EGF Cell Surface Receptor Quantitaiion on Ocular Cells by an Immunocytochemical Flow Cyfometry Technique

A method is presented for the rapid flow cytometric determination of epidermal growth factor (EGF) receptor densities on the surface of cultured ocular cells. The technique uses a biotinylated monoclonal antibody directed against the EGF receptor in conjunction with a streptavidin-bound fluorochrome and requires the specific fluorescence per cell to be measured as a function of ligand and receptor concentration. Because the measurement is noninvasive and restricted to cell surface-bound material, the cells can be kept in a physiologic environment, even at the moment of assay. Calculated receptor densities ranged from 5142/cell (infant human corneal endothelium) to 35,678/cell (infant human keratocytes) to >5 X 10 5 /cell for an A431 control cell line. Species and donor age differences were noted, as was transient receptor downregulation after EGF administration. Flow cytometry represents a valuable time saving procedure for large scale applications while providing the same level of sensitivity as standard radioimmunoassays. This technique is applicable to quantitation of other growth factor cell surface receptors and could greatly expand the use of flow cytometry in the research laboratory. Inves

A role for 12(S)- HETE in the response of human lens epithelial cells to epidermal growth factor and

Purpose. To determine whether the 12-lipoxygenase pathway of arachidonic acid metabolism is present in the human lens and whether 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) plays a role in regulating proto-oncogene expression and DNA synthesis in human lens epithelial cells (HLECs). Methods. Second-and third-passage primary cultures of HLECs were used for analysis. Human cataract epithelia were obtained from surgery. 12-lipoxygenase mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the PCR product was sequenced. The 12-lipoxygenase protein was detected by immunoblotting. 12(S)-HETE was detected in HLEC-conditioned medium by radioimmunoassay. For studies of growth factor-induced mitogenesis, HLECs were serum starved, then stimulated with 15 ng/ml epidermal growth factor (EGF) and 1 //g/ml insulin or with 0.3 pM 12(S)-HETE. The 12-lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-a-cyanocinnamate (CDC, 10 /xM) was used to block endogenous 12-lipoxygenase activity. Expression of c-fos mRNA was determined by RT-PCR, and DNA synthesis was measured by 3 H-thymidine incorporation. Results. 12-lipoxygenase mRNA and protein were detected in HLECs and in human cataract tissues. 12(S)-HETE was released into the medium by HLECs in the presence of EGF-insulin. Stimulation of c-fos mRNA expression and DNA synthesis by EGF-insulin was inhibited when the 12-lipoxygenase pathway was blocked by CDC. This inhibition was reversed completely by exogenously added 12(S)-HETE. However, exogenous 12(S)-HETE was unable to stimulate HLEC DNA synthesis in the absence of growth factors. Conclusions. The 12-lipoxygenase pathway of arachidonic acid metabolism is present in human lens epithelial cells. 12(S)-HETE does not stimulate HLEC DNA synthesis in the absence of growdi factors but enables the cellular response to EGF and insulin. Invest Ophthalmol Vis Sci. 1996;37:1411-1418 Arachidonic acid and its various metabolites are widely distributed in animal tissues and have important biologic roles in both pathologic and physiological conditions. 1 " 3 Arachidonic acid can be metabolized by three enzymatic pathways: the prostaglandin endoperoxide synthase pathway, the cytochrome P450 padiway, and the lipoxygenase pathways. In mammalian cells, three major lipoxygenases have been described-5-lipoxygenase, 12-lipoxygenase, and 15-lipoxygenase-which convert arachidonic acid to thei

Major Retinal Cell Components Recognized by Onchocerciasis Sera Are Associated With the Cell Surface and Nucleoli

Purpose. Cellular localization of the components recognized by onchocerciasis autoantibodies has not been investigated in any detail in cultured retinal cells. This study sought to examine, in cultured retinal cells, the subcellular localization of major components that cross-react with onchocerciasis sera. Methods. Immunofluorescence confocal laser scanning microscopy and Western blot analysis were carried out on adult pig retinal cells. Results. The onchocerciasis sera contain antibodies cross-reacting strongly with components of the surface and nucleoli in both the cultured retinal pigment epithelial and neural retinal cells. These epitopes are not recognized by the control sera obtained from noninfected individuals residing in an onchocerciasis hyperendemic area, and from those with or without ocular disease who have never been in any of the onchocerciasis hyperendemic countries. Double-labeling immunofluorescence microscopy does not detect any colocalization of a putative onchocerciasis autoantigen, calreticulin, and those cellular components recognized by onchocerciasis sera in either cell type. Furthermore, none of the onchocerciasis sera tested recognized recombinant calreticulin by Western blot analysis. Conclusions. Major epitopes for onchocerciasis anti-retinal autoantibodies are associated with the surface and nucleolus components of retinal cells. Interaction of the onchocerciasis antibodies with the retinal cell surface molecules may play an important role in the development of ocular diseases initiated by the damage of retinal cells. Furthermore, the finding that the cellular components recognized by onchocerciasis sera do not colocalize with calreticulin, taken together with the observation of lack of recognition of recombinant calreticulin by these sera on Western blots, suggests that calreticulin is not a major onchocerciasis autoantigen. Invest Ophthalmol Vis Sci. 1994; 35:1089-1099. V-lnchocerciasis (river blindness) is a tropical filarial disease caused by infection with the parasitic nematode Onchocerca volvulus. Eye diseases, along with dermatitis, sclerosing lymphadenitis, and subcutaneous nodules, are the major manifestations of human onchocerciasis, and have been well described clinically

Reports Genomic Organization of the Human TIMP-1 Gene Investigation of a Causative Role in the Pathogenesis of X-Linked Retinitis Pigmentosa 2

Purpose. To evaluate the role of TIMP-1 in inherited retinal degeneration. Methods. The genomic structure of the TIMP-1 gene was established and male patients with x-linked retinitis pigmentosa 2 from five families were screened for sequence alterations by direct sequencing in all exons, exon-intron boundaries, and the 5' upstream region of the gene. Results. TIMP-1 appears to be expressed in the retina at low levels and consists of six exons spanning a genomic region of approximately 4.5 kb on Xpll.23. No diseasespecific sequence alterations were identified. A site substitution in exon 5 was observed in samples from control subjects and patients, but it did not alter the amino acid sequence of the protein product. Conclusions. The results of this study exclude mutations in the TIMP-1 coding sequence, splice sites, and the 5' upstream region as a cause of retinal degeneration in x-linked retinitis pigmentosa 2. However, an as yet unidentified regulatory element that lies outside these intervals may be implicated. The role of this tightly regulated protein in the normal functioning of the retina has yet to be determined. Invest Ophthalmol Vis Sci. 1997; 38:1893-1896 L he turnover of the extracellular matrix is regulated by the synthesis of new components and the degradation of existing structures. Matrix catabolism is regulated by the action of matrix metalloproteinases, which degrade matrix components such as collagens, gelatins, fibronectin, and laminin. The activity of these From the

Laser Light Scattering Spectroscopy of In Vivo Human Lenses

Laser light scattering spectroscopy measures the thermal random movement of protein as characterized by the diffusion coefficient. This technique has been used in assessing cataract formation in animals. The changes detected appear to predict the later development of lens opacities. The sensitivity and quantitative aspects of this technique offer advantages over other presently available methods of detecting cataract formation. First studies in humans indicated a significant correlation between the diffusion coefficient and age (P < 0.05). The age adjusted mean diffusion coefficient for nondiabetics (4.60 ± 0.29; mean ± SEM) was significantly higher compared to diabetics without retinopathy (3.59 ± 0.41; P = 0.0473), diabetics with background or preproliferative retinopathy (2.73 ± 0.27; P = 0.0001), or to diabetics with preproliferative or proliferative retinopathy receiving laser photocoagulation within 1 year of measurement (3.02 ± 0.37; P = 0.0012). Diabetics with laser treatment more than 1 year prior to measurement (3.96 ± 0.51) did not differ significantly from nondiabetics. Invest Ophthalmol Vis Sci 25: [594][595][596][597][598] 198