Vijay K. Singh

Cellular Immune Responses of Patients with Uveitis to Retinal Antigens and Their Fragments

Of two patient populations totaling 82 patients, one in the United States and the other in Japan, we studied the cellular immune responses against S-antigen and interphotoreceptor retinoid binding protein as well as to fragments of each antigen. Behçets disease, birdshot retinochoroidopathy, pars planitis, ocular sarcoid, sympathetic ophthalmia, and the Vogt-Koyanagi-Harada syndrome were diagnosed in these patients. The response profile of both antigens paralleled each other. This profile was more commonly seen in patients suffering from diseases affecting the retina. Responders reacting to both antigens or to several fragments of an antigen were present. This pattern of response was seen in 26 of the patients tested. Patients with uveitis appeared able to recognize several autoantigens. This might be a consequence of the breakdown of the blood-retinal barrier and may help perpetuate the inflammatory process. Several patients were capable of responding to more than one epitope of the same antigen, which indicates that there are major differences between the experimental model and human autoimmune diseases in the response to autoantigens. Both of these findings may to help develop new immunotherapeutic strategies in the treatment of uveitis.

Molecular mimicry between uveitopathogenic site of retinal S-antigen and Escherichia coli protein: induction of experimental autoimmune uveitis and lymphocyte cross-reaction.

Experimental autoimmune uveitis (EAU) is caused by the immunization of microgram amounts of a soluble retinal protein, known as S-antigen, in susceptible animal strains including primates. The disease serves as an animal model of ocular inflammation. We induced EAU and pinealitis in Lewis rats with small synthetic peptides, corresponding to the amino acid sequence in Escherichia coli protein, which contains six consecutive amino acids identical to a uveitopathogenic site in human S-antigen (peptide M). EAU and pinealitis induced in rats by synthetic peptide derived from E. coli was indistinguishable from those induced by native S-antigen or other uveitopathogenic synthetic peptides corresponding to the amino acid sequence of S-antigen. Furthermore, lymph node cells from animals immunized with either peptide M or peptide derived from E. coli protein showed significant proliferation in the presence of either peptide when tested in vitro for lymphocyte mitogenesis using [3H]thymidine. Thus, molecular mimicry, a process by which an immune response directed against a nonself protein cross-reacts with a normal host protein, may play a role in autoimmunity.

Suppression of experimental autoimmune uveitis in rats by the oral administration of the uveitopathogenic S-antigen fragment or a cross-reactive homologous peptide

The oral administration of S-antigen fragment (a synthetic peptide designated as peptide M and known to be uveitopathogenic for rat, guinea pig, and monkey) to Lewis rats prior to challenge with an emulsion of peptide M and CFA resulted in either a total or partial suppression of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease studied as a model for human uveitis and experimental autoimmune pinealitis (EPA). Both the clinical and histopathologic manifestations of the disease were suppressed in a dose-dependent manner. Pinealitis associated with EAU was also suppressed by the oral administration of peptide M. Additionally, ingestion of a fragment of bakers yeast (Saccharomyces cerevisiae) histone H3, which has five consecutive amino acids identical to peptide M and which has been found to be uveitopathogenic in Lewis rats, induced tolerance to either peptide M or synthetic histone H3 peptide. In addition, the proliferative response to peptide M was inhibited in peptide M-fed rats. The suppression of EAU and in vitro lymphocyte proliferative responses to peptide M were observed to be antigen specific, since oral feeding of a control protein (BSA) exerted no suppressive effect. Furthermore, the T cells isolated from the spleen and lymph nodes of animals rendered tolerant by oral administration of peptide M can transfer protection against EAU adoptively. These results demonstrate that the oral administration of an autoantigen or its homologous peptide initiates an antigen-specific cellular mechanism which may ameliorate EAU.

S-antigen: From gene to autoimmune uveitis

Retinal S-antigen (S-Ag) is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals. EAU may serve as an animal model for studying human uveitis. As a first step we have determined the nucleotide sequence of an S-Ag gene and its cDNAs. The amino acid sequences were deduced from the cDNAs of various animals and human. Four uveitopathogenic sites in bovine S-Ag were characterized. One of the sites (peptide M) has sequence homology with non-self proteins from bakers yeast, potato, E. coli, hepatitis B virus, moloney murine leukemia virus, Moloney murine sarcoma virus, AKR murine leukemia virus and baboon endogenous virus. Mononuclear cells from animals immunized with peptide M showed significant proliferation when incubated with synthetic peptides corresponding to the amino acid sequences of the above-mentioned foreign proteins. In addition, all the peptides induced EAU in Lewis rats with a dose of 10-2000 micrograms. Moreover, native histone H3 from bakers yeast histone H3 induced EAU in Lewis rats. Thus, we found several examples of antigenic mimicry between self and non-self proteins. These findings establish a base to study further the mechanism of autoimmune inflammation.

Identification of a uveitopathogenic and lymphocyte proliferation site in bovine S-antigen

S-antigen is a well-characterized retinal protein that is highly pathogenic for the induction of experimental autoimmune uveitis (EAU), a severe inflammatory disease of the eye and the pineal gland. EAU was observed following the immunization of Lewis rats with various doses (50 to 200 micrograms) of a small synthetic peptide, peptide N (22 amino acids in length), which corresponds to amino acid positions 281 to 302 in bovine S-antigen. Peptide N 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 chamber and vitreous. Histopathologically, a severe inflammatory response was observed that resulted in the complete destruction of the photoreceptor cell layer of the retina. In addition, animals with ocular inflammatory disease had an associated pinealitis characterized by a lymphocytic infiltration of the pineal gland. Furthermore, draining lymph node cells of rats immunized with peptide N showed strong in vitro proliferative responses toward peptide N as measured by [3H]thymidine uptake. Our results indicate that several synthetic peptides, which correspond to the amino acid sequence of bovine S-antigen, are capable of inducing an EAU and, as such, suggest that multiple uveitopathogenic sites may be present in the molecule.

Rat pineal S-antigen: Sequence analysis reveals presence of α-transducin homologous sequence

S‐antigen (S‐Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S‐Ag from three species has been sequenced. In this study rat pineal S‐Ag was sequenced. Clones were isolated from a rat pineal λgt11 cDNA library by probing with a 300 bp fragment of mouse retinal S‐Ag cDNA containing the 5′‐coding region. The largest clone isolated (RPS‐118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S‐Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (≌44 992 Da). Comparison of the rat pineal and mouse retinal S‐Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S‐Ag indicates that expression of the S‐Ag gene in both tissues is similar. Further analysis of the rat pineal S‐Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S‐Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S‐Ag, rat pineal S‐Ag contains the same consensus phosphoryl‐binding site present in many GTP/ GDP‐binding proteins and a homologous sequence found in the C‐terminus of α‐transducin. These sequences may play a role in the action of pineal S‐Ag in transmembrane signal transduction.

S-antigen: experimental autoimmune uveitis induced in guinea pigs with two synthetic peptides

Experimental autoimmune uveitis (EAU) was observed in Hartley guinea pigs following immunization with two small synthetic peptides, peptide M and peptide M15L, which correspond to the amino acid sequence of a well-characterized region of bovine S-antigen. Groups of guinea pigs were immunized with 100 micrograms of each peptide in complete Freunds adjuvant and examined at regular intervals for the development of disease. Approximately two weeks later, an EAU was present which was characterized clinically by iris and pericorneal hyperemia. Histopathologically, a severe inflammatory response involving the uveal tract and retina was observed. In these eyes the photoreceptor cell layer of the retina was destroyed. A subretinal exudate containing mononuclear cells and polymorphonuclear leukocytes was also present. In addition, animals with EAU showed an associated pinealitis characterized by a lymphocytic infiltration of the subcapsular and central area of the pineal gland. Furthermore, draining lymph node cells of guinea pigs immunized with peptide M showed strong in vitro proliferative responses towards peptide M as measured by 3H thymidine uptake. These results demonstrate the existence of at least one common pathogenic epitope in bovine S-antigen for the induction of EAU in Hartley guinea pigs and Lewis rats.

Sequence homology between yeast histone H3 and uveitopathogenic site of S-antigen: Lymphocyte cross-reaction and adoptive transfer of the disease

Experimental autoimmune uveitis (EAU) serves as an animal model of ocular inflammation. The disease is caused by the immunization of microgram amounts of a soluble retinal protein, designated S-antigen, in susceptible animal strains, including primates. We induced EAU and experimental autoimmune pinealitis (EAP) in Lewis rats with a small synthetic peptide corresponding to amino acid positions 106-121 in yeast histone H3. This peptide contains five consecutive amino acids identical to a uveitopathogenic site (peptide M) in human S-antigen. Lymph node or mononuclear cells from different species of animals immunized either with histone H3 or with peptide M showed significant cross-reaction as measured by in vitro lymphocyte mitogenesis assay using [3H]thymidine. Also, we adoptively transferred the EAU and EAP in naive rats by immune lymph node cells. These findings support the fact that selected bacterial, viral, or fungal proteins with amino acid sequence homologies to normal retinal proteins are uveitopathogenic and, as such, provide a basis for autoimmune inflammatory diseases.

Uveitopathogenic Sites in Bovine S-Antigen

Experimental autoimmune uveitis (EAU) was observed in Hartley guinea pigs following immunization with high doses (200 micrograms) of four synthetic peptides designated peptide 3, K, N and M which correspond to amino acid positions 221-240, 241-260, 281-302, and 303 to 320 respectively in bovine S-antigen. Histopathologically, a moderate inflammatory response involving the uveal tract and retina was observed using peptide N and peptide M as immunogens, whereas peptide 3 and peptide K caused only a mild inflammatory response with very few inflammatory cells in the retina. In contrast, animals with EAU showed an associated pinealitis (EAP) characterized by an extensive lymphocytic infiltration of the central and subcapsular areas of the pineal gland. Lymph node cells of guinea pigs immunized with peptide 3, peptide K, peptide N or peptide M showed strong in vitro proliferative responses against the respective immunizing peptide as measured by [3H] thymidine uptake. The results suggest that under appropriate experimental conditions S-antigen may contain multiple pathogenic sites. The relevance of these studies in the pathogenesis of EAU is discussed.

Determination and Uncertainty Analysis of Inorganic Arsenic in Husked Rice by Solid Phase Extraction and Atomic Absorption Spectrometry with Hydride Generation

This study enables the selective determination of inorganic arsenic (iAs) with a low detection limit using an economical instrument [atomic absorption spectrometer with hydride generation (HG)] to meet the regulatory requirements as per European Commission (EC) and Codex guidelines. Dry rice samples (0.5 g) were diluted using 0.1 M HNO3-3% H2O2 and heated in a water bath (90 ± 2°C) for 60 min. Through this process, all the iAs is solubilized and oxidized to arsenate [As(V)]. The centrifuged extract was loaded onto a preconditioned and equilibrated strong anion-exchange SPE column (silica-based Strata SAX 500 mg/6 mL), followed by selective and sequential elution of As(V), enabling the selective quantification of iAs using atomic absorption spectrometry with HG. In-house validation showed a mean recovery of 94% and an LOQ of 0.025 mg/kg. The repeatability (HorRatr) and reproducibility (HorRatR) values were <2, meeting the performance criteria mandated by the EC. The combined standard measurement uncertainty by this method was less than the maximum standard measurement uncertainty; thus, the method can be considered for official control purposes. The method was applied for the determination of iAs in husked rice samples and has potential applications in other food commodities.