Biomedical subjects

J H Rockey

Publications and source records attributed to J H Rockey.

At least 19 recordsLinked to original sources

PubMed1995

Effect of interferon-gamma on the expression of transforming growth factor-beta by human corneal fibroblasts: role in corneal immunoseclusion.

Human corneal fibroblasts (HCF) inhibit T cell alloresponse in mixed leukocyte response-human corneal fibroblast coculture. The inhibition is contact independent, insensitive to indomethacin, and is enhanced by pretreatment of HCF with interferon-gamma (IFN-gamma). To investigate cytokine-dependent mechanisms of inhibition of T cell alloresponse by HCF, the capacity of cultured HCF to produce transforming growth factor-beta (TGF-beta) and the modulatory role of IFN-gamma on their TGF-beta production were investigated by radioreceptor binding inhibition assay (RRA) and the standard mink cell bioassay (BIA). The net total TGF-beta concentration of 4 day culture supernatants from IFN-gamma-treated HCF, measured by RRA, was 11.5 ng/ml. The net total bioactive TGF-beta concentrations of 4 day culture supernatants from HCF, before and after treatment with IFN-gamma, measured by BIA, were 2.0 and 4.8 ng/ml, respectively. These findings indicate that HCF produce TGF-beta and increase their TGF-beta output under the influence of the proinflammatory cytokine IFN-gamma. Media-borne TGF-beta binding proteins appeared to be primarily responsible for the discrepancy between the TGF-beta values measured by RRA and BIA. Active exclusion of TGF-beta binding proteins from intraocular fluids may have an important role in the maintenance of TGF-beta-dependent ocular immune privilege. Corneal fibroblasts may utilize TGF-beta-dependent mechanisms to maintain the immunosecluded environment of the cornea and to preserve the homeostasis of corneal optical competency. Interferon-gamma may enhance corneal immunoseclusion by upregulating the TGF-beta output of the corneal fibroblasts.

Biological Assay↗

PubMed1994

Interferon-gamma induces high-affinity transforming growth factor-beta receptor expression on human corneal fibroblasts.

The effect of interferon-gamma (IFN-gamma) on the expression of transforming growth factor-beta (TGF-beta) receptors on cultured human corneal stromal fibroblasts was examined. Scatchard analysis of specific saturable TGF-beta 1 binding data indicated that corneal fibroblasts expressed TGF-beta receptors with an average association constant of 6 x 10 M-1, before and after IFN-gamma treatment. An additional population of higher affinity TGF-beta receptors, with an average association constant of 4 x 10(12) M-1, was demonstrated only on IFN-gamma-treated corneal fibroblasts Interferon-gamma may alter the response of corneal fibroblasts to transforming growth factor-betas by upregulating their higher affinity TGF-beta receptors. The induction of higher affinity TGF-beta receptors by an immune cytokine and an associated autocrine elevation of TGF-beta output by the corneal fibroblasts may be a transient compensatory mechanism that maintains the homeostasis of corneal optical competency through enhancement of corneal immunoseclusion.

Cells, Cultured↗

PubMed1993

A soluble product of human corneal fibroblasts inhibits lymphocyte activation. Enhancement by interferon-gamma.

Corneal stromal fibroblasts expressed HLA-DP, -DQ and -DR Class II MHC antigens in response to interferon-gamma, but did not induce proliferative responses by allogeneic peripheral blood mononuclear cells in vitro when used as stimulator cells in a mixed leukocyte-type reaction. Furthermore, corneal stromal fibroblasts inhibited mixed leukocyte reactions between peripheral blood mononuclear cells of allogeneic donors, even when the corneal stromal fibroblasts were separated from the peripheral blood mononuclear cells by a 0.4 micron pore membrane. Pretreatment of the corneal stromal fibroblasts with interferon-gamma increased the inhibitory activity. Both [3H]thymidine incorporation and interleukin-2 production were inhibited, and the inhibition appeared to be mediated by a soluble factor whose production required protein synthesis. The inhibitory activity was not abolished by including 1-10 micrograms ml-1 indomethacin in the culture media. No inhibition was observed in the proliferation dose-response curves of responder peripheral blood mononuclear cells that had been cultured with corneal stromal fibroblasts for 3 days, prior to culture with allogeneic stimulator peripheral blood mononuclear cells. Thus, the ability of corneal stromal fibroblasts to interfere with alloimmune responses in vitro was dependent upon the continued presence of the fibroblasts and their continued production of a soluble inhibitory factor or factors. Inhibitors of allogeneic reactions that are produced by corneal stromal fibroblasts stimulated by immune cytokines (e.g. interferon-gamma) may play a role in prolonging corneal allograft survival.

Cell Communication↗

PubMed1991

Histamine and prostacyclin. Primary and secondary release in allergic conjunctivitis.

The relationship between the release of histamine, a major mast cell mediator of conjunctival type I reactions, and the production of a prostanoid, prostacyclin (prostaglandin I2, PGI2), was examined in a guinea pig model of allergic conjunctivitis. Guinea pigs were sensitized topically and challenged by repeated conjunctival instillation of fluoresceinyl ovalbumin. Histamine and 6-keto-PGF1 alpha, the stable product of the spontaneous degradation of PGI2, were measured in tears by radioimmunoassays. Clinical type I reactions and tear histamine appeared by 8 days and increased up to 22 days during the initial sensitization, with notable variations between animals. The kinetics of histamine and 6-keto-PGF1 alpha release in tears were examined over a 24-hr period after the antigen challenge. Histamine release was maximal during the first 10 min and returned to baseline values by 1 hr in all instances. The 6-keto-PGF1 alpha release also peaked during the first 10 min but continued for an extended period. The ratio of tear 6-keto-PGF1 alpha to histamine increased more than 16-fold over the 2 hr after antigen challenge. Late-phase reactions with second peaks of histamine or 6-keto-PGF1 alpha in the tears were observed in two different guinea pigs 4-8 hr after antigen challenge. Histamine applied to the eyes of naive guinea pigs also induced the release of 6-keto-PGF1 alpha in tears. Histamine appeared to act as a primary mediator, stimulating the secondary production and release of PGI2 by constitutive (eg, vascular) and possibly infiltrating inflammatory cells during an allergic conjunctival reaction.

6-Ketoprostaglandin F1 alpha↗

PubMed1990

Class II alloantigen induced on corneal endothelium. Role in corneal allograft rejection.

The expression of Class II major histocompatibility complex (MHC) antigens on corneal cells can be increased in vitro by (gamma-interferon) and in vivo in inflammatory reactions. The expression of Class II MHC by corneal endothelium of New Zealand White (NZW) rabbits during the rejection of corneal allografts was demonstrated by immunoperoxidase staining. Class II MHC expression by corneal endothelial cells may facilitate rejection of corneal allografts.

Animals↗

PubMed1990

Experimental ocular onchocerciasis: local and systemic antibody and cell-mediated immune responses.

Hartley guinea pigs injected subconjunctivally with Onchocerca lienalis (OL) microfilariae (Mf) develop punctate corneal opacities resembling the punctate keratitis of human onchocerciasis. Antibody production and antigen-induced proliferative responses were studied in conjunctival-associated lymphoid tissues (CALT), spleens (SL) and peripheral blood lymphocytes (PBL) from experimentally infected guinea pigs. Cultured single cell suspensions of CALT, SL and PBL were assayed for IgG1, IgG2, IgA and IgE antibody production. IgG1, IgG2, and IgA Onchocerca-specific antibodies were found in culture supernatants of CALT, SL and PBL. When initiated 10 days after a challenge injection of OL, CALT cultures produced antibody levels equal to or less than those produced by the corresponding SL cultures. When initiated 66 days after the last injection of Mf, CALT cultures produced significantly more antibody than the corresponding SL cultures. Blastogenic responses to OL Mf antigen were observed in peripheral and splenic lymphocytes of OL-infected guinea pigs. Animals given subconjunctival injections of Mf followed by treatment with a microfilaricide had greater responses to OL antigen than those given Mf alone, while responses to phytomitogens were similar in drug-treated and non-treated animals. The CALT was locally immunologically responsive against the subconjunctivally injected OL Mf, with the capacity for localized memory responses. The local immunologic responses to conjunctival Onchocerca microfilariae may play a significant role in the immunopathological reactions of ocular onchocerciasis.

Animals↗

PubMed1989

Massive follicular lymphoid hyperplasia in experimental allergic conjunctivitis. Local antibody production.

Acute and recurrent allergic conjunctival reactions were induced in guinea pigs by repeated conjunctival applications of fluoresceinyl ovalbumin (FL-OA) for up to 30 months. Early type I conjunctival reactions developed 11 to 25 days after the initial conjunctival exposure to FL-OA. Continuous topical challenges during a six- to 30-month period caused a variety of reactions, including papillary changes and massive hyperplasia of the conjunctival-associated lymphoid tissues. Hyperplasia of lymphoid tissues was induced during a shorter period (two to five months) with a mixture of FL-OA and phorbol ester. Culture fluid from hyperplastic conjunctival lymphoid tissue showed a ratio of IgG1/IgG2 antibody production of up to 15. A low level of recurrence of type I reactivity, after an initial desensitization phenomenon due to a loss of reactive mast cells, correlated with prominent follicular hyperplasia of the conjunctival-associated lymphoid tissue.

Animals↗

PubMed1989

Activation of Mg-ATPase of skeletal-muscle myosin by bovine retinal pigment epithelial actin.

The morphology and function of actin in cultured bovine retinal pigment epithelial (RPE) cells were studied. Filamentous actin was identified with a fluorescent mushroom toxin, nitrobenzoxadiazole (NBD)-phallacidin, specific for actin. Dark-field microscopy of cultured RPE cells revealed numerous pigment granules; fluorescent microscopy identified scattered lipofuscin granules. One-dimensional SDS polyacrylamide gel electrophoresis of urea-soluble proteins extracted from RPE cells showed a 46,000-dalton protein band which comigrated with authentic muscle actin. Densitometric scanning showed that this protein band comprised 7.6% of the total urea-soluble proteins. An actin-activated skeletal-muscle myosin Mg-ATPase assay, using skeletal-muscle heavy meromyosin as enzyme and [gamma-32P]-ATP as substrate, demonstrated functional actin in RPE cell extracts after DEAE-cellulose anion exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.19 and 0.36 M KCl. The activation of myosin ATPase by actin in RPE cells provides a molecular basis for the phagocytic activity which is important in maintaining the integrity of retinal photoreceptor cells.

Actins↗

PubMed1988

Effect of human corneal fibroblasts on lymphocyte proliferation in vitro.

Cocultivation of human corneal fibroblasts (CFB) with allogeneic peripheral blood mononuclear cells (PBL) induced a minimal lymphocyte proliferative response, even when the fibroblasts had been pre-treated with interferon-gamma (IFN-gamma) and were HLA-DR positive. IFN-gamma-treated CFB did not express detectable HLA-DQ alloantigens. Cocultivation of CFB with PBL in the presence of Concanavalin A (Con A) or Phytohemagglutinin inhibited mitogen-induced lymphocyte proliferation by 40-90% relative to PBL cultured without CFB. Induction of HLA-DR expression on the CFB did not alter their inhibitory properties. Addition of indomethacin to cultures reversed the effect of CFB on Con A responses. However, no difference between proliferative responses to HLA-DR positive or negative CFB was seen in the presence of indomethacin. This weak response to induced Class II alloantigens on CFB suggests that induction of Class II alloantigens on corneal stroma alone may be insufficient to sensitize a recipient for Class II alloantigen-driven corneal graft rejection.

Antibodies, Monoclonal↗

PubMed1988

Inhibitory effects of pyridoxal phosphate, ascorbate and aminoguanidine on nonenzymatic glycosylation.

Nonenzymatic glycosylation of serum albumin was studied in the presence of naturally occurring metabolites, pyridoxal, pyridoxal phosphate and ascorbate/dehydroascorbate, and a hydrazine compound, aminoguanidine. Pyridoxal, pyridoxal phosphate, ascorbate and dehydroascorbate, at concentrations of 0.1 mM or greater, significantly inhibited the nonenzymatic glycosylation of albumin. Aminoguanidine was the most potent inhibitor of nonenzymatic glycosylation and 54% or 85% inhibition occurred when 5 or 50 mM aminoguanidine, respectively, was present in the incubation mixture containing 20 mM glucose. A major effect of aminoguanidine was to lower the free glucose concentration in the incubation mixture by a direct reaction with glucose as judged by thin layer chromatography. The present studies suggest that vital metabolites such as pyridoxal phosphate and ascorbate may be potentially important in controlling glucose-induced nonenzymatic glycosylation of proteins. Pyridoxal phosphate forms a Schiff base with proteins as does glucose and therefore may be a preferable drug, over aminoguanidine which is a hydrazine, for inhibiting the effects of glucose-induced nonenzymatic glycosylation.

Ascorbic Acid↗

PubMed1988

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells.

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.

Aldehyde Reductase↗

PubMed1988

Prostacyclin is the major prostaglandin synthesized by bovine retinal capillary pericytes in culture.

Prostaglandin synthesis by bovine retinal pericytes was investigated using high pressure liquid chromatography to separate and identify 3H-labeled prostaglandins released from 3H-arachidonic acid labeled pericyte monolayers. A dominant peak activity corresponding to 6-keto-PGF1 alpha was observed. This peak was eliminated when monolayers were pretreated with cyclooxygenase inhibitors and was augmented when monolayers were stimulated by the calcium ionophore A23187. Suspensions of pericytes and the cell-free media of monolayers incubated with arachidonic acid inhibited adenosine diphosphate-, collagen-, and arachidonic acid-stimulated platelet aggregation in a bioassay for prostacyclin-like activity. This inhibitory activity was unstable at room temperature. Cultures of 7.5 to 10 x 10(5) pericytes (7th passage near-confluence) released nanogram quantities of 6-keto-PGF1 alpha as measured by radioimmunoassay. These results are evidence that prostacyclin is the main prostaglandin synthesized by bovine retinal capillary pericytes in culture. Pericytes may influence the microcirculation via their production and release of this potent vasoactive arachidonic acid metabolite.

Animals↗

PubMed1988

Experimental ocular onchocerciasis in cynomolgus monkeys. III. Roles of IgG and IgE antibody and autoantibody and cell-mediated immunity in the chorioretinitis elicited by intravitreal Onchocerca lienalis microfilariae.

Intravitreal injection of small numbers (10-500) of microfilariae (Mf) of Onchocerca lienalis in eyes of cynomolgus monkeys induces posterior segment lesions which resemble those occurring in human onchocerciasis in some respects. In order to determine the potential roles of antibody and cell-mediated immune responses in the formation of these lesions, anti-Onchocerca and anti-retinal IgG and IgE antibody and peripheral lymphocyte blastogenic responses were followed in eight monkeys, for one week to five months after intravitreal injection of Mf. Significant ocular inflammation occurred well before any antibody or peripheral lymphocyte blastogenic responses could be detected. It may be that direct effects of Mf, e.g., by activation of complement via the alternative pathway fulfill important roles in the pathogenesis of the posterior segment lesions.

Animals↗

PubMed1988

Experimental ocular onchocerciasis in cynomolgus monkeys. II. Chorioretinitis elicited by intravitreal Onchocerca lienalis microfilariae.

Chorioretinitis due to onchocerciasis is a major cause of blindness, and the pathogenesis is poorly understood. We have developed an experimental model for onchocercal chorioretinitis using cynomolgus monkeys (Macaca fascicularis). Two normal monkeys and two monkeys which had received prior sensitization with subcutaneous injections of live Onchocerca lienalis microfilariae were given intravitreal injections of either 0, 10, 50 or 500 live microfilariae. Posterior segment changes included disc edema, venous engorgement, retinal vasculitis, intraretinal hemorrhage, and progressive retinal pigment epithelial (RPE) disturbances. Histopathological findings included perivascular infiltrates with eosinophils, eosinophilic choroiditis, and RPE hypertrophy, hyperplasia and loss of pigment. Microfilariae in the retina had no surrounding inflammation but were found adjacent to areas of RPE alterations. Overall the inflammatory reaction in the two unsensitized monkeys was more severe than that seen in the sensitized monkeys. The retinal appearance of the monkeys resembled that found in human onchocerciasis, and this model appears to be a promising one for future investigations.

Animals↗

PubMed1988

Autoantibody induced by experimental Onchocerca infection. Effect of different routes of administration of microfilariae and of treatment with diethylcarbamazine citrate and ivermectin.

Hartley guinea pigs were injected with microfilariae (Mf) of Onchocerca lienalis as a model for acute inflammatory responses to Mf in human Onchocerca volvulus infection. IgG autoantibody reactive with a 3 M KCl extract of guinea pig cornea was detected by ELISA in the serum of guinea pigs injected with O. lienalis Mf three or more times sub-conjunctivally, or two or more times subcutaneously. Administration of the microfilaricides diethylcarbamazine citrate and ivermectin did not alter the proportion of animals expressing autoantibody or the mean autoantibody titer. The severity of acute corneal inflammatory reactions to Mf was similar in animals with and without circulating autoantibody. Although autoantibody responses did not correlate with acute corneal inflammatory reactions to dead Mf, the ability of Mf to induce formation of an antibody reactive with a component of autologous cornea suggests that autoimmune mechanisms might participate in chronic onchocercal lesions in the cornea, eg, sclerosing keratitis.

Animals↗

PubMed1987

Glycosaminoglycan synthesis and secretion by bovine retinal capillary pericytes in culture.

The synthesis and secretion of glycosaminoglycans (GAGs) was characterized in subcultures of bovine retinal capillary pericytes. The GAGs were metabolically labeled with [3H]glucosamine and 35SO4 for 3 days, and then precipitated from the cell layer or media by cetylpyridinium chloride and ethanol, separated by cellulose acetate electrophoresis and further identified by their susceptibility to degradative procedures. The predominant radioactively labeled GAG associated with the pericyte-cell layer was heparan sulfate (HS). Radioactively labeled chondroitin sulfate (CS) and hyaluronic acid (HA) were also present in the pericyte-cell layer. No radioactively labeled dermatan sulfate (DS) was detected. The profile of radioactively labeled GAGs secreted by pericytes into the media differed considerably from that associated with the cell layer. Equal amounts of radioactivity were incorporated into HS and CS. Small quantities of radioactively labeled HA were also present in the media. Although no radioactively labeled DS was detected in the pericyte-cell layer, it was present in the media. The total pericyte-cell layer GAG profile was determined by scanning densitometry of the three bands resolved after cellulose acetate electrophoresis and Alcian Blue staining. The slowest band was identified as HS, and accounted for 17% of the total GAGs. The middle band was identified as DS, and accounted for 34% of the total GAGs. The fastest band was tentatively identified as either DS or chondroitinase AC-resistant CS, and constituted 49% of the total GAGs. The GAGs associated with the fibroblast-cell layer and secreted into the media by fibroblasts also were characterized and compared with those produced by pericytes. The major differences were in the secretion of large amounts of HA into the media by fibroblasts, and the presence of radioactively labeled DS in the cell layer of fibroblasts.

Animals↗

PubMed1987

Synergistic activation of retinal capillary pericyte proliferation in culture by inositol triphosphate and diacylglycerol.

Inositol triphosphate (IP3) and diacylglycerol (DG) are second messengers which control ionic events implicated in cell proliferation in a variety of tissues. In order to determine if these two second messengers control the proliferation of bovine retinal capillary pericytes (BRCP) or feline retinal pigment epithelial cells (FRPE) in culture, both intact BRCP or FRPE or BRCP or FRPE made permeable by saponin were used to study the effects of IP3 and DG on [3H]thymidine incorporation into DNA. [3H]Thymidine incorporation by BRCP made permeable to saponin showed specific IP3 dose-dependence; the apparent Km was 0.3 microM of IP3. Similar effects of A23187, a Ca2+ ionophore, or synthetic DG (1-oleoyl-2 acetyl-glycerol) were also observed. The combination of synthetic DG (0-,2-,4-, 8 micrograms ml-1) and 1 microM A23187 produced greater stimulation of [3H]thymidine incorporation by intact BRCP than was seen with DG or A23187 alone. In contrast to BRCP. [3H]thymidine incorporation by FRPE was not stimulated by IP3, A23187 or synthetic DG. The synergistic activation of IP3 and DG provided direct evidence to support the view that BRCP proliferation in vitro were regulated by the levels of the two second messengers.

Animals↗

PubMed1987

Immune-mediated adherence of eosinophils to Toxocara canis infective larvae: the role of excretory-secretory antigens.

The participation of Toxocara canis larval excretory-secretory antigens in immune-mediated adherence was determined in vitro. Adsorption of immune sera with excretory-secretory antigens removed some complement components, removed IgG antibody directed against larval surfaces, and abrogated all adherence observed with untreated immune serum. At least four antigens could be implicated in adherence, by Western blot analysis of adherence mediating sera. Scanning and transmission electron microscopic examination of larval-eosinophil interactions revealed that eosinophils adhered to a membranous sheath-like layer that was frequently detached from the larval epicuticle. The layers appeared to be composed of surface antigens and antibody, and may provide larvae with protection against antibody and eosinophil toxins by preventing their contact with the epicuticle. The release of surface antigens also may be important in allowing larvae to evade the host's immune response by facilitating the removal of antibody and eosinophils from the larval surface.

Animals↗