rat peritoneal mast cells were treated with UVB irradiation and challenged by SP. Histamine release from the cells and intracellular calcium concentrations ([Ca2+]i) in the cells were measured. Results UVB irradiation at doses used in the present study did not induce histamine release from mast cells. UVB irradiation at doses from 25 to 200mJ/cm2 inhibited 10–5mol/L SP-induced histamine release in a dose-dependent manner. On the other hand, SP-induced increase of [Ca2+]i was inhibited by UVB irradiation only at doses of 100 and 200 mJ/cm2. Conclusion These data suggest that UVB irradiation inhibits histamine release from SP-activated rat peritoneal mast cells partially through the suppression of an increase in [Ca2+]i.

(+)-Methyl [2-[(2R,3aS,4R,5R,6aS)-octahydro-5-hydroxy-4-[(E)-(3S,5S)-3-hydroxy-5-methyl-1-[3- 14 C]nonenyl]-2-pentalenyl]ethoxy]-acetate ([nonenyl-3- 14 C]SM-10902) (1) was labeled with carbon-14 for use in mammalian metabolic studies. The synthesis was achieved in 7 steps from [ 14 C]carbon dioxide: including : 1) Grignard reaction, 2) esterification, 3) condensation with dimethyl methylphosphonate, 4) Wittig-Horner reaction, 5) separation of isomers by HPLC. Stereoselective reduction of the protected ketone with sodium borohydride in the presence of cerium (III) chloride and subsequent desilylation produced 1. The overall yield was 11.1% from Ba[ 14 C]CO 3

Clinically apparent panniculitis is rare in dermatomyositis. The common histopathological findings are infiltration of lymphocytes, epithelioid cells and plasma cells in the fat lobules, along with varying degrees of fat degeneration and fibrosis. We report a 65-year-old woman with dermatomyositis who developed panniculitis with a characteristic histological change known as a membranocystic lesion. Although this change has been observed in various diseases affecting the subcutaneous fat tissue, it has rarely been reported in dermatomyositis. Dermatomyositis should be included in the diseases showing a membranocystic lesion.

We studied the effect of intravenous administration of platelet-activating factor (PAF) on airway responsiveness to histamine and airway wall thickening in guinea pigs. Guinea pigs were killed and the lungs were fixed in formalin. Slides from paraffin-embedded sections of the lungs were stained and the airways that were cut in transverse section were measured by tracing enlarged images using a digitizer. Moreover, airway resistance (Raw) was determined by a pulmonary mechanics analyzer and we calculated two indices, an index of airway wall thickening and one of airway hyperresponsiveness to histamine, from changes of baseline-Raw and peak-Raw following intravenous administration of histamine before and after PAF administration. The infusion of 600 ng/kg PAF for 1 h induced an increase in the thickness of the airway wall in main bronchi demonstrable by histological examination. In analysis of airway function, the infusion of 600 ng/kg PAF for 1 h induced airway hyperresponsiveness to histamine with airway wall thickening. The PAF antagonist CV6209, which was given intravenously 30 min prior to PAF administration, inhibited these effects of PAF in a dose-dependent manner, suggesting PAF may induce airway wall thickening and airway hyperresponsiveness through PAF receptors in the airways.

(2S,3R)-2-[3-(4-Fluorophenyl)]propylamino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid pyrrolidine amide hydrobromide (SM-11044) was labeled with carbon-14 for use in mammalian metabolic studies. The synthesis was achieved according to the scheme shown in Fig. 5. Grignard reaction of 3,4-methylenedioxy-phenylmagnesium bromide with [14C]carbon dioxide liberated from barium [14C]carbonate ((UNDERLINE)6(/UNDERLINE)) gave the acid ((UNDERLINE)5(/UNDERLINE)). Reduction of (UNDERLINE)5(/UNDERLINE) with lithium aluminum hydride followed by oxidation of the resulting benzyl alcohol ((UNDERLINE)11(/UNDERLINE)) with chromium oxide-pyridine complex afforded the aldehyde ((UNDERLINE)3(/UNDERLINE)). Condensation of (UNDERLINE)3(/UNDERLINE) with the optically active (R)-oxazolidinone ((UNDERLINE)10(/UNDERLINE)) yielded the alcohol ((UNDERLINE)12(/UNDERLINE)). Catalytic hydrogenation of (UNDERLINE)12(/UNDERLINE) and subsequent hydrolysis produced the optically active β-hydroxy-α-amino acid ((UNDERLINE)2(/UNDERLINE)), which was treated with N-carbomethoxyphthalimide to give the hydroxy acid ((UNDERLINE)13(/UNDERLINE)). Reaction of (UNDERLINE)13(/UNDERLINE) with pivaloyl chloride gave the corresponding mixed anhydride, which was allowed to react with pyrrolidine to provide the amide ((UNDERLINE)14(/UNDERLINE)). Deprotection of (UNDERLINE)14(/UNDERLINE) with hydrazine hydrate afforded the hydroxyamide (15). Condensation of (UNDERLINE)15(/UNDERLINE) with the aldehyde ((UNDERLINE)16(/UNDERLINE)) and subsequent reduction of the resulting imine with sodium cyanoborohydride produced the fluoride ((UNDERLINE)17(/UNDERLINE)). Cleavage of the methylenedioxy group of (UNDERLINE)17(/UNDERLINE) gave the free base of (UNDERLINE)1(/UNDERLINE), which was treated with hydrobromic acid to afford (UNDERLINE)1(/UNDERLINE). The overall yield was 11.1% from (UNDERLINE)6(/UNDERLINE).

A synthetic route for 14 C-labeling of a phosphonooxyethyl (PE) analogue of E. coli lipid A was established by cold experiments. The PE analogue is chemically more stable than natural lipid A but shows practically identical biological activity. The glycosidation of ethylene glycol unit as the 14 C-labeled position was effected with a-disaccharide thioglycoside by use of a hypervalent iodine reagent, PhIO-SnClO 4 -AgClO 4 , as an activator. p -Methoxybenzyl group used for the protection of the distal hydroxyl group of ethylene glycol was selectively removed by trimethylsilyl triflate in CH 3 CNCH 2 Cl 2 followed by phosphorylation and deprotection gave the target compound.

Summary We studied the effect of intravenous administration of leukotriene (LT) C4 or LTD., on airway responsiveness to histamine and airway wall thickening in guinea-pigs. Guinea-pigs were killed and the lungs were fixed in formalin. Slides from paraffin-embedded section of the lungs were stained and the airways that were cut in transverse section were measured by tracing enlarged images using a digitizer, Moreover, airway resistance (Raw) was determined by a pulmonary mechanics analyser and we calculated two indices, an index of airway wall thickening and the one of airway hyperresponsiveness to histamine, from changes of baseline-Raw and peak-Raw following intravenous administration of histamine before and after the intravenous administration of LTC4 or LTD4. The infusion of LTC4 or LTD4 induced an increase of the relative thickness of the airway wall in peripheral bronchi demonstrable by the histological examination. In analysis of airway function, intravenous administration of LTC4 or LTD4 induced airway hyperresponsiveness to histamine with airway wall thickening. The LTC4 and LTD4 receptor antagonist ONO-1078 inhibited these effects of LTC4 and LTD4, suggesting LTC4 and LTD4 may induce airway wall thickening and airway hyperresponsiveness through LTC4 and LTD4 receptors in the airways.

Four compounds corresponding to partial structures of Re-type lipopolysaccharide (LPS) were synthesized for precise investigation of immunological properties of the 3-deoxy-D- manno-2-octulosonic acid (Kdo)-containing inner core and lipid A regions of LPS. The synthetic compounds lack all the ester-bound acyl groups which are present in natural LPS but known to be not essential for the antigenicity. The simplest compound is N,N'-diacylated glucosamine β1-6 disaccharide 4'-monophosphate 2 which corresponds to the des- O-acyl derivative of 1-dephospho lipid A. The other three contain one or two Kdo moieties linked to 2. The most complex one is a tetrasaccharide equivalent to the des- O-acylated 1-dephosphorylated derivative of LPS from Escherichia coli Re-mutant. Syntheses were performed by stepwise condensation of appropriately protected sugar moieties and final hydrogenolytic deprotection. The synthetic des- O-acyl LPS analogs were effectively purified by means of reversed phase HPLC to give homogeneous preparations.

New bifunctional reagents, 2-amino-6-carboxyethylpyridine and 2-amino-6-cyanoethylpyridine, were designed and synthesized in order to provide a novel procedure for preparation of neoglycoconjugates from fluorescence-labeled and purified sugar chains. Labeling of model sugar chains with these reagents was effected by reductive amination using BH3.Me2NH to give corresponding 6-carboxyethylpyridylaminated (CEPA-) and 6-cyanoethylpyridylaminated (CNEPA-) derivatives, which were readily purified by reversed phase HPLC. The reagent parts of the labeled products possess the functional groups which then serve as linkers for coupling with matrices such as proteins and polymers. A CEPA-derivative of glucose was directly coupled with the e-amino group of a Lys derivative to give a neoglycoprotein model. CNEPA-sugars were hydrogenated to give 6-aminopropylpyridylaminated (APPA-) derivatives, which can then be readily used for the preparation of various types of neoglycoconjugates by use of appropriate spacer...

Prostaglandin F2 alpha (PGF2 alpha) has been suggested to play a role in the pathogenesis of bronchial asthma. In this study, the effects of intravenous administration of 13,14-dihydro-15-keto-PGF2 alpha, a stable metabolite of PGF2 alpha, on bronchial smooth muscle in guinea pigs were investigated by measuring dynamic respiratory resistance using a formula that excludes the effects of differences in airway wall thickness. With this formula, the ratio of bronchial smooth muscle constriction by histamine can be estimated as an index of bronchial hyperresponsiveness. Administration of 13,14-dihydro-15-keto-PGF2 alpha did not induce airway wall edema. The ratio of bronchial smooth muscle constriction by histamine was significantly enhanced by the administration of 13,14-dihydro-15-keto-PGF2 alpha. Moreover, TXA2 antagonists, ONO-NT-126 and ONO-8809, inhibited the effect of 13,14-dihydro-15-keto-PGF2 alpha administration. These results suggest that 13,14-dihydro-15-keto-PGF2 alpha can be important mediators affecting bronchial hyperresponsiveness, and TXA2 may play a part in the 13,14-dihydro-15-keto-PGF2 alpha-induced bronchial hyperresponsiveness.

Peripheral blood eosinophils from patients with atopic dermatitis and normal healthy controls were isolated on a Percoll gradient and incubated with [gamma 32P]ATP in the presence of Mg2+. After stopping the reaction, lipid extraction was performed with acidic medium and phospholipids were separated by thin-layer chromatography on 1% (w/v) oxalic acid-impregnated silica gel plates. In the autoradiographs considerable amounts of the radioactivity were found to be incorporated into phosphatidylinositol-4-phosphate. 32P incorporation into phosphatidylinositol-4-phosphate in hypodense eosinophils from atopic dermatitis patients was less than in normodense eosinophils from the same patients and controls.