Biomedical subjects

T M Redmond

Publications and source records attributed to T M Redmond.

At least 55 records · Page 3Linked to original sources

PubMed1989

Rat T-cell lines specific to a nonimmunodominant determinant of a retinal protein (IRBP) produce uveoretinitis and pinealitis.

Rat lymphocyte lines were established, with specificity toward two synthetic peptides derived from the interphotoreceptor retinoid-binding protein (IRBP), which specifically localizes in the retina and pineal gland. One of the peptides, R4, is immunopathogenic, producing experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in immunized rats, while the other peptide, R3, exhibits no detectable immunopathogenicity in rats. The cell lines carry surface markers specific for the helper/inducer subset of T-lymphocytes. When tested by the proliferation assay, the line cells demonstrated major histocompatibility-restricted vigorous responses against the immunizing (homologous) peptide, but failed to recognize the intact IRBP molecule. This finding is in line with other data indicating that peptides R3 and R4 are nonimmunodominant determinants of IRBP for the Lewis rat. Yet, the cell lines specific for R4 were highly immunopathogenic, producing EAU and EAP in naive rats at numbers as low as 0.25 x 10(6), with histopathological changes similar to those induced by active immunization with this peptide. The immunological capacity of the cell lines was further demonstrated by the finding that spleen cells from recipient rats of these lines responded well against the homologous peptides. The uniqueness of this system, in which lymphocytes specific toward a nondominant determinant are immunopathogenic, is underscored and the possible mechanisms of disease induction are discussed.

Animals↗

PubMed1989

mRNA for interphotoreceptor retinoid-binding protein (IRBP): distribution and size diversity in vertebrate species.

Northern blots of total retinal RNA from a number of different vertebrate species were probed with a cDNA fragment corresponding to translated portions of bovine IRBP mRNA. A hybridizing band was detected in normal human retina (4.6 kb), Y-79 human retinoblastoma cells (4.4 kb), and in retinas of monkey (4.6 kb), guinea-pig (4.9 kb), mouse (6.1 kb), rat (two bands at 5.4 kb and 6.6 kb), rabbit (6.3 kb), cow (6.5 kb), and hamster (7.6 kb). Thus, the IRBP gene is expressed in the retinas of a wide variety of mammalian species. The mRNAs could be readily detected in about 20 micrograms of total RNA, suggesting that, in these species, IRBP message is relatively abundant. In contrast, only very weak hybridization was detected on northern blots of the three bird species examined (chicken, duck, quail). IRBP mRNA is thus relatively well expressed in mammals, but not in birds.

Animals↗

PubMed1989

Immune responses to peptides derived from the retinal protein IRBP: immunopathogenic determinants are not necessarily immunodominant.

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein specific to the retina and pineal gland, induces in immunized rats inflammatory changes in these organs (EAU and EAP, respectively). We report here on the immunological activities in Lewis rats of 10 IRBP-derived peptides. Only one of these peptides (R3) was found to induce high levels of antibodies in immunized rats, as detected by ELISA. On the other hand, the majority of the tested peptides stimulated substantial cellular immune responses, measured by the lymphocyte proliferation assay. None of the tested peptides were recognized, however, by antibodies or lymphocytes from rats immunized with the whole IRBP molecule, thus indicating that these synthetic peptides are nonimmunodominant in the Lewis rat. Two of these peptides, R4 and R9 (which contains R4), were previously found to be immunopathogenic, producing EAU and EAP in immunized Lewis rats. The immune responses to peptide R4 were further examined and the data show that it induces measurable lymphocyte responses only when injected at remarkably high doses (greater than or equal to 67 microgram/rat). Yet, peptide R4 was highly antigenic when tested for stimulation of specifically sensitized lymphocytes in culture. Furthermore, lymphocytes sensitized against R4 exhibited high capacity to adoptively transfer EAU and EAP to naive recipients. The finding of immunopathogenic but nonimmunodominant peptides is discussed.

Amino Acid Sequence↗

PubMed1988

Cyanogen bromide fragments of bovine interphotoreceptor retinoid-binding protein induce experimental autoimmune uveoretinitis in Lewis rats.

Interphotoreceptor retinoid-binding protein (IRBP), a retinal specific antigen, induces experimental autoimmune uveoretinitis (EAU) when injected into Lewis rats. Here we report that certain cyanogen bromide fragments of IRBP are capable of inducing EAU. Bovine IRBP, reduced and S-carboxymethylated, was subjected to cyanogen bromide cleavage. This CNBr digest was subjected to reversed-phase high performance liquid chromatography. Three fragments were purified to apparent homogeneity. These three fragments were subjected to gas-phase amino-terminal sequencing analysis. All three yielded single sequences, confirming their purity. On the basis of this amino-terminal sequencing and sequencing of cDNAs encoding bovine IRBP, two of these sequences, named CB-58 and CB-71, were localized to the C-terminal one-third of the IRBP molecule, whereas the third, a subfragment thought to result from cleavage at a tryptophan residue and named CB-47, was localized to the N-terminal one third of the protein. CB-71 and CB-47 shared a strong homology, suggesting a putative internal gene duplication event in the evolution of IRBP. All three of these fragments when injected into Lewis rats caused moderately severe EAU with early onset at relatively low doses. The histopathologic changes induced were indistinguishable from those caused by the intact protein. It would seem, therefore, that bovine IRBP contains multiple uveitogenic sites.

Amino Acid Sequence↗

PubMed1988

Lymphocyte responses to retinal-specific antigens in uveitis patients and healthy subjects.

A modified procedure for measuring human lymphocyte responses to retinal antigens was introduced and found to be highly sensitive. The main modifications are the use of round bottom culture wells, high antigen concentrations (up to 100 micrograms/ml) and extended incubation time (up to 9 days). Using this procedure, the majority of patients with uveitis, as well as a large proportion of healthy donors were found to respond positively (S.I. greater than or equal to 2.0) toward S-antigen and interphoto-receptor retinoid-binding protein. The responses to the retinal antigens were further enhanced by enriching the cultured lymphocytes for the helper/inducer subset. The responses to the retinal antigens were inferior to those against tuberculin (PPD), in particular in subjects who had been inoculated with BCG. The "secondary" responses to PPD were always higher in magnitude, were stimulated by markedly lower concentrations of antigen and were detected earlier in culture. The notion that the responses to retinal antigen in healthy donors are "primary" in nature was further supported by the findings that (a) lymphocytes from cord blood samples resembled adult lymphocytes in their response to S-Ag and (b) healthy donor lymphocytes which were prestimulated in vitro with S-Ag reacted to this protein by a specific "secondary" fashion. The possible involvement of lymphocytes with reactivity toward retinal antigens in pathogenic autoimmune processes in the eye is discussed.

Adult↗

PubMed1988

Synthetic peptides derived from IRBP induce EAU and EAP in Lewis rats.

In an earlier study we isolated three cyanogen bromide cleavage fragments of bovine IRBP that exhibited high levels of immunopathogenicity, producing inflammatory changes in the eyes (EAU) and pineal gland (EAP) of Lewis rats. These fragments have been localized within the IRBP sequence. In order to identify these putative immunopathogenic epitopes of IRBP, nine selected peptide sequences were synthesized and tested for the induction of disease in Lewis rats. Seven of the peptides were found inactive in producing disease while two closely related peptides, designated R4 (23-mer) and R9 (27-mer) were found to reproducibly induce EAU and EAP in immunized rats. No good correlation was found between the immunopathogenicity of the nine tested peptides and their amphipathicity: peptides R4 and R9 were not predicted to form strong amphipathic helices, while peptides selected for their high predicted helical amphipathicity were not immunopathogenic. EAU induced by peptides R4 and R9 was less severe and had a longer onset time than the disease induced by whole IRBP. In addition, the inflammatory changes induced by R4 and R9 in the posterior segment of the eye were less acute than those induced by whole IRBP and included granuloma formation and perivasculitis, features which are not generally seen in rats immunized with whole IRBP. Thus, the changes induced by R4 and R9 more closely resemble those which are characteristically found in human eyes affected by certain uveitic diseases than do changes produced by the intact protein.

Amino Acid Sequence↗

PubMed1987

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis.

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.

Animals↗

PubMed1987

Experimental autoimmune uveoretinitis (EAU) induced by retinal interphotoreceptor retinoid-binding protein (IRBP): differences between EAU induced by IRBP and by S-antigen.

Rats immunized with the retinal interphotoreceptor retinoid-binding protein (IRBP) develop an inflammatory eye disease, "experimental autoimmune uveoretinitis" (EAU). The ocular changes which characterize the EAU induced by IRBP resemble those seen in rats which develop EAU by immunization with another retinal protein, S-antigen (S-Ag). Yet, the two antigens do not cross-react antigenically and the two diseases differ by several features: At low doses (less than or equal to 4 micrograms/rat), IRBP was more uveitogenic in Lewis rats than was S-Ag, inducing disease more reproducibly and with earlier onset time. On the other hand, at higher doses (greater than or equal to 20 micrograms/rat) the disease induced by S-Ag was more severe than that induced by the same doses of IRBP. Rats of various inbred strains differed in their susceptibility to EAU induced by these two antigens. In particular, BN rats were more susceptible to IRBP-induced EAU than to the S-Ag-induced disease, while WF and RCS-rdy+ rats developed severe EAU when immunized with S-Ag but showed minimal or no ocular change when immunized with IRBP.

Animals↗

PubMed1987

Uveitis induced in primates by IRBP: humoral and cellular immune responses.

In a previous publication, we have reported that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) produces severe uveitis in monkeys. The present study further examined the uveitogenicity of IRBP and analysed the immune responses in the immunized monkeys. Uveitis developed in monkeys immunized with bovine IRBP at doses as low as 10 micrograms Kg-1 body wt. In contrast, no disease was detected in monkeys immunized with monkey IRBP at the total dose of 100 micrograms Kg-1. Serum antibodies were measured in the immunized monkeys by the enzyme-linked immunosorbent assay, while cellular immunity was determined by skin hypersensitivity and the lymphocyte mitotic reaction in culture. No humoral or cellular immune responses were detected in monkeys immunized with monkey IRBP. Monkeys immunized with bovine IRBP produced antibodies which cross-reacted well with monkey IRBP. On the other hand, these monkeys did not react with monkey IRBP by skin hypersensitivity and their lymphocytes responded in culture to this protein only when tested by a highly sensitive procedure. In contrast, significant responses of the same cells to bovine IRBP were obtained at all tested conditions. These data are interpreted to suggest that the pathogenic cellular immune responses in vivo detect cross-reactivity with this autologous ocular antigen more efficiently than most conventional laboratory tests.

Animals↗

PubMed1987

IRBP from bovine retina is poorly uveitogenic in guinea pigs and is identical to A-antigen.

Retinal interphotoreceptor retinoid-binding protein (IRBP) is a potent uveitogen in Lewis rats, producing experimental autoimmune uveitis (EAU) reproducibly at doses lower than those of S-antigen (S-Ag). In contrast, IRBP was found to be poorly uveitogenic in three strains of guinea pigs, inducing only minor changes in a small proportion of these animals. On the other hand, S-Ag was found to induce EAU in the majority of immunized guinea pigs, with changes more severe than those induced by IRBP. Unlike the difference in their uveitogenicity, IRBP and S-Ag induced similar levels of specific immune responses in the immunized guinea pigs. The poor uveitogenicity of IRBP in guinea pigs resembles that of A-antigen (A-Ag). The two proteins are also similar in other features and were found in this study to be antigenically identical. It is proposed that IRBP and A-Ag are one and the same protein.

Animals↗

PubMed1987

Immunohistochemical analysis of experimental autoimmune uveoretinitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) in the rat.

Experimental autoimmune uveoretinitis (EAU) was induced in rats by immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) and studied by immunohistochemistry. In general, the IRBP induction of inflammatory cellular components and expression of immune-related antigens on various non-lymphoid cells resembled those provoked by S-antigen (S-Ag). However, differences were found between the two diseases, including: 1) The increase in T suppressor/cytotoxic cells occurred in IRBP EAU more rapidly than in S-Ag EAU. 2) Fewer numbers of non-lymphoid cells expressed major histocompatibility complex class II surface antigens in IRBP EAU than in S-Ag EAU. The immunopathogenic mechanism of EAU induced by these two retinal antigens are discussed.

Animals↗

PubMed1986

Interspecies conservation of structure of interphotoreceptor retinoid-binding protein. Similarities and differences as adjudged by peptide mapping and N-terminal sequencing.

Structural properties of the retinal extracellular-matrix glycolipoprotein interphotoreceptor retinoid-binding protein (IRBP) from human, monkey and bovine retinas have been compared. SDS/polyacrylamide-gel-electrophoretic analysis of limited tryptic and Staphylococcus aureus-V8-proteinase digests show virtually identical patterns for the monkey and human proteins, whereas both sets differ considerably from the bovine protein pattern. Time-course digestion shows monkey IRBP to be more readily cleaved than bovine IRBP and also cleaved to smaller fragments. Also, reversed-phase h.p.l.c. of complete tryptic digests of the IRBPs indicate that, although they have in common a similar preponderance of hydrophobic peptides, all three proteins differ extensively in their fine structure. The N-terminal sequences of monkey and bovine IRBPs have been extended beyond those presented in our previous report [Redmond, Wiggert, Robey, Nguyen, Lewis, Lee & Chader (1985) Biochemistry 24, 787-793] to over 30 residues each. The sequences yet show extensive homology, differing at only two positions, although the major monkey sequence has an additional five amino acid residues at its N-terminus ('n + 5' sequence) not observed with bovine IRBP ('n' sequence). The newly determined N-terminal sequence of human IRBP demonstrates the presence of equal amounts of the 'n' and 'n+5' sequences that are qualitatively identical with those of the monkey. The presence of the five-amino-acid-residue extension in primate, but not bovine, IRBP may indicate variation in post-translational processing.

Amino Acid Sequence↗

PubMed1986

Uveitis induced in primates by interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein was found to be highly uveitogenic in primates. All six monkeys immunized with bovine interphotoreceptor retinoid-binding protein developed ocular inflammation. The main clinical changes were sheathing of retinal vessels and deep well-circumscribed yellow-white lesions. Histologic changes in the retina included shortening of the outer segments and foci of inflammation, which extended through all retinal layers. Particularly profound were the choroidal changes, such as severe inflammatory infiltration, which often formed focal granulomas with epithelioid and giant cells. Some granulomas had the structure of Dalen-Fuchs nodules. These structures and other histologic changes in the monkeys closely resemble those in uveitic conditions in humans, such as sympathetic ophthalmia or Vogt-Koyanagi-Harada syndrome. These findings show that antigen that localizes specifically in the retina may initiate an immunopathogenic process affecting mainly the choroid.

Animals↗

PubMed1986

Uveoretinitis and pinealitis induced by immunization with interphotoreceptor retinoid-binding protein.

Rats immunized with microgram amounts of interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein which localizes specifically in the eye and pineal gland, developed uveoretinitis and pinealitis. The severity and onset of changes were found to be dose-related and to be enhanced by B. pertussis bacteria. In general, the inflammatory changes induced by IRBP resembled those provoked by S-antigen (S-Ag), but significant differences were noted between the two diseases. The possible usefulness of the new experimental autoimmune disease is discussed.

Animals↗

PubMed1986

Immunochemical distribution of interphotoreceptor retinoid-binding protein in selected species.

An enzyme-linked immunosorbent assay (ELISA) was used to quantitate interphotoreceptor retinoid-binding protein (IRBP) in tissues of monkey and other species using rabbit antiserum against monkey IRBP. A 1:7500 antiserum dilution was found optimal with the linear range extending to 250 micrograms IRBP/ml. The highest IRBP concentration was found in cannulation fluid of the monkey interphotoreceptor space, although vitreous and aqueous humors also contained IRBP. The presence of IRBP in the vitreous was confirmed by Western blot and 3H-retinol binding studies. The pineal gland of monkey and rat also was found to contain IRBP, as assessed by ELISA and immunocytochemistry; IRBP was below the limits of detection in turtle and chicken retina. IRBP levels were uniformly low in retinas of human cases with hereditary retinal degeneration, including retinitis pigmentosa (three cases) and choroideremia (two cases). The presence of IRBP in pineal as well as in the vitreous and aqueous humors may indicate a broader role for this putative retinoid-transport protein than previously suspected.

Animals↗

PubMed1985

Endogenous fatty acids are covalently and noncovalently bound to interphotoreceptor retinoid-binding protein in the monkey retina.

Interphotoreceptor retinoid-binding protein (IRBP) purified from monkey interphotoreceptor matrix contains relatively high concentrations of endogenous fatty acids, 6.51 mol/mol of protein. Sixty-five percent of the total fatty acid bound to IRBP was found to be noncovalently attached, with the remainder covalently bound. The fatty acids are not residual components of phospholipids or neutral lipids, as judged by microchemical methods. The major fatty acids bound to IRBP are: palmitic (35%), stearic (21%), palmitoleic (7%), oleic (29%), linoleic (6%) and docosahexaenoic acids (2%). These fatty acids account for about 90% of the total fatty acid bound to interphotoreceptor matrix proteins extracted with organic solvents. Thus, IRBP may function as an intercellular fatty acid carrier and may depend on the covalently bound fatty acids for anchoring in the outer leaflet of cell membranes.

Animals↗

PubMed1985

cDNA clones encoding bovine interphotoreceptor retinoid binding protein.

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.

Amino Acid Sequence↗

PubMed1985

Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas.

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.

3',5'-Cyclic-GMP Phosphodiesterases↗