Immunohistochemical localization of epidermal growth factor receptor and epithelial antigen in tumors of the human conjunctiva,eyelid, lacrimal gland,and orbit

Background: Increased numbers of epidermal growth factor (EGF) receptors are observed in squamous cell carcinomas of human lung, head, neck, and cervix. We studied the presence of EGF receptors and epithelial antigen in some ophthalmic lesions. Methods: Immunohistochemical staining for EGF receptors was assessed in tumors of human conjunctiva, eyelid, lacrimal gland, and orbit with monoclonal antibodies (EGF-R1 and clone 29.1). Reactivity of Ber-EP4, which reocgnizes epithelial antigen, was also examined. Results: Strong staining of EGF-R1 and clone 29.1 and weak to moderate staining of Ber-EP4 were demonstrated in conjunctival squamous cell carcinomas. Cell membranes of conjunctival papilloma were moderately or strongly stained with these antibodies. Ductal components in sebaceous gland adenoma of the eyelid and pleomorphic adenoma of the lacrimal gland were positively stained. The antibodies did not bind to reactive lymphoid hyperplasia of the orbit and Wegener's granulomatosis. Relatively good correlation for immunostaining reaction was observed among EGF-R1, clone 29.1, and Ber-EP4 in each tumor. Conlcusion: Immunostaining using EGFR1, clone 29.1, and Ber-EP4 may be useful in differentiating epithelial tumors from non-epithelial lesions. Strong immunostaining for EGF receptor may be the hallmark of epidermoid malignancy.     

Expression of glucose transporter protein-1 (Glut-1) in ocular surface squamous neoplasia

PURPOSE: To examine the expression of glucose transporter protein-1 (GLUT-1) in ocular surface squamous neoplasia and to study its relationship with degree of neoplasia and cell proliferation index (Ki-67 labeling index). METHODS: Twelve cases diagnosed as ocular surface squamous neoplasia (4 invasive and 8 intraepithelial tumors) at Inonu University Faculty of Medicine, Department of Pathology, were included in this study. There were 3 squamous cell carcinomas, 1 basosquamous cell carcinoma, and 8 conjunctival intraepithelial neoplasms. Immunohistochemically, GLUT-1 and Ki-67 antibody staining were performed. RESULTS: GLUT-1 membranous immunoreactivity was seen in all tumors except in 1 case. GLUT-1 immunostaining was observed in all layers of the neoplastic epithelium of squamous cell carcinoma. Intense staining for GLUT-1 was determined in the upper two thirds of the severe dysplastic squamous epithelium. Although immunoreactivity for Ki-67 nuclear antigen was present throughout the epithelium, it was higher in the lower two thirds. Ki-67 labeling index ranged between 6% and 80%, and the mean value was 35% for invasive tumors and 20% for intraepithelial tumors. CONCLUSIONS: Marked GLUT-1 and Ki-67 immunoreactive cells throughout the neoplastic epithelium of ocular surface squamous neoplasia were observed. In most cases, it was observed that GLUT-1 expression was severe in cases having >10% Ki-67 labeling index. These findings indicate that glucose uptake was increased in dysplastic cells, especially by GLUT-1. To our knowledge, this is the first study on the subject in the literature, and further studies with more cases are needed with GLUT-1 and other GLUT members.     

MIB-1 and PC-10 immunostaining for the assessment of proliferative activity in primary acquired melanosis without and with atypia

AIMS—To compare the proliferative activity of intraepithelial melanocytes in primary acquired melanosis (PAM) without atypia and PAM with atypia by immunohistochemical staining for the Ki-67 antigen and the proliferating cell nuclear antigen (PCNA).
METHODS—Formalin fixed, paraffin embedded sections from 35 archival specimens of PAM without atypia (n=19) and with atypia (n=16) were studied by immunostaining with MIB-1 and PC-10 monoclonal antibodies that react with the Ki-67 antigen and PCNA respectively. The results were calculated as the mean number of positive cells per eyepiece grid. All specimens were evaluated by two masked observers, and the interobserver reproducibility was assessed.
RESULTS—The means of the positive cell count in PAM with atypia were significantly higher compared with PAM without atypia for both observers, in both the PC-10 and the MIB-1 stained sections. In a linear least square model that estimated the interobserver and between group variation, the difference of MIB-1 and PC-10 positive cell count between PAM without and with atypia remained highly significant. The difference between the observers was not significant.
CONCLUSIONS—Immunostaining with MIB-1 and PC-10 demonstrated that PAM with atypia has higher proliferative activity than PAM without atypia. This method was found to be reproducible between different observers.

Keywords: primary acquired melanosis; immunostaining; atypia     

Immunohistochemical features of carcinoma ex pleomorphic adenoma and pleomorphic adenoma in the lacrimal gland

AIM: To investigate C-myc, Ki-67, pan-cytokeratin, and vimentin immunohistochemical features of carcinoma ex pleomorphic adenoma (Ca-ex-PA) and pleomorphic adenoma (PA) in the lacrimal gland in order to find some clues in the differential diagnosis between them. METHODS: We reviewed microscopic slides and clinical records of 64 cases of PA and 15 cases of Ca-ex-PA in the lacrimal gland. Immunohistochemical antibodies for C-myc, Ki-67, pan-cytokeratin, and vimentin were employed. RESULTS: Median age of PA was 43.2y (from 21 to 75). The 35 patients (54.7%) were male and 29 patients (45.3%) were female. For the PAs, the average positivity of C-myc was 4.6%; the average proliferation index of Ki-67 was 3.2%; pan-cytokeratin was positive in ductal cells, and vimentin was positive in myoepithelial cells. Median age of Ca-ex-PA was 54.3y (from 26 to 76). There were 7 male patients (46.7%) and 8 female patients (53.3%). Among 15 Ca-ex-PAs, there were 6 myoepithelial carcinomas, 4 adenocarcinomas, 3 epithelial-myoepithelial carcinomas, and 2 squamous cell carcinomas. For the Ca-ex-PAs, the average positivity of C-myc was 36.4%; the average proliferation index of Ki-67 was 29.2%; pan-cytokeratin was positive in all cases, and vimentin was positive in myoepithelial carcinomas. CONCLUSION: PA has a lower positivity of C-myc and Ki-67, while Ca-ex-PA had a higher positivity of these two biomarkers. These four biomarkers as a set could provide valuable clues in the differential diagnosis between Ca-ex-PA and PA. Our results indicate that the activation of C-myc could play an important role in the pathogenesis of Ca-ex-PA and PA.     

VEGF、SDF-1、Ki-67、PCNA及Survivin与翼状胬肉的关系分析

目的：分析血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质细胞衍生因子-1(stromal cell-derived factor 1,SDF-1)、肿瘤增殖抗原(Ki-67)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)及生存素(Survivin)与翼状胬肉的关系。

方法：选取2013-01/2015-05于本院进行诊治的79例106眼翼状胬肉患者为观察组,同时期的79例正常结膜者为对照组,然后将两组的组织VEGF、SDF-1、Ki-67、PCNA及Survivin阳性细胞数分级及染色强度分级进行比较,并比较其中不同性别、分期及分型患者的检测结果,同时以Logistic分析上述指标与翼状胬肉的关系。

结果：观察组的组织VEGF、SDF-1、Ki-67、PCNA及Survivin阳性细胞数分级及染色强度分级均高于对照组,不同分期及分型患者的检测结果也存在一定差异(均P<0.05),而不同性别患者间的检测结果则无明显差异(均P>0.05),经Logistic分析显示,上述组织指标均与翼状胬肉有密切的关系。

结论：翼状胬肉组织中的VEGF、SDF-1、Ki-67、PCNA及Survivin表达呈现异常状态,上述指标均与翼状胬肉有密切的关系。     

脉络膜恶性黑色素瘤中Ki—67的表达和肿瘤主要组织病理特性的关系

目的利用抗Ki-67单克隆抗体对石蜡包埋的葡萄膜黑色素瘤组织作免疫组化染色,研究细胞增殖活性和肿瘤重要的组织病理特性之间的关系.方法对1988～1997年问57例葡萄膜黑色素瘤眼球摘除眼作组织病理检查和Ki-67免疫组化染色.所有患眼在接受眼球摘除术前均没有接受过其他治疗.结果Ki-67指数分布范围为0～4.89,平均(0.75±1.02).含有上皮样细胞肿瘤的Ki-67指数高于梭形细胞肿瘤,差异具显著性(P=0.037).大肿瘤组的Ki-67指数比中等大肿瘤组高,两者比较差异有高度显著性(P=0.007).肿瘤大小和细胞类型问存在内在的相关性(X2=4.528,P＜0.05),大肿瘤多为上皮样细胞肿瘤而中等大肿瘤则多为恶性度较低的梭形细胞肿瘤.未发现Ki-67表达与肿瘤位置;肿瘤巩膜外蔓延及年龄有关.结论Ki-67表达与肿瘤的组织细胞类型及大小有关.眼科学报2001;17114～117.     

培养人视网膜色素上皮细胞增生的抑制与Ki-67表达

目的 探讨柔红霉素对视网膜色素上皮(retinal pigment epithelium,RPE)细胞增生的抑制及其与Ki-67表达的关系。 方法 用180μg/L柔红霉素作用培养的人RPE细胞12h，之后24 h用氚-标记脱氧胸苷(tritium-labelled thymidine deoxyribose,³H-TdR)掺入法测定DNA合成抑制率，免疫细胞化学染色和定量分析观察Ki-67表达，流式细胞术检测细胞周期变化。 结果 培养人RPE细胞DNA合成抑制率与柔红霉素剂量成正比，对照组和柔红霉素组Ki-67阳性细胞率分别为89.3%和45.6%(P＜0.01)，两组中阳性细胞胞核积分光密度值分别为68.1±6.2和27.3±5.5 (P＜0.01)。柔红霉素组^G₂期细胞比例由8.9%增至29.5%。 结论 柔红霉素使培养人RPE细胞阻滞于^G₂期，抑制了细胞增生；Ki-67表达可以反映RPE细胞的增生抑制。（中华眼底病杂志，2000，16：1-70）     

High intraocular pressure-induced ischemia and reperfusion injury in the optic nerve and retina in rats

• Purpose: The purpose of this paper is to describe the damage caused to the retina and the axons of the optic nerve by acute ischemia-reperfusion injury and the extent to which optic nerve damage correlates with the duration if ischemia due to high intraocular pressure (IOP). • Methods: Acute ischemia in the retina and optic disc was induced in albino rats by increasing the IOP to 110 mmHg for a period of 45–120 min. Thereafter, the eyes were reperfused at normal IOP after 7 days. The retina and optic nerve were examined by light and electron microscopy, and morphometrical counts of the optic nerve axons were performed. • Results: After 45 min of ischemia, electron microscopic examination revealed swelling of mitochondria and degeneration of neurotubules on axons in cross sections of the optic nerve. The axonal counts in eyes subjected to 45 min of ischemia were 29% lower than in control eyes. After 60 min of ischemia, there were distinct disruptions of mitochondria and degeneration of the axons. After 90 min of ischemia, numerous axons showed degeneration with disordered myelin sheaths. Neuronal cell death was seen in the retina, mainly in the ganglion cell layer. • Conclusion: Damage to the retinal ganglion cell layer and the optic nerve was evident after only 45 min of ischemia in normal eyes. This experiment suggests that seriously injured eyes must be protected from high IOP; if IOP elevation is required during vitrectomy, it is essential to reduce the duration of interruption of blood flow to a minimum.     

Increased Ki-67 proliferative index and absence of P16INK4 in CIN-HPV related pathogenic pathways different from cervical squamous intraepithelial lesion

BACKGROUND/AIM: It is generally assumed that similar pathways are involved in human papillomavirus (HPV) induced pathogenesis of cervical squamous intraepithelial lesions (SILs) and cancers and a subset of conjunctival intraepithelial neoplasm (CIN)-that the malignancies or pre-cancerous lesions arise through HPV oncoproteins E6 and E7, which disrupt the pathways of p53 and the product of the retinoblastoma (Rb) gene and, in turn, increase the protein product of gene p16INK4 through the mechanism of positive feedback. Several cell cycle molecules are detected to test this hypothesis. METHODS: Nine cases of CIN and eight non-CIN cases were analysed for the expression of Ki-67, pRb, p53, and p16INK4 via immunohistochemistry. Nine cases of cervical high grade squamous intraepithelial lesion (HSIL), and 10 cases of cervical low grade squamous intraepithelial lesion (LSIL) were included for stain control of p16INK4a, and comparison of p16INK4a expression to CIN cases. A nested polymerase chain reaction and a genechip HPV typing were used to detect HPV infection and types in the CIN and non-CIN samples RESULTS: HPV positivity was demonstrated in all of the CIN lesions but in none of the non-CIN lesions. The Ki-67 proliferative index (Ki-67 PI) was statistically higher in the CIN group than the non-CIN group; however, there were no differences of expression of pRb and p53 between the two groups and no expression of p16INK4 in all cases. All nine cases of HSIL, and seven out of 10 cases of LSIL used for stain control were immunoreactive for p16INK4a. There were statistically significant differences in overexpression of p16INK4a between the CINs and SILs CONCLUSIONS: The Ki-67 proliferative index may be a sensitive marker for CIN lesions and these results, with significant differences in overexpression of p16INK4a between CINs and SILs, may provide new evidence that HPV related mucosal dysplasia in different anatomical locations may lead to dissimilar molecular pathways.     

Ki-67 Labeling Index as a Marker of Malignancy in Ocular Surface Neoplasms

Purpose To evaluate the relationships among histopathological type, clinical malignant grade, and Ki-67 labeling index (LI) in sebaceous gland carcinoma (SGC), conjunctival squamous cell carcinoma (SCC), and conjunctival intraepithelial neoplasia (CIN), with pterygium and normal conjunctiva as controls.Methods This retrospective study was conducted at the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. We used tissue specimens obtained from 20 patients (four SGC, four SCC, four CIN, four pterygium, and four normal conjunctiva). Ki-67 immunohistochemical analysis was performed in all 20 cases.Results The Ki-67 labeling index (LI) was 46.1 ± 3.0% (average ± SD) in SGC, 28.4 ± 4.5% in SCC, 20.0 ± 7.2% in CIN, 9.0 ± 2.2% in pterygium, and 6.8 ± 2.3% in normal conjunctiva. Ki-67 LI was significantly (Mann-Whitney U test, P < 0.05) higher in SGC than in SCC, and higher, but not significantly, in SCC than in CIN. Ki-67 LI was significantly (P < 0.05) higher in SCC and CIN than in pterygium.Conclusions These results suggest that Ki-67 LI may be a sensitive marker for ocular malignant tumor grading. Jpn J Ophthalmol 2004;48:524–529 © Japanese Ophthalmological Society 2004     

Perioperative analysis of vitreous cell components by immunoimpression cytology

• Background: Perioperative analysis and classification of vitreous cell components needs a quick method sufficient to prepare both single cells and cellular membranes. • Methods: In an attempt to clarify the usefulness of immunoimpression cytology we examined 82 eyes with proliferative vitreoretinopathy, proliferative diabetic retinopathy, perforating injuries and contusions. • Results: With this method it is possible to perioperatively obtain cells from surfaces of vitreous membranes as well as single cells in suspension. After staining, various cell types could be differentiated morphologically and on the basis of antibody response to cell markers. We found single cells reacting with the antibodies 27E10 (4 positive of 14 tested), vimentin (2/12), RM 3/1 (2/15), LCA (1/11) and, particularly,with anti-proliferating cell nuclear antigen (9/18). • Conclusion: With immunoimpression cytology it is possible to determine cell surface markers rapidly and accurately from specimens obtained at the time of vitrectomy. The clinical utility of this test will be ascertained by future studies.     

视网膜母细胞瘤PAX6、Ki-67和MMP-9的表达及其与组织病理学的关系

目的 检测PAX6、Ki-67及MMP-9在视网膜母细胞瘤（RB）中的表达,探讨其与RB临床组织病理学特征之间的关系及临床意义。设计实验性研究。研究对象石蜡包埋RB组织40例,其中〈3岁组27例,≥3岁组13例。方法 采用免疫组织化学Elivision法检测PAX6、Ki-67及MMP-9在40例RB组织中的表达情况,分析该3种蛋白表达与患者性别、眼别、年龄、视神经受侵情况以及是否化疗等临床组织病理学特征的关系,并分析3种蛋白表达之间的相关性。主要指标RB组织中PAX6、Ki-67、MMP-9的表达及临床组织病理学特征。结果 40例RB组织中,PAX6、Ki-67及MMP-9的阳性表达率分别为52.5%、55.0%、57.5%。Ki-67在年龄大于3岁组的阳性表达率高于年龄小于3岁组（χ^2=6.825,P=0.016）,PAX6（χ^2=0.631,P=0.511）及MMP-9（χ^2=0.129,P=1.000）的表达与年龄无显著相关性。3种蛋白的表达均与性别及眼别无显著相关性。PAX6、Ki-67及MMP-9在球后视神经受侵袭组的阳性表达率均高于未受侵袭组（χ^2=14.401,P=0.017;χ^2=11.831,P=0.046;χ^2=13.961,P=0.038）。PAX6及Ki-67在未化疗组的阳性表达率均高于化疗组（χ^2=8.120,P=0.010;χ^2=6.465,P=0.025）。PAX6和Ki-67的表达呈正相关（r=0.347,P=0.028）。结论 PAX6的高表达促进了RB细胞的增生;PAX6、Ki-67和MMP-9的高表达与RB的侵袭有一定的相关性;化疗对RB中PAX6和Ki-67的表达有一定的抑制作用,在一定程度上可以降低RB侵袭转移的风险。     

Cellular proliferation and leukocyte infiltration in the rabbit cornea after photorefractive keratectomy

PURPOSE: To map the proliferative activity of corneal cells during wound healing following photorefractive keratectomy (PRK) and to compare two markers for proliferation. METHODS: PRK, 5- mm in diameter with a -6 D setting, was performed in one eye of 28 New Zealand White Rabbits. The rabbits were sacrificed at time points between 12 hours and three months after surgery. The treated and fellow corneas were fixed in 10% formaldehyde, paraffin embedded, and immunohistochemically stained for proliferate cell nuclear antigen (PCNA) and at one time point, 1 week, also for Ki-67. RESULTS: Following initial sliding of the epithelial cells, the proliferative activity in the wound area starts in the leading edge (24 hours) and is spread towards the periphery. The proliferative activity peaks after one week and subsides during the following two weeks. Early (24 hours) proliferative activity is also seen in the limbal epithelium which peaks after three days. The keratocytes express PCNA in the peripheral stroma 48 hours after injury. They then also migrate to repopulate the stroma under the wound area. The expression period lasts 1 week and subsides the following week. Leukocytes are found in the wound as early as 12 hours after injury. The cells disappear around the time of epithelial wound closure, i.e. after 3 days. The two proliferative markers PCNA and KI 67 show a similar distribution after surgery. CONCLUSION: Epithelial proliferative activity starts earlier after injury, and is preceded by leukocyte presence in the wound. The PCNA expression starts later in the keratocytes but lasts somewhat longer (3 weeks). PCNA expression appears more efficient than Ki-67 to show proliferative activity of slow cycling cells in the cornea     

The Rho-kinase inhibitor H-1152P suppresses the wound-healing activities of human Tenon's capsule fibroblasts in vitro

PURPOSE: To analyze the role of Rho-kinase signaling in the wound-healing activities of human Tenon's capsule fibroblasts by using H-1152P, a potent inhibitor of this kinase, in vitro. METHODS: The optimal concentration of H-1152P was determined by MTT test. Cell proliferation was measured by BrdU incorporation and Ki-67 immunostaining, whereas cell viability was investigated by ethidium homodimer-1 dye exclusion. The actin cytoskeleton organization was demonstrated by alpha-smooth muscle actin (SMA) immunostaining and Alexa 488-phalloidin staining. Cell migration was studied on restrained collagen gels and in a scratch-wound assay followed by Ki-67 and fibronectin immunostaining. The effect of H-1152P on contraction was analyzed in floating collagen gels populated with fibroblasts, which were subsequently processed for fibronectin immunostaining. The levels of adducin and the protein kinase A (PKA)-dependent phosphorylation of this protein were detected by immunoblot analysis, to rule out interference with PKA. RESULTS: Incorporation of BrdU and upregulation of Ki-67 were reduced by 80% to 90% in cells incubated with 10 microM of this inhibitor for 4 days (P < 0.01). H-1152P caused the disassembly of stress fibers in a dose-dependent manner without exerting toxic effects and without a considerable interference with the PKA-pathway. H-1152P also significantly suppressed cell migration 3- to 3.5-fold and the contraction of collagen lattices fivefold with a dose-dependent impairment in the assembly of the fibronectin network. CONCLUSIONS: These findings imply a role for Rho-kinase in the wound-healing activities of human Tenon's capsule fibroblasts and show the potential of H-1152P as a safe and specific means to suppress these events.     

Lacquer crack lesions in experimental chick myopia

• Background: Lacquer crack lesion (LCL) is a complication of human pathologic myopia, accompanied by loss of retinal pigment epithelium (RPE) and break of Bruch’s membrane. The present paper describes comparable lesions occurring in prolonged experimental myopia in the chick. • Methods. Form-deprivation myopia was induced by unilateral eyelid suturing on the 1st day after hatching. Bruch’s membrane in NaOH hydrolyzed preparations and vascular corrosion casts of the choroidal vasculature were examined with scanning electron microscopy. Histological changes in the retina and choroid were also examined with light and transmission electron microscopy. • Results: Pale and linear lesions were found in the myopic chick eyes at the age of 8 weeks. In the lesion area, Bruch’s membrane was totally broken up and the network of choriocapillaries was totally ruptured with highly atrophied marginal capillaries. The retina was continuous, but was depressed to form a groove in the lesion with the apparently intact inner retina and degenerated photoreceptor cells. Attenuated fibroblasts encompassed the outer circumference of the lesion. RPE cells were scattered in the tissue space inside the fibroblastic investment and also in the choroidal stroma without their polarity. • Conclusion: The formation of LCL was suggested to be a result of passive stretch exerted upon Bruch’s membrane and the capillary network due to abnormal enlargement of the myopic eyes. These results may promote further understanding of the mechanism regarding the development of human lacquer cracks. Received: 2 May 1997 Accepted: 30 July 1997     

The correlation among different immunostaining evaluation methods for the assessment of proliferative activity in uveal melanoma

PURPOSE: Proliferative index of uveal melanoma cells serves as a prognostic factor. However, different methods are being used to determine proliferative index using immunostaining for proliferative markers. The major differences among assessment methods are whether the mean proliferative activity of all tumor cells in a section or for areas of rapidly proliferating cells is determined, and whether 10 or 20 fields are being evaluated. We aimed to assess the correlation among proliferative indexes obtained by different immunostaining evaluation methods. METHODS: Sections from 60 formalin-fixed paraffin-embedded uveal melanomas were immunostained with MIB-1 antibody. Immunostaining was assessed by counting immunoreactive cells in semi-randomly selected fields (non-selective method) and in areas with maximal immunoreactivity (selective method). Proliferative activity indexes according to the two methods in 10 and 20 high power fields (one high power filed = 0.785 mm(2)) were calculated and compared. RESULTS: The mean positive cell counts per mm(2) (MPCC/mm( 2)) according to the selective and the non-selective methods were 29.8 +/- 8.1 and 10.7 +/- 2.4 respectively (p = 0.004, paired t-test). Despite these different values, there was a good correlation between the MPCC/mm( 2) obtained by the non-selective and the selective methods for each tumor (r = 0.737, p < 0.0001, Pearson correlation). In addition, according to both methods, the readings of the first 10 fields and those of fields 1-20 correlated well. CONCLUSIONS: Mean proliferative activity of uveal melanoma cells correlates with the proliferative activity in localized areas of the tumor with rapidly proliferating cells. Therefore, uveal melanomas are classified similarly by the selective and non-selective methods of immunostaining evaluation. However, the two methods yield different proliferative index values for the same tumors, a fact that should be taken into account when comparing results of studies in which different techniques were used for immunostaining evaluation.     

The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes

• Background: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. • Methods: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-β or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-β and bFGF in conditioned media. • Results: EGF and bFGF increased, while TGF-β decreased, the proliferation of both cell types in a dosedependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-β and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-β levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. • Conclusions: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways.     

HLA class II antigen expression in conjunctival precancerous lesions and squamous cell carcinomas

PURPOSE: The expression of human leukocyte antigen (HLA) class II molecules on the cell surface is necessary for the presentation of peptide antigens to helper CD4 T lymphocytes of the immune system. We studied the immunoexpression of HLA class II antigen in conjunctival precursor lesions and conjunctival squamous cell carcinomas. METHODS: HLA class II antigen expression was analyzed in 8 conjunctival dysplasias, 6 carcinomas in situ and in 7 conjunctival squamous cell carcinomas, by immunoperoxidase staining with monoclonal antibody to HLA class II antigen on the archival clinical samples. Immunoanalysis was done by a semi quantitative method based on the intensity of staining and the percentage of stained cells. RESULTS: HLA class II antigen immunoexpression was heterogeneous in 8 conjunctival dysplasias and in 6 carcinoma in situ and negative in 7 conjunctival squamous cell carcinomas. CONCLUSIONS: Human leukocyte class II antigen immunoexpression is decreased in conjunctival precancerous and squamous cell carcinomas.     

Effects of different perfluorochemicals on dorsal root ganglion cells in vitro

• Background: Investigation of the effects of different perfluorochemicals (PFC) on cultured dorsal root ganglion (DRG) cells. • Method: DRG cell cultures from 9- to 11-day-old chicken embryos were exposed to emulsified perfluorodecalin (PFD; C₁₀F₁₈; 0.5%, 1% and 10%) or perfluorooctylbromide (PFO; C₈F₁₇Br; 0.5%, 1% and 10%). The cells were evaluated under phase-contrast optics after 30 h and 120 h for 0.5 and 1% and after 5 h for 10%. To study the integrity of neuronal cells, immunohistochemical labelling for neurofilaments (NF) and tubulin (TUB) was performed. • Results: Concentrations of 0.5% and 1% of PFD or PFO did not change immunohistochemical labelling of DRG cells. Co-cultured macrophages showed a foam cell response, presumably representing ingested PFC. At both concentrations PFD induced a weaker foam cell response than PFO. A concentration of 10% led to the death of DRG cells and macrophages within 5 h. • Conclusion: PFC caused a dose-dependent damage of neuronal cells. Co-cultured macrophages developed a foam cell response similar to that observed in vivo after prolonged presence of PFC in the vitreous body. These observations indicate that PFD and PFO may not be suitable for long-term vitreous replacement in vitreoretinal surgery. However, the model is limited by several factors: (1) there are physiological differences between DRG cells and retinal ganglion cells; (2) in vivo retinal ganglion cells are protected by the overlying tissues; (3) the PFC used in tissue culture must be emulsified. Received: 30 May 1996 Revised version received: 26 February 1997 Accepted: 7 May 1997     

Apoptosis of photoreceptor cells in ornithine-induced retinopathy

• Background: The intravitreal injection of ornithine produces selective damage to the retinal pigment epithelium (RPE) and results in a loss of RPE, choriocapillaris and photoreceptor cells. To elucidate the mechanism of secondary retinal atrophy, we investigated the presence of apoptotic cells in a rat model of ornithine-induced retinopathy. • Methods: At 6 and 12 h and 1, 2, 4, 7, 14 and 28 days after an intravitreal injection of L-ornithine hydrochloride in rat eyes, we removed the eyes and subjected them to histopathological examination. We detected apoptotic cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end labeling (TUNEL) assay, which stains the 3′-OH ends of fragmented DNA. We used electron microscopy to detect the apoptotic cells morphologically. • Results: RPE cells were selectively damaged immediately after ornithine administration. TUNEL-positive photoreceptor cells appeared exclusively in the photoreceptor cell layer 12 h after ornithine administration. The number of TUNEL-positive cells increased throughout the 2 days following the injection, then decreased markedly. TUNEL-positive cells remained until 28 days, when the photoreceptor cells had disappeared. The ganglion cell layer, inner nuclear layer and damaged RPE cells were negative for TUNEL staining during all stages. The electron microscopic study also revealed the pyknotic nuclei of apoptotic photoreceptor cells. • Conclusion: An intravitreal injection of ornithine caused primary damage to the RPE, and subsequently some of the photoreceptor cells revealed apoptosis by TUNEL assay. These findings suggest the dysfunction of the RPE causes photoreceptor cell death according to the intrinsic program of an apoptotic mechanism. Received: 16 April 1997 Revised version received: 7 July 1997 Accepted: 18 July 1997