Theodore W. Sery

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.

PHOTODYNAMIC SENSITIZERS FROM CHLOROPHYLL:PURPURIN–18 AND CHLORINp6

Abstract— Two easily prepared derivatives of chlorophyll,purpurin–18 and chlorin p6, are potent sensitizers of cell killing by low‐intensity red light. The internal anhydride group inpurpurin–18 provides the potential of covalently linking in one step the chlorin to cell targeting agents such as antibodies.

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.

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.

Activation of macrophages by ether analogues of lysophospholipids.

SummaryInflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L-α-lysophosphatidyl-DL-glycerol (lyso-Pg) resulted in an enhanced ingestion activity of peritoneal macrophages as did other lysophospholipids. However, lyso-Pg is rather toxic as indicated by a rapid decrease in macrophage activity 3 days after treatment while macrophage activity of lysophosphatidylcholine-treated mice continued to increase at least up to the 6th day after treatment. Alkyl-lysophospholipid derivatives, racemic 1-0-octadecyl-2-methylglycero-3-phosphocholine and -phosphoethanolamine stimulated mouse macrophages for Fc-mediated ingestion. Decomposed products of alkyl-lysophospholipids, alkylglycerols, were also found to be excellent activators of macrophages not only for ingestion of IgG-coated target cells but also antibody-mediated tumoricidal activity. Macrophages from mice treated with alkylglycerols developed superoxide generating capacity. Furthermore, alkylglycerols were found to be tumoricidal by direct contact with retinoblastoma cells. Therefore, the advantage of the potential application of alkylglycerols as chemotherapeutic agents is that they have dual beneficial effects: potentiation of macrophage activity and cytotoxicity to malignant cells.

Human esterase D gene: complete cDNA sequence, genomic structure, and application in the genetic diagnosis of human retinoblastoma.

SummaryThe gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilsons disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.

Photodynamic Therapy of Human Ocular Cancer

Photodynamic therapy (PDT), also known as photoradiation therapy, was employed in five patients with ocular tumors that had been photosensitized to hematoporphyrin derivative (HPD). In each case more conventional treatment had failed to control the tumor, or the patient was considered a poor candidate for surgical intervention because of advanced age or general health. Intravenous administration of 2.5-3.0 mg/kg HPD was followed by PDT 2-4 days later, using a dye laser tuned to a wavelength of 632 nm. Laser light was delivered either by a fiber optic probe maintained at a fixed distance, or via a slit lamp system, for intervals of up to 20 minutes. The levels of energy applied were mainly below 420 Joules/cm2, but for tumors less than 1 mm diameter energy levels were as high as 3000 J/cm2 with a conservative power value as low as 20 mW/cm2. Tumor response to PDT was disappointing. Although substantial superficial tumor necrosis occurred in several cases, it did not extend to the deeper levels of tumor tissue. In each instance surgical intervention became necessary. Deficiencies in the procedure are discussed.

EFFECTIVENESS OF PHOTOFRIN II IN ACIWATION OF MACROPHAGES AND in vitro KILLING OF RETINOBLASTOMA CELLS

Abstract 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.

Experimental disciform keratitis: I. Immune response of the cornea to herpes simplex virus☆

Abstract Large inocula of washed viable herpes simplex virus injected into the corneal stroma caused an acute inflammatory response in the cornea, conjunctiva, iris, and ciliary body. This reaction is due to an infection that reaches its peak in 7 days and subsides in 8 to 13 days. When virus was reinjected a month or more later, the majority of corneas did not suffer recurrence of inflammatory reactions. There to four corneal injections of virus given 2 to 5 weeks apart led to brief periods of corneal clouding which could also be clicited by noninfected chorioallantoic membrane (CAM) proteins. Only one cornea in 28 had a brief clouding response which could not be related to CAM protein. None of the corneal clouding reactions was permanent but cleared after 7 days and, apart from minor, trauma-induced scars, all corneas returned to full clarity. Corneas sensitized with heat-inactivated virus developed negative or very low titers of neutralizing antibody, but intracorneal or intra-aqueous injections of viable virus led to high titers of such antibody in corneal extracts as well as serum. A conclusion is drawn that corneal hypersensitivity to herpes simplex virus, for at lcast two widely divergent strains, could not be related to the clinical problem of disciform keratitis on an experimental basis.

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.