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Nobuyuki Ebihara

Publications and source records attributed to Nobuyuki Ebihara.

14 recordsLinked to original sources
PubMed2006

Bone marrow-derived cells in normal human corneal stroma.

OBJECTIVE: To examine the normal human corneal stroma for the presence of bone marrow-derived cells. METHODS: Thirty-four corneas from donors aged 56 to 71 years were used. The stroma of the donor corneas was examined immunohistochemically by fluorescent microscopy. CD45-positive and -negative cells were separated from collagenase-digested stroma by magnetic beads, and the expression of toll-like receptor 4 was analyzed. RESULTS: CD45-positive cells were mainly found in the anterior stroma of the central and paracentral cornea as well as all stromal layers of the peripheral cornea (n = 5). These cells uniformly expressed CD11b, CD11c, CD14, and HLA-DR antigen but not CD3, CD19, CD56, or CD66, indicative of bone marrow-derived monocyte lineage cells, which can include monocytes, macrophages, or dendritic cells. CD45-positive cells isolated with magnetic beads accounted for 6.0% of total stromal cells (n = 20). Stromal CD45-positive cells, but not CD45-negative cells, expressed toll-like receptor 4 by flow cytometry and reverse-transcriptase polymerase chain reaction. CONCLUSION: Our findings demonstrate that DR antigen-positive bone marrow-derived monocyte lineage cells exist in the anterior and peripheral posterior stroma of normal human cornea. Clinical Relevance These cells may play a role in the innate and adaptive immune responses in the human cornea.

Aged↗
PubMed2006

Reduced expression of laminin-5 in corneal epithelial cells under high glucose condition.

PURPOSE: Laminin-5 (alpha3, beta3, gamma2 chains) is a major component of corneal basement membrane and has a crucial role in corneal epithelial cell adhesion. On the other hand, diabetic keratopathy has a varied degree of adhesive disturbance in corneal epithelial cells. Therefore, in this study, we investigated whether a high glucose condition altered the expression of laminin-5 in corneal epithelial cells in vitro. METHODS: Human corneal epithelial (HCE) cells were cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium for 5 passages before being used in experiments. We first examined the effect of a high glucose condition on the expression of mRNA and proteins for 3 chains of laminin-5 in HCE cells by semiquantitative reverse transcriptase-polymerase chain reaction, Western blotting analysis, and immunofluorescence staining. Second, we tried a cell detachment assay. After 5 days of incubation in high or normal glucose medium, HCE cells were treated with a solution of 0.05% trypsin and EDTA (0.2 mmol/L), pH = 8. The number of detached cells at different times after treatment was determined using a cell count machine. RESULTS: We found that alpha3 chain expression was reduced at the mRNA level in a high glucose condition, whereas beta3 and gamma2 chains showed no change. The high glucose condition induced the inhibition in the synthesis of 190-kd, 160-kd alpha3 chain and 105-kd gamma2 chain proteins of laminin-5. The adhesion capacity of HCE cells in the high glucose medium was weaker than that of HCE cells in normal glucose medium. Soluble laminin-5 rescued the detachment of HCE cells in high glucose medium. CONCLUSION: The loss of homeostatic levels of laminin-5 under a high glucose condition may correlate to weaken epithelial cell adhesion, resulting in the clinical manifestation of diabetic keratopathy.

Blotting, Western↗
PubMed2006

[A clinical study of intraocular malignant lymphoma].

PURPOSE: We investigated whether vitreous cytology and the measurement of intravitreous cytokine were useful for the diagnosis of intraocular malignant lymphoma. SUBJECTS AND METHODS: 8 eyes of 5 patients with suspected intraocular malignant lymphoma during the past 15 months. 3 vitreous samples were collected from 3 patients at the time of pars plana vitrectomy. Polymerase chain reaction(PCR) amplification and flowcytometric analysis(FACS) of the vitreous samples were performed. Interleukin (IL)-10 and IL-6 concentrations were measured. RESULTS: Vitreous cytology showed increased atypical B lymphocytes. The vitreous IL-10/IL-6 ratio was higher than 1 in all cases. Monoclonal rearrangement of the immunoglobulin heavy chain gene and the light chain restriction of immunoglobulin were detected. CONCLUSION: The detection of the monoclonality of infiltrated cells into the vitreous by PCR amplification and FACS, and the measurements of IL-10 and IL-6 concentrations in the vitreous fluid may be useful in the diagnosis of intraocular malignant lymphoma.

Adult↗
PubMed2005

Chemokine receptor gene expression in giant papillae of atopic keratoconjunctivitis.

PURPOSE: Major chemokine receptors in the giant papillae of atopic keratoconjunctivitis (AKC) have yet to be determined. We evaluated chemokine receptor genes and their ligand expressions in upper tarsal conjunctival giant papillae of AKC with atopic dermatitis and/or asthma. METHODS: CC, CXC, and CX3C chemokine receptor (R) gene expression levels in giant papillae of five clinically active AKC patients and in three age matched non-allergic control conjunctiva were measured with a multi-probe ribonuclease protection assay (RPA) system. The ligands of abundant chemokine receptors in the giant papillae were examined by immunohistochemistry or reverse transcription-polymerase chain reaction. Interleukin (IL)-4 and IL-13 gene expression levels were measured with RPA. Ligand expression in cultured human conjunctival fibroblasts was examined by reverse transcription-polymerase chain reaction. RESULTS: High CXCR4 and CCR4 gene expression levels were detected in the giant papillae of all (CXCR4) and four out of five (CCR4) patients. As a CCR4-ligand, thymus and activation regulated cytokine (TARC/CCL17) rather than macrophage derived chemokine (MDC/CCL22), was predominant immunohistochemicaly in the giant papillae. Giant papillae with high CCR4 gene expression levels showed high IL-4 and IL-13 expression. Cultured human conjunctival fibroblasts express stromal cell derived factor-1 (SDF-1/CXCL12) in vitro. CONCLUSIONS: CXCR4 and CCR4 are the major chemokine receptor genes expressed in the giant papillae of AKC with atopic dermatitis and/or asthma. Our findings suggest a role for CXCR4 and CCR4 in the formation of giant papillae.

Adolescent↗
PubMed2005

Mast cell chymase decreases the barrier function and inhibits the migration of corneal epithelial cells.

PURPOSE: We investigated the in vitro effects of human mast cell chymase on corneal epithelial cells. METHODS: Human corneal epithelial cells were incubated with human chymase at activity levels that were likely to exist in the tears of patients with vernal keratoconjunctivitis. RESULTS: Incubation of chymase resulted in a decrease of barrier function of corneal epithelium. Occludin protein was cleaved by chymase. In the wound assay, incubation with chymase resulted in an inhibition of cell migration. CONCLUSION: Human chymase causes the proteolysis of occludin and fibronectin, resulting in a decrease of barrier function and inhibition of the migration of corneal epithelial cells.

Blotting, Western↗
PubMed2005

Mast cell chymase induces conjunctival epithelial cell apoptosis by a mechanism involving degradation of fibronectin.

PURPOSE: To determine the mechanism of conjunctival epithelial injury in vernal keratoconjunctivitis, we investigated the effects of human chymase on conjunctival epithelial cells in vitro. METHODS: Human conjunctival epithelial cells were incubated with human chymase for 24 or 48 hr at levels of activity that were likely to exist in the tear fluid of patients with vernal keratoconjunctivitis. Morphologic changes of the cells were observed by phase contrast microscopy. To determine the number of detached cells, we used an automated cell counter, while apoptotic cells were quantitated by flow cytometry. The level of soluble fibronectin in conditioned medium was measured by ELISA. RESULTS: Most of the cells in the incubation with chymase were detached by 24 hr. However, chymase-mediated apoptosis was a slower process and was only detected after incubation of cells with chymase for 36 to 48 hr. Both cell detachment and apoptosis were blocked when cells were incubated in fibronectin-coated plates. The increase of soluble fibronectin was dependent on the amount of chymase added and the exposure time. A caspase inhibitor (antiapoptotic agent) rescued cells from apoptosis but did not prevent cell detachment. These results indicate that chymase-induced apoptosis of conjunctival epithelial cells represents anoikis, which is a slowly occurring apoptotic process induced by lack of adhesion to an extracellular matrix. CONCLUSIONS: Human mast cell chymase caused conjunctival epithelial cell detachment by degrading fibronectin, and this led to secondary apoptosis.

Apoptosis↗
PubMed2005

Bone marrow-derived cells in mouse and human cornea.

Recently published experimental data on the distribution of bone marrow (BM)-derived cells in human and mouse corneas in comparison with in human skin/oral mucosa are reviewed. In mouse corneal epithelium, major histocompatibility complex (MHC) class II-negative dendritic cells (DC) are present. Immature MHC class II-negative and mature MHC class II-positive DC are present in the center and periphery of the anterior corneal stroma, respectively. Monocyte (Mo)/macrophage (MPhi) lineage cells including the MPhi marker F4/80-expressing cells reside in the posterior stroma. In human cornea, MHC class II (HLA-DR)-positive immature myeloid DC (CD11cCD16, CD11cCD16, and CD11cCD1c) and Mo/MPhi lineage cells are detectable in the corneal epithelium and stroma, respectively. Distribution of Mo/MPhi lineage cells (HLA.DRCD11bCD11cCD14) is predominant in the anterior stroma of the central cornea and all layers of the peripheral cornea. Both the phenotypes and distribution pattern of these cells in human cornea are different from those of human skin and nasal mucosa. These findings suggest that BM-derived cells in normal human cornea are present in situ in preparation for foreign antigen and pathogens and have critical roles in innate and acquired immunity of the ocular surface.

Animals↗
PubMed2005

Distinct populations of dendritic cells in the normal human donor corneal epithelium.

PURPOSE: To characterize dendritic cells (DC) in normal human corneal epithelium. METHODS: Normal human donor corneal epithelium was examined by fluorescence microscopy with single and double staining for multiple markers. Morphologic studies were also performed by confocal microscopy. HLA-DRa, CD1c, and CD16 mRNA expression in the corneal epithelium was examined by RT-PCR. CD45+ cells were separated from the corneal epithelium with magnetic beads and then were stimulated with TNF-alpha and lipopolysaccharide in vitro. RESULTS: CD45+ cells were mainly located in the basal-cell layer of the corneal epithelium and partly in the wing/surface layers. CD45-positive cell numbers were significantly higher in the peripheral cornea (3-6 mm from the center) than in the central cornea (0-3 mm from the center). All these cells expressed HLA-DR and CD11c but not CD3, CD11b, CD14, CD19, CD56, or CD66, suggesting that these were bone marrow-derived myeloid DC. Some DR+CD11c+ DCs from the periphery expressed CD1c and CD16. HLA-DRa, CD1c, and CD16 mRNAs were detected in normal corneal epithelium. These CD11c+ DCs did not express CD123, CD1a, DC marker (CMRF56), CD40, CD80, or CD86. When CD45+ cells were isolated from the corneal epithelium by magnetic cell sorting, CD40 and CD86 expression were detected after in vitro stimulation with TNF-alpha and lipopolysaccharide. CONCLUSIONS: These findings demonstrate that normal human corneal epithelium contains at least three DC phenotypes, with HLA-DR+ myeloid lineage CD11c+CD16- DCs as the main population plus a small number of CD11c+CD16+ DCs and CD11c+CD1c+ DCs. These cells can be discriminated from bone marrow-derived cells in the human corneal stroma.

Aged↗
PubMed2004

Tear chymase in vernal keratoconjunctivitis.

PURPOSE: To determine the levels of mast cell chymase and tryptase activity in the tears of patients with vernal keratoconjunctivitis (VKC). METHODS: Subjects were 38 VKC patients and 18 healthy controls whose chymase and tryptase activity in tears was measured by enzyme assay. VKC severity was quantified based on the following clinical signs: papillary hypertrophy, conjunctival hyperemia, edema, punctate keratitis, Trantas dots, and mucus production. Of the 38 VKC patients, the degree of disease severity was mild in 13, moderate in 18, and severe in 7. RESULTS: Mean chymase activity and standard deviation in tears was 0.23+/-0.07mU in mild VKC, 0.68+/-0.22mU in moderate VKC, 1.91+/-0.71 mU in severe VKC, and 0.11+/-0.05 mU in healthy controls. The increase in all VKC stages was statistically significant compared to that in healthy control. The degree of chymase activity in tears correlated significantly with VKC severity (r = 0.9245, p < 0.001). High tryptase activity was also detected in the tears of VKC patients, although increased tryptase activity in tears did not correlate with disease severity (r = 0.1999). CONCLUSIONS: Chymase activity in tears may thus be a sensitive marker for determining the severity of VKC.

Adolescent↗
PubMed2003

Smad7 suppresses the inhibitory effect of TGF-beta2 on corneal endothelial cell proliferation and accelerates corneal endothelial wound closure in vitro.

PURPOSE: The inhibitory activity of transforming growth factor-beta2 (TGF-beta2) on corneal endothelial cell proliferation is thought to be a cause of the limited regenerative capacity of corneal endothelial cells that may be related to impaired corneal transparency when many corneal endothelial cells are lost due to various stresses. We determined whether Smad7, an intracellular antagonist of TGF-beta signaling, regulated the inhibitory activity of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation. METHODS: The effect of Smad7 on TGF-beta2- or aqueous humor-mediated inhibition of corneal endothelial cell proliferation was evaluated using thymidine uptake assay with cultured rabbit corneal endothelial cells infected with adenovirus carrying Smad7. Expression of Smad or cell cycle-related proteins was detected by immunoblotting. In addition, a small scrape wound was made across a monolayer of Smad7-expressing cultured rabbit corneal endothelial cells to examine the effect of Smad7 on the wound-healing process in vitro. RESULTS: Overexpression of Smad7 abolished the inhibitory effect of TGF-beta2 or aqueous humor on the proliferation of cultured rabbit corneal endothelial cells associated with the inhibition of phosphorylation of Smad2 and downregulation of p27kip1. Smad7-overexpressing cultured rabbit corneal endothelial cells exhibited shorter wound closure time in the presence of aqueous humor than LacZ-expressing cells. CONCLUSION: Overexpression of Smad7 suppressed the inhibitory effect of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation and accelerated corneal endothelial wound closure in vitro. Modification of Smad7 expression in corneal endothelial cells may thus have applicability in the treatment of wounded corneal endothelium.

Adenoviridae↗
PubMed2003

[Gene transfer into corneal endothelial cells by Helios gene gun].

PURPOSE: To examine the efficiency of particle-mediated gene transfer into rabbit corneal endothelium with a Helios gene gun system. METHODS: Using gene gun, plasmid DNA coding for green fluorescent protein(GFP) was transferred into cultured rabbit endothelial cells and rabbit corneal grafts from their endothelial site. Then the transferred corneas were transplanted as autografts. The GFP expression was detected by fluorescence microscopy. Histological examination of corneas was performed by light microscope and scanning electron microscope. RESULTS: In cultured rabbit endothelial cells, the transfection efficiency without damage to cells was about 7% under optimized helium pressure of 120 psi. In ex vivo, the GFP expression was limited to endothelial cells of the grafts at 140 psi. The histological findings from light and scanning electron microscopy of the grafts showed no severe mechanical damage in endothelial cells penetrated by gold particles. CONCLUSIONS: The helios gene gun system successfully transferred genes into corneal endothelial cells. However, further studies will be required to obtain the stable results of the particle mediated gene transfer into the endothelial cells of corneal grafts for clinical practice.

Animals↗
PubMedDate not supplied

Disodium cromoglycate inhibits capsaicin-induced eosinophil infiltration of conjunctiva independent of mast cells.

PURPOSE: To investigate whether disodium cromoglycate (DSCG) inhibits capsaicin-induced eosinophil infiltration of the conjunctiva independent of mast cells. METHODS: We administered 5 microl of capsaicin solution (10(-5) M) into the conjunctival sacs of mast cell-deficient W/W(v) mice (12 animals) and wild-type mice (12 animals). As controls, the eyes of 12 wild-type and 12 W/W(v) mice were treated with phosphate-buffered saline. Following treatment, the eyelids and eyeballs were removed en bloc at 3, 9, or 24 h, and were histologically examined. The number of infiltrated eosinophils and the expression of vascular cell adhesion molecule-l (VCAM-1) in the conjunctiva were quantified by the staining method of Hansel and immunohistochemical analysis. We also investigated whether treatment by depletion of neuropeptides or by DSCG administration could suppress the capsaicin-induced eosinophil infiltration of the conjunctiva. RESULTS: In both W/W(v) and wild-type mice, eosinophil infiltration of conjunctival tissues was observed 3 h after capsaicin administration. In both strains of mice, the number of infiltrated eosinophils increased over time, with VCAM-1 expression on vascular endothelial cells peaking at 9 h after treatment, and decreasing gradually within 24 h after treatment. In both the neuropeptide-depleted and the DSCG-treated groups, eosinophil infiltration and VCAM-1 expression were suppressed in comparison with the nontreated group. CONCLUSION: DSCG can directly inhibit neuropeptide-induced eosinophil infiltration of the conjunctiva independent of mast cells.

Animals↗
PubMedDate not supplied

High level of Fc epsilon receptor I-bindable immunoglobulin E in the tear fluid and increased immunoglobulin E-saturated cells in the giant papillae of vernal keratoconjunctivitis patients.

PURPOSE: To investigate the concentrations of Fc epsilon receptor I (Fc epsilon RI)-bindable immunoglobulin E (IgE) in the tear fluid and the proportion of IgE-saturated cells among Fc epsilon RI-positive cells in giant papillae from vernal keratoconjunctivitis (VKC) patients. METHODS: The tear fluids and giant papillae were obtained from 8 VKC patients with their informed consent. To detect the quantitative difference between Fc epsilon RI-bindable IgE and total IgE in the tear fluid, we used a new enzyme-linked immunosorbent assay system we have developed. Next, to estimate the proportion of IgE-saturated cells among Fc epsilon RI-positive cells, we used two distinct monoclonal antibodies for Fc epsilon RI for the immunohistochemistry. One antibody recognizes all Fc epsilon RI regardless of whether it is with or without receptor-bound IgE. The other does not recognize IgE-bound Fc epsilon RI. RESULTS: The quantitative difference between Fc epsilon RI-bindable IgE and total IgE were detected in the tear fluid of VKC patients. Fc epsilon RI-positive cells were significantly increased in the giant papillae of VKC compared with normal conjunctivae. The proportion of IgE-saturated cells among Fc epsilon RI-positive cells in giant papillae was higher than that in normal conjunctivae. CONCLUSION: These results suggest that F epsilon RI-bindable IgE may be a critical factor to estimate the severity of VKC.

Antibodies, Monoclonal↗