Published papers

The reduction or absence of TCR ζ-chain (ζ) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of ζ mRNA containing an alternatively spliced 3′-untranslated region (3′UTR; ζmRNA/as-3′UTR) and a reduction in the expression of ζ mRNA containing the wild-type 3′UTR (ζmRNA/w-3′UTR) in T cells from SLE patients. Here we show that AS3′UTR mutants (MA5.8 cells deficient in ζ protein that have been transfected with ζmRNA/as-3′UTR) exhibit a reduction in the expression of TCR/CD3 complex and ζ protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab compared with that in wild-type 3′UTR mutants (MA5.8 cells transfected with ζmRNA/w-3′UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of ζmRNA/as-3′UTR in AS3′UTR mutants (3 h) was much shorter than that of ζmRNA/w-3′UTR in wild-type 3′UTR mutants (15 h). Thus, the lower stability of ζmRNA/as-3′UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including ζ protein, in SLE T cells.

PURPOSE: To verify the hypothesis that protein concentrations, such as lactoferrin, epidermal growth factor (EGF), and aquaporin 5 (AQP5), in tears are abnormal in patients with dry eye. DESIGN: Prospective case-control study. METHODS: One hundred three dry eye patients were divided into three groups: dry eye not associated with the Sjögren syndrome (non-SS; n = 71), Sjögren syndrome (SS; n = 23), and Stevens-Johnson syndrome (SJS; n = 9). Sixteen normal control subjects were also checked. The concentrations of lactoerrin, EGF, and AQP5 were measured by enzyme-linked immunosorbent assay. RESULTS: The concentration of lactoferrin was significantly decreased in tears of non-SS (P = .0001), SS (P = .00005), and SJS (P = .0006) patients compared with control subjects. The concentration of EGF was significantly decreased in non-SS (P = .0005), SS (P = .00002), and SJS (P = .0001) patients compared with control subjects. The concentration of AQP5 was significantly increased in tears of only SS patients (P = .01) compared with control subjects and increased in tears of only SS patients compared with non-SS patients (P = .007). CONCLUSIONS: The decrease in both lactoferrin and EGF was found not only in SS patients but also in non-SS patients, indicating that tear components in dry eyes differ in their quantity and quality. Quantification of AQP5 increased only in SS patients, suggesting that AQP5 protein leaks into the tears when acinar cells of the lacrimal gland are damaged by lymphocytic infiltration. © 2003 by Elsevier Inc. All rights reserved.

Serum soluble transferrin receptor (sTfR) has been reported to be higher in patients with iron deficiency or with elevated erythropoiesis. In the present study, serum sTfR was measured in various anemic diseases and their clinical significance was examined in a multi-institutional joint study. Serum sTfRs in patients with the following anemic diseases were markedly higher than those in normal healthy adults: non-treated iron deficiency anemia (IDA) (9.13 ± 7.04 mg/l, n = 52, p <0.0001), anemia of chronic disorders (ACD) (3.45 ± 1.38 mg/l, n = 20, p <0.0001), hemolytic anemia (HA) (5.57 ± 3.26 mg/l, n = 17, p <0.0001), and myelodysplastic syndrome (MDS) (4.03 ± 2.83 mg/l, n = 20, p <0.0001). There were significant differences between IDA and ACD (p <0.0001), between aplastic anemia (AA) (1.58 ± 1.26 mg/l, n = 16) and MDS (p <0.001), and between AA and MDS with refractory anemia (MDS-RA) (4.16 ± 3.40 mg/l, n = 9) (p <0.02). In patients with chronic renal failure (CRF), serum sTfR levels and serum sTfR/log serum ferritin ratios (sTfR/F index) were compared in the two classified groups according to Muirhead's criteria, as IDA and non-IDA groups with or without recombinant human erythropoietin (rHuEPO) treatment. Significantly high levels of both serum sTfR (p <0.0001) and the sTfR/F index (p <0.0001) were observed in IDA without rHuEPO treatment. Especially in CRF with rHuEPO treatment, the sTfR/F index showed marked elevation in the IDA group (p <0.0001) compared with serum sTfR (p <0.001), indicating more diagnostic efficacy of the sTfR/F index for CRF with IDA. In conclusion, the serum sTfR concentration is a useful diagnostic tool for discrimination between IDA and ACD, and between AA and MDS-RA, and for the detection of iron deficiency in CRF patients in the Japanese population.

Objective. To investigate whether Fas promoter polymorphisms show a genetic contribution to the development of systemic lupus erythematosus (SLE) in a Japanese population, and to study the functional difference in promoter activity of the polymorphisms. Methods. In 109 SLE patients and 140 controls, the frequencies of A/G polymorphisms at -670 nucleotide position and G/A at -1377 nucleotide position were determined by allele-specific polymerase chain reaction (PCR) or PCR-single strand conformation polymorphism analysis. The functional significance of the -670A/G polymorphism in the Fas gene was evaluated by a combination of Fas transcriptional activity in the reporter gene assay and binding activity of signal transducer and activator of transcription (STAT)1 protein in the electrophoretic mobility shift assay. Results. SLE patients exhibited significantly higher frequency of A allele at nucleotide position -670 (p = 0.004). There was no significant difference in the nucleotide position -1377 in Fas promoter gene between SLE patients and controls. The electrophoretic mobility shift assay demonstrated that the oligonucleotide with -670A in the Fas promoter had a higher binding ability to a GAS binding protein, STAT1, than that with -670G, although there was no statistically significant difference in the reporter gene assay. Conclusion. Fas promoter -670A/G polymorphism was significantly associated with SLE, suggesting a possibility that Fas promoter contributes, at least in part, to the pathogenesis of SLE.

We have reported that tyrosine phosphorylation and expression of the T cell receptor zeta chain (TCR ζ) was decreased in two systemic lupus erythematosus (SLE) patients with an abnormal TCR ζ lacking exon-7. To examine further the TCR ζ defect and any possible relationship with specific clinical features, we studied the expression of TCR ζ in peripheral blood T cells from 44 patients with SLE, 53 with other rheumatic diseases (30 rheumatoid arthritis (RA), 11 systemic sclerosis (SSc) and 12 primary Sjögren's syndrome(SjS)) and 39 healthy individuals. Flow cytometric analysis demonstrated a significant decrease in the expression of TCR ζ in SLE (P < 0.001), but not in the other rheumatic diseases. Immunoprecipitation experiments confirmed that the expression of TCR ζ in SLE T cells was decreased dramatically (normal: 111.4 ± 22.6%, SLE: 51.6 ± 37.4%,P < 0.0001). The decrease in TCR ζ did not correlate with disease activity, or with the dose of prednisolone (PSL). There were, however, three SLE patients in whom the level of TCR ζ expression normalized after treatment, suggesting that mechanisms responsible for the TCR ζ defect appear to be heterogeneous. These results confirm the defective expression and altered tyrosine phosphorylation of TCR ζ in a large proportion of SLE patients, suggesting that it may play an important role in T cell dysfunction in SLE.

The objective of our study was to investigate the possibility of Fas ligand protein abnormalities in certain types of Sjögren's syndrome patients with enlarged exocrine glands. Fas ligand expression by lymphocytes infiltrating the lachrymal glands and by peripheral blood monocytes in Sjögren's syndrome patients with enlarged exocrine glands was assessed immunohistologically and by immunoblotting. Cytotoxicity of peripheral blood monocytes and sensitivity to steroids in Sjögren's syndrome patients with enlarged exocrine glands were studied by functional assay. Minimal Fas ligand expression was detected in the lymphocytes of the lachrymal glands and a decreased level of Fas ligand was found in peripheral blood monocytes as assessed by immunoblotting. Functional assay confirmed the decreased cytotoxicity of lymphocytes in Sjögren's syndrome patients with enlarged exocrine glands, and that it is not affected by anti-Fas ligand antibody. By contrast, the sensitivity of lymphocytes in Sjögren's syndrome patients with enlarged exocrine glands to steroids was increased. These observations suggest that abnormal expression and function of Fas ligand occurs in Sjögren's syndrome patients with enlarged exocrine glands.

To investigate the mechanism of the downregulation of T-cell receptor ζ chain (TCRζ) expression in the peripheral blood T cells (PBTs) of systemic lupus erythematosus (SLE) patients, we analyzed the 3′ untranslated region (3′UTR) of TCRζ mRNA, because the 3′UTR in mRNA is responsible for posttranscriptional regulation. Use of the reverse transcriptase polymerase chain reaction (RT-PCR) to amplify the 917bp TCRζ 3′UTR cDNA demonstrated that the short variant cDNA (355bp), expressed as an alternatively spliced 3′UTR with 562-bp deletion, was predominated in the PBTs of 11 of 14 SLE patients, whereas mainly the wild-form cDNA (917bp) was detected in the PBTs of seven negative controls (two systemic sclerosis patients, five normal controls) and in two T-cell line hybridomas. Semiquantitative PCR also revealed the predominant expression of the TCRζ mRNA with alternatively spliced 3′UTR (TCRζ mRNA/as-3′UTR), and a decreased expression of TCRζ mRNA with the wild form 3′UTR (TCRζ mRNA/w-3′UTR) in SLE T cells. However, there was no difference in the expression of the open reading frame (ORF) TCRζ mRNA between the negative controls and SLE patients. The TCRζ protein expression level according to Western blot analysis correlated well with that of TCRζ mRNA/w-3′UTR (r = 0.931) and reversibly with TCRζ mRNA/as-3′UTR (r = -0.614), but not with ORF TCRζ mRNA (r = -0.296). It can be concluded that the reduced expression of TCRζ mRNA/w-3′UTR and the pre-dominant expression of TCRζ mRNA/as-3′UTR lead to downregulation of the TCRζ protein in SLE T cells.

Musculoskeletal and central nervous system infections caused by Bacillus cereus are very rare. Only a few cases have been reported, whose clinical courses strongly suggested that surgical procedures combined with appropriate antimicrobial therapy are necessary to cure these infections. A 60-year-old man with severe neutropenia due to myelodysplastic syndrome, developing necrotizing fasciitis and brain abscess caused by Bacillus cereus is reported. Without performing any surgical procedures, the patient was successfully treated with systemic antimicrobial therapy combined with granulocyte colony stimulating factor, which contributed to the increase in the neutrophil count.

PURPOSE. The object of this study was to evaluate improvement of lacrimal gland (LG) function after topical cyclosporin A (CyA). METHODS. Topical CyA (0.01% and 0.1%) was applied to two mouse models of Sjögren's syndrome, the NFS/sld after thymectomy and the nonobese diabetic (NOD) mouse, and the functional integrity of the lacrimal gland was evaluated by measuring basal and stimulated tear secretion and its histologic integrity by examining it for apoptosis and lymphocyte invasion. RESULTS. After treatment with CyA 0.1% in the NFS/sld mice, tear function increased, and there was a decrease in lymphocyte infiltration of the LG and a decrease in apoptotic figures among the acinar cells. In the NOD mice, tear function also improved, but there was no associated decrease in lymphocyte infiltration. However, the expression of Fas ligand (FasL) in NOD mice by infiltrating lymphocytes was suppressed with 0.1% CyA eye drops. CONCLUSIONS. CyA appears to improve tear secretion in mouse models of Sjögren's syndrome by preventing lymphocyte-induced apoptosis of acinar cells in one model this was achieved by preventing lymphocyte infiltration and in the other by reducing expression of FasL expression on infiltrating lymphocytes.

Objective. Systemic lupus erythematosus (SLE) in a multisystem autoimmune disorder characterized by multiorgan pathology and autoantibodies against a variety of autoantigens. The CD28 and CTLA-4 genes might be candidate genes for SLE, because costimulation signals from CD80/CD86 to CD28/CTLA-4 have been suggested to play an important role in the activation or inactivation of T lymphocytes. Methods. We investigated three polymorphic regions within the CTLA-4 gene, a C/T base exchange in the promoter region - 318 (CTLA-4 - 318C/T), an A/G substitution in the exon 1 position 49 (CTLA-4 49A/G), an (AT)n repeat polymorphism in the 3′ untranslated region of exon 4 [CTLA-4 3′ (AT)n], and a CD28 gene polymorphism, a T/C substitution in the intron 3 position + 17 (CD28 IVS3 + 17T/C), in SLE patients and controls. Results. SLE patients had significantly higher frequencies of the CTLA-4 49G allele (P = 0.003) and of the CTLA-4 (AT)n 106 bp allele (P = 0.0008) than controls. We also found a strong linkage disequilibrium between the A allele of CTLA-4 49A/G and the 86 bp allele of CTLA-4 3′ (AT)n. On the contrary, no association was found between SLE and CTLA-4 -318C/T or CD28 IVS3 + 17T/C. Conclusion. We conclude that the CTLA-4 gene appears to play a significant role in the development of SLE in the Japanese population.

Objective. Interleukin-18 (IL-18) is a proinflammatory cytokine that is involved in immunologically mediated tissue damage, but its bioactivity is regulated in vivo by its soluble decoy receptor, IL-18 binding protein (IL-18BP). This study was undertaken to determine levels of IL-18 and IL-18 binding inhibition in the blood of patients with adult-onset Still's disease (ASD). Methods. Serum concentrations of IL-18 in ASD patients were compared by enzyme-linked immunosorbent assay (ELISA) with those in patients with other systemic rheumatic diseases and healthy controls. The biologically active mature protein of IL-18 was detected by Western blot analysis with anti-IL-18 antibody and its induction of interferon-γ (IFNγ) secretion from IL-18-responding human myelomonocytic KG-1 cells. The inhibitory activity on IL-18 binding to its receptor was determined by 125I-IL-18 binding inhibition assay using the Chinese hamster ovary cell line transfected with a murine IL-18 receptor (CHO-K1/mIL-18R). Results. Concentrations of serum IL-18 were extremely elevated in patients with active ASD compared with those in patients with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, polymyositis/dermatomyositis, Sjögren's syndrome, or healthy individuals. Levels of IL-18 were found to correlate with serum ferritin values and disease severity in ASD. Western blot analysis revealed that serum samples from patients with active ASD contained an 18-kd polypeptide of IL-18, corresponding in size to the mature form. Accordingly, the samples were able to induce IFNγ secretion from KG-1 cells, which was largely abolished by neutralizing anti-IL-18 antibody. However, the level of IL-18 bioactivity was more than 10-fold weaker than the concentration of IL-18 protein measured by ELISA. Serum samples from patients with active ASD showed an inhibitory effect on the binding of 125I-IL-18 to CHO-K1/mIL-18R cells, and this activity was associated with elevation of IL-18. Conclusion. These data indicate that systemic overproduction of IL-18 may be closely related to the pathogenesis of ASD, despite the restriction on its inflammatory activity by IL-18 binding inhibitors such as IL-18BP. The disease activity appears to be determined on the basis of the relative levels of IL-18 and its specific inhibitors.