Taku Kokubo

CKD Accelerates Development of Neointimal Hyperplasia in Arteriovenous Fistulas

Arteriovenous (AV) access failure resulting from venous neointimal hyperplasia is a major cause of morbidity in patients with ESRD. To understand the role of chronic kidney disease (CKD) in the development of neointimal hyperplasia, we created AV fistulae (common carotid artery to jugular vein in an end-to-side anastomosis) in mice with or without CKD (renal ablation or sham operation). At 2 and 3 wk after operation, neointimal hyperplasia at the site of the AV anastomosis increased 2-fold in animals with CKD compared with controls, but cellular proliferation in the neointimal hyperplastic lesions did not significantly differ between the groups, suggesting that the enhanced neointimal hyperplasia in the setting of CKD may be secondary to a migratory phenotype of vascular smooth muscle cells (VSMC). In ex vivo migration assays, aortic VSMC harvested from mice with CKD migrated significantly greater than VSMC harvested from control mice. Moreover, animals with CKD had higher serum levels of osteopontin, which stimulates VSMC migration. When we treated animals with bone morphogenic protein-7, which promotes VSMC differentiation, before creation of the AV anastomosis, the effect of CKD on the development of neointimal hyperplasia was eliminated. In summary, CKD accelerates development of neointimal hyperplasia at the anastomotic site of an AV fistula, and administration of bone morphogenic protein-7 neutralizes this effect.

Tyrosinase epitope recognized by an HLA-DR-restricted T-cell line from a Vogt-Koyanagi-Harada disease patient

Abstract Human T-cell-mediated autoimmune diseases are often genetically linked to particular alleles of HLA class II genes. Vogt-Koyanagi-Harada’s (VKH) disease, which is regarded as an autoimmune disorder in multiple organs containing melanocytes, has been found to be associated with HLA-DR4 (DRB1*0405) and HLA-DR53 (DRB4*0101). Tyrosinase is a melanoma antigen (Ag) expressed by normal melanocytes as well as melanoma cells against which responses by autologous T cells have been detected. We established a T-cell line from the peripheral blood of a patient with VKH disease which responded to synthetic peptides corresponding to tyrosinase. The T-cell line was generated which recognized the tyrosinase p188 – 208 peptide when presented by the HLA-DR4 (DRB1*0405) molecule on the surface of HLA class II-expressing L-cell transfectants. The minimal antigenic peptide which induced T-cell responses was an 11-amino-acid sequence and located at tyrosinase p193 – 203 (E-I-W-R-D-I-D-F-A-H-E). This peptide contained the DRB1*0405-binding peptide motif (hydrophobic residues (Y, F, W) at position 1 as an anchor residue, and negatively charged residues (D, E) at position 9), which corresponded to the W at p195 and the D at p203. These observations demonstrate that tyrosinase peptides are immunogenic, and may be a candidate for an autoantigen in VKH disease, suggesting that probing the T-cell responses against synthetic peptides is a productive approach for identifying the autoantigenic peptides associated with autoimmune diseases including VKH disease.

ANALYSIS OF ANCHOR RESIDUES IN A NATURALLY PROCESSED HLA-DR53 LIGAND

The peptide motif of the HLA-DR53 (DRB4*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Haradas syndrome, was determined by peptide binding assay using human L plastin p581–595 peptide and its substituted analogues. L plastin p581–595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1*0901/DRB4*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylate peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. binding peptides in combination with functional analyses, by alignment of sequenced endogeneous peptides, and by the use of an M13 display library (Rammensee et al. 1995; Hammer et al. 1993, 1992). No sequence information has been reported for naturally occurring HLA-DR53 (DRB4*0101)-associated peptides partly because their expression on the cell surface is relatively low for sequencing endogeneous self-peptides (Kinouchi et al. 1995). It has been shown that HLA-DR53 is positively associated with Vogt-Koyanagi-Haradas Syndrome in Japanese subjects (Moriuchi et al. 1979). The identification of a peptide motif for HLA-DR53 may help in understanding the mechanisms of this disease. We have previously reported that naturally processed peptides bound to HLA-DR9/DR53 molecules (Futaki et al. 1995). In this report, we determined the peptide which could bind to the HLA-DR53 molecule and we identified a putative HLA-DR53-specific binding motif by a peptide binding assay using L-cell transfectants expressing HLA-DR molecules.

Epitope analysis of birch pollen allergen in Japanese subjects

Birch pollen is a very common cause of nasal allergy (pollinosis) not only in Scandinavia, Europe, Canada, and the northern part of the United States but also in Hokkaido, Japan. We have previously reported a positive association between the HLA-DR9 phenotype and the development of birch pollen allergy in Japanese subjects. However, there is little information about T cell epitopes of birch pollen which are presented by HLA class II molecules other than HLA-DR9. Therefore, we analyzed the difference in T cell epitope usage in patients who had HLA-DR9 versus those who did not. Seven Japanese patients with birch pollinosis were studied. Some groups of peptides representing T cell epitopes (Betula verrucosa; Bet VI peptides, p7–33, p23–46, p138–160) appeared to be shared by the majority, while another peptide (Bet VI p72–95) was recognized predominantly by patients who expressed HLA-DR9 and/or HLA-DQ3 molecules. Moreover, seven T cell clones and eight T cell lines were generated from two patients who did not have HLA-DR9 or HLA-DQ3. Using some of these T cell clones/lines, we investigated the relationship between HLA class II molecules and antigenic peptides. One of these T cell clones recognized antigenic peptides in the context of the HLA-DQ1 molecule. To our knowledge, this is the first indication that the epitope on Bet VI can be presented by the HLA-DQ molecule.

Clinical Results of Cystic Excision for Popliteal Artery Cystic Adventitial Disease: Long-term Benefits of Preserving the Intact Intima

Surgical treatment for popliteal artery cystic adventitial disease (PACAD) is still controversial. PACAD often occurs in young or middle-aged adults. Therefore, the maintenance of graft patency for very long periods is a concern if a prosthesis is used. Because the intima is intact in PACAD patients with popliteal artery stenosis, a treatment that preserves the healthy intima is ideal. We describe the cases of 3 patients who underwent cystic excision for PACAD with severe stenosis. No recurrence was observed for up to 11 years, and these long-term results revealed that cystic excision could be reconsidered as one of the first-line therapeutic methods.