Mitsuo Okubo

HLA class I deficiencies due to mutations in subunit 1 of the peptide transporter TAP1.

The transporter associated with antigen processing (TAP), which is composed of two subunits (TAP1 and TAP2) that have different biochemical and functional properties, plays a key role in peptide loading and the cell surface expression of HLA class I molecules. Three cases of HLA class I deficiency have previously been shown to result from the absence of a functional TAP2 subunit. In the present study, we analyzed two cases displaying not only the typical lung syndrome of HLA class I deficiency but also skin lesions, and found these patients to be TAP1-deficient. This defect leads to unstable HLA class I molecules and their retention in the endoplasmic reticulum. However, the absence of TAP1 is compatible with life and does not seem to result in higher susceptibility to viral infections than TAP2 deficiency. This work also reveals that vasculitis is often observed in HLA class I-deficient patients.

NOS2 polymorphisms associated with the susceptibility to pulmonary arterial hypertension with systemic sclerosis: contribution to the transcriptional activity.

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis. One of several complications of SSc, pulmonary arterial hypertension (PAH) can be refractory to treatment, both novel and established. In the present study we investigated the ratio of circulating nitric oxide to endothelin-1 in patients with both SSc and PAH, and determined whether polymorphisms in NOS2 (the nitric oxide synthase 2 gene) are associated with susceptibility to PAH. Endothelin-1 in plasma and nitric oxide metabolites (nitrate and nitrite) in serum were measured. The nitric oxide/endothelin-1 ratio was significantly lower in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. We confirmed the presence of two single nucleotide polymorphisms at positions -1,026 and -277 and a pentanucleotide repeat (CCTTT) at -2.5 kilobases. There were significant differences in single nucleotide polymorphisms between patients with SSc who had PAH and those who did not, and between patients with both SSc and PAH and healthy control individuals. The CCTTT repeat was significantly shorter in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. Transcriptional activity were analyzed using the luciferase reporter assay. The transcriptional activity of NOS2 was much greater in fibroblasts transfected by a vector with a long allele of the CCTTT repeat than in those transfected by a vector with a short allele. Polymorphisms in the NOS2 gene are associated with transcriptional activity of the NOS2 gene and with susceptibility to SSc-related PAH.

Human papillomavirus type 16 E6 variants and HLA class II alleles among Japanese women with cervical cancer

The enhanced oncogenicity of particular human papillomavirus type 16 (HPV16) E6 variants is population‐dependent, implying the involvement of additional genetic cofactors. This study was designed to investigate the association between E6 variants and human leukocyte antigen (HLA) polymorphism within a Japanese population. Fifty‐seven women with HPV16‐positive cervical cancer were analyzed for E6 sequence variation and its relationship to HLA class II alleles. Compared with local controls (n = 138) and published controls (n = 916), DRB1*1501 and DQB1*0602 frequencies were significantly increased among patients with HPV16 E6 prototype (n = 11). Additionally, DRB1*1502 was positively associated with a particular E6 variant designated D25E (n = 25), although we could not find a significant association between HLA class II alleles and L83V variants (n = 16). Our observations suggest that a specific match between E6 variant proteins and HLA types may contribute to HPV16‐related cervical carcinogenesis.

Cloning of novel soluble gp130 and detection of its neutralizing autoantibodies in rheumatoid arthritis

In an attempt to isolate disease-associated autoantigens in rheumatoid arthritis (RA), we cloned a new autoantigen named gp130-RAPS, which is a novel soluble form of the IL-6 signal-transducing molecule gp130. gp130-RAPS is a 50-kDa protein translated from alternatively spliced mRNA and has a truncated form of gp130 with a unique sequence, Asn-Ile-Ala-Ser-Phe (NIASF), in its COOH-terminus. We observed serum antibodies to this NIASF sequence frequently in patients with RA, but not in those with other systemic rheumatic diseases or in healthy subjects. In RA, detection of those antibodies was significantly associated with disease activity indices such as serum C-reactive protein (CRP) levels, erythrocyte sedimentation rate, blood platelet counts, and serum IL-6 concentration. In vitro experiments revealed that gp130-RAPS inhibited IL-6 activity, and this inhibition was neutralized by antibodies to the COOH-terminus of gp130-RAPS derived from patients with RA. Thus, autoantibody to gp130-RAPS may play an important role in the progression of RA by promoting IL-6 activity. Inspection of autoantibodies to gp130-RAPS may become a practical clinical test for RA. gp130-RAPS and its autoantibody provide a new clue to the complicated pathogenesis of RA.

Gold sodium thiomalate selectivity inhibits interleukin-5–mediated eosinophil survival

Gold sodium thiomalate (GST) has been used for treatment of patients with bronchial asthma. In this study we investigated the effects of GST on interleukin (IL)-5-mediated eosinophil survival in vitro. Blood cells were obtained from patients with bronchial asthma (n = 18). Eosinophils were purified from the blood samples by a Percoll discontinuous method and negative selection of neutrophils. Eosinophils (10(6) cells/ml) were incubated for 96 hours in 10% fetal bovine serum-RPMI 1640 (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) with various concentrations of recombinant human IL-5 (2 x 10(-9) to 2 x 10(-5) mmol/L) and with GST (0.01 to 100 mumol/L) in 96-well, flat-bottomed microtiter plates at 37 degrees C in a humidified 5% carbon dioxide atmosphere. Eosinophil viability was determined by trypan blue exclusion. IL-5 enhanced eosinophil survival in a dose-dependent manner. Higher concentrations of GST inhibited IL-5-mediated eosinophil survival. Moreover, GST induced apoptosis of eosinophils by antagonizing the effects of IL-5. However, GST did not inhibit IL-3- or granulocyte-macrophage colony stimulating factor-mediated eosinophil survival, suggesting that GST selectivity inhibits IL-5-mediated eosinophil survival. These results suggest that IL-5-mediated eosinophil survival is inhibited by GST and that GST blocks eosinophil apoptosis by IL-5. It is possible that GST may be capable of controlling eosinophil functions regulated by IL-5 in patients with bronchial asthma.

Characterisation of T cell clonotypes that accumulated in multiple joints of patients with rheumatoid arthritis

OBJECTIVE To investigate whether identical T cell clonotypes accumulate in multiple rheumatoid joints, the clonality of T cells that had infiltrated into synovial tissue (ST) samples simultaneously obtained from multiple joints of patients with rheumatoid arthritis (RA) was analysed. METHODS T cell receptor (TCR) β gene transcripts, amplified by reverse transcription-polymerase chain reaction from ST and peripheral blood lymphocytes of five RA patients, were subjected to single strand conformation polymorphism analysis and DNA sequencing. RESULTS Approximately 40% of accumulated T cell clonotypes found in one joint of a patient were found in multiple joints in the same patient. Furthermore, identical amino acid sequences were found in TCR β junctional regions of these clonotypes from different patients with at least one HLA molecule match. CONCLUSIONS The T cell clonotypes accumulating in multiple rheumatoid joints may be involved in the perpetuation of polyarthritis by reacting to antigens common to these multiple joints.

Unique Phenotypes of C1s Deficiency and Abnormality Caused by Two Compound Heterozygosities in a Japanese Family

A deficiency in the early components of complement is associated with an increased susceptibility to pyrogenic infections and multiple autoimmune diseases. We previously reported a Japanese case of selective C1s deficiency resulting from a compound heterozygosity for a 4-bp deletion in exon X and a nonsense mutation Glu597X in exon XII of the C1s gene. In this previous case, the patient suffered from unique symptoms including virus-associated hemophagocytic syndrome and died after a long period of loss of consciousness. In the present study, we report another patient from the same family, with C1s abnormality caused by a distinct compound-heterozygous genotype and who had a novel missense mutation Gly630Glu transmitted from the mother’s side and a previously identified nonsense mutation Glu597X from the father’s side. Thus three distinct mutations of the C1s gene were clustered and resulted in two distinct genotypes for C1s deficiency and C1s abnormality within this one family. The present patient showed symptoms that were similar in part to our previous patient, which were different from those of the cases reported in other families. The biochemical properties of C1s in the patient’s serum and the recombinant form were closely related to the undetectable or very low activity of complement activation. These results suggested that the uniqueness and severity of the symptoms observed here in the two patients might be under the control of a common C1s allele and distinct counterparts, respectively.

Analysis of HLA‐DRB1*0901‐binding HPV‐16 E7 helper T cell epitope†

Aim:  This study sought to determine the human papillomavirus (HPV)‐16 E7 epitopes that would be presented by HLA‐DR molecules to CD4‐positive T cells in patients with cervical carcinoma.

Association of human leukocyte antigen and T cell message with human papillomavirus 16–positive cervical neoplasia in Japanese women

To investigate whether an association exists between human leukocyte antigen (HLA) haplotype and cervical neoplasia within the Japanese population, we analyzed the human papillomavirus (HPV) genotypes, the HLA class I specificities and class II alleles, and the T-cell responses in the lesions of patients with cervical neoplasia. Eighty-one patients, consisting of 62 cervical intraepithelial neoplasia (CIN) lesions and 19 invasive cervical cancers (ICC), were examined. The frequencies of HPV infection in the CIN I/II and CIN III/ICC groups were 68.0% (17/25) and 80.4% (45/56), respectively. All patients and 138 local Japanese controls were analyzed for HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1. For major histocompatibility complex (MHC) class II HLA-DRB1 alleles, the frequency of DRB1*0901 was significantly elevated in HPV 16–positive CIN III/ICC patients compared with controls (59.3% versus 29.7%, P= 0.0031, OR = 3.44). Similarly for the HLA-DQB1 alleles, a significant increase in the DQB1*03032 frequency was observed in HPV 16–positive CIN III/ICC patients compared with controls (59.3% versus 28.3%, P= 0.0018, OR = 3.69). In the analysis of the T-cell responses in the lesions, Fas ligand was detected at a decreased frequency in HPV 16–positive CIN III/ICC patients with the HLA-DRB1*0901–DQB1*03032 haplotype. The presence of helper T cell–specific messenger RNAs in the cervical lesions supports an association among MHC class II, helper T cells, the immune response to HPV, and the development of cervical carcinoma. Accordingly, a specific MHC class II haplotype, DRB1*0901–DQB1*03032, may be a risk factor for cervical carcinoma in the Japanese population.

Synovial Mononuclear Cells Consist with T Cells Which Produce High Levels of Tumor Necrosis Factor α

To determine whether synovial mononuclear cells include a population of tumor necrosis factor α‐produeing T cells, we measured tumor necrosis α levels in culture supernatants of synovial mononuclear cells by ELISA and analyzed tumor necrosis α mRNA‐positive cell frequencies. There were no significant differences in the spontaneous levels of TNF α between synovial mononuclear cells and peripheral mononuclear cells. The frequency of tumor necrosis factor α mRNA‐positive cells in synovial mononuclear cells was higher than that of peripheral mononuclear cells. When stimulated with a superantigen, mononuclear cells from the synovial fluid of rheumatoid arthritis patients showed higher levels of tumor necrosis factor α production (1,035 ± 817 pg/ml) than did mononuclear cells from their peripheral blood (236 ± 180 pg/ml). In addition, we observed that a few T cell clones were resistant to superantigenic restimulation in vitro. We conclude that when these types of T cells persist in the synovium, they play a role in the development of rheumatoid arthritis via a mechanism involving tumor necrosis factor α production.