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Kohji Nishida

Publications and source records attributed to Kohji Nishida.

34 records · Page 2Linked to original sources
PubMed2005

Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes.

For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 degrees C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.

Cell Culture Techniques↗
PubMed2005

Rheumatoid arthritis-associated corneal ulceration complicated by bacterial infection.

We report two cases of rheumatoid ocular disease complicated by infection of methicillin-sensitive Staphylococcus aureus (MSSA) in one case, and methicillin-resistant Staphylococcus aureus (MRSA) in the other. In both cases, punctal occlusion and immunosuppressive therapy were presumed to be major risk factors of the infections. In addition, the characteristic feature was corneal melting, which is probably accelerated by infection. To avoid infectious progression and melting, potent antibiotics followed by immunosuppressive therapy were necessary.

Journal Article↗
PubMed2005

Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface.

PURPOSE: Autologous stem cell transplantation for total limbal stem cell deficiency is immunologically preferable, to avoid allograft rejection. This study was undertaken to investigate the possibility of a novel tissue engineering approach for ocular surface reconstruction, using autologous oral mucosal epithelial stem cells expanded ex vivo on temperature-responsive cell culture surfaces. METHODS: Rabbit oral mucosal epithelial cells cultured on temperature-responsive culture surfaces with mitomycin-C-treated 3T3 feeder cells for 2 weeks produced confluent epithelial cell sheets. Putative progenitor cell populations were estimated by colony-forming assays. Autologous transplantation of these cell sheets to surgically manipulated eyes was performed, and ocular surface reconstruction and cell phenotypic modulation were examined. RESULTS: All cultured oral epithelial cells were nonenzymatically harvested as transplantable intact cell sheets by reducing culture temperature to 20 degrees C. Oral epithelial cells were stratified in three to five cell layers more similar to corneal epithelium than to oral mucosal epithelium. Colony-forming assays and immunofluorescence for p63, beta1-integrin, and connexin 43 indicated retention of viable stem and/or progenitor cell populations in cell sheets. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface, with restoration of transparency. Four weeks after transplantation, epithelial stratification was similar to that in the corneal epithelium, although the keratin expression profile retained characteristics of the oral mucosal epithelium. CONCLUSIONS: Cell sheet harvest technology enables fabrication of viable, transplantable, tissue-engineered epithelial cell sheets that retain putative progenitor cells from autologous oral mucosal epithelial cells. Promising clinical capabilities for autologous tissue-engineered epithelial cell sheets for ocular surface reconstruction are indicated.

Animals↗
PubMed2005

Atypical composition and ultrastructure of proteoglycans in the mouse corneal stroma.

PURPOSE: Recently, gene-targeted strains of mice with null mutations for specific proteoglycans (PGs) have been used for investigations of the functional role of these molecules. In the present study, the corneal stroma of the mouse was examined to provide some baseline PG morphologies in this species. METHODS: Monoclonal antibodies to specific glycosaminoglycan (GAG) chain sulfation patterns were used to characterize PG composition in corneal extracts by SDS-PAGE and Western blot analysis and to identify their tissue distribution by immunofluorescence microscopy. PGs were also visualized by transmission electron microscopy after contrast enhancement with cationic dye fixation. RESULTS: Western blot analysis of pooled corneal extracts and immunofluorescence of tissue sections identified 4-sulfated, but not 6-sulfated, chondroitin sulfate/dermatan sulfate (CS/DS). Keratan sulfate (KS) was present only as a low-sulfated moiety. Electron microscopic histochemistry disclosed a complex array of corneal PGs present as (1) fine filaments radiating from collagen fibrils, and (2) elongate, straplike structures, running either along the fibril axis or weaving across the primary fibril orientation. These large structures were digested by chondroitinase ABC, but not by keratanase. CONCLUSIONS: KS in the mouse is predominantly undersulfated and generates an immunostaining pattern that differs from that observed in corneas of other mammalian species thus far investigated. The mouse cornea resembles other mammalian corneas in the presence of filamentous arrays of small, collagen-associated stromal PGs visualized by cationic dye staining. However, large dye-positive structures with a CS/DS component are also present and appear to be unique to the cornea of this species.

Animals↗
PubMed2004

Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.

BACKGROUND: Ocular trauma or disease may lead to severe corneal opacification and, consequently, severe loss of vision as a result of complete loss of corneal epithelial stem cells. Transplantation of autologous corneal stem-cell sources is an alternative to allograft transplantation and does not require immunosuppression, but it is not possible in many cases in which bilateral disease produces total corneal stem-cell deficiency in both eyes. We studied the use of autologous oral mucosal epithelial cells as a source of cells for the reconstruction of the corneal surface. METHODS: We harvested 3-by-3-mm specimens of oral mucosal tissue from four patients with bilateral total corneal stem-cell deficiencies. Tissue-engineered epithelial-cell sheets were fabricated ex vivo by culturing harvested cells for two weeks on temperature-responsive cell-culture surfaces with 3T3 feeder cells that had been treated with mitomycin C. After conjunctival fibrovascular tissue had been surgically removed from the ocular surface, sheets of cultured autologous cells that had been harvested with a simple reduced-temperature treatment were transplanted directly to the denuded corneal surfaces (one eye of each patient) without sutures. RESULTS: Complete reepithelialization of the corneal surfaces occurred within one week in all four treated eyes. Corneal transparency was restored and postoperative visual acuity improved remarkably in all four eyes. During a mean follow-up period of 14 months, all corneal surfaces remained transparent. There were no complications. CONCLUSIONS: Sutureless transplantation of carrier-free cell sheets composed of autologous oral mucosal epithelial cells may be used to reconstruct corneal surfaces and can restore vision in patients with bilateral severe disorders of the ocular surface.

Aged↗
PubMed2004

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2.

Many types of organ-specific stem cells have been recently shown to exhibit a side population (SP) phenotype based on their ability to efflux Hoechst 33342 dye. Because stem cells from corneal epithelium reside in the basal layer of the limbal epithelium, the purpose of this study was to examine whether the limbal epithelium contains SP cells. The ATP-binding cassette transporter Bcrp1/ABCG2 is reported to contribute to the SP phenotype in cells from several diverse sources. Here we show data from fluorescence-activated cell sorting and real-time quantitative RT-PCR analysis showing that harvested limbal epithelial cells contain SP cells expressing ABCG2. Immunofluorescence revealed that a portion of limbal epithelial basal cells expressed ABCG2. Data indicate that ABCG2 positive limbal epithelial cells are putative corneal epithelial stem cells.

Adenosine Triphosphate↗
PubMed2004

Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface.

BACKGROUND: Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and visual loss. Recent attempts have been made to fabricate corneal epithelial graft constructs, but the technology is still evolving. We have developed a novel cell-sheet manipulation technology using temperature-responsive culture surfaces to generate functional, cultivated corneal epithelial cell sheet grafts. METHODS: Human or rabbit limbal stem cells were cocultured with mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes at 37 degrees C. Cell sheets were harvested from the dishes after 2 weeks by reducing temperature to 20 degrees C. Histologic analyses, immunoblotting, and colony-forming assay were performed to characterize the cell sheets. Autologous transplantation was undertaken to reconstruct the corneal surfaces of rabbits with experimentally induced limbal stem cell deficiencies. RESULTS: Multilayered corneal epithelial sheets were harvested intact simply by reducing the temperature, without the use of proteases. Cell-cell junctions and extracellular matrix on the basal side of the sheet, critical to sheet integrity and function, remained intact. A viable population of corneal progenitor cells, close in number to that originally seeded, was found in the sheets. Harvested sheets were easily manipulated, transplantable without any carriers, and readily adhesive to corneal stroma so that suturing was not required. Corneal surface reconstruction in rabbits was highly successful. CONCLUSIONS: Cell sheet engineering technology allows us to create intact, transplantable corneal epithelial cell sheets that retain stem cells from limbal stem cells expanded ex vivo. Our research indicates highly promising clinical capabilities for our bioengineered corneal epithelial sheet.

3T3 Cells↗
PubMed2004

A spectrum of clinical manifestations of gelatinous drop-like corneal dystrophy in japan.

PURPOSE: Typical manifestations of gelatinous drop-like corneal dystrophy (GDLD) have been documented in past studies. In practice, however, we see few typical cases and encounter many with atypical clinical manifestations. Moreover, there have been no detailed reports about the various clinical presentations. The purpose of this study is to describe a clinical subclassification for GDLD. DESIGN: Observational case series. METHODS: A retrospective, observational case series study was conducted at a single institution, Osaka University Hospital. Examined were 18 eyes of ten patients with GDLD with no prior surgery. All were found to have M1S1 mutations. Using slit-lamp examinations we performed a phenotypic classification. RESULTS: We classified GDLD patients into four groups. They included band keratopathy type (seven eyes of four patients), stromal opacity type (five eyes of three patients), kumquat-like type (four eyes of two patients), and typical mulberry type (two eyes of two patients). CONCLUSIONS: Gelatinous drop-like corneal dystrophy is thought to be the result of mutations in M1S1; however, it has various clinical manifestations. To our knowledge, this study is the first to report the range of clinical phenotypes of GDLD in a Japanese population. As most clinicians have no criteria for diagnosing GDLD, many cases are likely to be overlooked or be mistaken for other conditions. We believe that our present classification will be useful for the diagnosis of GDLD.

Adolescent↗
PubMed2003

Electron microscopy of the epithelial flap created by ethanol treatment in the rabbit corneal epithelium.

PURPOSE: To assess the effect of corneal debridement with 25% ethanol on rabbit corneal epithelium by electron microscopy. SETTING: Department of Ophthalmology, Osaka University School of Medicine, Osaka, Japan. METHODS: Rabbit corneas were deepithelialized by applying 25% ethanol for 3 minutes, and a hinged epithelial flap was created and repositioned. Ten corneas were evaluated immediately after the epithelial debridement procedure and at 1, 3, and 7 days. Histological changes were evaluated using scanning and transmission electron microscopy. RESULTS: Corneas treated with ethanol showed a decrease in microvilli, breaks in intercellular junctions, epithelial cell edema, and damage in basement membrane hemidesmosomes. These toxic changes recovered to an almost normal appearance after 24 hours and rapidly improved over the following week. An increase in desquamated cells was observed after debridement; this returned to normal after 1 week according to the normal cellular cycle. Wing cells showed no damage at any stage. CONCLUSIONS: Although ethanol appeared to have a toxic effect on rabbit epithelial cells, the effect did not persist over time. The first signs of recovery were observed 24 hours after debridement, and full recovery was observed over the following week. The ethanol path to the central basement membrane appeared to be centripetally from a round cut through the basement membrane, leaving the wing cells intact.

Animals↗
PubMed2003

Tissue engineering of the cornea.

Tissue engineering of the cornea represents a paradigm shift in medical treatment to overcome the present disadvantages of corneal transplantation, primarily immune rejection and the shortage of donor corneas. Transplantation of cultivated corneal epithelial cells expanded ex vivo from corneal epithelial stem cells has been developed and has already entered the clinical realm. However, there remain many hurdles to be overcome. The author and colleagues are developing a method to transplant cultivated cell sheets that uses a temperature-responsive culture dish. This article reviews the present situation regarding tissue-engineered corneal epithelium and introduces the results of this program of research.

Cornea↗
PubMed2002

Wavefront aberrations measured with Hartmann-Shack sensor in patients with keratoconus.

OBJECTIVE: To compare the ocular wavefront aberrations of normal and keratoconic eyes and to describe the characteristics of the higher-order aberrations in eyes with keratoconus. DESIGN: Prospective case control and observational study. PARTICIPANTS: Thirty-five keratoconic eyes and thirty-eight normal controls. METHODS: Higher-order aberrations in refraction were measured with a wavefront sensor, and those aberrations resulting from the cornea were evaluated by videokeratographic data. MAIN OUTCOME MEASURES: Coma-like (S(3 + 5)), spherical-like (S(4 + 6)), and total (S(3 + 4 + 5 + 6)) higher-order aberrations in both refraction and the cornea. RESULTS: The mean +/- standard deviation of S(3 + 5) (1.88 +/- 1.16), S(4 + 6) (0.70 +/- 0.55), and S(3 + 4 + 5 + 6) (2.03 +/- 1.23) in refraction (6-mm diameter, root mean square, micro m) were significantly higher in the keratoconic eyes than in normal controls (0.26 +/- 0.10, 0.19 +/- 0.10, 0.34 +/- 0.11, respectively; Mann-Whitney U test, P = 0.001). Coma-like aberrations were 2.32 times larger than spherical-like aberrations in keratoconic eyes. CONCLUSIONS: The increase of ocular higher-order aberrations in keratoconic eyes results from an increase of corneal higher-order aberrations. Coma-like aberrations were dominant compared with spherical-like aberrations in keratoconic eyes. Wavefront sensing will enable us not only to evaluate the quality of vision but also to differentiate keratoconic eyes from normal eyes by analyzing the characteristics of the higher-order aberrations.

Adult↗
PubMed2002

Topical administration of HSV gD-IL-2 DNA is highly protective against murine herpetic stromal keratitis.

PURPOSE: To evaluate the immunopreventive effect of eyedrops that contain gD-IL-2 DNA (a chimeric gene of the glycoprotein D gene of herpes simplex virus type I (HSV-1) and human interleukin-2 (IL-2) on murine herpetic keratitis. METHODS: A plasmid containing gD-IL-2 (pHDLneo1) was constructed. The eyedrops containing 90 microg/10 microL of the DNA was instilled bilaterally into the conjunctival sacs of BALB/c mice on days 0 and 7. Three weeks after the last administration, neutralizing antibody, delayed-type hypersensitivity (DTH), and 51Cr-release from infected targeted cells by lymphocytes from the cervical lymph nodes and spleen were determined. Immunized mice were challenged with HSV-1, after which the clinical signs of the corneal epithelia and stroma were scored. RESULTS: Specific neutralizing antibody was raised and prominent DTH reaction was elicited from immunized mice. Lymphocytes obtained from the local lymph nodes and spleen vigorously potentiated the cytotoxic activity against the virus-infected cells. Clinically, the development of stromal keratitis was completely inhibited, but prevention or reduction of HSV-1 epithelial lesions was not demonstrated statistically. CONCLUSION: Topical immunization with a DNA vaccine encoding gD-IL-2 totally prevented the development of herpetic stromal keratitis. This procedure is a simple and convenient method for possible clinical application in the future.

Administration, Topical↗
PubMed2002

Changes in conjunctival clusterin expression in severe ocular surface disease.

PURPOSE: Clusterin is a unique gene transcript in the human ocular surface epithelia and is synthesized by and localized in mucosal epithelia in general. It is not present, however, in keratinized epithelia, such as epidermis. In severe ocular surface disease, pathologic keratinization (squamous metaplasia) of the ordinarily nonkeratinized corneal and conjunctival mucosal epithelia results in severe visual loss. In the current study, the expression of clusterin was examined in conjunctivalized corneas with severe ocular surface disease. METHODS: We examined conjunctiva covering cornea in eight eyes with ocular surface disease (Stevens-Johnson syndrome and ocular cicatricial pemphigoid) in which pathologic keratinization was present. Normal conjunctiva from four age-matched individuals served as the control. Semiquantitative RT-PCR was used to investigate expression of the clusterin gene. Immunohistochemistry was used to study the distribution of clusterin protein. RESULTS: The level of clusterin mRNA was significantly lower than normal in the diseased ocular surfaces. Clusterin protein was also markedly decreased in keratinized conjunctiva compared with that in normal eyes. CONCLUSIONS: Clusterin expression is markedly reduced in the pathologic, keratinized ocular surface epithelium, suggesting its importance in maintaining the ocular surface epithelium as a nonkeratinizing epithelium.

Adult↗
PubMedDate not supplied

Renin-angiotensin system in proliferative diabetic retinopathy and its gene expression in cultured human müller cells.

PURPOSE: To determine if the renin-angiotensin system (RAS) is involved in diabetic retinopathy, we measured the levels of angiotensin-converting enzyme (ACE) and angiotensin II in the vitreous of patients with proliferative diabetic retinopathy (PDR). We also investigated whether the genes of the RAS factors were expressed by cultured human Müller cells. METHODS: Vitreous samples were collected during vitreous surgery from patients with PDR, and from patients with idiopathic macular hole, who served as controls. The concentration of ACE was analyzed, and the level of angiotensin II was quantitatively determined by double antibody radioimmunoassay. In addition, the cDNA, prepared from the mRNA extracted from cultured human Müller cells, was used as a template for reverse transcriptase-polymerase chain reaction with primers selected for renin, angiotensinogen, ACE, and angiotensin receptor type I. RESULTS: The mean concentration of ACE was 0.82 +/- 0.73 IU/L at 37 degrees C in the PDR group, which was significantly higher than the 0.05 +/- 0.07 IU/L at 37 degrees C in the controls. The mean concentration of angiotensin II was 8.77 +/- 4.57 pg/mL in the PDR group, which was significantly higher than the 5.1 +/- 1.7 pg/mL in the controls. mRNA for renin, angiotensinogen, ACE, and angiotensin receptor type I was detected in cultured human Müller cells. CONCLUSIONS: The RAS is locally activated in eyes with PDR, and Müller cells may play a role in this local activation.

Angiotensin II↗
PubMedDate not supplied

Gene expression of neurotrophins and their high-affinity Trk receptors in cultured human Müller cells.

PURPOSE: To investigate the gene expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and their high-affinity receptors (TrkA, TrkB and TrkC) in cultured human Müller cells. METHODS: The polymerase chain reaction was performed using specific primers for NGF, BDNF, NT-3, TrkA, TrkB and TrkC with complementary DNAs as the template, which were generated from poly-A+ RNA of cultured human Müller cells. RESULTS: We detected the precursor mRNA transcripts for NGF, BDNF, NT-3, TrkB and TrkC, but not TrkA. CONCLUSION: Human Müller cells can direct mRNA expressions of a number of neurotrophins which may have a neurotrophic function in the retina. An autocrine mode of action is suggested, since Müller cells not only synthesize neurotrophic factors, but also express their specific receptors.

Cells, Cultured↗