Biomedical subjects
Publications and source records attributed to R Manabe.
Bilateral corneal edema occurred in a schizophrenic patient receiving tranquilizers. This particular corneal edema was rapidly resolved after tranquilizer discontinuation. Specular microscopic examination disclosed marked enlargement of the corneal endothelium. This endothelial impairment may have been due to long-term administration of tranquilizers.
The protective effects of passive immunization with two kinds of anti-glycoprotein D (anti-gD) monoclonal antibodies, having different antiviral activities, were investigated in murine herpetic keratitis. One monoclonal antibody, designated M1, had high virus-neutralizing antibody titers, along with undetectable levels of complement-dependent cytolysis (CDC) and antibody-dependent cellular cytotoxicity (ADCC); the other, designated M12, exhibited extremely low titers of virus-neutralization with high level of CDC and ADCC. When systemically administered 24 hours prior to virus inoculation to the cornea, both M1 and M12 almost completely prevented the development of stromal keratitis. The protective efficacy of both was observed to be dose-dependent. Pepsin-treated M1 retained its efficacy in suppressing stromal keratitis, whereas pepsin-treated M12 did not. When the administration of M1 and M12 were delayed, both provided significant (but less complete) protection, up to 24 hours after virus inoculation. These results suggest that both virus neutralization and CDC/ADCC play an important role in preventing virus growth in the corneal stroma during the early stage of corneal infection.
With a view toward possible future clinical application, we investigated whether recombinant human epidermal growth factor (hEGF) could induce corneal neovascularization. Ethylene-vinyl-acetate copolymer slow-release pellets containing either 250 ng, 500 ng or 1 microgram of hEGF or 250 ng of bovine serum albumin (BSA) were implanted into rabbit corneal stroma, and the corneas were examined by slitlamp biomicroscope for 3 weeks. The results indicated that less than 1 microgram of hEGF per pellet did not induce neovascularization in the cornea. However, when pellets containing 250 ng of basic fibroblast growth factor (bFGF) were implanted, corneal neovascularization toward these pellets occurred within 10 days. The same phenomenon occurred toward the 250 ng hEGF pellets embedded in the ipsilateral or contralateral cornea of the same animal, but was significantly less severe. When a pellet containing 250 ng bFGF was intramuscularly implanted in the animal's back, similar neovascularization was observed toward the pellets containing hEGF implanted in the same animal's cornea, but not toward implanted pellets containing BSA. These results suggest that less than 1 microgram of hEGF cannot initiate corneal angiogenesis, but can promote new vessel formation if the limbal vasculature is activated by a sprouting signal from a certain substance as trace amounts of bFGF.
The authors investigated the vitreous oxygen tension in 30 eyes of 29 cases of proliferative diabetic retinopathy patients in order to determine the distribution of oxygen tension and the possible role of neovascular tissue in tissue oxygenation. Vitreous oxygen tension was measured using a polarographic oxygen electrode and a PO2 monitoring system (PO-2080). Prior to pars plana vitrectomy, the oxygen electrode was inserted into the vitreous cavity under microscopic observation with dim illumination transmitted fiberoptically. The respective oxygen tension at the mid-vitreous cavity, above the optic disc, above the macula, above the neovascular tissue, in the peripheral vitreous, above the photocoagulated retina and above the non-photocoagulated retina were 15.8 +/- 4.7 mmHg, 31.2 +/- 10.0 mmHg, 17.1 +/- 4.0 mmHg, 32.0 +/- 9.9 mmHg, 15.6 +/- 5.1 mmHg, 16.5 +/- 5.5 mmHg and 18.6 +/- 4.9 mmHg. The oxygen tension values above the neovascular tissue and above the optic disc showed statistically significantly higher values than that of midvitreous cavity. We assume this to be due to differences between the oxygen demand and supply on the neovascular tissue, because in these tissues there are large amounts of vessels and blood flow compared to oxygen consumption. Therefore residual oxygen causes oxygen flow from the neovascularization to the mid-vitreous. This outcome is one of the facts which supports the hypothesis that neovascular tissues develop in order to compensate for retinal ischemia by releasing oxygen.
To investigate the effects of trigeminal denervation on the corneal epithelium, left postganglionic trigeminal neurotomy via intracranial approach was performed in 22 rabbits. Among 16 rabbits survived successfully, 13 denervated eyes (81%) showed corneal epithelial abnormalities that included 4 epithelial defects and 9 epithelial opacities. The remaining 3 eyes were normal. Histological examination showed many atrophic epithelial cells and the thinning of the corneal epithelial layer in the denervated eyes with corneal opacities. There was no abnormality in corneal stroma or endothelium. The ratio of epithelial to total corneal thickness calculated by a computer-assisted image analyzer was 6.5 +/- 2.1 (%) in control eyes and 3.8 +/- 1.9 (%) in the denervated eyes, the two values being significantly statistically different (p less than 0.001).
A 56-year-old man developed idiopathic corneal endotheliopathy. The lesion consisted of severe stromal edema at the lower half of the cornea along with a number of associated keratic precipitates and steadily progressed to the upper half of the cornea. By polymerase chain reaction, herpes simplex virus DNA was demonstrated in the aqueous humor of this patient. Corneal stromal edema was resolved in response to treatment with topically applied and systemic acyclovir. Herpes simplex virus DNA was repeatedly demonstrated in the aqueous humor when the endothelial lesion recurred later. This evidence strongly indicates that this unique endothelial disorder is of viral origin.
A consecutive series of 20 eyes with Mooren's ulcer was treated by a new surgical approach: keratoepithelioplasty alone or in combination with corneoscleral lamellar graft. Eighteen (90%) eyes showed complete remission promptly after surgery; the other two also healed with the administration of additional systemic and topical steroids. During the follow-up periods (mean, 3.1 years), minor recurrence was found in 5 (25%) eyes for the first 6 months, in none (0%) for the second 6 months, in 2 (12%) for the third 6 months, and none thereafter. All seven eyes with recurrence were cured by additional steroid treatment, alone or with conjunctival excision and/or keratoepithelioplasty. In the 18 (90%) eyes, the lenticules, constantly observed, remained silent on the scleras, which were free from conjunctival invasion. These results strongly support the authors' theory that keratoepithelioplasty using donor corneal lenticules can heal Mooren's ulcer via blockage of infiltrated conjunctival invasion.
The presence of histamine H1-receptors in the bovine retinal blood vessels was studied with a [3H]mepyramine binding assay. The membranes of purified vessels obtained from bovine retinas showed specific [3H]mepyramine binding sites with a dissociation constant (KD) of 2.78 +/- 0.32 nM. This was similar to values obtained from the retinal neuronal fractions. The binding capacity (Bmax) was 53.8 +/- 1.7 fmol/mg protein, which was about a half that of the retinal neuronal fractions (108.9 +/- 3.1 fmol/mg protein). Some H1-antagonists proved to be potent competitors for [3H]mepyramine binding sites in bovine retinal blood vessels. These results indicate that histamine H1-receptors exist in the retinal blood vessels which may be involved in the physiological and the pathological responses of blood circulation in retinas.
The protective role of T cell subsets in corneal herpes simplex virus type 1 (HSV-1) infection has been studied. However, the relative contribution of, and the role played by, each particular T cell subset still remain a controversial issue. We studied sequentially the appearance of major histocompatibility (MHC) and viral antigens in HSV-1 infected corneas of Balb/C mice and related them to induction of T cell subsets in local lymph nodes and corneal lesions. Immunohistochemical study has revealed a marked increase of expression of class II MHC antigen in the corneal stromal cells, while class I MHC antigen gradually increased in the corneal epithelium and stroma. Further immunohistochemical survey has revealed that L3T4 antigen bearing and Lyt 2 antigen bearing cells were induced to a similar extent with an equal rapidity in the local lymph nodes as well as in the corneal stroma. Transfer of these subpopulations to syngeneic nude mice showed that they played a role to prevent severe outcome of corneal herpetic infection. These results indicate that the corneal stroma is a major site of the host's immunological activities and both L3T4 and Lyt 2 bearing cells are equally important for the prevention of corneal herpetic infection.
The effects of bunazosin hydrochloride, a new highly selective alpha 1 adrenergic antagonist, on the intraocular pressure (IOP) and aqueous humor dynamics in healthy human volunteers were studied. Unilateral topical administration of 0.1% bunazosin significantly lowered IOP from 1 to 10 hrs in bunazosin-treated eyes, and from 2 to 8 hrs in contralateral placebo-treated eyes, with its maximum reduction at approximately 3 hrs, when the IOP decreased 5.0 mmHg in treated eyes, and 3.5 mmHg in contralateral eyes from the baseline, respectively. Fluorophotometrically measured aqueous humor flow rates did not change significantly either in treated or contralateral eyes. Bunazosin caused significant reduction of systolic and diastolic blood pressure 5 hr after application. The difference in pupil diameter between treated and contralateral eyes was found to be statistically significant from 1 to 5 hrs. A small but significant increase in anterior chamber depth also was observed from 2 to 6 hrs. Pulse rate and refraction were not substantially altered. There was moderate conjunctival vessel dilation, however, no serious complications were encountered. The results suggest that bunazosin hydrochloride appears to have great clinical potential for ocular hypotensive therapy.
Recent advances in genetic engineering techniques have enabled large-scale manufacture of human epidermal growth factor (hEGF), making possible the clinical use of this particular agent in treating a variety of corneal epithelial disorders. In view of future application to humans, it has to be determined whether hEGF could induce neovascularization in the cornea upon topical instillation, since the angiogenic effect of mouse EGF on the cornea in vivo has been reported. For this, a sheet of slow-release form polymer (EVA) containing hEGF was surgically implanted into the rabbit corneal stroma in search for subsequent corneal neovascularization. EVA sheets contained one of the following agents: (1) 250ng hEGF, (2) 500ng hEGF, (3) 250ng bFGF (positive control), (4) vehicle alone (negative control). On 5 and 14 days after implantation, the corneas were excised, sectioned, and stained with hematoxylin and eosin for histological evaluation. Slit lamp examination revealed that marked neovascularization developed in the corneas when EVA sheets containing bFGF were implanted. A number of polymorphonuclear leukocytes were accumulated around the implants. However, neovascularization did not occur in the corneas when EVA sheets containing either concentrations of hEGF or vehicle alone were implanted. Only a few polymorphonuclear leukocytes infiltrated. This result indicates that as much as 500ng hEGF does not induce corneal neovascularization.
Histamine (HA) content and histidine decarboxylase (HDC) activity were studied in the sensory retinas of the rat, guinea-pig, rabbit, dog, monkey, bovine and pig. Using a highly sensitive and specific method developed for the determination of histamine using high-performance liquid chromatography, HA was detected in the retinas of all species examined. HA content ranged from 11 (bovine) to 540 pmole/g wet tissue (dog), and HDC activity ranged from 2 (rabbit) to 150 (dog) fmol HA synthesized/min/mg protein. With the exception of the dogs, the retinal values were one order lower than those in the brain in all species examined. The canine retinas had the high HA and HDC values among the species examined, and were approximately equal to those in the brain. However, the ratios of HDC activity to HA content ranged from 0.04/min to 0.51/min. These values resembled those in the brain and were much higher than those in mast cells. This suggests that histamine in the retinas exists mainly in the neurons even though the actual amounts are small and may be involved in the physiological and the pathological responses in mammalian retinas.
We used immunocytochemistry to locate two calcium binding proteins, calbindin (CaB) and parvalbumin (PV), in the retina of goldfish, frog, chick, rat, guinea pig, dog, and man. The location of CaB depended on the type of dominant photoreceptor cells in birds and mammals. In cone-dominant retinas such as those of the chick, CaB-like immunoreactivity was found in the cones, cone bipolars, and ganglion cells. Amacrine cells 5-12 microns across were also labeled. In rod-dominant retinas, such as those of the rat, guinea pig, and dog, horizontal cells, small amacrine cells (about 6 microns across), and cells in the ganglion cell layer were labeled. In the human retina, which has both cones and rods in abundance, cones, cone bipolars, ganglion cells, horizontal cells, and small and large amacrine cells were labeled. In the frog and goldfish, the level of CaB-like immunoreactivity was low. In the frog, a few cones, amacrine cells, and cells in the ganglion cell layer were labeled. No immunoreactive structures were seen in the goldfish retina. PV-like immunoreactivity was found in chicks, rats, and dogs. No such immunoreactive structures were seen in the other species. In the chick, only amacrine cells were labeled. In the rat, amacrine cells and several displaced amacrine cells were labeled. In the dog, in addition to amacrine cells and displaced amacrine cells, horizontal cells were strongly labeled. Thus, PV-like immunoreactivity was found in those elements relating to the modulation of the main pathway of the visual transmission system.
%$%%protective effect of glycoprotein D (gD) immunization against murine herpetic keratitis was investigated. gD was purified by affinity chromatography using anti-gD monoclonal antibodies. Prior immunization with gD was shown to be effective in protecting mice from both the development of stromal keratitis and the spread of the virus to the central nervous system. The level of serum antibodies for virus neutralization, as well as for complement-dependent cytolysis (CDC), was significantly elevated in gD-immunized animals. Cellular immunity, however, was not detected. These results indicate that two antibody-mediated defense mechanisms--virus neutralization and CDC--were responsible for the protective effect observed in our study.
Conjugation reaction of methazolamide with glutathione and its subsequent reactions were studied in vitro. Glutathione, cysteinylglycine, and cysteine conjugates of methazolamide were chemically synthesized. All of the three compounds showed absorbance below 330 nm, with maximal absorbance at approximately 300 nm. At the wavelengths below 220 nm, absorbance was proportional to the number of the amino acids each compound had. Amino acid analysis of the glutathione conjugate showed that the conjugation reaction involved the cysteine residue of glutathione. In order to identify the chemical structure of the reaction product, cysteine conjugate was subjected to infrared, proton nuclear magnetic resonance, and mass spectral analyses. These studies indicated that the cysteine conjugate was S-(5-acetylimino-4-methyl-delta 2-1,3,4-thiadiazolinyl)cysteine. The reaction with glutathione was not catalyzed by glutathione S-transferases, but proceeded in the absence of the enzyme. The glutathione conjugate was degraded by bovine ciliary body homogenate to the cysteinylglycine conjugate and then to the cysteine conjugate.
Monoclonal antibodies were prepared against a putative cell-cell adhesion glycoprotein, with an apparent molecular mass of 64,000 (gp64), of the cellular slime mold, Polysphondylium pallidum. Five monoclonal antibodies obtained by means of an enzyme-linked immunoadsorbent assay did not bind to the antigens which were subjected to gel electrophoresis and blotting method in the presence of a reducing agent, but they did bind specifically to the antigens prepared in unreducing conditions of samples and then processed by the same blotting method. To solubilize gp64 in a sodium dodecyl sulfate (SDS)-sample buffer without mercaptoethanol (heated) or SDS-sample buffer with 2-mercaptoethanol (nonheated) was critical for the antibody binding onto gp64 on a membrane. Hence the antibodies seem to bind to a surface portion(s) of the localized protein structure folded up by disulfide cross-linkages. One of the antibodies obtained blocked cell-cell adhesion by about 20%.
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Thirty-five clinical isolates of herpes simplex virus type 1 (HSV-1) from 34 patients (35 eyes) with herpetic keratitis were examined in vitro for 5-iodo-2'-deoxyuridine (IDU) and acyclovir (ACV) sensitivity. In addition, the effect of clinical treatment with these two drugs in herpetic keratitis was also investigated. The viral effective dose50 (ED50) was defined as the concentration that inhibited the plaque count by 50% compared to the count of the no drug controls. The viral ED50 of IDU ranged from 0.073 to 0.77 micrograms/ml (0.33 +/- 0.16; Mean +/- SD) and that of ACV from 0.0032 to 0.33 micrograms/ml (0.13 +/- 0.11). No virus with markedly diminished sensitivity to IDU and ACV was found. These results suggest that all HSV-1 strains isolated from patients have good sensitivity in vitro to antiviral agents.