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Biomedical subjects

A D Dick

Publications and source records attributed to A D Dick.

At least 37 records · Page 2Linked to original sources
PubMed2001

Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium.

AIM: To investigate the efficacy of "ex vivo" adenoviral vector mediated gene transfection of human conjunctival epithelial cell as a possible route for gene therapy for the distribution of anti-inflammatory agents for the potential treatment of immune mediated ocular inflammatory disorders. METHODS: Human conjunctival cells (HCs) were cultured with various concentrations of recombinant adenoviral vectors carrying a reporter gene LacZ, GFP, or an immunomodulating cytokine vIL-10. vIL-10 in culture supernatant was detected by sandwich ELISA and biological activity was assessed by suppression of ConA stimulated splenocyte proliferation. X-gal and GFP expression was assessed by histochemistry. RESULTS: The extent of adenoviral vector mediated transfer of both reporter genes and vIL-10 was dose dependent. LacZ expression could be detected for at least 50 day after infection with multiple of infection (MOI) 200. Following AdCMVvIL-10 transduction, vIL-10 protein expression occurred between 4-6 days post-transduction, and was maintained at a detectable level for at least 1 month. Secreted vIL-10 showed biological activity, significantly inhibiting Con A induced splenocyte proliferation. Additionally, transfection of HCs with two Adv vectors, one carrying LacZ and the other carrying GFP, resulted in co-expression within a single cell. CONCLUSION: These results confirm previous successful adenoviral vector mediated gene transfer to HCs and further show that expression can be maintained. Furthermore the data show HCs can secrete biologically active vIL-10 that could be developed as a strategy to suppress immune mediated disorders. The successful co-transduction of HCs as described for other tissues, opens avenues to develop a multiple target gene therapy locally.

Adenoviridae↗
PubMed2001

Single dose intranasal administration of retinal autoantigen generates a rapid accumulation and cell activation in draining lymph node and spleen: implications for tolerance therapy.

BACKGROUND/AIMS: A single intranasal delivery of retinal autoantigen suppresses effectively experimental autoimmune uveoretinitis (EAU). To further unravel underlying mechanisms the authors wished to determine, firstly, the kinetics of antigen delivery and, secondly, the early cellular responses involved in the initial stages of nasal mucosal tolerance induction. METHODS: Flow cytometry, cell proliferation assays, and microscopy were used to track antigen following a single, intranasal dose of Alexa-488 labelled retinal antigen. RESULTS: A rapid accumulation of antigen within both superficial cervical lymph nodes (SCLN) and spleen was observed after 30 minutes. Significant proliferative responses to IRBP were elicited by 48 hours indicating that systemic priming of naive T cells to retinal antigen had occurred. Cell activation was further confirmed by immunoprecipitation studies, which demonstrated phosphorylation of STAT4 but not STAT6 in both lymph nodes and spleen. However, at 24 hours, STAT4 heterodimerisation with STAT 3 was only observed in spleen. CONCLUSIONS: The results provide novel evidence that following a single intranasal application rapid transfer of antigen occurs. Resulting T cell proliferation develops consequent to differential cell signalling in SCLN and spleen. Further understanding of these underlying cellular mechanisms, in particular as is inferred by the results the contribution of local versus systemic tolerance induction, may assist in strategies to clinically apply mucosal tolerance therapy successfully.

Animals↗
PubMed2001

Distribution of OX2 antigen and OX2 receptor within retina.

PURPOSE: OX2 is a member of the immunoglobulin superfamily expressed on a broad range of tissues including neurons of the central and peripheral nervous systems, thymocytes, and endothelium. The recently identified OX2 receptor (OX2R) is restricted to the surfaces of myeloid lineage cells, including microglia. Functional data have implicated the OX2-OX2R interaction as a myeloid downregulatory signal. The purpose of this study was to determine the distribution and extent of expression of OX2 and its receptor within the retina, a tissue developed to restrain immune-mediated inflammatory damage. METHODS: OX2 and OX2R monoclonal antibodies (mAbs) were used to determine OX2 and OX2R protein expression, respectively, by flow cytometry of isolated myeloid-derived cells from normal and inflamed rat retina and by immunohistochemistry of serial sections of rat retina. For comparison, distribution of OX2 was documented using species-specific monoclonal antibodies in mouse and human retina. No OX2R mAbs are available for mouse or human detection. RESULTS: OX2 was expressed on retinal vascular endothelium and glial fibrillary acidic protein (GFAP)-negative neurons in retina and optic nerve and on a subpopulation of CD45(+) perivascular and juxtavascular cells. Within normal retina, OX2R was not detected on myeloid-derived cells. During experimental autoimmune uveoretinitis (EAU), expression of both OX2 and OX2R was noted on infiltrating leukocytes. CONCLUSIONS: Taking these results of the distribution of OX2 in normal and OX2R in inflamed retina with other functional data of OX2-OX2R interaction, it is suggested that the OX2-OX2R interaction has the potential to contribute to a novel pathway that suppresses and limits immunologic inflammatory damage within the retina.

Animals↗
PubMed2001

CD69 expression on peripheral CD4+ T cells parallels disease activity and is reduced by mycophenolate mofetil therapy in uveitis.

PURPOSE: To assess the effects of mycophenolate mofetil (MMF) therapy on T helper cell activation status, using CD69 expression and cytokine profile with flow cytometry in relation to clinical activity in uveitis. METHODS: Patients with posterior or intermediate uveitis treated with MMF (n = 10), patients with active uveitis not treated with MMF and receiving no or minimal therapy (n = 10), and healthy volunteers (n = 21) had peripheral blood lymphocyte immunofluorescence analysis for T helper cell (CD4, CD3) markers, activation status (CD69), and intracellular cytokine (interleukin [IL]-2, interferon [IFN]-gamma, and IL-4) levels. Patients were compared before and during MMF therapy in relation to T helper cell activation and clinical activity. RESULTS: Patients with active uveitis not treated with MMF and receiving no or minimal therapy had increased frequency of CD69-positive CD4 T cells (10.5% +/- 4.6%, P = 0.0007) compared with healthy volunteers (3.3% +/- 2.7%). Of all patients receiving MMF therapy, only patients with moderate to severe uveitis activity in the pre-MMF treatment group (n = 5; 15.5% +/- 5.0%, P = 0.004) had increased frequency of CD69-positive CD4 T cells compared with healthy volunteers. During MMF therapy, a significant reduction in frequency of CD69-positive CD4 T cells occurred in patients with prior moderate to severe uveitis activity (to 8.9% +/- 3.8%, P = 0.04). Levels of CD69-positive CD4 T cells in patients who had had inactive or mildly active disease (n = 5) before and during MMF therapy were comparable with levels in healthy volunteers. No significant changes in cytokine levels were found between the patient and control groups. A significant association between changes in frequency of CD69-positive CD4 T cells and changes in visual acuity (P = 0.008) and changes in vitreal haze (binocular indirect ophthalmoscopy score; P = 0.01) was observed in MMF-treated patients with prior moderate to severe uveitis activity. CONCLUSIONS: Reduction in uveitis activity during MMF therapy correlates with reduction in frequency of peripheral blood CD69-positive CD4 cells. The frequency of CD69-positive CD4 T cells is a measure of activity in posterior uveitis and may guide adequate immunosuppression.

Adult↗
PubMed2000

Campath-1H therapy in refractory ocular inflammatory disease.

BACKGROUND: Standard therapy for severe, immune mediated, ocular inflammation has significant side effects, and may fail to control the disease. T cell directed monoclonal antibody (mAb) therapy can provide long term remission of inflammatory disease in experimental models. The Campath-1H mAb was administered to patients with severe, refractory, ocular inflammation. METHODS: 10 patients with severe, refractory, non-infectious ocular inflammatory disease were treated with Campath-1H mAb. This is a fully humanised mAb which recognises the pan-lymphocyte antigen CD52. RESULTS AND DISCUSSION: Following Campath-1H therapy, all 10 patients showed an initial resolution of their ocular symptoms and signs. Long lasting remissions were achieved in eight patients, in whom baseline immunosuppression could subsequently be reduced to minimal levels. The possible mechanisms of action of Campath-1H therapy are discussed.

Adult↗
PubMed2000

Prospective surveillance of sympathetic ophthalmia in the UK and Republic of Ireland.

AIMS: To establish current epidemiological data, risks, and interventional outcomes of newly diagnosed sympathetic ophthalmia (SO). METHODS: Prospective surveillance took place of all permanently employed ophthalmologists in the UK and Republic of Ireland by a monthly reporting card through the British Ophthalmological Surveillance Unit. Case ascertainment was made of newly diagnosed SO from July 1997 and questionnaire data were returned at baseline, 6 months, and 1 year after diagnosis. RESULTS: 23 patients with newly diagnosed SO were recruited over 15 months, corresponding to a minimum estimated incidence of 0.03/100 000. Baseline data were available on 18 patients, in whom SO occurred after surgery in 11 patients, after retinal surgery alone in six patients, and after accidental trauma in seven patients. 12 of the 16 patients with 1 year follow up had a visual acuity of 6/12 or better. Good visual outcome was related to prompt and adequate systemic immunosuppressive therapy. CONCLUSIONS: The incidence of sympathetic ophthalmia is very low. The main current risk is surgery, particularly retinal surgery, but visual prognosis is good if early diagnosis is made and rapid, adequate immunotherapy is commenced.

Adolescent↗
PubMed2000

Early treatment with cyclosporin in serpiginous choroidopathy maintains remission and good visual outcome.

AIMS: To describe management and clinical outcomes of serpiginous choroidopathy treated primarily with cyclosporin at a tertiary uveitis referral centre METHODS: A case series of 14 eyes of seven patients with serpiginous choroidopathy with follow up ranging from 1.3 to 13 years is described. All patients had fundus lesions consistent with serpiginous choroidopathy, were investigated for systemic disease, had fluorescein angiography, and were treated with combined immunosuppressive therapy including cyclosporin. RESULTS: No patients suffered significant loss of acuity after starting systemic immunosuppression with cyclosporin as the primary agent. All but one patient achieved remission and were able to stop medications with no recurrences in the follow up period. Side effects from cyclosporin were well tolerated and there were no serious complications from immunosuppression. CONCLUSIONS: Cyclosporin is a safe and effective option with which to manage serpiginous choroidopathy. Significantly, adequate immunosuppression can result in clinical remission and cessation of therapy in some patients.

Adult↗
PubMed2000

IFN-gamma and LPS-mediated IL-10-dependent suppression of retinal microglial activation.

PURPOSE: Human retinal microglia (MG) express constitutively major histocompatability complex (MHC) class II molecules and have thus been highlighted as potential immunocompetent antigen-presenting cells (APCs). This study was undertaken to characterize microglial expression of coaccessory molecules and the functional changes in antigen expression, cytokine production, migration, and phagocytosis after proinflammatory stimulation. METHODS: Fresh donor retinal MG were obtained and isolated using a percoll density gradient technique. Phenotypic characteristics used for isolating rodent microglia were applied and modified. Coaccessory molecule expression and intracellular cytokine production were assessed using three-color flow cytometric analysis in both freshly isolated and interferon (IFN)gamma-lipopolysaccharide (LPS)-stimulated MG. Using five-millimeter retinal explants in culture, microglial migratory behavior, changes in cell surface antigen expression and phagocytic activity were documented. RESULTS: MG could be clearly defined by the flow cytometric phenotype CD45low CD11b+ MHC class II+ CD86low CD40low. Freshly isolated MG showed mannose receptor-mediated uptake of dextran-FITC. MG migrated from explants, were adherent, and upregulated MHC class II expression. After IFNgamma-LPS stimulation of single-cell suspension of MG isolates, MHC class II expression was reduced, with an increase occurring in MG intracellular interleukin (IL)-10 and IL-10 production. Microglial migration from explants was reduced after IFNgamma-LPS stimulation. CONCLUSIONS: These results highlight both phenotypic and behavioral characteristics that support an antigen-processing and -presenting capability of freshly isolated MG. However, proinflammatory stimulation with IFNgamma-LPS induces an IL-10-mediated downregulation of cell surface antigen expression and loss of migratory and phagocytic activity. Therefore, although equipped to act as APCs, MG are able to rapidly modulate their own function and behavior and as a result may have the potential to limit inflammation.

Aged↗
PubMed2000

Analysis of the cellular infiltrate in the iris during experimental autoimmune encephalomyelitis.

PURPOSE: Previous studies have shown that experimental autoimmune encephalomyelitis (EAE) and anterior uveitis (AU) develop in Lewis rats immunized with myelin basic protein (MBP). The purpose of this study was to characterize the dynamics, distribution, and phenotype of infiltrating cells in the iris during EAE-associated AU. METHODS: Lewis rats were immunized with MBP emulsified in complete Freund's adjuvant (CFA) or with CFA alone. Cellular infiltration of the iris was analyzed at various time points by immunohistochemistry of wholemounts, flow cytometry, and immunoelectron microscopy, by using monoclonal antibodies specific for monocytes/macrophages (ED1), T lymphocytes (R73, W3.25, OX8), T-cell activation markers (OX39, OX40), granulocytes (HIS48), major histocompatibility complex (MHC) class II (OX6), and neurofilament (2H3). RESULTS: MBP-immunized rats showed development of characteristic monophasic EAE, followed, after resolution of paralysis, by mild self-limited AU. Initially, focal infiltrates of round MHC class II(+) and ED1(+) cells were found in the iris. During the course of AU, the midiris became massively infiltrated with ED1(+) monocytes-macrophages, R73(+) T cells, granulocytes (HIS48(+)), and MHC class II(+) cells. The influx of T cells consisted of CD4(+) and CD8(+) cells, of which only a small fraction (<14 and 11%, respectively) expressed activation markers. The infiltrating cells accumulated in proximity to myelinated and nonmyelinated nerve bundles and in the vicinity of blood vessels in the iris. No evidence was found for demyelination or nerve degradation. Neither EAE nor AU developed in CFA-treated control rats. CONCLUSIONS: These data show that EAE-associated AU is characterized by a transient mixed cellular infiltrate consisting of monocytes-macrophages, granulocytes, and CD4 and CD8 T cells. The preferential accumulation of inflammatory cells in the vicinity of nerve fibers suggests that AU in this model may result from autoreactivity to nerve antigens.

Animals↗
PubMed1999

Intranasal administration of retinal antigens induces transient T cell activation and apoptosis within drainage lymph nodes but not spleen.

Mechanisms of mucosal tolerance induction, including anergy/deletion and active suppression are frequently described as mutually exclusive; dependent upon nature, dose and route of antigen administration. We have previously described induction of low-dose tolerance with administration of retinal autoantigens via the nasorespiratory tract which is antigen-specific, suppresses both cell mediated immunity and ultimately tissue destruction in experimental autoimmune uveoretinitis (EAU) and is mediated by splenic-derived regulatory cells. The present data further shows that splenocytes or fractionated splenic T cells, which secrete IL-4 and IL-10 when stimulated with retinal antigen in vitro, and not regional drainage lymph node cells transfer tolerance to naïve animals. Analysis of apparent mechanistic differences shows that during intranasal antigen administration, the proportion of CD4(+)T cells within drainage lymph nodes increases, concurrent with a burst of IFN-gamma. Following subsequent antigen challenge, T cells downregulate alphabetaTCR expression and undergo apoptosis in regional drainage lymph nodes. An increase in functional Th2 cytokine activity was noted in both Con-A and retinal antigen stimulated lymph node cultures in tolerized animals. T cells from tolerized animals secreted IL-4, whereas IL-10 was secreted predominantly by the non-T cell population present equally in control and tolerized animals. Therefore, spleen derived regulatory cells which suppress Th1 responses and T cell deletion/apoptosis in regional drainage lymph nodes are mechanisms which co-exist in tolerant rats. Th2 cytokine production after immunization appears consequential to tolerance-induced Th1 suppression.

Administration, Intranasal↗
PubMed1999

Modulating phenotype and cytokine production of leucocytic retinal infiltrate in experimental autoimmune uveoretinitis following intranasal tolerance induction with retinal antigens.

BACKGROUND/AIM: Nasal administration of retinal antigens induces systemic tolerance which results in suppression of experimental autoimmune uveoretinitis (EAU) when subsequently exposed to antigen. The aim was to establish if tolerance induction alters retinal infiltrating leucocyte phenotype and cytokine profile in tolerised animals when there is significantly reduced tissue destruction despite immunisation with retinal antigen. METHODS: Female Lewis rats were tolerised by intranasal administration with retinal extract (RE) before immunisation with RE to induce EAU. Control animals were administered phosphate buffered saline (PBS) intranasally. Post immunisation, daily clinical responses were recorded and at the height of disease, retinas were removed and either infiltrating leucocytes isolated for flow cytometric phenotype assessment and intracellular cytokine production, or chorioretina processed for immunohistochemistry. Fellow eyes were assessed for cytokine mRNA by semiquantitative RT-PCR. RESULTS: Flow cytometric analysis showed that before clinical onset of EAU there is no evidence of macrophage infiltration and no significant difference in circulating T cell populations within the retina. By day 14 a reduced retinal infiltrate in tolerised animals was observed and in particular a reduction in numbers of "activated" (with respect to CD4 and MHC class II expression) macrophages. Immunohistochemistry confirmed these findings and additionally minimal rod outer segment destruction was observed histologically. Cytokine analysis revealed that both IL-10 mRNA and intracellular IL-10 production was increased in tolerised eyes 7 days post immunisation. Although by day 14 post immunisation, IL-10 production was equivalent in both groups, a reduced percentage of IFN-gamma + macrophages and IFN-gamma + CD4+ T cells with increased percentage of IL-4+ CD4+ T cells were observed in tolerised animals. CONCLUSIONS: Leucocytic infiltrate is not only reduced in number but its distinct phenotype compared with controls implies a reduced activation status of infiltrating monocyts to accompany increased IL-10 and reduced IFN-gamma production in tolerised animals. This modulation may in turn contribute towards protection against target organ destruction in EAU.

Animals↗
PubMed1999

Fas-Fas ligand-mediated apoptosis within aqueous during idiopathic acute anterior uveitis.

PURPOSE: Despite ocular immune privilege, (auto)immune-mediated acute anterior uveitis (AAU) is relatively common. However, although relapses of AAU are usually self-limiting, possible regulatory mechanisms remain undefined in humans. Experimentally, Fas-Ligand (FasL)-mediated apoptosis of Fas+ inflammatory cells contributes to the immune privilege within the anterior chamber and provides an explanation for the success of corneal allograft transplantation. Therefore, whether such mechanisms regulate the immune response in AAU was investigated. METHODS: Aqueous and peripheral blood samples from consecutive patients presenting with idiopathic AAU were obtained with consent. Leukocytic phenotype was analyzed by flow cytometry, and apoptosis was determined by both flow cytometry and TdT-dUTP terminal nick-end labeling analysis. Presence of soluble Fas and FasL was determined by western blot analysis and enzyme-linked immunosorbent assay and compared with control aqueous from patients undergoing cataract surgery. The ability of the aqueous to induce apoptosis in a Fas+ Jurkat cell line was also determined. RESULTS: During AAU aqueous-infiltrating Fas+ cells included CD3+ T cells and granulocytes, whereas FasL+ cells comprised predominantly of non-CD3+ T cells. Higher levels of functional soluble FasL were found in aqueous of AAU patients than in normal aqueous, capable of inducing apoptosis in 68.9% +/- 7.6% of Fas+ lymphoid cells. Compared with peripheral blood, the CD4+ T cells infiltrate within aqueous showed significantly increased CD69 and CD25(IL-2r) expression. Flow cytometric analysis of aqueous showed that 9.32% +/- 1.2% of infiltrating non-granulocyte CD45+ cells were apoptotic, confirmed as T cells on subsequent three-color flow cytometric analysis. CONCLUSIONS: Taken together with published experimental data, the present study provides evidence for FasL-mediated apoptotic cell death contributing to the local immune regulation of ocular inflammatory disease and provides a mechanism to account for the self-limiting clinical course of AAU.

Acute Disease↗