Junichiro Morioka

Quantitative Analysis of Mast Cells in Benign and Malignant Breast Lesions

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.

Development of a Novel Enzyme-Linked Immunosorbent Assay for Blood and Urinary Eosinophil-Derived Neurotoxin: A Preliminary Study in Patients with Bronchial Asthma

Background: Eosinophil-derived neurotoxin (EDN), also called eosinophil protein X (EPX), has been suggested to be a useful marker of eosinophilic inflammation. However, no commercial enzyme-linked immunosorbent assay (ELISA) kit for EDN is available yet. Methods: EDN was purified from pooled urine from healthy male volunteers. Polyclonal and monoclonal anti-EDN antibodies were subsequently raised, and a sandwich ELISA for EDN was established. EDN levels in serum, plasma and urine from asymptomatic patients with bronchial asthma were measured by the ELISA method. Some of the blood samples were also measured by a commercial radioimmunoassay (RIA) kit. Results: The ELISA method detected human EDN with a minimum detection limit of less than 0.62 ng/ml and did not cross-react with other eosinophil granule cationic proteins including eosinophil cationic protein. The intra- and interassay coefficients of variation of the ELISA method ranged from 2.6 to 3.6% and from 6.5 to 9.4%, respectively. Good linearity was observed with serially diluted different samples, and the recoveries of the purified EDN added to serum samples ranged from 85 to 110%. Median EDN concentrations in serum (36.9 vs. 19.1 ng/ml), plasma (23.0 vs. 14.5 ng/ml) and urine (118.2 vs. 72.1 μg/mmol Cr) were significantly raised in asymptomatic asthmatic patients compared with healthy control subjects. EDN levels in serum, plasma and urine from the patients significantly correlated with the number of peripheral blood eosinophils, but not total serum IgE levels. A significant relationship between EDN values measured by the EPX-RIA kit and the EDN-ELISA method was observed. Conclusions: We have developed a novel efficient ELISA method to specifically measure blood and urinary EDN, which may be useful to study the role of eosinophils in allergic diseases including bronchial asthma.

Thromboxane A2 receptor +795T>C and chemoattractant receptor-homologous molecule expressed on Th2 cells -466T>C gene polymorphisms in patients with aspirin-exacerbated respiratory disease.

It is well known that aspirin-exacerbated respiratory disease (AERD) is more common in women than in men, however, whether gene polymorphisms of the thromboxane A2 receptor (TBXA2R) and chemoattractant receptor-homologous molecules expressed on Th2 cells (CRTH2) are associated with the susceptibility of AERD remains unknown. In this study, we examined the gene polymorphisms in a Japanese population. DNA specimens were obtained from the following three groups: 96 patients with AERD, 500 patients with aspirin-tolerant asthma (ATA) and 100 normal controls. The target DNA sequence of each gene was amplified, and an allelic discrimination assay for single nucleotide polymorphisms relating to expression of each gene was carried out. The frequencies of the CC/CT genotype of TBXA2R +795T>C were higher than those of the TT genotype in AERD patients compared to ATA patients (P=0.015). In female AERD patients, but not in males, frequencies of the CC/CT genotype were higher than those of the TT genotype of TBXA2R +795T>C compared to female ATA patients (P=0.013). Also, frequencies of the TT genotype of CRTH2 -466T>C were higher than those of the CC/CT genotype in AERD patients compared to ATA patients (P=0.034). In female AERD patients, but not in male, frequencies of the TT genotype were higher than those of the CC/CT genotype of CRTH2 -466T>C in AERD patients compared to female ATA patients (P=0.046). Based on our investigations, no significant relationship was found between the genotype and the clinical characteristics according to these gene polymorphisms in AERD patients. Our results suggest that an association between the TBXA2R and CRTH2 gene polymorphisms with AERD may exist in the Japanese population.

Arg16Gly β2-Adrenergic Receptor Gene Polymorphism in Japanese Patients with Aspirin-Exacerbated Respiratory Disease

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.