研究者詳細 - 竹内　勤

論文 - 竹内　勤

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Flow cytometric analysis of surface antigens on human conjunctival epithelial cells

Fujihara Tsutomu, Takeuchi Tsutomu, Saito Keiko, Tsubota Kazuo

Ophthalmic Research 29 （ 2 ） 103 - 109 1997年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0030-3747

We analyzed the levels of expression of different surface adhesion molecules on normal human conjunctival epithelial cells with cytology and flow cytometry. The levels of β₁ (CD29), VLA-1 (CD49a), VLA-2 (CD49b), and VLA-3 (CD49c) integrins were high, whereas those of VLA-5 (CD49e) and VLA-6 (CD49f) were faint, and that of VLA-4 (CD49d) was undetectable. Among the β₂ integrins, the levels of Mac-1 (CD11b) and p150.25 (CD11c) were positive, while LFA-1 (CD11a) was not detectable. HLA class I and class II were also expressed. However, CR2 (CD21), CD57, CD44 and ICAM-1 (CD54) were not detected. Our study suggests that these techniques may aid in identifying changes in the expression levels of various surface antigens on conjunctival epithelial cells that could occur in ocular surface disease.

Spontaneous establishment of an Epstein-Barr virus-infected fibroblast line from the synovial tissue of a rheumatoid arthritis patient

竹内勤

Journal of Virology 71 （ 3 ） 2478 - 2481 1997年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-538X

Flow cytometric analysis of surface antigens on human conjunctival epithelial cells

竹内勤

Ophthalmic research 29 （ 2 ） 103 - 109 1997年01月

研究論文（学術雑誌）, 共著, ISSN 0030-3747

Elevated levels of substance P in tears of patients with allergic conjunctivitis and vernal keratoconjunctivitis

Fujishima H., Takeyama M., Takeuchi T., Saito I., Tsubota K.

Clinical and Experimental Allergy 27 （ 4 ） 372 - 378 1997年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0954-7894

Background - Recent studies have suggested that the nervous system may participate in inflammatory processes. Substance P (SP) acts as a chemical mediator as well as a neurotransmitter. Objective - In order to clarify the pathogenesis of ocular allergic diseases, we assessed the concentration of SP in tears. Methods - Using a highly sensitive and specific double-antibody enzyme immunoassay (EIA), we determined the SP concentration in tears of 10 patients with seasonal allergic conjunctivitis, 10 with atopic dermatitis without keratoconjunctivitis (AD), 13 with vernal keratoconjunctivitis (VKC) and 65 normal controls. Giemsa's staining for brush cytology samples and histocytological study by immunocytochemical staining of giant papillary conjunctival cells from VKC and normal controls was conducted. Results - The mean SP level was low in the normal controls anti AD, whereas patients with seasonal allergic conjunctivitis and VKC showed significant elevation of SP (P < 0.01). Brush cytology samples showed conjunctival epithelial cells with lymphocytes, neutrophils and eosinophils that were not seen in normal subjects. Histocytological examination demonstrated SP positive cells in the conjunctiva of patients with VKC, but not in normal controls. Conclusion - This study suggests that the increased level of SP in tears may contribute to the pathogenesis and severity of ocular allergic diseases.

Characterization of cytokine mRNA transcripts in conjunctival cells in patients with allergic conjunctivitis

Fujishima Hiroshi, Saito Ichiro, Takeuchi Tsutomu, Shinozaki Naoshi, Tsubota Kazuo

Investigative Ophthalmology and Visual Science 38 （ 7 ） 1350 - 1357 1997年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0146-0404

Purpose. The host response to allergens appears to be regulated by specific patterns of local cytokine production. More than 20,000 conjunctival superficial cells were collected with a special brush, a smaller version of the Cytobrush used in cervical cytology, from the upper palpebral conjunctiva. Methods. Samples were obtained by cytology brush from seven patients with allergic conjunctivitis and from seven healthy volunteers. Giemsa staining, immunocytochemistry, and flow cytometric analysis were performed. Cytokine gene expression was assayed by the reverse- transcription-polymerase chain reaction method. Results. Giemsa staining of cytocentrifuged preparations from patients with allergic conjunctivitis showed conjunctival epithelial cells with lymphocytes, mast cells, and eosinophils. In an immunohistochemically study, a few CD3- and CD4- bearing cells, but not CD20- and CD14- bearing cells, were seen in patients. In 82.6 ± 17% of the samples obtained from allergic patients, HLA-DR was present, but it was present in only 34.2 ± 17.8% of samples from control subjects (P = 0.0001) using flow cytometric analysis. Steady state transcripts of mRNA for cytokines were analyzed with RT-PCR in conjunctival cell samples, and results showed that samples from allergic conjunctivitis expressed increased transcripts of interleukin 4 and interleukin 13 but virtually no interleukin 2 or interferon-γ; six samples from seven healthy subjects expressed no interleukin 2, interleukin 4, interleukin 13, or interferon-γ transcripts. Conclusions. These results suggest that the clinical features of allergic conjunctivitis in humans are associated with a specific local pattern of proinflammatory cytokine expression.

Evaluation of human conjunctival epithelium by a combination of brush cytology and flow cytometry

Fujihara Tsutomu, Takeuchi Tsutomu, Saito Keiko, Kitajima Yasuo, Kobayashi Tadao K., Tsubota Kazuo

Diagnostic Cytopathology 17 （ 6 ） 456 - 460 1997年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 8755-1039

Cytology using the brush technique is readily available and is a rapid means of establishing a presumptive diagnosis of ocular surface changes. However, those techniques have some limitations when obtaining cells from certain localized areas particularly if using the Cytobrush-S®. We have described here a new type of brush (Accellon-M®), which can collect the local cells using its spherical tip, and evaluated them by comparing with Cytobrush-S®. Furthermore, we differentiated epithelial cells from non- epithelial cells that were collected by brushing, and by a combination of brush cytology and flow cytometry using an anti-keratin antibody, AE-3, which reacts with all basic epithelial keratins. Accellon-M® could collect the epithelial cells from conjunctiva as effectively as with the Cytobrush-S®, and there were no statistical differences between both groups. AE-3, which is reported as the marker of epithelial cells, were detected quantitatively by a combination of brush cytology and flow cytometry techniques. The result of the present study emphasizes that both the Cytobrush-S® and the Accellon- M® are valuable for conjunctival brush cytology. An additional positive feature of the Accellon-M® may be improved cell collection from the conjunctival epithelium especially when the target cells are in a limited area. The combination of the brush cytology and the flow cytometry technique appears to be a useful adjunct as an additional diagnostic or research tool for use in the detection of various antigens in the conjunctival epithelium.

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Lacrimation and salivanon is not relatedtothe cd4 positive lymphoctteinfiltration in lacrimaland salivary gland in

Fujihara T., Takeuchi T., Saito I., Tsubota K.

Investigative Ophthalmology and Visual Science 38 （ 4 ） 1997年

研究論文（国際会議プロシーディングス）, 共著, 査読有り, ISSN 0146-0404

Purpose: We measured the secretion volume of tear and sauva in MRL and BALB/c mice to confirm that lacrimationand salivation is related to lymphocyte infiltration in these glands. Methods: To quantify the tear secretion volume. Schirmer test with cotton thread test was performed in MRL/ipr, MRL/+ and BALB/c mice. Saliva secretion volume was measured with capillary tube. Lymphocytic infiltration into the exocrine glands was graded, and infiltrated lymphocytes were characterized by immunohislochemistry. The apoptotic cells in the exocrine glands were detected by TUNEL method. Results: There were no significant differences in tear secretion between BALB/c and MRL/lpr or MRL/+ mice. In MRL/lpr the saliva secretion was reduced in the comparison with BALB/c mice. Immunohistochemical analysis revealed that the CD4 positive T lymphocytes were located in the focus region in both of the glands in MRL/lpr and MRL/+ mice. In the salivary gland, the CDS positive T lymphocytes were observed in the focus region and around acini and duct in MRL/lpr mice. In the salivary glands the apoptotic cells were detected at the ductal and acinar cells only in MRL/lpr mice. Conclusion: In this study, we found that the CD4 positive T lymphocyte dose not necessarily result in the decrease of the secretion. In salivary gland. CDS positive lymphocytes around acini and duct may cause the apoptosis of the acinar and ductal epithelial ceils and the dysfunction of the salivary secretion.

Interleukin-4 and IgE in seasonal allergic conjunctivitis

Fujishima Hiroshi, Shimazaki Jun, Takeuchi Tsutomu, Saito Ichiro, Tsubota Kazuo

Ophthalmologica 210 （ 6 ） 325 - 328 1996年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0030-3755

We measured interleukin-4 (IL-4) and IgE to study their presence in patients with seasonal allergic conjunctivitis. Twenty patients and 10 normal subjects were enrolled in this study, and their brush cytology samples and peripheral blood lymphocytes (PBL) were cultured for 3 or 24 h. IL-4 levels were measured using ELISA in the supernatants. Measurements of serum IgE were also performed. IL-4 was detected in 4 of 40 brush cytology samples and 10 of 40 PBL samples, but not from the controls. IL-4 positivity rate of patients was significantly higher than normal in 24-hour cultured PBL (p = 0.03). Patients' serum IgE was also significantly higher (p = 0.006). IL-4 may contribute to the development of seasonal allergic conjunctivitis.

Measurement of interleukin-4 and histamine in superficial cells of conjunctiva in patients with allergic conjunctivitis

竹内勤

Current Eye Research 15 （ 2 ） 209 - 213 1996年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0271-3683

Signal transduction through CD16 in human conjunctival epithelial cells

竹内勤

Investigative Ophthalmology and Visual Science 37 （ 3 ） 1653 - 1653 1996年02月

研究論文（国際会議プロシーディングス）, 共著, 査読有り, ISSN 0146-0404

Interleukin-4 and IgE in seasonal allergic conjunctivitis

竹内勤

Ophthalmologica 210 （ 6 ） 325 - 328 1996年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0030-3755

Biopsy of labial salivary glands and lacrimal glands in the diagnosis of sjögren's syndrome

Xu Ke Ping, Katagiri Shigeo, Takeuchi Tsutomu, Tsubota Kazuo

Journal of Rheumatology 23 （ 1 ） 76 - 82 1996年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0315-162X

Objective. To determine the diagnostic value in Sjögren's syndrome (SS) of evaluating the presence of focal adenitis in labial salivary gland (LSG) and lacrimal gland (LG) biopsy specimens. Methods. We studied 105 cases with suspected SS and performed biopsies prospectively. The diagnostic criteria of Fox, et al were followed. The Chisholm-Mason grading standard and Greenspan focus score were used for evaluation. Results. Epimyoepithelial islands and severe lymphocyte infiltration with germinal centers were observed only in LG biopsy specimens (5.9 and 3.5%, respectively). The lymphocytic focus scores of LG (2.5 ± 2.1/4 mm²) were significantly higher than those of LSG specimens (1.3 ± 1.1/4 mm²) in 23 cases with both biopsy results (p < 0.05). Six of the 32 LSG biopsies were normal while their LG biopsies were significantly higher. Conversely, 5 of the 32 LG biopsies were aided by corresponding LSG biopsies, since in LG biopsies no glandular tissue was obtained. Evaluation of both specimens for the same patient was significantly more effective in diagnosing SS than using either specimen alone (p < 0.01). Conclusion. LG biopsy specimens have a more evident histopathology than LSG biopsy specimens. We recommend that both LSG and LG biopsies be performed in patients with suspected SS to reduce false negative results and improve diagnostic accuracy.

Decreased reflex tearing is associated with lymphocytic infiltration in lacrimal glands

Tsubota Kazuo, Xu Ke Ping, Fujihara Tsutomu, Katagiri Shigeo, Takeuchi Tsutomu

Journal of Rheumatology 23 （ 2 ） 313 - 320 1996年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0315-162X

Objective. To investigate whether reflex tearing measured by the Schirmer test with nasal stimulation is better correlated with lymphocytic infiltration of the lacrimal gland and is thus a more appropriate test for Sjogren's syndrome (SS). Methods. 272 patients with dry eye (age 55.5 ± 13.5 years) with regular Schirmer test results <10 mm were recruited for the study. They were divided into 2 groups depending on the results of the Schirmer test with nasal stimulation. 24 age and sex matched patients from each group were selected for lacrimal gland biopsy and 10 patients for salivary gland biopsy, and the levels of systemic autoantibodies and degrees of ocular surface staining and lymphocytic infiltration of the lacrimal and salivary glands were compared. Results. Of the 272 patients, 187 (68.8%) had good reflex tearing (GT) and 85 (31.2%) had poor reflex tearing (PT). There were no significant differences in the age or sex of the 2 groups. Lymphocytic infiltration of the lacrimal gland was observed in 7 GT cases and 22 PT cases out of 24 cases. Lymphocytic infiltration of the salivary gland was also higher in the PT (6 of 10) than the GT group (2 of 10). Conclusion. Evaluation of reflex tearing by the Schirmer test with nasal stimulation identifies 2 groups of patients with dry eye. Those with poor reflex tearing were more likely to have autoantibodies and lymphocytic infiltration of the exocrine glands consistent with SS.

SYSTEMIC LUPUS-ERYTHEMATOSUS WITH NECROTIZING VASCULITIS AND UP-REGULATED EXPRESSION OF VLA-4 ANTIGEN

竹内勤

Clinical Rheumatology 14 （ 3 ） 370 - 374 1995年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0770-3198

Systemic lupus erythematosus with necrotizing vasculitis and upregulated expression of VLA-4 antigen

Takeuchi T., Aoki K., Koide J., Sekine H., Abe T.

Clinical Rheumatology 14 （ 3 ） 370 - 374 1995年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0770-3198

We report on a patient (S.A.), with well-defind SLE who developed a perforation of the ileum due to pathologically confirmed necrotizing vasculitis. No anti-DNA antibody was detected at the ileal perforation, and the serum complement level was normal. These findings rais ethe alternative possibility of a cellmediated immune mechanism as a cause of necrotizing vaculitis. Upregulateed expression and function of VLA-4 antigen on peripheral blood T cells were oberved, suggesting that T cells with VLA-4 antigen may participate in the onset and/or perpetuation of vascular inflammation. © 1995 Acta Medica Belgica.

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Measurement of IL-4 in tears of patients with seasonal allergic conjunctivitis and vernal keratoconjunctivitis

Fujishima H., Takeuchi T., Shinozaki N., Saito I., Tsubota K.

Clinical and Experimental Immunology 102 （ 2 ） 395 - 398 1995年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0009-9104

To elucidate the mechanism of ocular surface allergic disease, we focused on IL-4, which is one of the key factors in regulating IgE production, and thus determined the concentration of IL-4 in tears. IL-4 concentration was determined in the tears of 15 patients with seasonal allergic conjunctivitis, 15 vernal keratoconjunctivitis (VKC), 10 giant papillary conjunctivitis (GPC), 10 patients with non-allergic conjunctivitis and post-cataract surgical conjunctivitis as intermediate conjunctivitis, and 10 normal subjects using a highly sensitive sandwich ELISA. The mean level of IL-4 in normal controls was low, and seasonal allergic conjunctivitis, VKC and GPC showed a significant elevation (P < 0.05), respectively. IL-4 of VKC and GPC were also significantly higher than allergic conjunctivitis, and non-allergic conjunctivitis and post-cataract surgical conjunctivitis were not higher than normal. These results raise the possibility that the increased level of IL-4 in tears could play a role in allergic disease and its severity in patients.

An unusual variant of Wegener's granulomatosis and the role of integrin adhesion receptors for pulmonary lesion

Amano K., Sekine H., Itoh T., Hosono O., Koide J., Takeuchi T., Abe T.

Japanese Journal of Rheumatology 5 （ 3 ） 217 - 226 1994年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0169-1163

We report a patient with Wegener's granulomatosis (WC) whose initial manifestation was acute renal failure. The open renal biopsy specimen revealed periglomerular granulomatosis with crescentic glomerulonephritis. Pulmonary lesion appeared late period of his illness. Both renal and pulmonary disease responded to prednisolone and cyclophosphamide treatment. Serial determination of adhesive receptors on the patient's peripheral blood lymphocytes showed upregulated expression and function when his pulmonary lesion had appeared and returned to normal when the pulmonary lesion subsided. We think that the patient represents an unusual variant of WG and discuss the role of upregulated adhesive receptors for the development of the pulmonary lesion.

Upregulated expression and function of integrin adhesive receptors in systemic lupus erythematosus patients with vasculitis

Takeuchi Tsutomu, Amano Kouichi, Sekine Hiromi, Koide Jun, Abe Tohru

Journal of Clinical Investigation 92 （ 6 ） 3008 - 3016 1993年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-9738

Upregulation of integrin adhesive receptors has been implicated in various pathological conditions. We examined expression and function of integrin adhesive receptors on peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE), particularly those with the complication of vasculitis, and found that VLA-4 and LFA-1 expression was increased in SLE patients with vasculitis, while LFA-1 but not VLA-4 expression was increased in those without vasculitis. These results suggested a role of VLA-4 in the pathogenesis of vasculitis in SLE. Functional studies further demonstrated that adhesion to cytokine-activated human umbilical cord vein endothelial cells and to the CS-1 alternatively spliced domain of fibronectin was significantly increased in SLE patients with vasculitis. Analysis of the functional epitopes on the α₄ chain demonstrated that antigen densities of all the functional epitopes were increased in those with vasculitis, indicating that the increased expression of VLA-4 resulted from the increased number of VLA-4 molecules, and was not secondary to an increase in one particular functional epitope. Immunoprecipitation studies further support these results. Interestingly, high molecular weight bands associated with VLA-4 were observed in about half of the SLE patients with vasculitis. These results introduce a possibility that upregulation of integrin adhesive receptors has a potential role in the pathogenesis of vasculitis in SLE.

Integrin VLA-5 negative primary plasma cell leukemia.

Ohtake K., Takeuchi T., Saito K., Ito T., Ito I., Aoki K., Takano Y., Amano K., Hosono O., Koide J.

Internal medicine (Tokyo, Japan) 32 （ 7 ） 565 - 568 1993年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0918-2918

We present a case of primary plasma cell leukemia with Bence Jones proteinuria. After combination chemotherapy, leukemic cells and the urinary levels Bence Jones protein were decreased. Small lytic bone lesions were detected only in the skull. Typical plasma cells were rarely seen in peripheral blood on the hyperleukocytic phase, however they were increased in the advanced stages. The most important diagnostic sign was persistent expression of CD38 antigen on leukemic cells throughout the entire course of the illness and these leukemic cells expressed very late antigen-4 (VLA-4) but not VLA-5.

Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjogren's syndrome

Saito I., Terauchi K., Shimuta M., Nishiimura S., Yoshino K., Takeuchi T., Tsubota K., Miyasaka N.

Journal of Clinical Laboratory Analysis 7 （ 3 ） 180 - 187 1993年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0887-8013

We attempted to determine whether cell adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule- 1 (ICAM-1), and E-selectin (endothelial-leukocyte adhesion molecule-1; ELAM- 1), are involved in the lymphoid cell infiltration of the salivary and lacrimal glands in Sjogren's syndrome (SS) patients. Both immunohistochemical analysis and the reverse-transcripts polymerase chain reaction (RT-PCR) were used to analyze the expression of VCAM-1, ICAM-1, ELAM-1, very late antigen 4 (VLA-4 [α4, β1]), lymphocyte function-associated antigen-1 (LFA-1), interferon-γ(IFN-γ), tumor necrosis factor (TNF), and interleukin-1 β (IL- 1β). Immunohistochemical analysis of salivary gland biopsies from SS patients showed a marked expression of VCAM-1 and ICAM-1 in the venules surrounded by infiltrated CD4⁺ CD45RO⁺ T cells. E-selectin was expressed on vascular endothelium with weak intensity. Increased levels of VCAM-1, ICAM- 1, IFN-γ, and IL-1β mRNA were demonstrated by RT-PCR, whereas E-selectin mRNA were weakly expressed in SS lacrimal and salivary gland tissues. This is in contrast with strong expression of ELAM-1 in IL-1β-stimulated human umbilical vascular endothelial cells (HUVEC) in vitro. Cytokine-mediated up- regulation of VCAM-1 and ICAM-1 that facilitates the recruitment of VLA-4 and LFA-1 expressing T cells might contribute to lymphoid cell infiltration in the salivary and lacrimal glands in SS.

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