Auxiliary production of antibodies to ocular antigens in experimental autoimmune uveoretinitis

In experimental autoimmune uveoretinitis (EAU), there are concurrent autoreactive humoral and cellular immune responses. Many studies have recently focused on T-cell reactivities in EAU, while analysis of autoantibody responses to uveitogenic epitopes has been less well characterized. In this study, a defined 16-mer uveitogenic interphotoreceptor retinoid binding protein (IRBP) peptide, designated #896, was used to induce EAU. Histological analysis of eyes at day 10 demonstrated extensive leukocyte infiltration of the anterior segment, with mononuclear and plasma cells in the posterior segment. The presence of plasma cells suggests local production of antibodies within the eye. ELISA analyses of serum, aqueous, and vitreous from rats with IRBP-induced, severe EAU revealed the presence of antibodies against peptide #896. There was little difference between the serum and intraocular antibody titers, presumably due to breakdown of the blood-ocular barriers. In addition to the anti-#896-IRBP antibodies, there were detectable antibodies reactive against separate and distinct IRBP epitopes, as well as, against epitopes on retinal-S antigen. These results indicate that in #896-peptide-induced severe EAU, humoral immune responses are induced to the primary immunogen and that there is also auxiliary production of autoreactive antibodies against other epitopes present on intraocular antigens. These auxiliary responses may contribute to the immunopathogenesis of severe EAU.     

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.     

Localized increase of GABA levels in brain areas of the rat and inhibition of the plasma LH rise following orchidectomy

Previous studies have suggested that gamma-aminobutyric acid (GABA) exerts inhibitory actions on luteinizing hormone (LH) secretion that are likely to be mediated by modifications in noradrenergic transmission. To explore further this hypothesis we have studied the effect of increasing GABA contents in discrete areas of the brain on plasma LH levels in short-term orchidectomized rats. GABA accumulation was produced by the GABA transaminase inhibitor, gamma-vinyl-GABA (GVG). The locus coeruleus area (LC), where the noradrenaline (NA) cells projecting through the dorsal noradrenergic bundle are located, and several hypothalamic areas that are innervated by NA-containing fibers were microinjected with GVG. Most of these areas are known to be related to the neural control of LH secretion. GVG microinjected in the LC and medial preoptic area increased the GABA content and blunted significantly the acute increase of plasma LH produced by castration. Bicuculline prevented these effects. Delayed effects of GVG were observed when applied in the anterior hypothalamic area and ventromedial-arcuate nucleus area. In these latter areas, a single injection of GVG did not augment the GABA concentrations and was unable to prevent LH release, but a clear inhibitory effect took place after a second injection of GVG between 24 and 48 h after orchidectomy. Unresponsive areas to GVG treatment were the lateral preoptic area, the median eminence and the dorsal raphe. These results add support to the view that GABA inhibits LH release in rats, at discrete areas of the brain.     

External quality control assessment in PCR diagnostics of dengue virus infections

BACKGROUND: Increased travelling to countries endemic for dengue fever (DF) demands efficient laboratory diagnostics. Nucleic acid amplification techniques (NAT) are now frequently used for rapid diagnosis of imported viral diseases. Different PCR systems are available. OBJECTIVES: In order to assess the quality of molecular diagnostics of dengue virus infections, an external quality assurance (EQA) in PCR diagnostics was conducted. Study design: A panel of 10 human plasma samples was prepared and spiked with dengue virus types DEN-1 to DEN-4. In addition, a 10-fold dilution series (1:10-1:10(4) ) of DEN-3 virus was included. The panel was pre-tested by nested RT-PCR, in-house real-time PCR, and a commercial real-time PCR kit. The samples were inactivated by gamma irradiation and shipped in freeze dried state. Thirteen laboratories, within the European network for the diagnostics of imported viral diseases (ENIVD) took part using either single-round, nested, or real-time RT-PCR methods. Two laboratories used two methods in parallel, summarising up to 15 comparable results. RESULTS: 33-100% correct results were achieved. All laboratories detected DEN-2 correctly, followed by DEN-1 (14 positive results of 15), DEN-3 (12/15) and DEN-4 (11/15). Testing of the serial dilution revealed low sensitivity in many labs, with results ranging from 33 to 80% of correctly tested samples. CONCLUSION: The EQA gives a feedback of the quality of the RT-PCR system used by each respective laboratory. The different test systems and amplification conditions demonstrate the importance of external quality control measures.     

Release of prolactin and luteinizing hormone by histamine agonists in ovariectomized,steroid-treated rats under ether anesthesia

Responses to histamine agonists administered intraventricularly under ether anesthesia were analyzed to evaluate receptor mediation in histamine stimulation of prolactin and LH release in ovariectomized, estradiol-progesterone-treated rats (OVX-E2P-treated rats). Prolactin release was markedly increased by the H2-histamine agonists, 4-methyl histamine and Dimaprit. These effects were antagonized by metiamide, an H2-blocking agent. The H1-histamine agonist, 2-(2-pyridyl)ethylamine (PEA) in high doses released prolactin and its effect was partially prevented by metiamide. Mepyramine, and H1-antagonist, did not exert any effect on the release of prolactin enhanced by the histamine agonists. LH release was significantly increased after 4-methyl histamine administration. Its effect was weak and was blocked by metiamide. Neither Dimaprit nor PEA exhibited action on plasma LH levels. The results obtained with histamine agonists suggest that histamine evokes prolactin release in OVX,E2P-treated rats through H2-receptors. At present, conclusions on H2-receptor mediation in LH release induced by histamine cannot be drawn from these results. The above-mentioned data, however, conclusively discard a significant participation of H1-receptors.     

Neuroblastic differentiation potential of the human retinoblastoma cell lines Y-79 and WERI-Rb1 maintained in an organ culture system. An immunohistochemical, electron microscopic, and biochemical study

The differentiation potential of the human retinoblastoma cell lines Y-79 and WERI-Rb1 was evaluated in vitro for up to 120 days in a matrix system and in rotary suspension for 30 days. Matrix cultures were grown with 10% fetal calf serum (FCS), with and without differentiation-promoting agents. The latter were applied for a total of 5-45 days (usually 30 days) and included 7S nerve growth factor, dibutyryl cyclic AMP, sodium butyrate, retinoic acid, hydrocortisone, and ascorbic acid. Fully defined, serum-free medium and medium containing 5 or 15% FCS were also used for matrix cultures, and medium with 5 or 10% FCS for suspension cultures. By immunoperoxidase (performed on matrix cultures, both untreated and treated for 30 days with differentiation-promoting agents), the cells of both lines were positive for neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), class III beta-tubulin (human h beta 4) isotype, and synaptophysin. In addition, the WERI-Rb1 cells expressed 200 kd neurofilament protein (NFP-H) and retinal S-antigen. Both lines were invariably negative for glial fibrillary acidic (GFA) protein, myelin-associated glycoprotein, myelin basic protein, the epitope recognized by the Leu-7 monoclonal antibody, opsin, and hydroxy-indole-O-methyltransferase. In the Y-79 line the presence of NSE and the absence of NF proteins-H, -M and -L, of GFA protein, and of retinal S-antigen were confirmed biochemically. No differentiated features were found by electron microscopy in either line. Thus, in the matrix system employed, both lines exhibited solely a potential for neuroblastic differentiation, which was more advanced in the WERI-Rb1 line, as reflected by the antigenic expression of NFP-H and of retinal S-antigen.     

Intracellular acetylcholinesterase of adult rat myofibers is more concentrated in endplate than non-endplate regions

The intracellular distribution of acetylcholinesterase (AChE) was determined in adult rat anterior gracilis muscles. Echothiophate iodide (ECHO), a water-soluble cholinesterase inhibitor, was applied to muscles in situ to eliminate extracellular and/or extracellularly oriented enzyme. Control and ECHO-treated muscles were either cut into 1-mm segments and assayed for AChE activity or cytochemically stained for AChE. Subsequent analysis by light and electron microscopy showed that the AChE stain inside myofibers was highly localized and clearly visible only in the zone immediately underlying the point of nerve-muscle contact. Biochemical assay of muscle segments showed intracellular AChE to be most highly concentrated in regions containing large numbers of endplates (approximately twice the activity of endplate-free areas). Since such "endplate-rich" segments are in fact mostly extra-synaptic tissue, we conclude that intracellular AChE of adult rat gracilis myofibers, although present along the length of the cell, is more than two times as concentrated in sub-synaptic areas as compared to extra-synaptic areas. This result must be carefully considered when attempting to identify "endplate-specific" AChE activity of mammalian muscle, and further points to the importance of neural influences on AChE metabolism/regulation.     

Epitope discovery using bacteriophage display: the minimum epitope of an anti-IRBP antibody.

The purpose of this study was to determine, using random peptide library (RPL) technologies, the minimal epitope requirements of the mouse monoclonal anti-interphotoreceptor-retinoid-binding protein antibody, H3B5. This previously characterized antibody is used as an example to examine whether RPL's offer a relatively easy and rapid route to obtaining detailed epitope mapping data.A pentadecamer random peptide library (RPL) displayed on the major coat protein (gene 8) of filamentous bacteriophage (F88-4-15) was used as a target for selection by the anti-IRBP monoclonal antibody, H3B5. Three rounds of library selection were performed, and 90 of the resultant RPL clones were examined for affinity to H3B5 by enzyme-linked immunosorbent assay (ELISA). DNA sequencing of ELISA positive clones provided sequence of the region encoding the random peptide.After three rounds of selection of the RPL, 76.7% of clones examined interacted with H3B5, 17.7% did not show significant binding and 6.6% bound to control antibody also. The essential elements of the peptide epitope were determined by sequence comparison of 24 clones to be the four amino-acid sequence (Aspartic or glutamic acid)-Proline-Arginine-(Leucine, Isoleucine or Valine). This motif [(D/E) PR (L/I/V)] is in agreement, but at greater resolution, than previous synthetic peptide studies where the motif AASEDPRL was identified. Other motifs were found which bound to H3B5 but did not share primary structure similarities (peptidomimetics). Selection from a RPL has rapidly defined the minimal requirements for the H3B5 epitope in fine detail. Such a process offers great potential for investigating antibody-antigen interactions and core sequences of an epitope, and enables the identification of motifs in other proteins which may be recognized by the antibody, providing information on possible cross-reactivity.     

Autosomal dominant Stargardt-like macular dystrophy: I. Clinical characterization, longitudinal follow-up, and evidence for a common ancestry in families linked to chromosome 6q14

PURPOSE: Characterize the phenotype of autosomal dominant Stargardt-like macular dystrophy in two families linked to chromosome 6q14 and determine whether they share a common ancestry. METHODS: Two families spanning 10 generations were identified and studied independently. Participating members were examined and genetic linkage and genotyping performed. RESULTS: Presenting symptoms included decreased vision, hemeralopia, and mild photophobia. The subjective onset of visual loss ranged from age 3 to 50 with a mean of 14 years. A Snellen acuity of 20/200 occurred at a mean age of 22 years. Over decades, the macular lesion enlarged and visual acuity decreased to 20/300 to 20/800. The typical phenotype was well-circumscribed, homogenous atrophy of the retinal pigment epithelium and choriocapillaris in the macula, with surrounding yellow flecks and temporal optic nerve pallor. The phenotypic spectrum included a pattern dystrophy-like appearance, diffuse geographic atrophy, and extensive fundus flecks. Genotyping revealed that the two families were linked to chromosome 6q14 and shared a common haplotype spanning 21 cM between D6S430 and D6S300. The two families were subsequently shown by genealogic investigation to represent different branches of a common kindred. CONCLUSIONS: Families with autosomal dominant Stargardt-like macular dystrophy linked to chromosome 6q14 share a common phenotype and in some cases can be distinguished from similar dystrophies by inheritance pattern and clinical features. The finding that these two families shared a common ancestor suggests the existence of a founder effect. Characterization of the gene for autosomal dominant Stargardt-like macular dystrophy may enable better understanding of this condition and elucidation of its potential role in other forms of macular degeneration.     

A high‐fat plus fructose diet produces a vascular prostanoid alterations in the rat

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