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1016 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1985 after age 30 and most pronounced after age 50), and into the layer of rods and cones (only after age 40, most common after age 50). Accompanying the loss of nuclei in the ONL, they noted shrunken and deformed rods and cones, and a diminution in the numbers of photoreceptors. They also stated, however, that nuclear displacement was rare in the center of the fovea. Thus, although receptor cells are increasingly lost with age, no conclusive evidence is available whether or not foveal cones are significantly affected before the age of 50. The disks in the outer segments of the photoreceptors which contain the photopigment are known to be subject to a continuous process of renewal.8 The efficacy of this mechanism might be the reason why we find that the cones remain in fine shape up to the age of about 50 yr. Key words: cones, pigment density, age effects, pigment regeneration From the Royal Netherlands Eye Hospital,* Dondersstraat 65, 3972 JE Utrecht, and the Institute for Perception TNO,t Kampweg 5, 3769 DE Soesterberg, The Netherlands. Submitted for publication: September 24, 1984. Reprint requests: Dirk van Norren, PhD, Vol. 26 Royal Netherlands Eye Hospital, Dondersstraat 65, 3972 JE Utrecht, The Netherlands. References 1. Kilbride DE, Hutman LP, Read JS, and Fishman M: The aging human eye and cone pigment density difference in the fovea. ARVO Abstracts. Invest Ophthalmol Vis Sci 25(Suppl): 198, 1984. 2. Van Norren D and van der Kraats J: A continuously recording retinal densitometer. Vision Res 21:897, 1981. 3. Smith VC, Pokorny J, and van Norren D: Densitometric measure of human cone photopigment kinetics. Vision Res 23:517, 1983. 4. Weale RA: Senile changes in visual acuity. Trans Ophthalmol Soc UK 95:36, 1975. 5. Kjlbride PE, Read JS, Fishman GA, and Fishman M: Determination of human cone pigment density difference spectra in spatially resolved regions of the fovea. Vision Res 23:1341, 1983. 6. Baker HD and Kuyk TK: In vivo densitometry of cone pigments after repeated complete bleaching. In The effects of constant light on visual processes, Williams TP and Baker BN, editors. New York, Plenum Press, 1980, pp. 347-353. 7. Gartner S and Henkind P: Aging and degeneration of the human macula. 1. Outer nuclear layer and photoreceptors. Br J Ophthalmol 65:23, 1981. 8. Young RW: Biogenesis and renewal of visual cell outer segment membranes. Exp Eye Res 18:215, 1974. Retinal 5-Antigen Epitopes in Vertebrate and Invertebrate Photoreceptors Mossoud Mirshohi, Cloude Doucheix, Germoine Collenor, Brigitte Thillaye, and Jean-Pierre Faure Monoclonal antibodies specific for the retinal S-antigen were obtained by hybridization of spleen cells from a BALB/c mouse immunized with bovine S-antigen and NS1 myeloma cells. Five selected antibodies specifically labeled the photoreceptor cells of the retina by immunofluorescence. Whereas antibody S9E2 only reacted with bovine S-antigen, the other antibodies showed interspecies cross-reactivity. They were used for the characterization of specific epitopes of S-antigen in photoreceptors from a wide range of species representative of various classes of vertebrates and invertebrates. The presence of S-antigen in distant species (vertebrates, Amphioxus, nemerteans, annelids, molluscs) indicates a high phylogenetic stability and suggests an important role for this protein in photoreceptor function. Invest Ophthalmol Vis Sci 26:1016-1021, 1985 The retinal "S-antigen" is a specific component of photoreceptor cells that has been isolated and purified from the retina of several mammals. 1 " 3 The immunologic properties of this protein include organ specificity, interspecies cross-reactivity and immunopathogenicity. Most work with S-antigen has dealt with its ability to induce experimental autoimmune uveoretinitis (EAU) in laboratory animals. This an- Downloaded From: http://iovs.arvojournals.org/ on 06/16/2017 tigen is also involved in ocular autoimmune disease in man. 45 EAU and circulating antibodies are produced after immunization with xenogenic or allogenic S-antigen and even with autologous retina.6 These antibodies have been used to localize S-antigen in the retinal photoreceptors of guinea pigs by immunofluorescence.1 Cross reactivity between S-antigen from various mammals has been demonstrated by immunodiffusion,2 enzyme immunoassay (ELISA),7 immunofluorescence1 and pathogenic activity.2 The use of monoclonal antibodies to bovine S-antigen in ELISA studies showed the presence of two types of epitopes in the protein. Some epitopes were specific to bovine S-antigen, while others were common to Santigen from various mammals. These nonspeciesspecific epitopes were recognized in the retinas of other classes of vertebrates by immunofluorescence.8 In this article, we present an immunofluorescent analysis of the distribution of S-antigen epitopes in the photoreceptors of selected species representative of various classes of vertebrates and invertebrates. Materials and Methods. Details of hybridization and of the specificities of the antibodies analyzed by No. 7 ELISA have been reported elsewhere.8 Briefly, monoclonal antibodies were obtained after fusion of NS-1 myeloma cells and spleen cells from a BALB/c mouse immunized with purified bovine S-antigen. Thirty anti-bovine S-antigen hybridomas were detected by ELISA. ELISA was performed by using microELISA plates coated with purified bovine (or other mammalian) S-antigen (1 fig/m\ in 0.05 M carbonate buffer, pH 9.6, at 4°C overnight).7 Hybridoma supernatants and peroxidase-labeled goat antimouse IgG (EY laboratories; San Mateo, CA) were diluted in phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 0.5% bovine serum albumin. Enzymatic activity was detected by orthophenylenediamine 0.04% in 0.1 M citrate buffer, pH 5, with 0.01% H 2 O 2 . Out of 12 cloned hybridomas, three were specific for the bovine S-antigen and the others cross-reacted with S-antigen from various mammals. Five antibodies were selected for this study (Table 1). They were strongly reactive with S-antigen and showed no reactivity with other proteins or tissues in several preliminary tests, including ELISA with various proteins and immunofluorescence on various tissues.8 For the immunohistologic study, all tissues were fixed in Bouin solution for 18 hr, followed by standard paraffin embedding. Five-/um sections were immediately prepared and kept at —20°C. Dissection of the tissues varied from one species to another depending on the size of the animal. Sections included either a part of the retina and neighboring tissues (bovine), or the whole eye (other vertebrates); and eventually, a large part or the totality of the animal (Amphioxus, invertebrates). The test was performed after elimination of paraffin by a 15-min incubation in toluene and 5 min in ethanol followed by two washings in PBS. The slides were covered with monoclonal antibodies as undiluted culture supernatants, then with FITC-labeled goat antimouse IgG antibody (Nordic; Tilburg, Netherlands), diluted 1/40. All washings were made in PBS. A rat antiserum was used as positive control for the presence of S-antigen. This was a pool of 10 sera obtained after four injections of 50 /ng of bovine S-antigen in complete Freund's adjuvant. This serum only revealed the S-antigen precipitation line in immunodiffusion against crude retinal extracts. Unrelated monoclonal antibodies, antifibrinogen9 and antidifferentiation10 antigens prepared in the laboratory of one of us (CB) were used as negative controls. Results. Testing of a wide range of tissues demonstrated that the five antibodies were specific for the photoreceptor cells. In vertebrate retinas, antibodies S8D8, S7D6, S2D2, S6H8 labeled different parts of the visual cells: the reaction was strongly positive in the outer segment, but even more in the inner Downloaded From: http://iovs.arvojournals.org/ on 06/16/2017 1017 Reporrs Table 1. Properties of 5 monoclonal antibodies to retinal S antigen Reactivity in ELISA with purified S-antigen Antibody S9E2 S8D8 S7D6 S2D2 S6H8 Isotype IgG IgG IgG IgG IgG Cattle Other mammals 2a 2a 1 2b 2a * Man, swine, guinea pig, mouse. segment; there was always a weaker labeling of the cell body without staining of the nucleus; the outer plexiform layer, containing the axonal and synaptic parts of the cell, was also strongly reactive (Fig. 1). The pattern of labeling was different with S9E2, which is specific for bovine S-antigen and stained predominantly the perinuclear area and less intensively the inner and outer segments. In all mammals of this study, both cones and rods bound monoclonal or polyclonal antibodies. In the chicken, not all photoreceptor cells were labeled, the highest percentage of positive cells was in the visual axis, whereas the number of positive cells decreased in the periphery of the retina. In reptiles, all photoreceptor cells were positive (viper, lizard), or, in the turtle retina, all photoreceptors located in the visual axis were positive, whereas progressive decrease of intensity was observed towards the periphery of the retina. Urodeles and frog showed a different pattern of labeling with monoclonal antibodies between cones and rods in that cones were positive and rods were negative, whereas in Xenopus all photoreceptors were labeled. However, in every amphibian, the polyclonal serum against S-antigen stained both rods and cones. In fishes, all photoreceptor cells were positively stained. The distribution of S-antigen epitopes in the various species is shown in Table 2. The epitope S9E2 was the most restricted since it was present only in bovine photoreceptors. The epitopes recognized by antibodies S8D8, S7D6, S2D2, and S6H8 were widely expressed and were found as a group in all vertebrate retinas, with the exception of the frog, where only S7D6 gave a positive staining. The four epitopes were also present in the protochordate Amphioxus, in the nemertean Lineus and the annelid Nereis (Fig. 1). Three epitopes were found in the mollusc Pecten and two in the mollusc Helix. None of the antibodies stained the photoreceptors in the compound eyes of three arthropods, or in the simple eyes of a planarian and a starfish. In protochordates, molluscs, and annelids we found species which were positive with several antibodies as well as species that were negative. The polyclonal serum yielded similar results as the mono- 1018 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1985 Vol. 26 'If 1 Mi Downloaded From: http://iovs.arvojournals.org/ on 06/16/2017 Fig. 1. Immunofluorescent staining of photoreceptor cells by monoclonal antibody S6H8. In vertebrates, man (A) or turtle Pseudemys (B), the labeling is limited to the outer and inner segment, the perinuclear and synaptic parts of visual cells. In Amphioxus (C), the antibody strongly reacted with the Hesse cells, photoreceptors scattered along the neural tube and capped by crescent-shaped pigment cells. In the annelid Nereis (D), the brightly labeled photoreceptor structures form a continuous layer located in the inner part of the ocellus wall. The label can also be observed on the other side of the pigmented layer. 1019 Reports No. 7 Table 2. Immunofluorescent reactivity of 5 monoclonal antibodies and a rat polyclonal antiserum (AS) to S-antigen on photoreceptors from various species S9E2 VERTEBRATES Mammals Man Cat Ox Rat, mouse, guinea pig, rabbit Birds Gallus Reptiles Lizard Snake (Vipera) Chelonia (Pseudemys) Amphibians Anurans (Xenopus) (Rand) Urodeles (Pleurodeles, Ambystoma) Teleostei (Salmo, Carassius) Selachians (Scylliorhinus, Torpedo) PROTOCHORDATES Amphioxus Ascidia (Ciona) INVERTEBRATES Echinodermata Asterias (Marthasterias) Nemertea (Lineus) Platyhelminthes Planaria (Dugesia) Annelida Nereis Hinido Mollusca Lamellibranchia {Pecten) Gastropodia (Helix) Cephalopodia (Loligo) Arthropoda Insects (Sarcophaga, Aeschna) Arachnida (Scodra) S8D8 S7D6 S2D2 S6H8 AS NT NT NT: not tested. clonal antibodies; in all species in which at least one monoclonal antibody was positive, there was staining with the polyclonal rat serum; the other species were negative. However, the staining of photoreceptors was less intense and the background staining was heavier with the polyclonal serum. The unrelated monoclonal antibodies used as controls did not give any staining of photoreceptor cells. Discussion. Our investigation shows that in addition to the strong organ specificity and autoantigenicity characteristic of the retinal S-antigen, there exists a large interspecies cross-reactivity. Such properties are shared by only a limited number of proteins in the body and allow an immunologic approach to the taxonomic distribution of the epitopes of homologous proteins using monoclonal antibodies. Compared to the study of protein homology by amino acid sequence Downloaded From: http://iovs.arvojournals.org/ on 06/16/2017 analysis, the use of monoclonal antibodies presents a major difference: they may detect conformational epitopes; ie, molecular arrangements not necessarily linked to an amino acid sequence but consisting of the juxtaposition of amino acids, carbohydrates, or both, which are brought together by the folding of the protein, but which are not due to their proximity in the sequence. Such conformational epitopes may be closely related to the function of the molecule. Their identification by monoclonal antibodies could be the basis for a simple method suitable for analyzing phylogenetic relationships between species. The five monoclonal antibodies used in this study can be considered highly specific for S-antigen. They do not react with other proteins in extracts of the retina or other tissues in ELISA, and they only stain photoreceptor cells in sections of vertebrate eyes in 1020 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1985 immunofluorescence.8 The rat hyperimmune serum, though directed at purified S-antigen,3 is probably less specific and gave a higher background on sections. The interspecies distribution of the S-antigen epitopes recognized by the five monoclonal antibodies indicates that these epitopes are different, since they are not found together in all positive species. Only S6H8 and S2D2 were always found together in this study. However, in a recent study performed on human retinoblastoma, we have observed different patterns for these two antibodies (data not shown), suggesting that they also recognize different epitopes. With the immune serum and four monoclonal antibodies, we found in all vertebrates the same localization of labeling, previously described by Wacker et al1 in guinea pig retina using guinea pig serum against bovine S-antigen. The pattern outlined the entire photoreceptor cell except the nucleus. The reason for the difference of labeling between S9E2, which stained predominantly the perinuclear area, and the other antibodies is not known. It is hypothesized that the epitope S9E2 is present on the native molecule but disappears or is masked during further processing of the molecule towards its site of action. S-antigen epitopes were found in all vertebrates and in some species of protochordates, nemerteans, annelids, and molluscs. They were not detected in other groups of invertebrates, such as arthropods. Since only a few species were investigated in the various invertebrate phyla, this study does not allow a definitive conclusion on the absence of the Santigen in those groups. Therefore, we can only state that S-antigen is present in species belonging to both protostomia (that include annelids and molluscs) and deuterostomia (echinoderms and all chordates). Considering the theoretical age of divergence between protostomia and deuterostomia, the four epitopes S8D8, S7D6, S2D2, and S6H8 should have appeared at the early Cambrian era or even earlier, as invertebrate phyla evolved during the preCambrian era and were well differentiated by the earliest paleozoic period (ie, Cambrian). This indicates a high phylogenetic stability suggesting that the structure of S-antigen has been well suited to its unknown function, very early in evolution. It may be of interest to consider the possibility of a relationship between the distribution of S-antigen and the ciliary or rhabdomeric type of photoreceptors.'' Indeed, among the most perfect eyes, the photoreceptors of vertebrates are ciliary and positive for Santigen; whereas those of arthropods and cephalopods are rhabdomeric and negative for S-antigen. However, in protochordates and most invertebrates, the presence of S-antigen epitopes are not clearly related to the existence of ciliary structures in photoreceptors. It Downloaded From: http://iovs.arvojournals.org/ on 06/16/2017 Vol. 26 should be pointed out that the dichotomy between the two types of photoreceptors is not strict and the presence of S-antigen epitopes could be linked to some functional difference in photoreceptors rather than anatomic differences. Despite major variations in the architecture of photoreceptor organs and the structure of photoreceptor cells between vertebrate and invertebrate phyla, they are directed and adapted for a common function: light captation, and visual function for the most elaborated photoreceptor organs. It is, therefore, not surprising that some common molecular mechanisms are shared by these morphologically different structures as it is the case for the visual pigments. It would be likely that other proteins involved in photoreceptor function, eg, in the amplification of the light stimulus, are common to different photoreceptor cells. The localization and wide interspecies distribution of Santigen suggest that it could belong to such molecules. Until now only S-antigen extracted from mammals (man, cattle, swine, guinea pig) has been shown to induce EAU in laboratory animals. It will be of interest to examine the ability of the S-antigen from more distant species to induce disease in rat or guinea pig models to determine if the immunopathogenic property is linked to transevolutionary antigenic determinants. Key words: retina, retinal S-antigen, monoclonal antibodies, interspecies cross-reactivity, evolution, immunofluorescence Acknowledgments. The purification of S-antigen was carried out by Mrs. C. Dorey. The authors acknowledge A. Bernadou, Y. de Kozak, J. Y. Perrot, J. Sebag, P. Krief, and J. Soria for their helpful comments and J. Bierne, M. L. Celerier, J. Dorchen, J. C. Lacroix, J. Reperant, J. Taxi, G. Vernet, the Station Biologique de Roscoff, the laboratoire Maritime de Concarneau, and the Laboratoire Maritime de Banyuls for providing animal specimens. From the Laboratoire d'Immunopathologie de 1'Oeil, CNRS ER 227, INSERM U 86, Universite de Paris VI, Hotel-Dieu, Paris, the Unite INSERM U 253, Villejuif, the Equipe de Recherche Biologie du Developpement, Universite de Paris VII, Paris, France. Presented at the ARVO meeting, Sarasota, Florida, May 1984. Submitted for publication: July 2, 1984. Reprint requests: Jean-Pierre Faure, MD, PhD, Unite de Recherche d'Ophtalmologie, Hotel-Dieu, 1 Parvis Notre-Dame, 75181 Paris 04, France. References 1. Wacker WB, Donoso LA, Kalsow CM, Yankeelov JA, and Organisciak DT: Experimental allergic uveitis: isolation, characterization and localization of a soluble uveitopathogenic antigen from bovine retina. J Immunol 14:1949, 1977. 2. Dorey C and Faure JP: Isolement et caracterisation partielle d'un antigene retinien responsable de ruveo-retinite autoimmune experimentale. Ann Immunol (Inst Pasteur) 128c:229, 1977. 1021 Reports No. 7 3. Dorey C, Cozette J, and Faure JP: A simple and rapid method for isolation of retinal S antigen. Ophthalmic Res 14:249, 1982. 4. Nussenblatt RB, Gery I, Kuwabara T, de Monasterio FM, and Wacker WB: The role of the retinal S-antigen in primate uveitis. In Immunology of the Eye, Workshop II, Helmsen RJ, Suran A, Gery I, Nussenblatt RB, editors. Washington, DC, Information Retrieval, 1981, pp. 49-65. 5. Faure JP and de Kozak Y: Cellular and humoral reaction to retinal antigen; specific suppression of experimental uveoretinitis. In Immunology of the Eye, Workshop II, Helmsen RJ, Suran A, Gery I, and Nussenblatt RB, editors. Washington, DC, Information Retrieval, 1981, pp. 33-48. 6. Faure JP: Autoimmunity and the retina. Curr Topics Eye Res 2:215, 1980. 7. Tuyen VV, Faure JP, Thillaye B, de Kozak Y, and Fortier B: Antibody determination by ELISA in rats with retinal S antigen-induced uvoretinitis. Curr Eye Res 2:7, 1982. 8. Faure JP, Mirshahi M, Dorey C, Thillaye B, de Kozak Y, and Boucheix C: Production and specificity of monoclonal antibodies to retinal S antigen. Curr Eye Res 3:867, 1984. 9. Boucheix C, Perrot JY, Mirshahi M, Giannoni F, Billard M, Bernadou A, and Rosenfeld C: A new set of monoclonal antibodies against acute lymphoblastic leukemia. Leukemia Res, in press. 10. Soria J, Soria C, Boucheix C, Mirshahi M, Perrot JY, Bernadou A, Samama M, and Rosenfeld C: Immunochemical differentiation of fibrinogen, fragment D or E and crosslinked fibrin degradation products using monoclonal antibodies. In Fibrinogen: Structuren, Functional Aspects, Metabolism, Haverkate E, Henschen A, Nieuwenhuizen W, and Straub, editors. Walter de Gruyter and Co, 1983, pp. 227-233. 11. Eakin RM: Structure of invertebrate photoreceptors. In Handbook of Sensory Physiology, VII/1, Photochemistry of Vision, Dartnall HJA, editor. Berlin, New York, Springer-Verlag, 1972, pp. 625-684. Reduction of Body Swoy by Stimuli Imoged within o Corticol Scotomo: A Cose Study Jane E. Raymond* and Herschel W. Leibowirz The reduction of body sway by visual stimulation was equally effective for stimuli imaged within a cortical scotoma or in the mirror image position in the normal visual field. The results are consistent with the concept of distinct visual orientation and discrimination modes of processing visual information, which suggests that spatial orientation functions do not necessarily involve awareness. Invest Ophthalmol Vis Sci 26:1021-1024, 1985 Using ablation techniques, Schneider1 demonstrated that visual discrimination and visually-guided spatial orientation can be selectively dissociated in the hamster. His finding that removal of either the visual cortex or the superior colliculus disrupted discrimination or orientation functions, respectively, suggested the existence of two reasonably separate and parallel visual systems. For humans, Held has proposed the term "two modes of processing" to differentiate object recognition, ie, "focal" vision, from spatial orientation, ie, "ambient" vision.2 Subsequent research has suggested that the focal mode, which addresses the question of "what," is cortically based and well-represented in consciousness. Alternately, the ambient mode, which concerns the question of "where," is thought to be mediated reflexively with little or no conscious concomitant. 3 Evidence for the dissociation of discrimination and orientation functions in humans has been found in the study of cortically blind individuals. Observations of visual functions in patients with unilateral cortical Downloaded From: http://iovs.arvojournals.org/ on 06/16/2017 lesions has shown that while pattern vision is probably absent in the scotomatous field,4 some residual visual functions (eg, flicker perception, motion perception, or the appearance of eye movements toward unseen objects in the blind hemifield) may remain. Although earlier views of cortically blind patients conservatively held that any reported residual vision resulted from surviving cortical tissue,5 more recent evidence suggests that some visual functions persist and may be mediated by subcortical pathways. Perenin and Jeannerod4 examined the capacity of hemianopic patients to point to a briefly flashed light presented in their blind field. By comparing patients with pre- and postgeniculate lesions, they were able to demonstrate that elimination of cortical inputs only (ie, postgeniculate lesions) did not impair pointing, whereas removal of both cortical and subcortical pathways did. Similarly, accurate pointing to unseen objects by cortically blind patients was also reported by Bridgeman and Staggs.6 In a study involving patients with unilateral surgical removal of one hemisphere, it was reported that patients were able to make saccades towards objects placed in their hemianopic field,7 although the accuracy of such eye movements is quite poor.8 In the studies described above, the cortically blind observer is asked to make a conscious judgement regarding the position of unseen objects in space. Since residual visual functions in cortically blind individuals are most likely to be ambient and, therefore, primarily reflexive, their existence may be more