Photodynamic killing of retinoblastoma cells with hematoporphyrin and light.

Hematoporphyrin and white light exerted a lethal effect on two cell lines of retinoblastoma cells. The lowest values of dye concentration and light exposure capable of killing an entire cell population were, respectively, 2 X 10(-5) M and 6 min exposure to an irradiance of 6.0 microwatts/sq mm. Cells exposed to light in the presence of the dye did not require lengthy incubation periods but were rapidly killed with increasing periods of light exposure. Cells washed free of the dye, however, required a minimum sensitizing period of 3.5 hr to achieve a value close to 100% cell death. An inhibitor of this photodynamic process was demonstrated in normal serum. When the concentration of either fetal calf serum, rabbit serum, or rabbit plasma was increased to 25% from a standard 10%, there was as much as a 100-fold greater requirement of hematoporphyrin concentration to produce the same lethal response. The suggested explanation for this phenomenon is a porphyrin-binding plasma factor, hemopexin, which is a natural beta-glycoprotein believed to be responsible for the transport of circulating porphyrins to the liver for their elimination. Retinoblastoma cells grow in suspension and thereby provide an excellent tool for study of photosensitive dyes, especially in the case of the rapidly growing Y79 cell line.

Characterization of a new continuous cell line derived from a human retinoblastoma.

A new continuous cell line derived from a human retinoblastoma has been established. This cell line, WERI-RB1, has been maintained in vitro since December 1974. The purpose of this investigation was to characterize WERI-Rb1 on the basis of morphology, growth, tumorigenicity, cytogenetics, and to compare this cell line with Y79, a human retinoblastoma cell line established at another institution. Morphologically, both cell lines were similar; each spontaneously grew as a suspension of small round cells in grapelike clusters. Each exhibited growth of cells in rosettes, as well as unusual chain formations. Growth rates differed: the population-doubling times for WERI-Rb1 and Y79 were 96 and 33 hr, respectively. When the negative surface charge on a plastic tissue culture flask was changed, each cell line grew as a monolayer. Y79 could be cloned in soft agar; WERI-Rb1 could not. An inoculm of 10(7) WERI-Rb1 or Y79 cells produced a retinoblastoma in test rabbits. Karyological examination showed each cell line to have a stable, near diploid chromosome number. Although large markers were observed in each line, they shared no common marker.

Photodynamic immunopotentiation: in vitro activation of macrophages by treatment of mouse peritoneal cells with haematoporphyrin derivative and light.

Peritoneal macrophages treated in vivo with haematoporphyrin derivative (HPD) exhibited significant enhancement of Fc receptor mediated ingestion activity. To examine this process more rigorously, we studied photodynamic activation of macrophages by exposure in vitro of mouse peritoneal cell cultures (containing macrophages and B and T-lymphocytes) to HPD and red fluorescent light. A short (10 s) exposure of peritoneal cells in medium containing 0.03 ng HPD/ml produced the maximal level of ingestion activity of macrophages. A singlet oxygen quencher, DABCO, inhibited the effect of HPD. Photodynamic treatment of macrophages alone did not activate the cells and activation was only observed when macrophages were mixed with photodynamically treated non-adherent cells (B and T-lymphocytes). These results imply that activation of macrophage is a consequence of peroxidation of lymphocyte membrane lipids by photodynamically generated singlet oxygen.

Cell-mediated immunity in herpes corneal stromal disease.

Regional draining lymph nodes (RDLN) from rabbits with herpes virus disciform keratitis (defined as corneal edema or corneal edema with concomitant epithelial lesions) were tested for general as well as specific immune cap-city. Although general RDLN immune capacity did not seem to be impaired, specific reactivity to herpes antigens was found only in those rabbits which presented with disciform keratitis and concomitant epithelial lesions; specific reactivity to herpes antigens was not found in those animals which presented with disciform edema alone. Additionally, the unilateral nature of the RDLN immune response during ocular infections of unequal severity was demonstrated.

S-antigen. Experimental autoimmune uveitis following immunization with a small synthetic peptide.

Experimental autoimmune uveitis was observed following the immunization of Lewis rats with a small synthetic peptide, peptide M (18 amino acids in length), which corresponds to the amino acid sequence of a well-characterized region of S-antigen (404 amino acids in length). Rats were immunized with varying doses of peptide M ranging from 1 to 100 micrograms in complete Freund's adjuvant. As little as 5 micrograms of the synthetic peptide was sufficient for the induction of disease. Clinically, the disease that developed was characterized by iris and pericorneal hyperemia followed by exudates in the anterior chamber. Histopathologically, a severe inflammatory response was observed in animals immunized with high doses of the peptide (greater than 50 micrograms). In these eyes the photoreceptor cell layer of the retina was completely destroyed. A massive subretinal exudate containing mononuclear cells and polymorphonuclear leukocytes was also present. The inflammatory changes were generally less severe following immunization with low doses of the peptide (less than 50 micrograms), and in some eyes only occasional focal lesions were observed. In addition, animals with ocular inflammatory disease had associated pinealitis characterized by a lymphocytic infiltration of the subcapsular and central area of the pineal gland. Both clinically and histopathologically, the experimental autoimmune uveitis produced by the synthetic peptide was indistinguishable from the disease caused by native S-antigen. We comment on the significance of these findings and the relationship of S-antigen in the pathogenesis of experimental autoimmune uveitis.

S-antigen. Adoptive transfer of experimental autoimmune uveitis following immunization with a small synthetic peptide.

Experimental autoimmune uveitis was observed following the adoptive transfer of T cell lymphocytes (T cells) from Lewis rats previously immunized with a small synthetic peptide, peptide M, which corresponds to the amino acid sequence of a well-defined region of S-antigen. Prior to adoptive transfer, the T cells were restimulated in tissue culture with peptide M. Approximately five days following the intravenous administration of restimulated T cells, a severe uveitis was documented both clinically and histopathologically. Clinically, the disease was characterized by iris hyperemia followed by anterior chamber exudates and posterior iris synechiae. Histopathologically, the photoreceptor cell layer of the retina was completely destroyed. A subretinal exudate containing mononuclear cells and polymorphonuclear leukocytes was also present. In addition, the pineal glands of animals with experimental autoimmune uveitis showed inflammatory changes characterized by a lymphocytic infiltration of the subcapsular and central region of the gland. The clinical and histopathologic features of the experimental autoimmune uveitis were similar to those that develop following the adoptive transfer of T cells from Lewis rats previously immunized with S-antigen. Our results indicate that the amino acid sequence of the region of S-antigen corresponding to peptide M represents a distinct pathogenic site with the ability to adoptively transfer disease. We comment on the significance of this finding with regard to T cell-mediated immune mechanisms in certain forms of human uveitis.

Effectiveness of photofrin II in activation of macrophages and in vitro killing of retinoblastoma cells.

Administration of a small dose (300 ng/mouse) of photofrin II (PII) to mice, followed by 4 days of exposure to only ambient fluorescent light in animal quarters, induced Fc-receptor-mediated phagocytic and superoxide-generating capacities of peritoneal macrophages by five- and seven-fold, respectively. When these mice were kept in the dark for 4 days, no activation of macrophages was observed. These results suggest that macrophage activation is a consequence of photodynamic activation. Much higher doses (> 3000 ng/mouse) suppressed macrophage activity. However, 2 months after administration of 3000 ng PII/mouse, greatly enhanced phagocytic and superoxide-generating capacities of peritoneal macrophages were observed. In vitro photodynamic activation of macrophages was analyzed after white or red fluorescent light exposure of mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) in media containing PII. A short (10 s) white fluorescent light treatment of peritoneal cells in a medium containing 0.03 ng PII/mL produced the maximal level of phagocytic activity of macrophages. Illumination with the same total fluence of red fluorescent light requires a three-fold higher concentration of PII to achieve the same extent of enhanced phagocytic activity of macrophages. Thus, photodynamic activation of macrophages with PII by white fluorescent light was more efficient than by red fluorescent light. Similarly, photodynamic killing of retinoblastoma cells was more efficient with white than red fluorescent light. The concentration of hematoporphyrin (HP) or PII required for direct photodynamic killing of retinoblastoma cells was roughly four orders of magnitude greater than that required for activation of macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

The reverse Wessely phenomenon: immune corneal rings following systemic immunisation.

Rabbits were hyperimmunised systemically over a 4-week period with bovine serum albumin (BSA) in complete Freund's adjuvants and were subsequently challenged systemically with a massive dose of BSA designed to produce serum sickness. Without any ocular manipulation a spontaneous corneal immune reaction was observed which we termed a 'reverse Wessely phenomenon'. It was due to a reversed sequence of antibody becoming sequestered in the cornea and later responding to antigen entering via the limbal circulation. In the stroma, lymphocytes were prominent and only few polymorphonuclear cells were found, but this may have been due to late sectioning of the corneas tested. The corneal ring formed at the limbus and migrated centripetally ahead of peripheral clouding, which is typical of the Wessely phenomenon. Possible relationships of this reaction with some autoimmune disorders are discussed.

Scanning electron microscope study of herpes simplex virus experimental disciform keratitis.

Scanning electron microscopy of the endothelium of experimental disciform keratitis revealed corneal endothelial changes which distinguished disciform oedema from the more progressive stages of disciform keratitis. The endothelium of corneas with disciform oedema were wholly intact and characterised by subtle morphological alterations. In contrast, the more progressive stages of disciform keratitis were characterised by massive destruction and denudation of the endothelium. The significance of these observations is discussed.

Photoradiation of rabbit ocular malignant melanoma sensitized with hematoporphyrin derivative.

Photoradiation therapy (PRT) against the Greene-Harvey amelanotic malignant melanoma on the rabbit iris was effectively used to cause tumor regression. A dose of 2.5 mg/kg of hematoporphyrin derivative (HPD) given intravenously, followed by photoradiation at a wavelength of 632 nm and a power density as low as 71 mW/cm2 for 24 minutes (102 J/cm2) was found to be lethal for tumors 4 mm in diameter with an acceptable level of reversible toxicity to the surrounding tissues. This was best accomplished with a dye laser as the source of light because of its very narrow expanding cone of light, as emitted from a fiber optic. A 1000 Watt xenon arc lamp was also effective but not as efficient. Because of this tumor's exceptionally rapid growth rate, it was necessary to compromise one important variable - the 3 to 4 day period between injection of HPD and photoradiation to allow for HPD depletion from normal tissues. Thus, the best tumor death responses were achieved when the light was given 1 to 16 hours after administering HPD. It is surmised that with this rapidly growing tumor, new cell progeny possess insufficient concentrations of HPD to be killed by the radiant energy. At such a short delay period, toxicity to normal tissues was observed mainly as conjunctivitis and conjunctival chemosis. A dose level of HPD at 5 mg/kg was very close to the threshold where minor increases in light intensity would cause strong inflammatory reactions. Higher doses, at 7.5 and 10 mg/kg were excessive. A dose of 23 mg/kg accompanied by mild light energy exposures, even after 30 days, caused massive damage to normal tissues.

Superoxide anion radical as an indirect mediator in ocular inflammatory disease.

Intravitreal injection of a superoxide-generating reaction mixture of xanthine oxidase and xanthine, either with or without rabbit plasma, was shown to be a mediator of an intense uveal and retinal inflammation in pigmented and albino rabbits. Controls of heat-inactivated xanthine oxidase with or without rabbit plasma, or plasma by itself, was without effect on ocular tissues. Xanthine alone as a control exhibited little or no inflammatory response. Controls of active xanthine oxidase by itself, or with rabbit plasma, produced a very strong inflammatory response that may represent enzymic reaction with endogenous xanthine. When the superoxide generating reaction mixture was given intravitreally the reaction began in the anterior segment within 16 hours and reached its peak after 2 days. The response in the posterior segment was delayed and did not become evident until after at least 24 hours, and may be due to the close proximity of the anterior chamber to the ciliary processes where cellular exudates first appear. Anterior segment uveitis began to recede after 4 days but posterior segment inflammation persisted beyond 6 days, and in many instances, led to retinitis, and retinal detachment. Superoxide dismutase was effectively used in vitro to quench superoxide in the reaction mixture but it did not prevent inflammatory reactions in vivo because it was found to possess strong toxic qualities of its own in ocular tissues. Other free radicals of oxygen, as well as hydrogen peroxide, can develop with the breakdown of superoxide, and cause tissue damage. A known ability of superoxide to convert a plasma precursor into a factor chemotactic for neutrophils may also cause superoxide production in situ by accumulating neutrophils. Because phagocytes are potential sources of superoxide, this study provides a good experimental model for studying the influence of oxygen free radicals in ocular inflammatory disease.

Nonspecific local immune inhibition of herpes simplex virus keratitis.

Rabbit cornea, which had recovered from initial sensitization to bovine serum albumin were found to possess immune memory capable of responding to a totally different antigenic stimulus, i.e., viable herpes simplex virus. Two months after BSA sensitization these corneas were found to be extremely resistant to primary infection by the virus. The long term residence of mononuclear leukocytes in the cornea, following BSA sensitization, is apparently responsible for this non-specific immune phenomenon.

Human IRBP: characterization of uveitopathogenic sites.

Human interstitial or interphotoreceptor retinoid binding protein (IRBP) is a 136,000 molecular weight photoreceptor cell protein capable of inducing an experimental autoimmune uveitis (EAU) in susceptible animal strains. In order to determine specific sites in human IRBP responsible for its uveitopathogenicity, we synthesized 60 peptides, corresponding to its entire 1262 amino acid sequence, and tested each peptide for its ability to induce an EAU in Lewis rats. Three peptides with extensive amino acid sequence homology, designated HIRBP 715, HIRBP 730, and HIRBP 745, were uveitopathogenic when used at a 50 micrograms immunizing dose. The most potent peptide for the induction of EAU was HIRBP 715. Histopathologically a severe inflammatory response was present in the anterior and posterior segments of the eye. In these eyes the retina was infiltrated extensively with inflammatory cells. Focally the photoreceptor cell layer of the retina was destroyed. There was an associated subretinal exudate as well as an occasional subretinal granuloma. The clinical and histopathological changes in the eyes of rats immunized with peptides HIRBP 730 and HIRBP 745 were less severe as compared to HIRBP 715. One additional peptide, HIRBP 720, without extensive amino acid sequence homology to other regions of human IRBP, was also uveitopathogenic under our experimental conditions. Our study identifies multiple uveitopathogenic sites in the human IRBP molecule and, based on the primary amino acid sequence three of these sites are interrelated by several gene duplications which occurred some 600-800 million years ago in the native IRBP molecule.

Human S-antigen: characterization of uveitopathogenic sites.

Human S-antigen (HSA) is a 50,000 molecular weight photoreceptor cell protein capable of inducing an experimental autoimmune uveitis (EAU) in susceptible animal strains. In order to determine specific sites responsible for its uveitopathogenicity, we synthesized 39 overlapping peptides corresponding to its entire 404 amino acid sequence and tested each peptide for its ability to induce an EAU in Lewis rats. Two synthetic peptides designated peptide HSA 319 (amino acid positions 286 to 305) and peptide HSA 320 (amino acid positions 306 to 325) were uveitopathogenic when used at 50 and 100 micrograms immunizing doses. Smaller peptides corresponding to the amino, mid, and carboxy terminal portions of each peptide further refined each uveitopathogenic site to 12 amino acids. A computerized analysis of the amino acid sequence of S-antigen indicates that these uveitopathogenic sites are complex and may be related to a two-fold symmetry in the molecule. Our present and previous studies provide a basis for the uveitopathogenicity of human and bovine S-antigen in the pathogenesis of EAU as well as the pathogenesis of certain forms of uveitis in humans.

The use of synthetic peptides in the study of experimental autoimmune uveitis.

S-antigen is a highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis (EAU). EAU is predominantly a T cell mediated autoimmune disease of the uveal tract and retina of the eye and the pinealocytes of the pineal gland. Using synthetic peptides it has been possible to identify several B cell and T cell epitopes in the molecule. In addition, synthetic peptides derived from proteins of diverse origin with amino acid sequence homology to pathogenic regions of S-antigen induce an EAU which is indistinguishable from the disease induced by native S-antigen. These studies aid in the understanding of immune mechanisms in EAU and provide a basis for the pathogenesis of uveitis in humans.

IRBP: preparation and characterization of site-specific monoclonal antibodies.

Interstitial retinoid binding protein (IRBP) is a 136,000 molecular weight photoreceptor cell protein which is a highly pathogenic autoantigen for the induction of experimental autoimmune uveitis (EAU). In this study we produced a series of monoclonal antibodies (MAbs) which define different epitopes in the native molecule. These MAbs were further subdivided into three distinct groups based on a radioimmunoassay, and by ELISA assay using native IRBP and synthetic peptides corresponding to its entire amino acid sequence. Group I MAbs (MAbD7-B1 and MAbC6-B4) bound to native IRBP but not to any synthetic peptides, suggesting that their antigenic epitopes are strictly conformation dependent. Group II MAbs (MAbC7-D3 and MAbG8-H4) bound weakly to multiple peptides which shared amino acid sequence similarity located within each of four homology domains indicating that these epitopes are also conformation dependent. In group III (MAbH3-B5, MAbH7-A5, and MAbB6-D12) MAb binding was localized to a specific peptide. The MAbH3-B5 binding site was further refined to amino acid positions 361 to 367 in the native molecule. MAbH3-B5 was also useful in localizing IRBP in the mouse retina by immunohistochemical techniques. The application of these MAbs in the study of EAU and interphotoreceptor transport mechanisms is discussed.

S-antigen: characterization of a pathogenic epitope which mediates experimental autoimmune uveitis and pinealitis in Lewis rats.

S-antigen (48K protein) is a photoreceptor cell protein highly pathogenic for the induction of experimental autoimmune uveitis (EAU) and intimately involved in the visual process. EAU is characterized, in part, as a T-cell mediated autoimmune disease which results in a severe inflammation of the uveal tract, and pineal gland. In order to determine specific sites in S-antigen responsible for its pathogenicity we synthesized twenty-three peptides, corresponding to the entire 404 amino acid sequence, and tested each peptide for its ability to induce EAU in Lewis rats. One peptide, peptide M (18 amino acids in length), was found to be highly pathogenic and consistently induced an EAU that was identical to the disease caused by native S-antigen. Clinically, the disease that developed in the eye was characterized by iris and pericorneal hyperemia, followed by inflammatory exudates in the anterior and vitreous chambers. Histopathologically a severe inflammatory response was observed which resulted in the complete destruction of the photoreceptor cell layer of the retina. In order to more fully characterize this pathogenic site, 14 additional smaller peptides (eight to eighteen amino acids in length) corresponding to the left and right portions of peptide M were synthesized. Of these peptides, peptide M16L, M15L, and M12L induced EAU, further localizing this pathogenic site to a small well-characterized region of S-antigen consisting of twelve amino acids. In addition, animals with ocular inflammatory disease had an associated pinealitis characterized by a lymphocytic infiltration of the subcapsular and central area of the pineal gland. The significance of these findings and the relationship of S-antigen in the pathogenesis of EAU and other autoimmune diseases is discussed.

Characteristics of two new retinoblastoma cell lines: WERI-Rb24 and WERI-Rb27.

From 49 eyes enucleated for retinoblastoma, two new cell lines, WERI-Rb24 (W-24) and WERI-Rb27 (W-27), were established in long-term culture (greater than 5 years). The W-24 cell line was derived from a 22-month-old boy with sporadic retinoblastoma; the W-27 cell line was derived from a 24-month-old boy with bilateral retinoblastoma. Both cell lines show abnormal mRNA transcripts corresponding to the retinoblastoma gene. At the DNA level, one retinoblastoma allele was not present in the W-24 cell line. In the W-27 cell line a mutation was identified in one allele, while the other appears grossly normal. Since no normal retinoblastoma mRNA can be detected in either cell line, a subtle mutation must occur on the grossly normal allele. Comparison of DNA in W-27 lymphocytes and tumor cells with DNA probe p6NR-0.5 indicates that the observed mutation occurred somatically. The application of these two new retinoblastoma cell lines to the characterization of defects in the retinoblastoma gene and to gene replacement therapy in retinoblastoma is discussed.