Takafumi Etoh

Regulation of interleukin-5 production by peripheral blood mononuclear cells from atopic patients with FK506, cyclosporin A and glucocorticoid.

Upon stimulation with phorbol ester and ionomycin, peripheral blood mononuclear cells (PBMC) of atopic patients with moderate eosinophilia produced significantly higher amounts of IL-5 compared to that of normal subjects. This finding renders further support to the notion that T cell-eosinophilic inflammation plays a central role in allergic disorders. IL-5 induction in vitro was completely inhibited by immunosuppressant FK506, cyclosporin A and dexamethasone. FK506 applied in vivo effectively suppressed clinical symptoms of atopic dermatitis and IL-5 production of PBMC. FK506 and cyclosporin A may become a better therapeutic modality against allergic diseases.

Tacrolimus Has Antifungal Activities against Malassezia furfur Isolated from Healthy Adults and Patients with Atopic Dermatitis

SummaryTacrolimus (FK506), a new immunosuppressant used clinically in the prevention of allograft rejection, has been shown to be effective when used topically in atopic dermatitis, especially for face and neck lesions. Because Malassezia furfur, normally found in the seborrhoeic area, has been suggested to be one of the major allergens in atopic dermatitis and a provocative factor for face and neck lesions, the antifungal activity of tacrolimus against M. furfur was investigated. 26 strains of M. furfur were isolated from healthy adults and adult patients with atopic dermatitis. The growth of all the strains was inhibited by 0.5 to 32 mg/L of tacrolimus, whereas cyclosporin showed no such antifungal activity. This inhibition was found to be mediated by the fungicidal effects of tacrolimus. Microscopic examinations of M. furfur on the faces of patients with atopic dermatitis showed markedly diminished numbers of spores after 1 week’s treatment with topical tacrolimus ointment. The antifungal effects of tacrolimus against the causative allergenic yeast, M. furfur, may also be beneficial when treating face and neck atopic dermatitis with tacrolimus ointment.

Lack of primary association between transporter associated with antigen processing genes and atopic dermatitis

We examined polymorphisms of transporter associated with antigen processing (TAP) genes in 37 Japanese patients with atopic dermatitis and 52 control subjects. We have evaluated, again, the specificity polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method since our previous report and have also developed a mismatch PCR-RFLP method for discriminating two dimorphic sites of TAP1 gene and four dimorphic sites of TAP2 gene. Amplified products from genomic DNA were digested with restriction endonucleases: Sau3A1 for TAP1 codon 333 (Ile-Val), AccI for TAP1 codon 637 (Asp-Gly), and BfaI for TAP2 codon 687 (Stop-Gln). The other sites of TAP2 gene were analyzed by mismatch PCR-RFLP: AccII for TAP2 codon 379 (Val-Ile), RsaI for codon 565 (Ala-Thr), and MspI for codon 665 (Thr-Ala). We observed three TAP1 alleles and six TAP2 alleles. Infrequent allele TAP1 C was observed in one patient and one control subject. We did not identify any differences in TAP allele frequencies between those patients with atopic dermatitis and Japanese control subjects. Analysis of TAP gene polymorphisms will provide better understanding of susceptibility loci in HLA class II-associated disease because TAP genes are located between HLA-DQB1 and HLA-DPB1 loci.

Polymorphisms of transporter associated with antigen processing genes in atopic dermatitis

We investigated polymorphisms of transporter associated with antigen process (TAP) genes in atopic dermatitis. We developed a polymerase chain reaction-restriction fragment length polymorphism method for discriminating TAP alleles. Genomic DNA was obtained from 29 Japanese patients with atopic dermatitis and 35 control subjects. Dimorphic regions of TAP1 and TAP2 genes were amplified by polymerase chain reaction. Amplified products were digested with restriction endonucleases to determine TAP alleles: Sau3A1 for TAP1 codon 333 (Ile-Val), AccI for TAP1 codon 637 (Asp-Gly), and EcoRII for TAP2 codon 687 (Gln-Stop). We observed four alleles for TAP1 and dimorphism for TAP2 codon 687. Six of 35 controls had the TAP1 D allele, which has been reported to be a rare allele in Caucasian populations. Gene frequency of TAP1 637Asp exhibited a tendency to increase in the patients with atopic dermatitis. TAP1 637Asp and TAP1 A alleles were estimated to constitute a haplotype with DRB1* 1302-DQB1*0604 and DRB1*0803-DQB1*0601 in the Japanese population. Because TAP1 and TAP2 genes are located between HLA-DQB1 and -DPB1 loci, analysis of TAP gene polymorphisms will be useful for a better understanding of susceptibility loci in HLA class II-associated disease.

Analysis of disease-associated amino acid epitopes on HLA class II molecules in atopic dermatitis.

We investigated the association between HLA class II alleles and severe atopic dermatitis with high serum IgE levels (greater than 8000 U/ml). The frequencies of HLA-DRB1*1302 and DQB1*0604 were increased, whereas the frequency of HLA-DQB1*0302 was decreased. A strong haplotype, HLA-DRB1*1302-DQB1*0604, has been reported in the Japanese population, and this haplotype is conserved in patients with AD. Further analysis of the amino acid epitopes on the HLA-DR beta 1 and DQ beta 1 domains revealed that DR beta 1 71Glu (RR = 5.71, p < 0.05) and DQ beta 1 30His (RR = 3.25 p < 0.01), and 57Val (RR = 3.13, p < 0.05) were increased in frequency. DR beta 1 71Glu was exclusively unique to DRB1*1302. DQ beta 1 30His was shared by the following alleles: DQ beta 1*0501, *0502, *0503, and *0604, which were all increased in patients with AD. DQ beta 1 57Val was shared by DQB1*0501 and *0604. A well-known haplotype, HLA-DRB1*1302-DQB1*0604, contains DR beta 1 71Glu and DQ beta 1 30His and 57Val. Therefore HLA-DR beta 1 71Glu and/or DQ beta 1 30His/57Val are considered to play the most important role in the development of atopic dermatitis.

Enhanced Production and Gene Expression of lnterleukin-5 in Patients with Bronchial Asthma: Possible Management of Atopic Diseases by Down-Regulation of lnterleukin-5 Gene Transcription

Interleukin (IL)-5 was produced in vitro by peripheral blood mononuclear cells (PBMCs) of mite-sensitive atopic patients upon challenge with specific allergen, while PBMCs of healthy controls produced essentially no IL-5. Stimuli delivered by the combination of phorbol ester (PMA) and CA2+ ionophore (ionomycin) induced marked IL-5 production by PBMCs obtained from atopic as well as nonatopic asthmatics. CD2- or CD4-bearing-cell depletion almost completely removed IL-5 production and gene transcription while CD8 depletion did not. These findings indicated that CD4+ T cells are the principal source of IL-5 in asthmatic PBMCs. The capacity of PBMCs of atopic asthmatics, nonatopic asthmatics and healthy controls to produce IL-2, IL-3, IL-4, interferon-gamma and granulocyte/macrophage-colony-stimulating factor was comparable among the three groups. FK506 suppressed IL-5 production and gene expression in vitro in a dose-dependent manner.

The effects of various growth factors on endothelial cell survival in vitro

The effects of various growth factors on endothelial cell survival in vitro were studied. Using rat heart endothelial cells, the cell survival curves were obtained; the cells were cultured until confluent, the medium was changed to serum-free medium with or without growth factors, and the cells were counted after 3, 6, 9, and 12 days. Transforming growth factor-beta, which is known as a potent growth inhibitor for vascular endothelial cells, shortened the rat heart endothelial cells survival period, while epidermal growth factor or transforming growth factor-alpha prolonged survival. Insulin did not affect the rat heart endothelial cells survival. Our data indicate that growth factors play a role not only in cell proliferation but also in cell survival in vitro. In addition, elevated levels of growth inhibitors such as transforming growth factor-beta may cause tissue damage in vivo by affecting cell survival.

A Case of Neurofibrosarcoma Associated with Neurofibromatosis: Light Microscopic, Ultrastructural, Immunohistochemical and Biochemical Investigations

A case of neurofibrosarcoma (NFS) with neurofibromatosis was studied by light microscopic, ultrastructural, immunohistochemical and biochemical methods. Histologically, spindle‐shaped tumor cells with atypical hyperchromatic nuclei were arranged in a fascicular or sheet‐like fashion. Electron microscopic examination revealed discontinuous basement membrane‐like structures. Immunohistochemical study revealed S100 protein α chains in tumor cells.

A Case of Subcutaneous Malignant Fibrous Histiocytoma Circumscribed by Fibrous Tissue

We report a case of malignant fibrous histiocytoma (MFH) located in the subcutaneous tissue on the right axilla. We excised the tumor sufficiently beyond the clinical margin. It was pathologically diagnosed as a storiform‐pleomorphic type of malignant fibrous histiocytoma almost completely circumscribed by fibrous tissue, including fascicles of fibroblasts; this is a rare histological picture. The tumor has not recurred for three years. Although MFH frequently undergoes metastasis, the circumscribed‐type subcutaneous MFH characteristic of superficiality and of histologically well‐defined structure seems to have a relatively more favorable prognosis after adequate radical excision.

The effects of scleroderma sera on endothelial cell survival in vitro

SummaryThe effects of sera and of platelet-poor plasma from patients with scleroderma on endothelial cell survival in vitro were studied. The survival ratio of rat heart endothelial cells was studied both in 10% test serum and in 10% platelet-poor plasma. Sera from patients with scleroderma decreased the survival ratio significantly when compared with sera from normal controls. In contrast, there was no significant difference between platelet-poor plasma from patients with scleroderma and that from normal controls. Our data indicate that platelets in the patients with scleroderma may cause vascular damage by affecting endothelial cell survival.