Assessment of liposomal transfection of ocular tissues in vivo.

A possible route to the treatment of inherited retinal degenerations such as retinitis pigmentosa (RP) is the application of somatic gene therapy by the transfer and expression of corrective functional genes in ocular tissue. Cationic liposomes are established vehicles for the delivery and expression of exogenous genes in mammalian cells both in vitro and in vivo1. We report here a preliminary assessment of liposome-mediated transfer of a plasmid carrying the reporter gene lacZ (encoding the enzyme beta-galactosidase) into tissues of the adult rabbit eye.

Parathyroid hormone related peptide gene expression in human fetal and adult heart.

OBJECTIVE: The aim was to investigate the expression of parathyroid hormone related peptide (PTHrP) gene in the human fetal and adult heart. METHODS: Molecular biological techniques were employed as well as immunocytochemistry and western blot analysis using rabbit polyclonal anti-PTHrP(1-34) and anti-PTHrP (56-86) on normal human fetal and adult heart tissues. Northern blot analysis of both normal human fetal and adult heart total RNA, using a human full length cDNA probe, and polymerase chain reaction analysis of normal human fetal and adult heart cDNAs with exon specific oligonucleotides were carried out. RESULTS: Positive staining was detected with both anti-PTHrP(1-34) and anti-PTHrP(56-86) in fetal heart at 12 weeks of gestation. In both fetal and adult hearts, multiple putative PTHrP proteins were observed with apparent molecular mass of 14-125 kDa. Multiple hybridising PTHrP mRNA isoforms (1.4, 2.1, 3.2, and 4.5 kb) were detected in both fetal and adult heart total RNAs. The fetal and adult heart cDNAs amplified from the cDNA libraries showed the presence of the 5' non-coding exon II and coding exons III-IV but not the 5' non-coding exon Ic. CONCLUSIONS: PTHrP is expressed in normal human fetal and adult hearts suggesting that it has a function as an endogenous modulator of the cardiovascular system.

The antigenic specificity of the humoral immune response to primary and repeated ocular infections of the guinea pig with the GPIC agent (Chlamydia psittaci).

The antigenic specificity of the humoral immune response in guinea pigs to primary and repeated ocular infections with the guinea pig inclusion conjunctivitis (GPIC) chlamydial agent was analysed using microbiological, serological and Western blotting techniques. The results indicate that although there was a response to many minor polypeptide antigens, there was a marked lack of reactivity to the major outer membrane protein (MOMP), particularly following reinfection of guinea pigs. It is suggested that, lack of a good antibody response to the MOMP, may be one of the reasons why guinea pigs are susceptible to repeated ocular infections with this chlamydial agent.

Neural cell adhesion molecule (N-CAM) in fetal and mature human heart.

Using Northern blot analysis and immunoblotting techniques we report for the first time, that the neural cell adhesion molecule, N-CAM, is expressed in human heart. We found several different N-CAM transcripts in human fetal (13-17 weeks gestation) and mature heart (left ventricle from a 5-year-old child). Northern blotting showed that a 5.2 kb transcript was the most abundant and progressively increased with age, both in fetal and mature heart. These transcripts may correspond with the different protein isoforms shown by immunoblotting. We also confirmed the presence of N-CAM in fetal and mature myocytes by immunohistochemical techniques, using a monoclonal antibody to human N-CAM. Results demonstrated that N-CAM is mainly confined to the myocyte cell surface.

Experimental autoimmune uveoretinitis and pinealitis induced by interphotoreceptor retinoid-binding protein and S-antigen: induction of intraretinal and subretinal neovascularization.

Experimental autoimmune uveoretinitis (EAU) and pinealitis were induced in Lewis rats following hind foot pad injection of interphotoreceptor retinoid-binding protein (IRBP) or S-antigen. A comparison is made in this study of the in vivo and histological changes in uveoretinitis and pinealitis induced by administering similar doses of highly-purified IRBP and S-antigen emulsified in complete Freund's adjuvant (CFA). The time of onset of ocular inflammation after inoculation was slightly later in S-antigen (14-18 days) as compared with IRBP-inoculated animals (10-14 days), while the severity of the inflammation was lower in the latter group. The distribution of inflammation in the anterior segment was similar in both the S-antigen and IRBP sensitized animals but there was major variation in the location of the posterior segment disease. Vasculitis was a predominant feature of IRBP induced disease while chorioretinitis and photoreceptor destruction was more prominent in the S-antigen sensitized animals. A striking feature of this study is that both antigens induced intraretinal and subretinal neovascularization, an observation which has not been reported previously. Inflammation was induced in all pineal glands and as with EAU the severity was closely related to the type of antigen inoculated.

D-[3H]galactose incorporation into glycogen in retinal cone cells.

Previous studies have shown that bovine retinas incubated with [3H]galactose incorporated it, unmodified, into large molecules. Light and electron microscope autoradiography showed a significant proportion of the label to be in cone inner segments, and pulse-chase studies showed it was subsequently transported to the synaptic pedicles. In this report, evidence is presented to show that the galactose-labelled macromolecules are resistant to hydrolysis by proteolytic enzymes, testicular hyaluronidase, chondroitinase ABC, beta-glucosidase and beta-glucuronidase, but are readily degraded by alpha-amylase and beta-galactosidase, and to a lesser extent by beta-amylase. Treatment with alpha-amylase also leads to specific removal of radioactivity from cone inner segments and pedicles, as judged by light-microscopic autoradiography. These studies appear to indicate that the cone-specific galactose label is in glycogen or glycogen-like molecules.

A specific ELISA using purified opsin, for studying autoimmunity in retinal diseases.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed to measure nanogram quantities of rhodopsin or its apoprotein, opsin, in bovine retinal rod outer segment (ROS) preparations. Anti-opsin anti-sera could detect as little as 4 ng of purified opsin or of opsin in ROS preparations. The purified opsin was prepared by quantitative elution from a preparative polyacrylamide gel, and showed higher immunoreactivity with anti-opsin than did ROS when the same amount (per weight) of protein was allowed to bind in the wells of the ELISA plates. The effect of the ionic detergent SDS (sodium dodecyl sulphate) on the immunoreactivity and antigen binding to the ELISA wells was studied. Concentrations of 0.1% SDS and above reduced the apparent binding of opsin with anti-opsin when examined by ELISA. This may have been because the negatively charged SDS reduced the efficiency of the antigen coating process, or because changes in the epitopes' conformations made them less recognisable by the corresponding antibodies. A similar ELISA system using a specific anti-S-antigen anti-serum allowed the detection of even very small amounts (nanograms) of S-antigen in ROS preparations. The presence of S-antigen in ROS preparations was confirmed by immunoblotting. Thus purified opsin is preferable to ROS for ELISA tests of autoimmunity to rhodopsin in retinal diseases. These sensitive ELISA techniques could be used to examine the presence of minute amounts of rhodopsin, opsin or S-antigen in different retinal preparations.

Immunochemistry of the outer retina.

The immunochemistry of the outer retina is discussed with particular reference to photoreceptor cells, the retinal pigment epithelium and the interphotoreceptor space. The antigens identified and the techniques utilised are summarised.

A monoclonal antibody (HNo-g7) with specificity for a human nucleolar protein.

A murine monoclonal antibody which reacts strongly with the nucleoli of human epithelial cells has been isolated. The antibody is of the IgM class and the antigen has a molecular weight of 45 000. The antibody appears to react with interphase chromatin only and to have specificity for epithelial cells.