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Biomedical subjects

K Tsubota

Publications and source records attributed to K Tsubota.

At least 127 records · Page 7Linked to original sources

Dry eye and Meige's syndrome.

AIMS: To determine the relation between dry eye and Meige's syndrome. METHODS: 325 patients with dry eye were divided into those responsive to topical and other forms of treatment (n = 276) and those who were not (n = 49). A neuropsychiatric examination was performed to check for Meige's syndrome in the latter group. RESULTS: Twenty eight (57%) of the treatment unresponsive patients were diagnosed with Meige's syndrome. CONCLUSIONS: There is a subgroup of patients with dry eye who do not respond to simple therapy. More than half of these patients have Meige's syndrome and need psychiatric, as well as ophthalmic, care.

Adult↗

Flow cytometric analysis of surface antigens on human conjunctival epithelial cells.

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 beta 1 (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 beta 2 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.

Adult↗

Effect of retinol palmitate as a treatment for dry eye: a cytological evaluation.

Vitamin A is known to regulate the proliferation and differentiation of corneal epithelial cells and preserved conjunctival goblet cells and has been used in the treatment of disease of the eye such as dry eye and superior limbic keratoconjunctivitis for some time. This study was undertaken in order to evaluate the efficacy of retinol palmitate aqueous ophthalmic solution under development for the treatment of dry eye failing to respond to the conventional therapy with artificial tears or cornea-protective drugs. Retinol palmitate ophthalmic solution was applied repeatedly for 4 consecutive weeks. Before and after instillation therapy, brush cytology (Cytobrush-S) was performed and cytodiagnosis was made for keratinized cells, nonkeratinized cells, goblet cells and inflammatory cells on samples prepared using an automated smear apparatus (ThinPrep). In dry eye, an increase in goblet cells (1.3+/-2.6-->2.1+/-1.8 cells/slides), a decrease in keratinized cells (11.2+/-16.5-->5.2+/-10.9 cells/300 cells) and, hence, an increase in nonkeratinized cells (287.3+/-16.6-->293.4+/-11.4/300 cells) were found after treatment with retinol palmitate. As to inflammatory cells, there was no change from the pretreatment baseline (1.4+/-1.4-->1.4+/-1.3 cells/300 cells). These results demonstrate that brush cytology suggests the efficacy of retinol palmitate ophthalmic solution in dry eye treatment.

Administration, Topical↗

Ultraviolet B-induced mitochondrial dysfunction is associated with decreased cell detachment of corneal epithelial cells in vitro.

PURPOSE: To evaluate the effects of ultraviolet B light (UV-B) on mitochondrial inner membrane function, cell viability, and migration of cultured human corneal epithelial cells. METHODS: After UV-B exposure in SV-40 transfected human corneal epithelial cells (T-HCEC), mitochondrial function was assessed by digital microfluorography using the mitochondrial marker, rhodamine 123 (Rh 123). The oxygen consumption rate of T-HCEC suspensions (10(7) cells/ml) was measured by an O2 meter, and adenosine triphosphate contents were measured by luciferase-driven chemiluminescence. Cell viability and migration was observed by propidium iodide (PI) staining and migration assays. RESULTS: UV-B exposure caused an immediate drop in O2 consumption by T-HCEC suspensions, whereas exposure of a monolayer culture of T-HCEC to UV-B at radiant exposures of 50 mJ/cm2 caused a reversible decrease in Rh 123 fluorescence (22.4%) and a significant decrease in adenosine triphosphate contents (1.52 +/- 0.05 nmol/10(6) cells) compared to control (2.93 +/- 0.12 nm/10(6) cells) after 10 minutes. The effects on Rh 123 fluorescence were irreversible at 100 mJ/cm2, which approximately corresponded with the threshold dose at which cells positive to PI staining (PI+) appeared. UV-B doses of 50 mJ/cm2 caused detachment of T-HCEC, primarily PI-, whereas higher doses (100 mJ/cm2) resulted in PI+ cells that did not detach from the dish. These PI+ cells hindered the migration of surrounding viable cells; detachment of PI- cells allowed cells to migrate and to cover a trough created by a 500 microns wide beam of UV-B. CONCLUSIONS: Threshold levels of UV-B (100 mJ/cm2) are associated with irreversible mitochondrial dysfunction and with the loss of the ability for cultured corneal epithelial cells to detach in vitro.

Adenosine Triphosphate↗

Soluble interleukin-2 receptors and serum autoantibodies in dry eye patients: correlation with lacrimal gland function.

PURPOSE: Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by decreased lacrimal and salivary gland function causing dry eye and dry mouth. We examined several parameters as measures of lacrimal function and compared the serum soluble interleukin-2 receptor (sIL-2R) and serum autoantibodies as measures of systemic autoimmune condition. METHODS: Serum levels of sIL-2R, antinuclear antibody (ANA), and rheumatoid factor (RF) in 128 patients (16 men, 112 women; average age, 54.3 +/- 12.5 years) were measured. The lacrimal function was evaluated clinically. RESULTS. There was no correlation between systemic condition and the cotton-thread test, Schirmer test with or without anesthesia, tear-clearance test, tear-function index, or the BUT. However, the Schirmer test with nasal stimulation and rose bengal and fluorescein staining was positively correlated with systemic parameters. CONCLUSION: We show the correlation of serum sIL-2R, ANA, and RF to lacrimal function, which is good evidence that systemic immune conditions directly affect local lacrimal gland function.

Antibodies, Antinuclear↗

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

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 and 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.

Adolescent↗

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

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 conjuntiva. 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 immunohistochemical 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-gamma; six samples from seven healthy subjects expressed no interleukin 2, interleukin 4, interleukin 13, or interferon-gamma 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.

Adolescent↗

RANTES production in a conjunctival epithelial cell line.

PURPOSE: Although corneal tissue damage in allergic ocular diseases is thought to be induced by inflammatory cells that infiltrate from conjunctival tissue, the mechanisms of recruiting these cells remain unclear. The objective of this study was to demonstrate whether conjunctival epithelial cells have the ability to produce "regulated on activation, normal T-cell expressed and secreted" (RANTES). To test this hypothesis, we investigated RANTES expression in the conjunctival tissue and also RANTES production by cytokine stimulation in a human conjunctival epithelial cell line. METHODS: We investigated the expression of the chemokine RANTES in conjunctival epithelium from two patients with atopic keratoconjunctivitis (AKC) and one patient with vernal keratoconjunctivitis (VKC) by using immunohistochemistry. We also investigated the production and suppression of RANTES from a human conjunctival epithelial cell line, Wong-Kilbourne-derived human conjunctiva (WK-hC) by using enzyme-linked immunosorbent assay (ELISA). RESULTS: Conjunctival epithelium from a patient with AKC stained positively for RANTES. We found that tumor necrosis factor-alpha (TNF-alpha) induced de novo production of RANTES, and interferon-gamma (IFN-gamma) synergistically increased the TNF-alpha-dependent production of RANTES from WK-hC cells. Dexamethasone suppressed the RANTES production from the cell line. CONCLUSION: Taken together, human conjunctival epithelial cells were capable of producing RANTES in response to inflammatory stimuli such as TNF-alpha and may play a role in recruiting inflammatory cells such as eosinophils and T lymphocytes toward the ocular surface.

Adolescent↗

Suppression of nuclear factor kappa B and CD18-mediated leukocyte adhesion to the corneal endothelium by dexamethasone.

PURPOSE: To demonstrate that leukocyte adhesion to cultured corneal endothelial cells is mediated by the CD18 antigen, and to determine whether dexamethasone directly suppresses adhesion by inhibiting activation of nuclear factor kappa B (NFkappaB). METHODS: Cultured bovine corneal endothelium was stimulated for 6 hours by 40 micron/ml tumor necrosis factor alpha (TNFalpha). Dexamethasone was added 1 hour before TNFalpha stimulation in the dexamethasone group. After stimulation, neutrophils separated from a healthy human volunteer were added with or without anti-CD18 antibody. The culture plate was settled for 15 minutes at 37 degrees C, and then neutrophils were activated by N-formyl-methionyl-leucyl-phenylalanine for 5 minutes. Nonadherent neutrophils were removed by sealing and inverting the culture well. The intracellular localization of NFkappaB after TNFalpha simulation was determined by confocal immunocytochemistry using an anti-p65 antibody. RESULTS: Neutrophil adhesion to cultured corneal endothelial cells increased significantly on exposure to TNFalpha (451.4+/-45.4 cells/mm2, n = 16) compared to control (156.7+/-27.3 cells/mm2, n = 16, P < 0.01). This increased adhesion was suppressed by the addition of anti-CD18 antibody (157.6+/-25.1 cells/mm2, n = 8, P < 0.01) and by pretreatment with 10(-7) M dexamethasone (207.9+/-31.5 cells/mm2, n = 10, P < 0.01). Immunocytochemistry 60 minutes after stimulation revealed that NFkappaB was located in the cytoplasm in unstimulated cells; however, the addition of TNFalpha caused NFkappaB to translocate into the nucleus. Pretreatment with dexamethasone tapered NFkappaB translocation into the nucleus. CONCLUSIONS: Leukocyte adhesion to the corneal endothelium was shown to be mediated by CD18 expressed on activated leukocytes. Pretreatment of the endothelium with dexamethasone inhibited leukocyte adhesion; this may be due in part to the suppression of NFkappaB entry into the nucleus.

Animals↗

Effects of artificial tear temperature on corneal sensation and subjective comfort.

PURPOSE: Cooling reduces acute inflammation and local nerve sensation. We investigated the relationship between artificial tear temperature, ocular surface sensation, and patient comfort. METHODS: We placed preservative-free artificial tears and eye mask stored at four temperatures (36 degrees C, 25.2 degrees C, 4 degrees C, and -10 degrees C) in the right eyes of 24 normal subjects, whose left eyes served as controls. Corneal and conjunctival sensations were measured and corneal temperature was recorded. Comfort was reported on a 7-point scale. RESULTS: Corneal temperature was significantly lowered with all temperature artificial tears and frozen eye mask (p < 0.001 for each temperature relative to the previous one). Aesthesiometer readings were inversely correlated with corneal temperature (r = -0.45, p = 0.0005), decreasing with lower temperatures, reaching 2.0 +/- 1.3 g/mm2 (p = 0.001) for the mask. Conjunctival sensation reacted similarly and was well correlated with both corneal temperature (r = 0.43, p = 0.0009) and corneal sensation (r = 0.39, p = 0.006). Treatments provided relief, with the 4 degrees C tears being the most comfortable (p = 0.0001). CONCLUSION: Although there may still be some biases, cooled artificial tears provide relief to the eye by the mechanism of reduced corneal and conjunctival sensation.

Administration, Topical↗

Common T cell receptor clonotype in lacrimal glands and labial salivary glands from patients with Sjögren's syndrome.

Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single-strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS.

Amino Acid Sequence↗

Quantitative videographic analysis of blinking in normal subjects and patients with dry eye.

OBJECTIVE: To study patterns of eye blinking in normal subjects and patients with dry eye. METHODS: We developed an automated, noninvasive blink monitor that permits quantitative analysis of 6 parameters of blinking. We used this method under normal conditions and then examined the effects on the patterns of blinking in patients with dry eye; several steps in this method were designed to exacerbate or ameliorate ocular surface desiccation. RESULTS: The mean (+/-SD), maximum, and coefficient of variation of the interblinking time in normal subjects and patients with dry eye were 4.0 +/- 2.0 and 1.5 +/- 0.9 seconds, 8.9 +/- 4.0 and 4.2 +/- 2.4 seconds, and 55% +/- 21% and 65% +/- 24%, respectively. Those values for the blinking time were 0.20 +/- 0.04 and 0.27 +/- 0.16 seconds, 0.35 +/- 0.12 and 0.99 +/- 1.30 seconds, and 23% +/- 9% and 46% +/- 34%, respectively. The use of artificial tears or spectacles with moist panels and moist inserts tended to normalize the patterns of blinking in the patients with dry eye, whereas exposure to wind made them more abnormal. CONCLUSIONS: Our technique permitted a rigorous analysis of blinking that was previously unavailable. We have shown that local ocular surface conditions alone can significantly affect patterns of blinking. This method should be applicable to studying psychologic and any other factors that may influence blinking.

Adolescent↗

Increased expression of human thioredoxin/adult T cell leukemia-derived factor in Sjögren's syndrome.

OBJECTIVE: To determine the involvement of human thioredoxin/adult T cell leukemia-derived factor TRX/ADF) in Sjögren's syndrome (SS) and the correlation with Epstein-Barr virus (EBV). METHODS: Indirect immunohistochemical techniques and reverse transcriptase polymerase chain reaction were utilized to analyze TRX/ADF expression and the presence of EBV, using 6 normal tissues and 23 surgical specimens. The kinetics of expression of TRX/ADF induced by EBV was examined in vitro with peripheral blood B cells from EBV-seronegative donors. RESULTS: Marked expression of TRX/ADF was found in the infiltrating B cells and the epithelial cells of salivary gland tissues from patients with SS (11 of 12 cases), but not in those from patients with other salivary gland inflammatory conditions (0 of 11 cases) or those of normal individuals (0 of 6 cases). In immunohistologic analyses, a striking topographic correlation between TRX/ADF and EBV was found. The coexistence of TRX/ADF messenger RNA and EBV DNA was detected by polymerase chain reaction (r = 0.75, P < 0.01). Peripheral blood B cells from EBV-seronegative donors showed de novo synthesis of TRX/ADF following in vitro infection with EBV. EBV-infected B cell lines all expressed TRX/ADF. TRX/ADF was not detected in non-EBV-infected cells. Tumors in SCID mice reconstituted with mononuclear cells of salivary glands from SS patients, which were composed of human B cells carrying EBV DNA, were positive for TRX/ADF. CONCLUSION: These findings suggest that TRX/ADF expression closely reflects the intracellular event of EBV reactivation in SS. This is also the first report to show the ectopic in vivo expression of TRX/ADF in human autoimmune disease.

Adult↗