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D S Gregerson

Publications and source records attributed to D S Gregerson.

At least 55 records · Page 3Linked to original sources
PubMed1989

Identification of T cell recognition sites in S-antigen: dissociation of proliferative and pathogenic sites.

Experimental autoimmune uveoretinitis (EAU) is a predominantly CD4+ T cell-mediated autoimmune inflammatory disease of the retina and uveal tract of the eye and the pineal gland. S-antigen, a protein found in retinal photoreceptor cells and pinealocytes, is a potent agent for the induction of EAU in susceptible species and strains. In order to identify the T cell recognition sites of S-antigen responsible for its uveitogenicity and proliferative responses, cyanogen bromide (CB) fragments as well as synthetic peptides were used to test the proliferative responses of two uveitogenic T cell lines, R9 and R17, prepared against native bovine and human S-antigen, respectively. Two nonoverlapping synthetic peptides which are known to actively induce EAU, amino acid residues 286-297 and 303-314 of the bovine sequence, were unable to induce proliferative responses in either S-antigen-specific T cell line. However, both of these sites were adjacent to synthetic peptides, residues 273-292 and 317-328, respectively, which were unable to actively induce EAU, but elicited strong proliferative responses from T cell lines raised to bovine and human S-antigen. Repeated in vitro selection of the R9 T cell line with a synthetic peptide containing one of these proliferative sites, residues 317-328, gave rise to a transiently uveitogenic T cell line. Several species-specific T cell epitopes were identified, but none of these were found to be involved in a uveitogenic response. Our results indicate that spatially separated and distinct T cell epitopes are present in S-antigen which are responsible for the active induction of EAU, lymphocyte proliferation, and the ability to adoptively transfer EAU.

Amino Acid Sequence↗
PubMed1989

Selection of antibody epitopes in an immunopathogenic neural autoantigen.

Results with two well-characterized self-antigens, cytochrome c and myelin basic protein, have led to differing opinions regarding the predominant specificities of autoantibodies, whether regions of sequence diversity or 'structurally inherent features' of a protein determine favored antigenic sites. To further examine this question, 16 antibody epitopes have been mapped on a highly immunopathogenic autoantigen, retinal S-antigen (S-Ag). The epitopes were characterized for: (1) sequence diversity and cross-reactivity on S-antigens from several species; (2) conformational dependency; and (3) probability of their occurrence on the surface of S-antigen. A single C-terminal region containing sequence diversity was most frequently recognized, but no evidence for recognition of any other regions of sequence diversity was found. Thirteen of 16 monoclonal antibodies raised to native S-Ag bound epitopes strongly predicted to be on the surface of S-antigen. Conversely, only one of six antibody preparations raised to peptides or affinity-purified on peptides was found to recognize an epitope predicted to be on the surface, suggesting a good correlation between specificity for conformation-dependent sites and surface probability based on the surface prediction algorithm. Three of these six antibodies which preferred denatured epitopes bound sites which overlapped or coincided with T cell sites; two of these T cell sites are immunopathogenic. The epitopes recognized on denatured antigen and peptides were similar whether the antibodies were elicited with intact human or bovine S-antigen or with cyanogen bromide-cleaved peptides. Our data suggests that in the case of S-antigen, structural features are more significant factors in epitope selection than sequence diversity.

Amino Acid Sequence↗
PubMed1989

Identification of the main immunogenic region of retinal S-antigen: subordinate influence of MHC, IGH, species or strain differences on the specificity of the antibody response.

The factors which lead to selection of dominant antigenic sites concentrated in discreet regions of proteins and polypeptides are important to the development of antigen-specific immunotherapies for autoimmune diseases and for vaccine design. In this study, the main immunogenic regions of the immunopathogenic autoantigen, retinal S-antigen, have been identified by examination of the specificity of antibody responses of different species. Using cyanogen bromide and synthetic peptides in western blots and the ELISA, the specificities of antisera from rabbits, guinea pigs, rats and 19 inbred strains of mice were tested. All animals produced high titers of antibody to S-antigen with the exception of PL/J mice. Antibodies which bound epitopes contained in peptide CB46, a 46 amino acid-containing peptide located at the C-terminus of S-antigen, were dominant in all species and strains tested. The epitopes in CB46 were multiple, overlapping, and concentrated in a stretch of approximately 30 residues. Two overlapping synthetic peptides from that region substantially competed the anti-CB46 response of all animals. Antibodies which recognized peptide CB47, a 47 residue peptide from the N-terminus, comprised the next most common group. This epitope was similar in all mice and overlapped the epitope defined by rat antibodies. All anti-CB47 antibodies mapped to an 11 residue region of CB47. Eleven strains of mice did not respond to CB47 after one immunization with S-antigen; however, multiple immunizations readily converted all animals so tested to CB47 responders. Rabbits and guinea pigs exhibited very weak responses to CB47 following one immunization; multiple immunizations increased the response minimally. Rats produced a strong antibody response to peptide CB123, which contains the known uveitogenic sites, while very little activity to CB123 was raised in rabbits and guinea pigs. Only 3 murine strains, LP, LP.R3, and B10.R3-71, responded with antibodies to CB123 and the epitope was mapped to a 30 residue region which in rats also contains two distinct pathogenic sites and an antibody epitope. Only rats and rabbits made antibody to the CB35 peptide; the epitopes were contained within an 18 residue sequence. The results show that a main immunogenic region is located in S-antigen near the C-terminus and is independent of species or MHC. Less dominant, species and strain-dependent immunogenic regions were found in three other areas, i.e. peptides CB47, CB123 and CB35.

Animals↗
PubMed1988

Assignment of several epitopes to cyanogen bromide peptides of bovine retinal S-antigen by immunoblotting with peptide-specific antibodies.

Peptide fragments of bovine S-antigen, an immunopathogenic retinal autoantigen which mediates experimental autoimmune uveoretinitis, were produced by cyanogen bromide cleavage and used to study antibody-defined epitopes, primarily those defined by antibodies from Lewis rats immunized with the intact antigen or various peptide fragments purified from the digests by HPLC. Antibodies from the sera have been affinity-purified on several of the peptides and examined by western blot analysis and enzyme-linked immunosorbent assay on S-antigen, digests and purified fragments. By immunoblotting it could be shown that five of the purified peptides, CB46, CB47, CB67, CB74 and CB123 were immunogenic, eliciting antibodies which recognized the peptides to which they were prepared; all, except for CB67, elicited antibodies which also bound intact S-antigen. Two more peptides, CB14 and CB27 were not immunogenic and did not contain epitopes. An epitope was also found in CB35, a previously uncharacterized peptide. Using these procedures together with amino acid sequence and composition data, we have been able to determine the origins of the peptides which contain antibody epitopes, including those which we have previously determined to possess epitopes recognized by class II MHC-restricted T cell lines raised to S-antigen and several of the peptides. A T cell line specific for the non-uveitogenic CB47 peptide was unable to transfer the disease.

Animals↗
PubMed1988

Preparation of overlapping peptides of bovine retinal S-antigen and their localization by immunoblotting with peptide-specific antibodies.

Bovine retinal S-antigen was cleaved by three chemical cleavage procedures including o-iodosobenzoic acid (IBA), mile acid and cyanogen bromide. The resultant peptides were used to study antibody-defined epitopes. Treatment with IBA, which cleaves primarily at tryptophanyl peptide bonds, produced at least 4 major fragments and several minor fragments. The peptides have been identified by their migration on SDS-PAGE and tested for their immunoreactivity to several affinity-purified anti-CNBr-peptide antibodies and to affinity-purified anti-IBA peptide antibodies. The presence of a single tryptophan residue 194 residues from the amino-terminus should result in 2 fragments of approximately 23,000 and 26,000 molecular weight based on the known size of intact S-antigen. The additional fragmentation is due to the presence of acid labile bonds and cleavage at IBA-sensitive tyrosyl residues associated with a side reaction. Western immunoblots using affinity-purified antibodies against the various IBA and CNBr peptides have allowed location of these peptides within the intact molecule. Specifically, IBA23K and IBA21K are overlapping fragments on the carboxy end, mutally exclusive of all other peptides. IBA15K and IBA5.6K overlap, and IBA18K and IBA10K overlap within IBA26K which comprises the N-terminal half of S-Ag. Additionally, IBA10K contains an antibody epitope destroyed by CNBr cleavage of the methionyl residue between CB53 and CB56. Further characterization of these IBA peptides will expedite the location of possible additional uveitogenic epitopes in the amino-terminal half of S-Ag as well as epitopes lost by other peptide generating techniques.

Animals↗
PubMed1988

Epitope mapping of bovine retinal S-antigen with monoclonal antibodies.

We examined the binding of seven murine monoclonal antibodies raised to S-antigen, an immunopathogenic, 404 residue photoreceptor cell autoantigen which induces experimental autoimmune uveoretinitis. S-antigen has also been identified as arrestin, a protein involved in the regulation of phototransduction. One additional monoclonal antibody (C10C10), raised to a synthetic peptide (peptide N) corresponding to residues 281 to 302 in bovine S-antigen, was also studied. In preliminary studies we examined the specificity of the antibody response to bovine S-antigen in sera from Balb/c mice. Western blots of mouse sera on the cyanogen bromide digest of bovine S-antigen demonstrated that all animals produced antibody which recognized epitopes within the C-terminal cyanogen bromide peptide designated CB46. Mice of the H-2d haplotype, including the Balb/c strain often used to produce monoclonal antibodies, showed little activity to cyanogen bromide peptides other than CB46. Also, all seven of the monoclonals raised to S-antigen are specific for epitopes in the CB46 peptide. The epitopes recognized by the monoclonal antibodies could be grouped into four distinct sites defined by peptides AE-1 (A2G5), peptide AA (PDS-1), peptide 19-OV (A9C6), and peptide 199 (BDS-1,2,3 and 4). The mono-clonal antibody, C10C10, raised to peptide N recognizes an epitope in the N peptide and binds to a larger cyanogen bromide peptide designated CB123 as well as intact S-antigen. Fine mapping of these epitopes was done with various subpeptides. None of the antibodies bound the known immunopathogenic peptide, peptide M, which resides in CB123 although the C10C10 antibody binds a peptide adjacent to peptide M.

Amino Acid Sequence↗
PubMed1987

Identification of a uveitogenic cyanogen bromide peptide of bovine retinal S-antigen and preparation of a uveitogenic, peptide-specific T cell line.

Peptide fragments of bovine retinal S-antigen produced by cyanogen bromide (CB) digestion have been purified and tested for their ability to induce experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in Lewis rats. Following immunization with the various peptides in complete Freund's adjuvant, one of the peptides, CB123, was found to be potently uveitogenic. A CB123-specific, class II restricted T helper lymphocyte line (R208) prepared from one of the CB123 peptide-immunized animals by repeated in vitro selection with purified CB123 was able to transfer severe EAU and EAP to naive rats. Peptides CB36, CB46, CB51 and CB66 were immunogenic as assessed by the presence of antibodies detected in the enzyme-linked immunosorbent assay and sensitized lymphocytes found in [3H]thymidine incorporation assays using lymphocytes from the peptide-immunized rats, but they did not induce significant EAU or EAP. Antibodies induced by immunization with the peptides also bound intact S-antigen, unlike the lymphocytes which were only weakly responsive to S-antigen. Cross-reactivity of antibodies and lymphocytes from CB51 and CB66 immune animals suggested that these peptides overlap and contain a common epitope. No measurable immunity to any CB peptide or intact S-antigen was found in the animals immunized with peptide CB26. Although a T cell line (R17) raised to human S-antigen was also able to transfer EAU, it was weakly responsive to the CB123 peptide; also, the R208 line did not respond well to human S-antigen. Since both human and bovine S-antigen are uveitogenic, these results suggest that the uveitogenic epitope in CB123, which is of bovine origin, is not the same as the uveitogenic epitope of the human antigen.

Animals↗
PubMed1987

Uveoscleral outflow using different-sized fluorescent tracers in normal and inflamed eyes.

Sodium fluorescein and fluorescinated dextrans (FD) of selected molecular weights were combined and perfused into the anterior chamber of normal and inflamed eyes of cynomolgus monkeys. The eyes were dissected into iris, anterior and posterior uvea, anterior and posterior sclera, retina and intraocular fluids (excluding aqueous). Each tissue was homogenized and centrifuged and the supernatant was run through a gel-filtration column to separate the fluorescent tracers. Each of the resultant peaks was quantitated and facility of uveoscleral outflow was determined. In control eyes the calculated facility of uveoscleral outflow was very similar with all tracers (from 0.047-to 0.052 microliter min-1 mmHg-1) and each tracer was found in highest concentration in the anterior sclera and anterior uvea. In inflamed eyes the calculated facility of uveoscleral outflow increased two- to five-fold with each tracer (0.12-; 0.17-; 0.29-; and 0.24 microliter min-1 mmHg-1 with fluorescein, and the fluorescinated dextrans of MWs 4000, 40,000 and 150,000, respectively). Each tracer was found in the anterior sclera and uvea in inflamed eyes whereas the posterior sclera and uvea contained predominantly the higher molecular-weight tracers (MWs 40,000 and 150,000). It is concluded that iridocyclitis causes an increase in uveoscleral outflow by increasing the permeability of the anterior uvea to all tracers and fluid. Small tracers may then diffuse into uveal blood vessels or across the sclera, yielding lower values for uveoscleral outflow. Of the four tracers studied, the optimal tracer size for studying uveoscleral outflow in either normal or inflamed eyes is MW 40,000.

Animals↗
PubMed1986

Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea.

Various buffer systems were examined for their ability to resolve and provide molecular weight determinations of proteins and peptides over a wide size range using electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Sharp bands and high resolution were achieved in the 1300 to 100,000 molecular weight range using a modified Laemmli discontinuous buffer system with high concentrations of Tris in the resolving gel (0.75 M) and in the running buffer (0.05 M). Linear gradient gels (8 to 25% acrylamide) were tested with and without varying concentrations of urea and/or glycerol and/or sucrose. At this high molarity of Tris, the inclusion of urea, glycerol, or sucrose proved unnecessary for successful peptide electrophoresis. Gels run without these reagents showed superior resolution throughout the entire molecular weight range when run with Tris at 0.75 and 0.05 M, respectively, obviating the need for urea or other additives as used in other systems. A single gel is thus able to resolve an entire range from large proteins to small peptides.

Animals↗
PubMed1986

S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis.

Two S-antigen-specific rat T cell lines expressing the T helper cell surface phenotype (W 3/25+, OX 8-) have been isolated from the spleen and lymph node cells of retinal S-antigen-immunized Lewis rats, one of which displayed neither clinical nor histopathologic signs of experimental autoimmune uveoretinitis. The other rat had recovered from severe experimental autoimmune uveoretinitis for 2 mo before isolation of the cell line. Both lines are specific for S-antigen presented by histocompatible antigen-presenting cells, and also respond in vitro to several of the peptides produced by cyanogen bromide cleavage of bovine retinal S-antigen. The lesions induced by the i.v. transfer of from 1 to 10 X 10(6) viable line cells involve the retina and pineal gland, as is found when Lewis rats are immunized with immunopathogenic doses of S-antigen. Histologic examination of the eyes and pineal glands revealed pathologic lesions typical of experimental autoimmune uveoretinitis, and consisted of marked infiltration of the retina and surrounding tissues and the pineal gland by lymphocytes and inflammatory cells. T cells capable of mediating autoimmune disease are clearly present and readily isolated from both asymptomatic and convalescent animals. No significant differences in specificity for the cyanogen bromide peptides of S-antigen or cell surface phenotype were found in the T cell lines isolated from these two rats, nor was any difference found in the specificity or titer of serum antibodies taken from the original rats for the cyanogen bromide peptides of S-antigen.

Animals↗
PubMed1986

Characterization of immunologically active cyanogen bromide peptide fragments of bovine and human retinal S-antigen.

Peptides which account for most, if not all, of the cyanogen bromide (CNBr)-generated peptide fragments of bovine retinal S-antigen have been identified and examined for their immunoreactivity with antisera raised to bovine and human S-antigen and with immune lymphocytes further selected twice in vitro with either bovine or human S-antigen. Amino-acid sequencing of a large fragment of S-antigen missing a small N-terminal peptide revealed the location of three overlapping CNBr peptides near the N-terminus. Amino-acid sequencing of several other CNBr peptides has allowed their position in a partial DNA-predicted sequence of the carboxy terminal half of the antigen to be determined. The total CNBr digest of human S-antigen was also prepared and compared with the fragments of the bovine antigen. Sera from rats immunized with bovine or human S-antigen were similar in their specificity in the enzyme-linked immunosorbent assay (ELISA) for purified bovine peptides except for the CB21 peptide which was not significantly bound by anti-human S-antigen sera. All of the other bovine peptides recognized by anti-bovine S-antigen sera were also bound by antibodies in the sera raised to the human antigen. The CNBr peptides of human and bovine S-antigen were extracted from gel slices and also assayed in the ELISA. Peptides of bovine S-antigen purified by HPLC were tested for their ability to stimulate an in vitro proliferative response in lymphocytes from Lewis rats immunized with either bovine or human S-antigen. Only quantitative differences in the proliferative response to human vs. bovine S-antigen and CNBr peptides were found. Methodology for the purification and analysis of the peptides is presented as well as the properties of the peptides.

Amino Acid Sequence↗
PubMed1985

Ultrastructural localization of retinal S-antigen in the rat.

Using a peroxidase-antiperoxidase immunohistochemical technique, the light and electron microscopical localization of retinal S-antigen was demonstrated in the rat retina, using antisera to bovine and guinea pig S-antigen. Comparable localization of S-antigen was observed with both antisera, although in our hands, the antiserum to bovine S-antigen gave more intense immunostaining. By light microscopy, the most intense immunoreactivity to retinal S-antigen was seen in the outer segment of the photoreceptor layer. Some immunostaining was also demonstrated surrounding the cell bodies of the outer nuclear layer. The inner segment of photoreceptors was virtually devoid of immunoreactivity. Ultrastructurally, immunoreactivity to S-antigen was demonstrated mainly on the inner surfaces of the visual discs of the rods. The plasma membranes of the outer segment of the rods were stained as well. Immunoreactivity surrounding the cell bodies of the outer nuclear layer was ultrastructurally localized on plasma membranes. The inner segment of the photoreceptor layer, other parts of the retina, or any eye structures other than retina showed no immunoreactivity. The relationship of retinal S-antigen to other cell components in the retina and the possible mechanisms of the autoimmune uveal diseases are discussed.

Animals↗
PubMed1985

Serum antibody level to S-antigen in children with chronic uveitis.

Bovine S-antigen was purified by gel filtration and ion exchange chromatography according to previously described techniques. An enzyme linked immunosorbent assay (ELISA) using antiserum to bovine S-antigen raised in guinea-pigs was employed to detect S-antigen in the chromatographic fractions. The purity of the S-antigen was determined by SDS-PAGE electrophoresis, where a single band was found. The purified S-antigen in microgram quantities together with Freund's complete adjuvant induced uveitis in rats two weeks after injection into the foot pad. Serum samples from children suffering from chronic uveitis and healthy children were tested for antibodies to S-antigen by the ELISA. A statistically significant difference in the level of specific antibodies between patients and controls was found. There was no clear-cut correlation between the severity of uveitis and antibody titre, but cases with retinal involvement and aggressive uveitis all showed definite elevations of antibodies to S-antigen.

Antibodies↗
PubMed1984

Preparation, isolation, and immunochemical studies of the cyanogen bromide peptides from a retinal photoreceptor cell autoantigen, S-antigen.

Purified bovine retinal S-antigen (50,000 m.w.) was treated with cyanogen bromide, producing seven major and several minor fragments. Six of the major and one of the minor components were isolated by reverse phase high performance liquid chromatography. The peptides were characterized with respect to size by urea-SDS-gel electrophoresis, by amino acid composition, and by their ability to bind antibodies, raised in rabbits immunized with purified bovine S-antigen, in both competition and direct enzyme-linked immunosorbent assays. Four of the purified peptides were found, by the direct assay, to bind antibodies in immune sera raised to the intact antigen. Peptides that were negative, or only weakly bound, in the direct enzyme immunoassay were subsequently conjugated to a carrier, poly-L-Glu-Ala-Tyr, and were retested in the enzyme immunoassay in which a peptide of about 25 residues was also found to contain an antigenic determinant. The same five peptides were positive in the competition assays. Isolation of the peptides and gel electrophoresis under reducing and nonreducing conditions revealed that two of the peptides in the reaction mixture were joined by a disulfide linkage.

Animals↗
PubMed1983

Growth-stimulatory effects of retinoblastoma-derived growth factors and other mitogens on Nakano mouse lens epithelial cells.

A polypeptide growth factor (RDGF) secreted by a human retinoblastoma cell line (Y-79) grown in serum-free defined medium stimulated DNA synthesis up to 36-fold and cellular proliferation by 100% upon 3 days of exposure to quiescent Nakano mouse lens epithelial cells (NKR-11) deprived of serum supplements. The stimulation of DNA synthesis produced by RDGF in the absence of serum could be further potentiated by the presence of 0.1 microgram/ml of insulin or 0.1 microgram/ml of arginine vasopressin. 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on the other hand, produced a 30-40% decrease in RDGF activity at 0.01-1.0 microgram/ml of TPA. The stimulation of DNA synthesis produced by RDGF, and its potentiation by the simultaneous addition of insulin, could be sustained after transient exposure (30 min) to RDGF, or RDGDF plus insulin, respectively, whereas insulin alone as little effect after exposures of up to 6 h. Nerve growth factor (1.0 pg to 1.0 microgram/ml) did not stimulate DNA synthesis or cell division in NKR-11 cells, whereas fibroblast growth factor (FGF) produced an optimal 3-fold stimulation of DNA synthesis at 10 ng/ml. Epidermal growth factor (EGF) activity was optimal and showed a 5-fold increase in DNA synthesis at 100 pg/ml in NKR-11 cells. The early receptor-mediated events that might be responsible for the transition to a proliferative state in quiescent Nakano mouse lens epithelial cells are discussed.

Animals↗
PubMed1983

Application of monoclonal antibodies to detect intraocular mycoplasma antigens in Mycoplasma arthritidis-infected Sprague-Dawley rats.

Sprague-Dawley rats infected with Mycoplasma arthritidis by tail vein inoculation develop extensive disseminated joint inflammation, frequently accompanied by conjunctivitis and anterior uveitis. The intraocular inflammation is apparently directed at mycoplasmas localized within the stroma of the ciliary body, which have been detected with monoclonal antibodies by indirect immunofluorescence. The monoclonal antibodies are directed against an antigenic determinant on the enzyme arginine deiminase isolated from M. arthritidis, but they do not react with the same enzyme derived from Mycoplasma hominis. The antigen bound by the monoclonal antibodies can also be detected by immunofluorescence in M. arthritidis-infected tissue cultures and is not lost after glutaraldehyde fixation or paraffin-embedding procedures. The value in the application of monoclonal antibodies reactive with arginine deiminase lies in the fact that although this enzyme may be found in mycoplasmas and several other species of bacteria it is not a normal constituent of mammalian tissues.

Animals↗
PubMed1983

Longitudinal study of serum antibody responses to bovine retinal S-antigen in endogenous granulomatous uveitis.

Twelve patients with granulomatous uveitis were followed up longitudinally for as long as 20 months after their initial visit, and multiple serum antibody titres to bovine retinal S-antigen were determined and compared with the clinical activity at the time of each sampling. In those patients who presented with highly active lesions which then resolved during the course of the study without recurrences (7 toxoplasmosis and 1 pars planitis) the antibody titres reached a peak approximately 2 months after the initial visit and declined thereafter. No correlation of serum anti-S titres with clinical activity or predictable pattern of titres could be found in those patients who had recurrences during the course of the study (3 granulomatous iridocyclitis and 1 ocular sarcoidosis).

Antibodies↗