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PubMedDate not supplied

Multilayer primary epithelial cell culture from bovine conjunctiva as a model for in vitro toxicity tests.

OBJECTIVE: The purpose of this study was to obtain a primary cell culture of bovine origin similar to the conjunctiva in terms of morphology and cell types, which could be of use for in vitro toxicity studies. METHODS: After separation from the stroma by enzymatic treatment, conjunctival epithelial cells were dissociated and plated onto collagen-coated Transwell filters (1.13 cm(2) area). One group of plates was maintained in immersion and another was cultured under air-lifted conditions. Anti-epithelial keratin antibodies (AE1/AE3, K4) and antidesmoplakin 1 and 2 were used to characterize the cells by indirect immunofluorescence. The cell layer was examined after histological processing of the Transwell filter. Ultrastructural analysis was carried out by scanning electron microscopy (SEM). The bioelectric parameters transepithelial electrical resistance (TEER), potential difference (PD), short circuit current and paracellular permeability profile of carboxyfluorescein were monitored as indices of the functional characteristics of these cultures. Cytotoxicity was evaluated on morphological and functional (TEER) grounds after treating the cultures with several test substances. RESULTS: Morphological studies showed pure and homogeneous cell cultures. In the SEM analysis, we observed contiguous polygonal cells with numerous short microvilli, a characteristic proportion of light, medium and dark cells and a sparse population of rounded PAS-positive cells, i.e. resembling goblet cells. Air-lifted cultures also showed a tissue-like cellular organization (8-9 layers). Immersion cultures reached a maximum TEER value of around 2.95 kOmega x cm(2) 7 days after plating while in air-lifted cultures TEER peaked up to 5.59 komega x cm(2) 11 days after plating. With regard to the use of bovine conjunctival epithelial cells (BCECs) for cytotoxicity screening, the system responded finely to the insults and yielded morphological and functional results in accordance with data obtained in vivo. CONCLUSIONS: BCECs reproduce cell morphology and differentiation of the original tissue and should prove a useful tool for initial studies of drug toxicity.

Adjuvants, Immunologic↗
PubMedDate not supplied

The protective effect of alpha-lipoic acid against oxidative damage in rabbit conjunctiva and cornea exposed to ultraviolet radiation.

PURPOSE: The purpose of this study was to determine the protective effect of alpha-lipoic acid against oxidative damage in rabbit conjunctiva and cornea exposed to ultraviolet radiation. METHODS: 20 rabbits weighing 2,500- 3,000 g were used, and we divided them into 4 groups with 5 randomly selected rabbits. The rabbits were exposed to 2 J/cm(2)/h of ultraviolet A radiation (UVA) in the range of 320-405 nm for 12 h per day within 90 days. The control group did not undergo any procedure, the UVA group was only exposed to UVA radiation. The PUVA group was treated with 8-methoxypsoralen and UVA. The alpha-lipoic acid group was administered 8-methoxypsoralen + UVA + alpha-lipoic acid. At the end of 90 days, the rabbits were killed by decapitation, and the eyes were enucleated. Both eyes of each rabbit were used for biochemical evaluation. Conjunctival and corneal free malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) levels were compared among the groups. RESULTS: Conjunctival free MDA levels were lower in the alpha-lipoic acid group compared with the UVA and PUVA groups (p < 0.05, p < 0.001, respectively). Both conjunctival SOD levels (p < 0.05, p < 0.01, respectively) and conjunctival GSH-PX levels (p < 0.01, p < 0.001, respectively) were higher in the alpha-lipoic acid group compared with other groups. Corneal free MDA levels were lower in the alpha-lipoic acid group compared with the UVA and PUVA groups (p < 0.01, p < 0.001, respectively). Both corneal SOD levels (p < 0.01, p < 0.01, respectively) and corneal GSH-PX levels (p < 0.01, p < 0.01, respectively) were higher in the alpha-lipoic acid group compared with the other groups. CONCLUSION: alpha-Lipoic acid which is considered as potent antioxidant protects the eye from the damaging effect of ultraviolet exposure.

Animals↗
PubMed1992

Organization of capillaries in the primate conjunctiva.

Conjunctival capillaries in the monkey were observed in histological sections to organize approximately parallel and close to the basement membrane of the epithelium, while larger vessels including the lymphatics were disposed deeper in the stroma. This peculiar vascular organization was evident in the palpebral but not in the bulbar conjunctiva. The trilaminar relationship that existed between the conjunctival epithelium, the elastic lamina previously described [1] and the capillaries is discussed. It was argued that such subepithelial capillary organization enhances the exchange of nutrients and gases between the cornea and the conjunctival vessels across the palpebral conjunctival epithelium when the lids are shut during sleep.

Animals↗
PubMed1979

Effects of urea treatment in malignancies of the conjunctiva and cornea.

A series of 9 patients with extensive squamous cell carcinomas of the conjunctiva, affecting also the cornea in 5 of them, were treated with local applications of urea. 8 out of these patients were cured. In 1 patient with a concomitant extensive conjunctivitis, the treatment was ineffective. Urea treatment is very simple and without any complications, apart from a transient opacity of the cornea. Recurrences are easily and effectively retreated as in 1 of 2 of our cases. But if the eyeball has been destroyed, as in the second case, retreatment is impossible. At least 5 out of the treated patients would have had an enucleation of the globe or an exenteration of the orbit without the urea treatment.

Adult↗
PubMed1985

Light and electron microscopical study of the conjunctiva in sicca syndrome.

Conjunctival biopsies of 11 patients with sicca syndrome were studied under light and transmission electron microscopy. The notable findings were vasculitis of the conjunctival vessels. There were severe changes in the microvilli, namely a loss of microvilli and decrease in their height, measuring approximately 1,000-2,000 A, in comparison with microvilli of the control conjunctiva, measuring 4,000 A in height. There was no branching yet or fusion of microvilli.

Aged↗
PubMed2003

Characterization of a spontaneously immortalized cell line (IOBA-NHC) from normal human conjunctiva.

PURPOSE: To characterize a new nontransfected, spontaneously immortalized epithelial cell line from normal human conjunctiva (IOBA-NHC), both morphologically and functionally, to determine whether the differentiated phenotype of conjunctival epithelial cells is preserved. METHODS: Outgrowing cells from explanted conjunctival tissue were successively passaged and preliminarily characterized at passage 3 to assess epithelial origin. The cells were further characterized at passages 15 to 20, 40, 60, and 100 by analyzing (1) proliferation and in vitro behavior (viability, plating efficiency, colony forming efficiency and colony size, and Ki-67 protein expression), (2) karyotype and G-banding, (3) epithelial marker expression (cytokeratins, desmoplakins, EGF receptor), (4) absence of contaminating cell types, (5) expression of conjunctival differentiation markers (mucin gene expression), and (6) functional capability in response to proinflammatory stimuli. IOBA-NHC cells were analyzed by light and electron (transmission and scanning) microscopy, immunohistochemistry, electrophoresis and Western blot analysis, flow cytometry, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: IOBA-NHC cells showed high proliferative ability in vitro and typical epithelial morphology. Cytokeratins and GalNAc, GluNAc, mannose, and sialic acid residues were immunodetected in these cells. No contaminating cell types were found. MUC1, -2, and -4, but not -5AC or -7 mucin genes were expressed in every cell passage tested. Exposure of cells to inflammatory mediators (IFNgamma and/or TNFalpha) resulted in increased expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR. CONCLUSIONS: Morphologic and functional characterization of the nontransfected, spontaneously immortalized IOBA-NHC cell line shows that this new cell line may be a useful experimental tool in the field of ocular surface cell biology.

Biomarkers↗
PubMed1988

Scanning electron microscopy of experimental keratoconjunctivitis sicca in dogs: cornea and bulbar conjunctiva.

Keratoconjunctivitis sicca was produced experimentally in 16 beagles by bilateral surgical removal of the lacrimal and nictitans glands; four dogs were not treated, and 12 received tear-replacement therapy on post-operative days 7 through 28. Keratoconjunctivitis sicca was verified by reduction in Schirmer tear test values by post-operative day 6, and there was no response on day 28 to tear-replacement therapy. Corneas of both normal and tear-deficient dogs had polygonal squamous epithelial cells of light and dark electron density by scanning electron microscopy. Light cells had more microvilli and microplicae than dark cells. Conjunctivae were similar to corneas, except for numerous goblet cells on the surface. Corneal dark-cell density and goblet cell density were not different between groups. Goblet cells most often occurred singly in normal dogs, while they were in clusters in tear-deficient dogs. A hypothesis that petrolatum/mineral oil ointment should provide more effective artificial tear replacement than hydroxymethylcellulose drops for tear-deficient dogs could not be confirmed by objective analysis of corneal dark-cell density or conjunctival goblet cell density.

Animals↗
PubMed1989

Conjunctiva-associated lymphoid tissue (CALT) in normal and Bordetella avium-infected turkeys.

Conjunctiva-associated lymphoid tissue (CALT) was characterized in normal and Bordetella avium-infected turkey poults during the first 5 weeks of life. At 1, 5, 12, 19, 25, and 33 days post-hatching (DPH), upper and lower eyelids were examined by gross, histologic, and electron microscopic techniques. CALT was confined to the proximal part of the lower eyelid near the conjunctival fornix; it appeared by 5 DPH as individual lymphoid nodules and as dense masses by 19 DPH. In the upper eyelid, CALT was present only as isolated nodules. Histologically, CALT was composed of dense lymphocyte infiltrates within subepithelial connective tissue, intraepithelial lymphocytes, and flattened lymphoid-associated epithelium that lacked goblet cells. Germinal centers were in CALT by 19 DPH. By scanning electron microscopy, epithelial cells over lymphoid areas were flat and had short, irregular microvilli; non-lymphoid areas were covered by cells with tall, regular microvilli. Transmission electron microscopy revealed that with increasing age of birds, the epithelium over conjunctival lymphoid infiltrates became progressively flattened and infiltrated by lymphocytes. Some blood vessels in CALT had high endothelial cells; lymphocytes were in the lumen and between or beneath endothelial cells. In B. avium-infected poults, CALT was increased, developed earlier, and contained more germinal centers than in normal poults. We conclude that CALT of turkeys closely resembles other mucosal lymphoid tissues and may serve as a site for local antigen uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Upregulation of ICAM-1 expression in the conjunctiva of patients with chronic graft-versus-host disease.

PURPOSE: To correlate conjunctival intercellular adhesion molecule 1 (ICAM-1) expression with cytologic and clinical findings of chronic graft-versus-host disease (GVHD). METHODS: Seven patients with chronic GVHD-related keratoconjunctivitis and five age-matched normal controls were recruited for the study. Clinical examination included medical history, visual acuity, evaluation of ocular signs and symptoms (scored from 0 to 3), corneal fluorescein staining (scored from 0 to 5 on the basis of the number of corneal sectors involved), Schirmer test type I, and break-up time (BUT). Impression cytology samples were collected from the nasal and inferior bulbar conjunctiva of patients and controls. Goblet cells were counted in three randomly selected fields and averaged. Immunofluorescent staining for ICAM-1 was carried out and the percentage of cells expressing the marker was evaluated. RESULTS: All patients showed signs and symptoms of keratoconjunctivitis sicca. Schirmer test type I and BUT were reduced (4.8+/-6.7 mm/5 min and 3.9+/-2.7 seconds, respectively). Goblet cells were significantly reduced in GVHD eyes with respect to normal eyes (65+/-30.5 and 192+/-16.9 cells/field respectively; p<0.001). Goblet cell number was directly related to Schirmer test values (p<0.01, rho=0.817) and inversely related to total sign score (p<0.01, rho=-0.939). ICAM-1 expression was increased in GVHD eyes with respect to normal controls, in which no staining was observed. ICAM-1 expression showed an inverse relation to goblet cell number (p<0.01, rho=-0.852) and Schirmer test values (p<0.01, rho=-0.926), and was directly correlated to total sign score (p<0.01, rho=0.982). CONCLUSIONS: Conjunctival ICAM-1 expression is increased in GVHD patients. The severity of the disease is associated with tear parameters, goblet cell decrease, and inflammatory markers, such as ICAM-1.

Adolescent↗
PubMed2002

Hydrocortisone reduced in vivo, inflammation-induced slow rolling of leukocytes and their extravasation into human conjunctiva.

Hydrocortisone reduces the number of inflammatory leukocytes within tissues, but thus far the site of action on the multistep adhesion cascade leading to leukocyte extravasation has not been identified. We have recently developed a noninvasive in vivo reflected-light confocal microscopy technique to study this at sites of inflammation in human patients. In the present study, we evaluated the effect of preoperative intravenous hydrocortisone treatment on leukocyte trafficking after conjunctival inflammation induced by cataract surgery in human subjects in vivo. The surgery generated leukocyte rolling along the endothelial lining of conjunctival vessels. While preoperative hydrocortisone did not reduce the number of rolling cells, it significantly raised the velocity of individual rolling leukocytes and concomitantly reduced leukocyte emigration into conjunctival tissue. Immunohistology of conjunctival biopsies excised from the individuals studied provided circumstantial evidence that endothelial P-selectin might play a role in the surgery-induced up-regulation of the leukocyte rolling. Furthermore, hydrocortisone reduced surgery-induced P-selectin induction, suggesting a role for this selectin in the regulation of local leukocyte traffic into sites of inflammation in human conjunctiva. Taken together, these results suggest that control of the rolling velocity might be an effective way to adjust leukocyte traffic in vivo in human subjects.

Biopsy↗
PubMed1977

Malignant melanoma of conjunctiva metastatic to parotid gland. Reports of cases and discussion of surgical management.

Invasive malignant melanoma of the conjunctiva is a rare neoplasm fatal in 40% of cases. Accepted techniques of surgical management include wide local excision, conjunctivectomy, and exenteration. Recently lymphatic dissections have been proposed, but not described, for invasive conjunctival melanoma. We present five cases of invasive conjunctival melanoma, including two with intraglandular parotid metastases. The surgical management of these patients is presented, and a proposal is made for radical bloc dissection of the orbital contents and regional lymphatics. The histology of conjunctival melanoma is discussed, and a classification proposed to assist the head and neck surgical team in the management of this disease.

Aged↗
PubMed2002

Failure to detect human papillomavirus DNA in malignant epithelial neoplasms of conjunctiva by polymerase chain reaction.

To elucidate the putative role of human papillomavirus (HPV) infection in the etiology of conjunctival tumors, 44 formalin-fixed, paraffin-embedded specimens of conjunctival tumors (24 patients with papillomas and 20 patients with dysplastic and/or malignant tumors) were screened for HPV infection using 4 different polymerase chain reactions (PCRs). Of the 24 samples of papilloma, 14 (58%) displayed positive results by applying nested PCR using primer sets of HPV consensus L1 region. HPV type 6 or 11 was detected in 9 cases of papilloma by type-specific primer sets, but none of them were positive for HPV type 16 or 18. However, by using the highly sensitive PCR technique, we failed to demonstrate the HPV DNA of HPV types 6, 11, 16, and 18 in any of the 20 malignant epithelial tumors of conjunctiva. We conclude that HPV-6 or HPV-11 is present in a substantial percentage of conjunctival papillomas, which is in accordance with findings of previously reported studies. In contrast, malignant conjunctival carcinomas are not associated with HPV infection; other pathogenic mechanisms, such as UV light, probably are more important in the cause of these malignant lesions.

Carcinoma, Squamous Cell↗
PubMed1988

Temperature of the hyperemic bulbar conjunctiva.

The relationship between bulbar conjunctival hyperemia and temperature was investigated. Conjunctival redness was induced in eighteen volunteers by instilling hypertonic saline into the conjunctival sac. The degree of redness was estimated using a subjective grading scale. The subsequent changes in temperature of the nasal bulbar conjunctiva were monitored using an infra-red bolometer. Conjunctival hyperemia was significantly correlated with conjunctival temperature; the maximum response of a 3-grade change in redness was accompanied by an increase of 0.5 degrees C in temperature. These findings confirm the classic association between inflammation, rubor and calor.

Adult↗
PubMed1995

Localization of nerves adjacent to goblet cells in rat conjunctiva.

Neural stimulation of the cornea induces conjunctival goblet cell mucous secretion. Immunofluorescence microscopy was used to determine if nerves are present near conjunctival goblet cells and what types of nerves are present. In euthanized rats, the local anesthetic lidocaine (1%) was placed topically on the ocular surface for 10 min to prevent goblet cell mucous secretion. The ocular surface tissues were removed and either fixed in formaldehyde and then frozen, or frozen first and then post-fixed in formaldehyde. Tissue was sectioned and nerves localized by indirect immunofluorescence microscopy, using antibodies to synaptophysin (indicates nerve, independent of type), vasoactive intestinal peptide (VIP, indicates parasympathetic nerves), tyrosine hydroxylase (TH, indicates sympathetic nerves), dopamine beta-hydroxylase (DBH, indicates sympathetic nerves), phenylethanolamine-N-methyltransferase (PNMT, indicates sympathetic nerves), and calcitonin gene-related peptide (CGRP, indicates sensory nerves). Goblet cells were identified by phase-contrast microscopy. Synpatophysin-containing nerves were present in the basolateral region of conjunctival goblet cells clusters. Nerve fibers immunoreactive to VIP were found in the conjunctiva along the epithelial-stromal junction and around the basolateral aspect of goblet cell clusters. Nerve fibers immunoreactive to TH and DBH were detected surrounding goblet cells and in the conjunctival stroma. Nerve fibers immunoreactive to CGRP were detected in the epithelium and at the epithelial stromal junction, but were not localized near goblet cell clusters. CGRP-containing nerve fibers were also detected in the conjunctival stroma under the epithelium. We conclude that efferent parasympathetic and sympathetic, but not afferent sensory, nerves appear to be located adjacent to conjunctival goblet cell clusters. Activation of efferent parasympathetic and sympathetic nerves could directly stimulate conjunctival goblet cell mucous secretion. Antidromic activation of afferent sensory nerves releasing neurotransmitters could stimulate goblet cell secretion by a paracrine mechanism.

Animals↗
PubMed1999

Midterm follow-up of necrotic bleb excision and advancement of the fornical conjunctiva.

Mitomycin C has improved the success rate of glaucoma filtering surgery in patients at high risk for surgical failure. However chronic hypotony is marked by decreased vision and a late-onset leaking bleb after filtration surgery using mitomycin C. Bleb excision and conjunctival advancement is the method of choice to repair bleb leakage and chronic hypotony. Five eyes from five patients were received glaucoma filtration surgery with topical mitomycin C. All of the patients' blebs were avascular and transparent. The reasons for bleb excision were two spontaneous bleb leaks, two traumatic bleb leaks and one case of severe irritation. The mean follow-up period was 18.4 +/- 8.3 months (ten to 29 months). Cataract surgery was combined in one eye. Postoperative intraocular pressure (IOP) increased from 2.3 +/- 1.5 mmHg to 9.5 +/- 3.7 mmHg at nine months postoperatively in four eyes. It went from 28 mmHg to 40 mmHg in one patient with uveitis, for whom a second trabeculectomy with mitomycin C; 0.4 mg/ml for 3 minutes, was performed. After surgery, IOP decreased to 4 mmHg in three months. Postoperative visual acuity improved four snellen lines in three eyes. A partially avascular bleb recurred in three eyes, a corneal bleb in one eye and blepharoptosis, which disappeared spontaneously at four months postoperatively, in one eye. Necrotic bleb excision and advancement of fornical conjunctiva were useful methods to increase IOP and to improve visual acuity for the patient experiencing irritation symptoms, and for leaking blebs, and hypotonic maculopathy.

Adult↗
PubMedDate not supplied

Perioperative microbiologic profile of the conjunctiva in photorefractive keratectomy.

PURPOSE: Therapeutic soft contact lenses are used commonly as an adjunctive treatment after photorefractive keratectomy (PRK) to decrease postoperative pain caused by the movement of lids over the corneal epithelial defect and to facilitate epithelial healing. We assessed the microbiological profile of the conjunctiva of patients undergoing PRK for myopia, before and after the concurrent use of a therapeutic soft contact lens, and compared the effect on ocular bacterial colonization of prophylactic administration of topical tobramycin 0.3% versus ofloxacin 0.3%. METHODS: Forty-three consecutive eyes from 37 patients underwent PRK for myopia or myopic astigmatism. Eyes were assigned randomly to prophylactic antibacterial treatment with either topical ofloxacin 0.3% or tobramycin 0.3%, applied prior to surgery and three times daily after surgery until therapeutic soft contact lens removal. Material from the conjunctival sac was obtained for bacteriologic cultures prior to surgery. Clinical evaluation of all eyes was conducted prospectively. Three days after PRK, the therapeutic soft contact lenses were removed and cultured. Cultures from the conjunctival sac were then repeated. RESULTS: No statistically significant differences were observed in culture positivity between the two groups of eyes, in spite of some positive preoperative and postoperative cultures. Only one out of 43 eyes (assigned to the ofloxacin group) developed a peripheral corneal infiltrate. The corneal infiltrate healed completely without sequelae using antibiotic and corticosteroid therapy. CONCLUSIONS: The use of therapeutic soft contact lenses after PRK with either topical tobramycin 0.3% or ofloxacin 0.3% were well tolerated. However, perioperative positive conjunctival cultures were relatively frequent and prophylactic antibiotics should be used in the setting of an epithelial defect and therapeutic soft contact lens following PRK.

Adult↗
PubMedDate not supplied

Microvascular abnormalities in the bulbar conjunctiva of patients with type 2 diabetes mellitus.

OBJECTIVE: To study and quantify microvascular abnormalities objectively in vivo in patients with type 2 diabetes mellitus (T2DM). METHODS: The conjunctival microcirculation in 14 patients with T2DM and in age-matched healthy control subjects without diabetes was videotaped and objectively studied by using computer-assisted intravital microscopy (CAIM), a novel and quantitative real-time technology. RESULTS: Patients with T2DM (N = 14) had significantly (P<0.01) wider conjunctival vessel diameters (71.9 +/- 5.2 mm) than did healthy nondiabetic control subjects (54.0 +/- 4.4 mm). In the study patients, microvascular distribution was significantly (P<0.01) abnormal (36.7 +/- 18.2 versus 45.3 +/- 9.6 cm per unit area, patients versus control subjects), and vessel distribution was uneven on the surface of the bulbar conjunctiva. The arteriole:venule (A:V) ratio in patients with T2DM was extremely variable and differed significantly (P<0.01) from that in the nondiabetic control subjects (A:V approximately 1:2). In addition, a unique sinusoidal (hypertensive) vascular pattern frequently existed in some of the large veins of all study patients with T2DM but in none of the nondiabetic control subjects. CONCLUSION: We identified the presence of microvascular changes (abnormalities) in the conjunctival microcirculation of patients with T2DM. Although all these abnormalities did not appear in the same patient at the same time, the sum total of their presence in each patient correlated significantly with disease severity, as noted in the medical records. The severity of microvascular abnormalities, however, did not correlate with the duration of the disease since diagnosis. CAIM may be a useful objective and quantitative technique for assessing microangiopathy in patients with T2DM. The easy noninvasive accessibility of the conjunctival vessels and the ability to identify and locate the same vessels repeatedly for longitudinal evaluations further emphasize the usefulness of this real-time technology.

Adult↗
PubMed1984

Spider (Latrodectus hesperus) poisoning through the conjunctiva. A case report.

Spider poisoning is usually a result of the direct bite, envenomation through the chelicera, of the spider. Reported is an unusual case in which poisoning occurred through the conjunctiva when a fragment from a smashed black widow spider (Latrodectus hesperus) flew into a patient's eye. In addition to an immediate local reaction involving the periorbital tissues, the patient experienced systemic effects.

Adult↗