Changes in immunoreactivity in S-antigen-induced pinealitis.

Changes in immunoreactivity in rat pineal gland were investigated in S-antigen (S-Ag)-induced pinealitis. Anti-S antibodies were used to study the accessibility of pathogenic epitopes in the organ, and phosphatidylinositol 4,5-bisphospate (PI[4,5]P2) antibodies were chosen to study changes in functional phospholipid in membranes. Control pineal gland (without inflammation) did not show much reactivity toward these antibodies. Immunoreactivity toward both antibodies was markedly increased throughout the organ at the peak of inflammation and decreased almost to control level in the postinflammatory stage. The rise and fall of immunoreactivity were attributed to changes in the accessibility of antigenic sites rather than changes in the amounts of S-Ag and P(4,5)P2. The transient enhancement in immunoreactivity, probably consequential to tissue damage, suggests that the pineal gland, unlike the retinal photoreceptors, is capable of quick repair of tissue.

Immunochemical evidence for the light-regulated modulation of phosphatidylinositol-4,5-bisphosphate in rat photoreceptor cells.

Immunocytochemical localization of phosphatidylinositol-4,5-bisphosphate (PIP2) in the rat rod photoreceptor outer segments (OS) was investigated with rabbit antiPIP2 antibodies. The OS of the light-adapted rat eye showed little or no staining, whereas the OS of the dark-adapted eye were intensely stained for PIP2. The immunoreactivity of photoreceptor PIP2 in the eye exposed to a brief flash of light was markedly reduced. However, subsequent dark-adaptation of the flash-bleached eye resulted in a rapid recovery of PIP2 immunoreactivity; dark-adaptation for 5 min was sufficient for recovery to the fully dark-adapted level. In dark-adapted eyes exposed to graded light intensities, the PIP2 immunostaining varied with light levels and was correlated with unbleached rhodopsin concentrations. These results suggest that PIP2 in the rat photoreceptor cells is rapidly hydrolyzed upon light exposure and rapidly synthesized in the dark and that the decrease of PIP2 level is triggered by photic bleaching of rhodopsin.

Immunohistochemical localization of phosphatidylinositol-4,5-bisphosphate in the rat lens.

We have attempted to localize immunohistochemically phosphatidylinositol-4,5-bisphosphate (PIP2) in rat lens tissue using affinity purified rabbit anti-PIP2 antibodies. Evidence indicates that PIP2 is localized to the lens epithelial cells but appears to be absent from the lens fiber cells.

Immunocytochemical localization of phosphatidylinositol-4,5-bisphosphate in dark- and light-adapted rat retinas.

Light-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (TPI) to 1,2-diacylglycerol and D-myo-inositol 1,4,5-triphosphate (IP3) has been reported in the visual photoreceptor cells. We have investigated the localization of the TPI antigenic sites in dark- and light-adapted rat retinas using rabbit anti TPI antibodies (Ab). Sprague-Dawley rats were dark-, or light-adapted, or were exposed to a light flash. The eyes were fixed immediately and the tissue sections stained with the rabbit anti TPI Ab. The peroxidase-antiperoxidase method was used to find the localization of the TPI antigenic site. Image analysis of the sections was performed to obtain optical density profiles of the stain. Dark-adapted retinas showed intense staining of the rod outer segment (OS) layer but much less staining of the rod inner segment layer. Compared with the OS of dark-adapted retinas, those of the flash-bleached retinas were stained much less. The OS of fully bleached retinas showed little or no staining. The anti TPI Ab-protein A-gold conjugate intensely stained disks from dark-adapted retina but those from bleached retina much less. Our results suggest that rapid hydrolysis of TPI in rat rod outer segments occurs in vivo in response to light.

Roles of adroxazine, a new heterocyclic hormone of the adrenals, and leucogenenol, a thymothyroid hormone, in the formation of blood cells.

Treatment of normal rats with the thymothyroid hormone leucogenenol accelerates the rate at which the 'functional' cells, neutrophils, red blood cells and lymphocytes, develop in the bone marrow from their corresponding committed precursor cells. Following the initial treatment of normal rats with leucogenenol, there is a temporary elevation in the bone marrow of the relative concentrations of myeloblasts, rubriblasts and lymphoblasts. It has now been found that bilaterally adrenalectomized rats have a different response from normal rats to treatment with leucogenenol. Although treatment of bilaterally adrenalectomized rats with leucogenenol accelerates the rate of development of their functional cells, the initial injection of leucogenenol is followed by a temporary decrease in the relative concentrations of myeloblasts, rubriblasts and lymphoblasts in their bone marrow. Additionally, adrenalectomized rats treated concurrently with tritiated thymidine and leucogenenol show a significant lower percentage of labeled cells in their bone marrow than do correspondingly treated normal rats. These results indicate that adrenalectomized rats have a lower than normal concentration of stem cells in their bone marrow that can be committed to become functional cells. Treatment with adroxazine, a new recently isolated heterocyclic hormone of the adrenals, causes adrenalectomized rats to respond as normal rats to the injection of leucogenenol. There is a temporary elevation of myeloblasts, rubriblasts and lymphoblasts in their bone marrow as well as a normal increase in the percentage of cells that are labeled following concurrent treatment with tritiated thymidine and leucogenenol. It may be concluded that treatment with adroxazine increases the rate of replication of uncommitted bone marrow stem cells while treatment with leucogenenol increases the rate at which committed cells develop into their respective functional cells. A scheme is presented to show the suggested roles that leucogenenol and adroxazine play in regulating the formation of blood cells.

Preparation of a uveitogenic peptide by chymotryptic digestion of bovine S-antigen.

Limited proteolysis of bovine S-antigen with alpha-chymotrypsin resulted in the accumulation of three peptides of MW 24,000, 16,000, and 12,000 daltons, respectively. By ELISA (enzyme-linked immunosorbent assay), MW 24,000 peptide was found to react with anti-S antibodies, but the other two peptides did not react with the antibodies under the assay conditions. The reactive peptide was separated from the smaller peptides by gel filtration on Sephadex G-75 and Sephadex G-50. When the MW 24,000 peptide was injected into Lewis rats, severe to mild uveitis was produced in all injected animals. The results indicate that the pathogenic determinant is on the MW 24,000 peptide.

Localization and properties of an immunoreactive protein in bovine ciliary body similar to retinal S antigen.

Immunocytochemical studies showed that monoclonal antibodies to bovine retinal S-antigen recognize an immunoreactive protein present in the bovine ciliary body epithelium. This protein was purified by DEAE-agarose chromatography and found to have a M.W. of 28,000 daltons by SDS polyacrylamide gel electrophoresis. Crude and purified preparations of this protein cross-react with polyclonal antibodies to retinal S-antigen. The relative reactivity of S-antigen monoclonal antibodies to ciliary body protein was determined by ELISA. Lewis rats immunized with crude and purified ciliary body protein failed to develop experimental uveitis. The results indicate that the ciliary body protein contains an antigenic peptide domain which is similar to that of S-antigen but lacks the immunopathogenic domain.

Ornithine decarboxylase in developing neonatal rabbit ocular tissue.

The specific activity of ornithine decarboxylase (ODC) was assessed in rabbit ocular tissue from 2 to 15 days postnatal. ODC levels in cornea were low, but significant at 2 days, and decreased slightly with time. Activity was not detectable in lens tissue. On the other hand, in retina, ODC decreased from fairly high specific activity to nil in the 1st week after birth. Ciliary body paralleled retina but at a lower level. In contrast, ODC specific activity in the vitreous body rose 25-fold between day 5 and 15. These temporal changes are discussed in terms of neonatal ocular development.

Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies.

Two monoclonal antibodies (RSA1/83 and RSA2/83) were developed against a homogeneous preparation of bovine retinal S-antigen. The two hybridomas produced by mouse X mouse hybrid myeloma cells secrete immunoglobulin G. Indirect autoradiography on glutaraldehyde-fixed preparations of bovine explants was used to locate the antigenic site. Antibody RSA1/83 recognizes the antigen primarily in the apical region of the rod outer segment, while antibody RSA2/83 located the antigen both in the outer and inner segments of the rod photoreceptor cells. A distinct band of silver grains also appeared along the inner limiting membrane with both antibodies. Control explants showed no specific labeling pattern over the various retinal compartments.

Biometry and clinical characteristics of congenital cataracts and microphthalmia in the Miniature Schnauzer.

Forty-two Miniature Schnauzer pups and adults with congenital cataracts and microphthalmia were evaluated by serial ophthalmic examinations, slit lamp biomicroscopic photography, and A-scan ultrasonography. The cataracts were evident when the eyelids opened at 2 weeks, affecting predominantly the lens nucleus and posterior cortex. Lenticonus was evident in 19% of the cataractous lenses. Progression of the cataracts was variable and related to involvement of the equatorial and posterior cortices. Lens-induced uveitis developed in some adult dogs with advanced hypermature cataracts. The globe and lens were smaller than normal in the cataractous eyes, as ascertained by A-scan ultrasonography. Age-matched comparisons of clear lens carrier Miniature Schnauzers and normal Beagles with the cataractous Miniature Schnauzers indicated affected globes and cataractous lenses were reduced 10% to 20% in their anteroposterior lengths. The microphthalmia appeared related to the congenital microphakic cataract.

Inheritance of congenital cataracts and microphthalmia in the Miniature Schnauzer.

Congenital cataracts and microphthalmia in the Miniature Schnauzer were inherited as an autosomal recessive trait. Eighteen matings of affected X affected Miniature Schnauzers resulted in 87 offspring with congenital cataracts and microphthalmia (49 males/38 females). Two matings of congenital cataractous and microphthalmic Miniature Schnauzers (2 females) X a normal Miniature Schnauzer (1 male) yielded 11 clinically normal Miniature Schnauzers (7 males/4 females). Eighteen matings of congenital cataractous and microphthalmic Miniature Schnauzers (6 males) X carrier Miniature Schnauzers (9 females) produced 81 offspring; 39 exhibited congenital cataracts and microphthalmia (20 males/19 females) and 42 had clinically normal eyes (17 males/25 females).

Reduced, oxidized, and protein-bound glutathione concentrations in normal and cataractous lenses in the dog.

Amounts of reduced, oxidized, and protein-bound glutathione (GSH) were measured in normal and cataractous lenses of pups and adult dogs. Lenses from pups included normal lenses from clinically normal pups, clear lenses from Beagle pups bred for glaucoma, and congenital cataractous lenses from Miniature Schnauzer pups. Lenses from adults included normal lenses from normal mixed-breed dogs, congenital cataractous lenses from Miniature Schnauzers, and complete mature cataractous lenses from clinical patients of different breeds. Glutathione in the normal lenses from pups and adult dogs is predominantly reduced GSH; oxidized GSH is about 2.1% to 2.6% of the reduced GSH values. The reduced GSH values are lower in normal pups [7.08 mumoles/g (wet wt) of lens] than in adults [7.83 mumoles/g (wet wt) of lens]; reduced GSH values decrease further in cataract formation. The decrease in oxidized GSH values parallel those of reduced GSH, except in the advanced cataracts of clinical patients in which oxidized GSH [0.045 mumoles/g (wet wt) of lens] was 9% of the GSH values. The GSH bound to soluble and insoluble lens proteins of congenital cataractous Miniature Schnauzer pups was significantly (P less than 0.01 and P less than 0.02, respectively) lower per gram of protein than that in pups with normal lenses. However, the soluble and insoluble protein-bound GSH of congenital cataractous lenses of adult Miniature Schnauzers and lenses in clinical patients with mature cataracts [based on mumole of GSH/g (wet wt) of lens] were not significantly different (P greater than 0.05) from that in adult dogs with normal lenses.

Hybridoma antibodies to insoluble antigens of the corneal epithelium.

Human corneal epithelial water insoluble proteins were used to immunize mice. Immune splenocytes were fused with Sp 2/0-Ag14 mouse myeloma cells by 40% PEG. Hybridoma antibodies obtained by somatic cell fusion were tested by radioimmunoassay. Supernatants from antibody positive hybrid colonies were used in immunofluorescence and crossreaction assays to locate and characterize corneal epithelial antigens. At least three distinct epithelial cell antigens were detected, one of which cross-reacts with rabbit corneal epithelial cells.

Adherence properties of Pseudomonas pili to epithelial cells of the human cornea.

In vitro adherence of Pseudomonas fluorescens organisms to the human cornea is correlated with bacterial pili. The role of pili in the attachment to human corneal epithelial cells was studied in an in vitro adherence assay system. A homogeneous, purified pilin preparation showed a molecular weight of 16,500 on SDS-polyacrylamide gels. Within 5 minutes incubation, 5.5 pg of pilin adhere to 15,000 epithelial cells. When epithelial cells were preincubated with purified pilin, a subsequent decrease in adherence of labeled pilin was noted. It appears that pili mediate adherence of Pseudomonas organism to human cornea.

Production of hybridomas secreting antibodies to the cornea.

Major controversies exist in the literature on the presence of blood group antigens on the endothelial and stromal layers of the cornea, and the importance of major histocompatibility typing for keratoplasty. Antibodies were raised in BALB/C mice against water soluble proteins of corneal epithelium. Following fusion of spleen cells with myeloma cells (Sp2/0-Ag14) hybrid colonies were maintained in HAT selective medium. The supernates of each colony were measured and screened for antibody production by radioimmunoassay. Gel electrophoresis of the antigen showed nine major bands. The antibodies were partially characterized by cross reaction against other soluble corneal fractions.