Induction of hepatocyte growth factor in fibroblasts by tumor-derived factors affects invasive growth of tumor cells: in vitro analysis of tumor-stromal interactions.

Invasive and metastatic potentials of several types of carcinoma cells are regulated through interactions with host stromal cells, e.g., tumor-stromal interactions. Because hepatocyte growth factor (HGF), a ligand for the c-Met proto-oncogene product, is a mesenchymal- or stromal-derived factor that induces mitogenic, motogenic, and morphogenic responses, we examined the mechanisms involved in tumor-stromal interactions in vitro. The c-Met/HGF receptor was expressed in A431 human epidermoid carcinoma cells, A549 human non-small cell lung cancer cells, HuCC-T1 human cholangiocellular carcinoma cells, and SBC-3 human small cell lung carcinoma cells. HGF stimulated cell growth, scattering, and invasion of these cells. Although these cells did not produce biologically significant levels of HGF, these cells did secrete soluble factors that potently stimulated HGF production in human skin fibroblasts. These carcinoma cell-derived HGF inducers proved to be interleukin-1 (IL-1) in A431 cells, IL-1 plus basic fibroblast growth factor (bFGF) in A549 and HuCC-T1 cells, and bFGF plus platelet-derived growth factor in SBC-3 cells. When these carcinoma cells were cocultured with fibroblasts, HGF levels in the coculture system were much higher than the levels in fibroblasts alone, without cocultured carcinoma cells. Together with the increase in HGF levels, the number of invasive cells increased, but in vitro invasion of carcinoma cells in the coculture system was strongly inhibited by anti-HGF antibodies. Thus, there are mutual interactions between carcinoma cells and fibroblasts: IL-1, bFGF, and platelet-derived growth factor derived from tumor cells play a role in inducing HGF expression in stromal fibroblasts, whereas fibroblast-derived HGF, in turn, leads to invasive growth in carcinoma cells. The mutual interactions, as mediated by HGF and HGF inducers, may play a significant role in the occurrence of invasion and metastasis of carcinoma cells.

Differentiation in cultured limbal epithelium as defined by keratin expression.

The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AE1, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AE1. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AE1 binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AE1. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AE1. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells.

Recurrent HSV-1 corneal lesions in rabbits induced by cyclophosphamide and dexamethasone.

Herpes simplex virus type 1 (HSV-1) ocular shedding and recurrent corneal epithelial lesions were assessed following an intravenous injection of cyclophosphamide (75 mg/kg) and 24 hr later an intravenous injection of dexamethasone (4 mg/kg) in 24 eyes of 15 rabbits latently infected with HSV-1 strain McKrae. Sampling for HSV-1 ocular shedding and epithelial lesion began on the day after cyclophosphamide injection and continued for 8 consecutive days. Ocular tear film was collected on a Dacron swab with care taken to avoid swabbing the corneal epithelium. Slit-lamp biomicroscopic examination was used to observe and characterize induced HSV-1 corneal epithelial lesions as deep punctate keratitis, dendritic keratitis or geographic epithelial defects. The ratio of positive days of epithelial lesions per total days was 82/187 (44%). There were 32 deep punctate lesions, 17 dendritic lesions, and 33 geographic epithelial defects. The ratio of positive swabs per total swabs was 78/187 (42%). Of the 82 positive lesion days, 54 (66%) were associated with a positive swab. Of the 78 positive swabs, 54 (69%) were associated with an epithelial lesion. Of the 54 days of both positive lesion and swab, 16 (30%) were associated with a dendritic lesion. By chi-square analysis, there was a significant association between HSV-1 swabs and HSV-1 lesions (P less than 0.001). These results confirm that intravenous injections of cyclophosphamide and dexamethasone induce both HSV-1 ocular shedding and recurrent herpes simplex corneal lesions in rabbits latently infected with HSV-1 strain McKrae.

Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.

Goblet cell density in thermal and chemical injuries.

Abnormality of the conjunctival epithelium was evaluated in ten patients with severe thermal and chemical injuries at the scarred stage, eight patients with Stevens-Johnson syndrome, five with cicatricial ocular pemphigoid, and 13 normal subjects, based on goblet cell densities obtained using impression cytologic examination. The average and SDs of goblet cell densities were 197.4 +/- 264.4/mm2 in thermal and chemical injuries, 3.0 +/- 5.6/mm2 in Stevens-Johnson syndrome, 0.6 +/- 0.8/mm2 in cicatricial ocular pemphigoid, and 38.7 +/- 25.8/mm2 in normal subjects. There were statistically significant differences in goblet cell densities between thermal and chemical injuries and both Stevens-Johnson syndrome and cicatricial ocular pemphigoid. Moreover, conjunctival specimens from both eyes in six unilateral cases of thermal and chemical injuries revealed higher goblet cell densities in the injured than in the normal fellow eyes in all cases. These findings imply that ocular surface diseases with similar clinical manifestations may have different cell-biologic abnormalities in the conjunctival epithelium.

Idiopathic corneal endotheliopathy. A report of two cases.

Two male patients had corneal endotheliopathy of unknown origin. In both patients it was characterized by continuously progressing bullous keratopathy preceded by a line of keratic precipitates resembling a rejection line. The disease was progressive and did not respond to intensive corticosteroid therapy. No virus could be isolated from the aqueous humor, and the aqueous-antibody titers to herpes simplex, varicella zoster, and measles viruses were below detection level.

Expression of a mucin-like glycoprotein produced by ocular surface epithelium in normal and keratinized cells.

PURPOSE: We previously characterized a monoclonal antibody (H185) to a mucin-like glycoprotein produced by human ocular surface epithelium. In the current study, we used H185 to investigate the pattern of the mucin-like glycoprotein in normal and keratinized apical cells of the ocular surface epithelium. METHODS: We compared the cell characteristics and the pattern of H185 binding in cytologically and immunohistochemically stained samples of apical cells of conjunctival surface epithelium from 20 normal subjects and six patients before and after treatment for superior limbic keratoconjunctivitis. RESULTS: In the superior bulbar conjunctiva of normal subjects, the intensity with which H185 antibody bound to apical surface epithelial cells varied, with areas of high-, medium-, and low-intensity binding occurring in a mosaic pattern. This mosaic pattern, and presumably expression of the mucin-like glycoprotein, was absent or remarkably reduced in keratinized cells obtained from patients with superior limbic keratoconjunctivitis before treatment. However, the pattern of H185 binding was normal in samples obtained 2 months after the start of treatment for superior limbic keratoconjunctivitis, and cells had recovered their normal small, round appearance. CONCLUSION: When they are keratinized, apical cells of the ocular surface epithelium are altered in appearance and lack the normal mosaic pattern of expression of a mucin-like glycoprotein.

Acanthamoeba keratitis with symbiosis of Hartmannella ameba.

PURPOSE: To report a case of severe amebic keratitis in which both Hartmannella and Acanthamoeba were isolated simultaneously from the same lesion. METHOD: Case report. The deep corneal lesion was scraped for cytopathology and isolation of the pathogens. We tested the in vitro sensitivities of the pathogens to several drugs. RESULTS: Cultures of the corneal scrapings and of the solution in the patient's contact lens storage case were positive for Acanthamoeba E9 cysts and trophozoites. Hartmannella ameba coexisted with Acanthamoeba in the cornea. When tested in vitro, Acanthamoeba trophozoites were sensitive to both miconazole nitrate and natamycin, while cysts were sensitive only to natamycin. However, the patient did not respond to these antiamebic drugs. CONCLUSIONS: This case suggests that Acanthamoeba is not the only origin of amebic keratitis. Hartmannella may also cause severe drug-resistant keratitis.

Corneal guttata associated with the corneal dystrophy resulting from a betaig-h3 R124H mutation.

AIMS: To investigate the frequency of corneal guttata in patients with a corneal dystrophy resulting from an Arg124His (R124H) mutation of betaig-h3 gene. METHODS: Slit lamp examination was performed on 30 eyes with corneal dystrophy from a genetically confirmed betaig-h3 R124H mutation and on 50 age matched control eyes. The stage of the corneal dystrophy was classified as stage 0, I, or II and the degree of guttata was classified as none, mild, or severe. Specular microscopic examinations were performed to evaluate the morphology of the corneal endothelium. RESULTS: Slit lamp examination disclosed the presence of corneal guttata in 21 eyes (70%) of the 30 eyes with the corneal dystrophy, but in only one (2%) of the 50 eyes in the age matched control group (p<0.001, chi(2) with Yates's correction). Of the 12 eyes with stage I betaig-h3 R124H corneal dystrophy, seven had no corneal guttata and five had a mild degree of guttata. Of the 18 eyes with stage II, the degree of guttata was none in two, mild in nine, and severe in seven. The degree of corneal guttata was significantly related to the stage of the corneal dystrophy (p<0.0001, Kruskul-Wallis test ANOVA on ranks). There was no significant differences between eyes with betaig-h3 R124H corneal dystrophy and normal eyes in cell density, coefficient of variation, and cell hexagonality of corneal endothelium. CONCLUSION: Corneal guttata are one of the characteristics of the corneal dystrophy resulting from betaig-h3 R124H mutation.

Using a reference point and videokeratography for intraoperative identification of astigmatism axis.

PURPOSE: To estimate the misalignment of the astigmatism axis caused by intraoperative identification of the axis without using reference points. SETTINGS: Osaka University Medical School, Suita, Japan. METHODS: This study included 38 eyes of 19 patients with no ocular pathology except refractive error and 32 eyes of 16 patients with cataract. A point was marked on the nasal conjunctiva, on the "intraoperative" horizontal axis as estimated by the examiner using a surgical microscope while the patient lay on the operating table in the supine position. Videokeratography was performed with the patient seated, and the degree of axial misalignment was determined by measuring the angle between the conjunctival mark and the horizontal axis identified on the video image. RESULTS: Mean axial misalignment for all patients was 4.4 degrees +/- 2.8 (SD), which could theoretically cause about a 15% loss of surgical effect. The maximal misalignment was 14 degrees, which would correspond to a 48% loss of astigmatic correction. CONCLUSION: The results of this study suggest that intraoperative identification of the astigmatism axis without using reference points may reduce the surgical effect because of axis misalignment. The use of a reference point and preoperative videokeratography may increase the accuracy of identification of the astigmatism axis.

Endothelial decompensation in a schizophrenic patient receiving long-term treatment with tranquilizers.

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.

Double anterior chamber deep lamellar keratoplasty: case report.

PURPOSE AND METHODS: We report a case in which, although we planned to perform a penetrating keratoplasty for corneal stromal opacity with normal corneal endothelium, the host's Descemet's membrane became inadvertently detached and the operation resulted in double anterior chamber deep lamellar keratoplasty (DLKP). RESULT: After surgery, the patient's corrected visual acuity was 20/30. CONCLUSION: Double anterior chamber DLKP is safe and valuable.

A newly established cell line of rabbit lens epithelium.

Rabbit lens epithelial cells have been cultured continuously for more than 24 months (200 generations) in monolayers. Their morphology resembled cobblestones when confluent and was spindle-shaped during growth. The doubling time was 40 hours. The cells were capable of colony formation and their plating efficiency was about 6%. They had immunoreactivity to antiserum to the crystalline-rich supernatant of rabbit lens homogenate. Therefore, it was concluded that they were a permanent cell line and they were named TOTL-86 cells.

Dome formation in cell cultures of chick embryo retinal pigment epithelium and effects of ouabain and 2,4-dinitrophenol thereon.

Cells from chick embryo retinal pigment epithelium were cultured on glass slips. The primary culture cells formed confluent cell layers which were studded with a number of domes. The domes had usually appeared by the fourth day of culture and were susceptible to 3 X 10(-6) M ouabain and 10(-4) M 2,4-dinitrophenol, indicating that fluid was transepithelially transported from the apical to the basal side by means of an energy-requiring and ouabain-sensitive mechanism. Other than domes, small blisters appeared after 4-10 days of culture. They were also susceptible to the metabolic inhibitors.

Core vitrectomy preceding triple corneal procedure in patients at high risk for increased posterior chamber pressure.

We evaluated the effectiveness of performing a core vitrectomy to prevent intraoperative posterior chamber pressure evaluation in eyes at high risk for development of this complication, prior to penetrating keratoplasty, extracapsular cataract extraction and posterior chamber lens (IOL) implantation. Results in 10 cases with core vitrectomy were compared with 10 cases without (controls); in all eyes with vitrectomy, a posterior chamber IOL was easily implanted but four eyes of the control group developed vitreous complications. Our results indicate that core vitrectomy does facilitate IOL implantation during a triple corneal procedure in eyes at increased risk of elevated posterior chamber pressure.

Suppression of graft rejection in rat keratoepithelioplasty by anterior chamber inoculation of donor lymphocytes.

A newly developed model of keratoepithelioplasty (KEP) in the rat was used to analyze the effect of anterior chamber-associated immune deviation on the suppression of corneal allograft rejection. The right eyes of Fisher rats received an anterior chamber (AC) injection of lymphocyte suspension from Dark Agouti (DA) rats (DAL group) or phosphate-buffered saline (positive control group). Seven days later, DA corneal lenticules were grafted onto both eyes of the recipients. Delayed type hypersensitivity (DTH) was also assessed in another group of Fisher rats receiving an AC injection of DA lymphocytes in the right eye. Within 15 days, 88% of the eyes in the positive control group demonstrated vigorous epithelial edema, a large area of epithelial defect, and vascularization. By contrast, in the DAL group, only 37% of the eyes showed slight epithelial edema, a small area of epithelial defect, and rare vascularization; suppression of graft rejection was observed in both eyes. DTH assay demonstrated significant suppression in the recipients of the AC injection of donor-type lymphocytes. This study indicates that the AC injection of donor-type lymphocytes prior to corneal grafting suppresses allograft rejection in the rat KEP model, correlative with the suppression of DTH reaction.

[The effect of cultivation on the differentiated function of rabbit lens epithelial cells in vitro].

Changes in the characteristics of rabbit lens epithelial cells during long-term cultivation were examined. From the beginning of cultivation to the 24th generation, the cells grew exponentially, and then the growth stopped. This growth-arrested period lasted for 60 days, and then they grew exponentially again. The cells had a cobblestone-like appearance through the whole cultivation period except in the growth-arrested period. In an immunofluorescence examination with a monoclonal antibody against alpha-crystallin, all of the primary cells and the cells in the 20th generation showed specific fluorescence to alpha-crystallin throughout their cytoplasm. However, some of the cells in the 24th generation did not show this fluorescence, and all of the cells in the 35th and 70th generations showed fluorescence only around the nuclei and none in the cytoplasm. The primary cells and the cells in the 20th generation were capable of forming lentoid bodies, but the cells after the 24th generation were not. These results indicate that some of the characteristics of the rabbit lens epithelial cells are lost during the growth-arrested period.

[Tissue of origin of herpes simplex virus type 1 reactivated in tear film].

In the present study, we tried to detect the tissue of origin of herpes simplex virus type 1 (HSV-1) reactivated in tear film after artificial reactivation. The combined treatment consisted of iontophoresis on postinoculation day 35, followed by topical epinephrine on 2 days, after which the rabbits were killed. The ocular tissues and trigeminal ganglia were immediately dissected. Their cell-free supernatants were inoculated on CV-1 (African green monkey kidney cell) monolayers for infectious HSV-1 detection. The percentage of recovery from the cell-free supernatants was 50% (5 out of 10 samples) from the cornea, 0% from conjunctiva or lacrimal glands, and 20% (2 out of 10 samples) from trigeminal ganglia. The percentage of HSV-1 reactivation in the tear film was 50%. No infectious virus was detected from tissues or tear film in the control group. Four eyes showed HSV-1 reactivation simultaneously from the cornea and tear film, but only one eye from the trigeminal ganglion and tear film. These results demonstrate that the cornea might be the tissue of origin of HSV-1 reactivated in tear film.