Yasuhiro Osakabe

Effect of donor age on morphologic variation of cultured human corneal endothelial cells

Purpose. To examine the effect of donor age on the morphologic variation of cultured human corneal endothelial cells (HCEC). Methods. HCEC were obtained from the remaining corneoscleral rims of seven human corneas used for penetrating keratoplasty. The donor age ranged from 2 to 75 years. Primary cultures were established from explants of the endothelial cell layer, including the Descemets membrane, and were propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. At the fourth passage, frequency distribution of cell area in the confluent monolayer was calculated and the effect of donor age on cell area was analyzed. Results. The percentage of HCEC with cell area over 2000 &mgr;m2significantly increased with donor age (r = 0.935, p = 0.0007). Conclusion. Cultured HCEC established from older donor tissue display greater heterogeneity. The use of HCEC from younger donors may be preferable to maximize the benefits of HCEC transplantation.

Classic type of Kaposi's sarcoma and human herpesvirus 8 infection in Xinjiang, China

We report 17 cases of the classic type of Kaposis sarcoma in Xinjiang, which is located in the north‐western area of China surrounded by Mongolia in the east, Russia in the north and Kazakhstan in the west. Fifteen of the patients were of the Uygur people. All patients were male and did not have acquired immunodeficiency syndrome. Most of the lesions were found in the lower and/or upper extremities, with 16 patients showing multiple lesions. Immunohistochemical examination of the lesions revealed that human herpesvirus 8 (HHV‐8)‐encoded latency‐associated nuclear antigen was expressed in the nuclei of spindle‐shaped tumor cells. HHV‐8 DNA was detected by polymerase chain reaction in all seven cases examined. Phylogenetic tree analysis revealed that DNA sequences of the HHV‐8‐encoded K1 gene in the seven Kaposis sarcoma cases were classified as subtype C that was common in the Mediterranean, the Middle East and East Asian countries. In addition, using immunofluorescence we investigated the seroprevalence of HHV‐8 in 73 Uygur patients with diseases other than Kaposis sarcoma. Surprisingly, the serological study revealed that 34 of the patients (46.6%) were positive for antibodies against HHV‐8, suggesting that HHV‐8 infection is widespread in Xinjiang area. The occurrence of the classic type of Kaposis sarcoma with a high seropositivity rate implies that Xinjiang is the most endemic area for HHV‐8 infection in the world known to date. Considering that Xinjiang is located at the middle point of the Silk Road that used to extend from Rome to China, these data imply that the virus may have been in circulation in this area due to the migration of the people via the Silk Road.

Detection of cytomegalovirus DNA from cytomegalovirus corneal endotheliitis after penetrating keratoplasty.

Purpose: To report the detection of cytomegalovirus (CMV) DNA in the cornea of a CMV endotheliitis patient after penetrating keratoplasty (PKP). Case: A 71-year-old man without immunodeficiency developed corneal endotheliitis in the right eye. The patient had previously received PKP several times. Polymerase chain reaction (PCR) detected CMV-DNA in the aqueous humor in his affected eye, and we started administration of ganciclovir. There was resolution of the inflammation; however, bullous keratopathy was subsequently noted in the cornea. Additional PKP was performed with perioperative intravenous administration of ganciclovir. The failed graft obtained during the additional PKP was subjected to PCR analysis and histopathological examination. PCR analysis showed CMV-DNA in the failed graft. Little inflammatory change was noted in either the epithelial or stromal layers of the failed graft. With continued ganciclovir treatment, the graft remained clear and no recurrence or rejection occurred until 12 months after the last PKP. Conclusion: Our PCR analysis showed the presence of CMV-DNA within the cornea of the patient with corneal endotheliitis.

Evaluation of posterior capsule opacification using a new posterior view method in rabbits: single-piece acrylic versus 3-piece acrylic intraocular lens.

PURPOSE: To introduce a new procedure for evaluating posterior capsule opacification (PCO) in rabbit eyes and to perform a comparative study of the single‐piece and 3‐piece acrylic intraocular lenses (IOLs) on PCO using the new evaluation method. SETTING: Jinshikai Medical Foundation, Nishi Eye Hospital, Osaka, Japan. METHODS: A single‐piece or 3‐piece acrylic IOL was implanted in 1 eye and the other in the contralateral eye of 5 rabbits. Three weeks after surgery, PCO was scored by Evaluation of Posterior Capsule Opacification (EPCO) in posterior view. Before the posterior view was photographed, the anterior capsule was removed from the whole optic area to eliminate disturbing anterior capsule opacification (ACO) for the PCO evaluation. RESULTS: Posterior capsule opacification could be well observed and viewed in the posterior view so that it could be scored by EPCO without confusion with ACO. The mean PCO score of the single‐piece and 3‐piece acrylic IOLs was 3.12 ± 0.19 and 2.41 ± 0.70, respectively (P<.05 and P = .03, respectively). CONCLUSION: The removal of ACO allowed scoring of PCO by EPCO in rabbit eyes. The single‐piece acrylic IOL showed singnificantly more PCO than the 3‐piece acrylic IOL at least 3 weeks after surgery in rabbits.

Properties of Corneas Reconstructed with Cultured Human Corneal Endothelial Cells and Human Corneal Stroma

PurposeTo examine the properties of corneas tissue-engineered with cultured human corneal endothelial cells (HCEC) and human corneal stroma.MethodsPrimary HCEC cultures were established from endothelial cell layer explants and propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. A cell suspension of HCEC at the fifth passage was transferred onto human corneal stroma deprived of endothelial cells, and the corneas were gently centrifuged to enhance cell attachment. The cell density of the tissue-engineered corneas was examined after staining with alizarin red and trypan blue. The tissue-engineered corneas were histologically examined by light and electron microscopy. The pump function of the tissue-engineered corneas was measured using an Ussing chamber.ResultsThe mean endothelial cell density of four tissue-engineered corneas was 2380 ± 264 cells/mm2 (mean ± SD). HCEC on the tissue-engineered corneas had a morphology similar to HCEC in vivo. The pump function parameters of the tissue-engineered corneas were 55%–75% of those of normal corneas.ConclusionsHCEC on the tissue-engineered corneas have morphology and cellular density similar to HCEC in vivo, whereas the pump function of the tissue-engineered corneas was lower than in normal corneas. Jpn J Ophthalmol 2005;49:448–452

Intraoperative mydriasis by intracameral injection of mydriatic eye drops: in vivo efficacy and in vitro safety studies

Background:  This study investigated the efficacy and safety of intracameral injection of commercially available eye drops containing 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin‐P, Santen Pharmaceutical, Osaka, Japan).

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Classification of secondary corneal amyloidosis and involvement of lactoferrin.

PURPOSE To classify secondary corneal amyloidosis (SCA) by its clinical appearance, to analyze the demographics of the patients, and to determine the involvement of lactoferrin. DESIGN Retrospective, observational, noncomparative, multicenter study. PARTICIPANTS Twenty-nine eyes of 29 patients diagnosed with SCA by corneal specialists at 9 ophthalmologic institutions in Japan were studied. METHODS The clinical appearance of SCA was determined by slit-lamp biomicroscopy and was classified into 3 types. The demographics of the patients, for example, age, gender, and the duration of the basic disease (trichiasis, keratoconus, and unknown), were determined for each clinical type. Surgically excised tissues were stained with Congo red and antilactoferrin antibody. The postoperative prognosis also was determined. MAIN OUTCOME MEASURES Clinical appearance of the 3 types of SCA, along with the gender, age, and duration of the basic diseases were determined. RESULTS Classification of SCA into 3 types based on clinical appearance found 21 cases with gelatinous drop-like dystrophy (GDLD)-like appearance (GDLD type), 3 cases with lattice corneal dystrophy (LCD)-like appearance (LCD type), and 5 cases with the combined type. Patients with the GDLD type were younger (average age: 40.9 years for the GDLD type, 74.3 years for the LCD type, and 46.8 years for the combined type), predominantly women (85.7% for the GDLD type, 33.3% for the LCD type, and 60% for the combined type), and had the basic disease over a longer time (average duration: 22.1 years for the GDLD type, 14.0 for the LCD type, and 11.4 for the combined type). The distribution of the basic diseases (trichiasis vs. keratoconus vs. unknown) was not significantly different for each type. Surgical treatments, for example, phototherapeutic keratectomy, lamellar keratoplasty, and simple keratectomy, resulted in a good resolution in all surgically treated cases. One subject dropped out of the study. Spontaneous resolution was seen in one subject after epilation of the cilia. Amorphous materials in the excised tissues showed positive staining results by Congo red and by antilactoferrin antibody. CONCLUSIONS Secondary corneal amyloidosis can be classified into 3 clinical types based on its clinical appearance. Larger numbers of females and lactoferrin expression were seen in all 3 types. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Detection of Streptococcus species by polymerase chain reaction in infectious crystalline keratopathy.

Purpose: Investigation of a positive polymerase chain reaction (PCR) for Streptococcus species from an excised cornea of a patient with infectious crystalline keratopathy. Methods: Samples microdissected from corneal intrastromal aggregates present in infectious crystalline keratopathy were subjected to PCR for 3 primer pairs respectively specific for Streptococcus, Staphylococcus, and several kinds of fungi. Results: The PCR detected DNA from Streptococcus but not from Staphylococcus or fungi. Direct sequencing of the PCR products showed that the PCR products had the highest identity (99.8%) with Streptococcus mitis and Streptococcus gordoni, indicating that these species were the most probable causes of infectious crystalline keratopathy in this patient. Conclusion: Our findings suggest that PCR provides a good adjunct technique in the diagnosis of corneal infection.

Immunohistochemical, ultrastructural and molecular study of well differentiated adenocarcinomas of the lung predominantly composed of goblet cells.

In order to clarify the morphological and biological characteristics of well differentiated adenocarcinoma of the lung predominantly composed of goblet cells (WDAG), histopathological examinations, including some molecular biological procedures, were carried out using 42 surgical specimens of primary lung carcinoma which were predominantly (>50% of the total cell population) or totally composed of goblet cells. The subjects included 19 men and 21 women, ranging in age from 41 to 81 (mean 60 years old) with predominantly nodular, peripherally located lesions. Ultrastructural examination revealed characteristic apical microvillous filamentous core rootless (AFCR) in some, but not all, cases. Histologically, these AFCR corresponded well with structures stained by phosphotungstic acid hematoxylin (PTAH). The goblet cells of WDAG were divided into PTAH-positive (26 cases) and -negative (16 cases) groups. The PTAH-positive group had larger tumor size, greater number of intrapulmonary and extrapulmonary metastases and shorter disease-free interval. The immunoexpression of p53 protein (60%) and rate of K-ras point mutation (84%) were also higher in the PTAH-positive group. Therefore the goblet cell population of WDAG, though it may appear morphologically homogeneous under light microscopy, is actually composed of heterogeneous groups of cells with different histopathological characteristics and biological behavior.