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P Adamson

Publications and source records attributed to P Adamson.

At least 19 recordsLinked to original sources
PubMed2002

Long-term preservation of cortically dependent visual function in RCS rats by transplantation.

Cell transplantation is one way of limiting the progress of retinal degeneration in animal models of blinding diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Here we transplanted a human retinal pigment epithelial (RPE) cell line into the subretinal space of one such model, the Royal College of Surgeons (RCS) rat, and showed, using head tracking to moving stripes and pattern discrimination in conjunction with single-unit cortical physiology, that cortically mediated vision can be preserved with this treatment.

Animals↗
PubMed2001

Subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats.

Royal College of Surgeons rats are genetically predisposed to undergo significant visual loss caused by a primary dysfunction of retinal pigment epithelial (RPE) cells. By using this model, we have examined the efficacy of subretinal transplantation of two independent human RPE cell lines each exhibiting genetic modifications that confer long-term stability in vitro. The two cell lines, a spontaneously derived cell line (ARPE19) and an extensively characterized genetically engineered human RPE cell line (h1RPE7), which expresses SV40 large T (tumor) antigen, were evaluated separately. Both lines result in a significant preservation of visual function as assessed by either behavioral or physiological techniques. This attenuation of visual loss correlates with photoreceptor survival and the presence of donor cells in the areas of rescued photoreceptors at 5 months postgrafting (6 months of age). These results demonstrate the potential of genetically modified human RPE cells for ultimate application in therapeutic transplantation strategies for retinal degenerative diseases caused by RPE dysfunction.

Animals↗
PubMed2001

Cross-linking of brain endothelial intercellular adhesion molecule (ICAM)-1 induces association of ICAM-1 with detergent-insoluble cytoskeletal fraction.

Intercellular adhesion molecule (ICAM)-1 plays a vital role in the process of leukocyte transmigration through endothelial cell (EC) barriers and has been shown to mediate signal transduction events in ECs induced either by its cross-linking or by the binding of T lymphocytes. Immunoblotting of ICAM-1 of Triton X-100 detergent fractions demonstrated that the majority of ICAM-1 was contained within the detergent-soluble fraction (noncytoskeletal associated) under basal conditions. After cross-linking of endothelial ICAM-1 with monoclonal antibody or coculture with T lymphocytes, EC ICAM-1 was observed to partition with a Triton X-100-insoluble (cytoskeletal associated) fraction in a dose- and time-dependent manner. Redistribution of ICAM-1 was specific, inasmuch as no association with the Triton X-100-insoluble fraction was observed after cross-linking of vascular cell adhesion molecule-1, nor did cross-linking of ICAM-1 result in a redistribution of the platelet and EC adhesion molecule. ICAM-1 association with the endothelial cytoskeleton after cross-linking was unaffected after treatment of the cells with cytochalasin D, C3-transferase, removal of extracellular calcium ions, or chelation of intracellular calcium ions. These data show that ICAM-1 colocalizes with the endothelial cytoskeleton and associates with a detergent-insoluble fraction after cross-linking.

Actins↗
PubMed2000

ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines.

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)gamma1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC's activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.

Actins↗
PubMed2000

Dexamethasone regulation of matrix metalloproteinase expression in CNS vascular endothelium.

Matrix metalloproteinases (MMPs) have been implicated in the early breakdown of the blood-brain barrier in neuroinflammatory disease. Although expression of these enzymes by resident glial cells and recruited immune cells has been described, altered expression of MMPs by the CNS vascular endothelial cells may also contribute to barrier disruption. In the present study, the in vitro expression of MMP-2 and -9 as well as tissue inhibitor of metalloproteinase (TIMP)-2 by rat CNS microvascular endothelial cells has been determined and compared with that by endothelial cell lines derived from rat aorta and high endothelial venules. Primary cultures of rat brain microvascular endothelial cells as well as the rat brain (GP8/3.9) and rat retinal endothelial (JG2/1) cell lines constitutively expressed MMP-2, -9 and TIMP-2. In vitro activation of CNS endothelium with the pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta, resulted in selective upregulation of MMP-9 activity, whereas no significant changes were seen in MMP-2 or TIMP-2 levels at 24 h. The addition of dexamethasone partially inhibited the cytokine-induced upregulation of MMP-9. Treatment of GP8/3.9 brain endothelial cells with active MMP-9 caused subtle but distinct alterations in the expression of the junctional protein, ZO-1. Quantitative differences found between CNS and non-CNS endothelial cells in the expression of both MMP-2 and -9, and in the expression of TIMP-2 demonstrate that CNS vascular endothelium is functionally distinct from non-CNS endothelium. These results suggest that cytokine-induced upregulation of MMP-9 expression by the CNS vascular endothelium may play a role in the pathogenesis of blood-brain and blood-retinal barrier breakdown in vivo.

Animals↗
PubMed2000

Cyclic adenosine monophosphate regulates the expression of the intercellular adhesion molecule and the inducible nitric oxide synthase in brain endothelial cells.

The authors studied whether cyclic AMP (cAMP), a widespread regulator of inflammation, modulates the cytokine-mediated expression of the intercellular adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and the inflammatory nitric oxide synthase 2 (NOS-2), in primary and immortalized brain endothelial cell cultures (GP8.3 cell line). When measured by enzyme-linked immunosorbent assay (ELISA), ICAM-1 was constitutively expressed and was up-regulated twofold by interleukin-1beta, with no effect of interferon-gamma. The NOS-2 activity, assessed by nitrite accumulation, was absent from untreated cultures but was induced by interleukin-1beta and interferon-gamma acting synergistically. Stimulation of cAMP-dependent pathways with forskolin or dibutyryl cAMP decreased ICAM-1 protein expression, whereas it increased NOS-2 protein expression. For both ICAM-1 and NOS-2, mRNA expression correlated with protein expression. Blockade of NOS activity with L-N-monomethylargiuine (L-NMMA) did not alter ICAM-1 expression, indicating that the nitric oxide released by NOS-2 did not cause the down-regulation of ICAM-1. Analysis of NFKB activation indicated that cAMP acted through a mechanism other than inhibition of nuclear translocation of NFKB. The authors conclude that cAMP modulates the expression of proinflammatory molecules in brain endothelium. This suggests that inflammatory processes at the blood-brain barrier in vivo may be regulated by perivascular neurotransmitters via cAMP.

Animals↗
PubMed2000

Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR.

A nested PCR protocol has been developed for the detection of and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. First-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, was followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). All products were detected by gel electrophoresis. The sensitivity of the assay was between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative. In summary, this protocol has demonstrated potential as a rapid diagnostic test in confirming the diagnosis of infection and also determining the Gram status of bacteria with high specificity and sensitivity.

Aqueous Humor↗
PubMed2000

Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR.

A protocol for the rapid detection of fungal DNA in ocular samples, derived from three species, Candida albicans, Aspergillus fumigatus, and Fusarium solani, has been developed. Two novel panfungal primers complementary to 18S rRNA sequences present in all three species were designed. Panfungal PCR was followed by three nested PCRs utilizing species-specific primers. PCR sensitivity ranged from 50 to 100 fg of free DNA and between one and two C. albicans organisms. In addition, we also developed a rapid and reliable DNA extraction protocol. This protocol minimized DNA loss during extraction, whilst removing compounds from vitreous and aqueous fluids that have previously been shown to have inhibitory effects on PCR. Preliminary results obtained after testing the protocol on three patient samples support culture results and medical history. However, one patient was PCR positive but culture negative, suggesting that the sensitivity of this protocol may exceed that of traditional culture techniques. This system, therefore, constitutes an additional protocol that may significantly aid patient management in cases where fungal endophthalmitis is suspected.

Aspergillosis↗
PubMed2000

Interactions between brain endothelial cells and human T-cell leukemia virus type 1-infected lymphocytes: mechanisms of viral entry into the central nervous system.

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.

Animals↗
PubMed2000

Comparison of PCR detection methods for B1, P30, and 18S rDNA genes of T. gondii in aqueous humor.

PURPOSE: Comparison of polymerase chain reaction (PCR) amplification of three Toxoplasma gondii genes in aqueous humor. METHODS: Nested PCRs carried out using published methods were optimized for maximum sensitivity and specificity. Five pairs of oligonucleotide primers, directed against the B1, P30, and ribosomal genes, were used and compared to determine which sequences were most effective in detecting T. gondii DNA. Methods were developed with DNA templates in water and were subsequently applied to both normal and inflamed aqueous. RESULTS: After one round of PCR amplification, P30 and ribosomal primers were able to detect 1 pg genomic T. gondii DNA. However, those directed against the B1 gene were able to detect 50 fg (approximately single tachyzoite). This level of sensitivity was also achieved using the P30 primers after a second round of PCR; however, only primers based on the B1 gene maintained this level of sensitivity in both normal and inflamed aqueous. B1-specific primers did not amplify sequences from fungal, bacterial, or human lymphocyte DNA. The sensitivity of T. gondii detection using B1 gene-specific primers was not compromised when large amounts of human lymphocyte DNA were present, and application to an ocular sample or retinal section from patients with toxoplasma chorioretinitis was successful in confirming the presence of T. gondii DNA. CONCLUSIONS: The B1 PCR protocol appears to be the most sensitive protocol in the detection of T. gondii DNA and has been successful in identification of T. gondii DNA in ocular fluids and retinal sections. This provides direct evidence of the presence of T. gondii within the eye and may therefore help in the management of toxoplasma retinochoroiditis.

Animals↗
PubMed2000

PCR-RFLP-mediated detection and speciation of bacterial species causing endophthalmitis.

PURPOSE: To determine the usefulness of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the identification and speciation of bacteria causing endophthalmitis. METHODS: PCR-RFLP was performed on 53 strains of 14 bacterial species (eight Gram positive and five Gram negative) collected from both keratitis and endophthalmitis patients. Two pairs of oligonucleotide primers based on the 16S rDNA gene were used to PCR-amplify 1.2- and 1.0-kb fragments of bacterial genomic DNA. RFLPs within the PCR product were used to speciate the organisms. RESULTS: The sensitivity of the nested PCR amplification reaction was one organism. All bacteria tested could be identified and speciated using RFLP analysis except for Escherichia coli and Serratia marcescens, which could not be interdifferentiated using RFLP. Molecular analysis of two vitreous samples from two eyes with typical signs of bacterial endophthalmitis confirmed the presence of E. coli in the vitreous from a culture-positive case with E. coli endophthalmitis and revealed the presence of Staphylococcus epidermidis in the vitreous of a culture-negative case. CONCLUSIONS: It is expected that this technique will provide a useful laboratory tool for future microbiologic diagnosis of patients presenting with endophthalmitis, especially for those eyes that prove culture negative.

Bacterial Typing Techniques↗
PubMed2000

PCR-based evidence of bacterial involvement in eyes with suspected intraocular infection.

PURPOSE: To assess the usefulness of polymerase chain reaction (PCR) in detection of bacteria in ocular samples. METHODS: Thirty-seven samples (aqueous and vitreous) were collected from 25 eyes showing typical symptoms and clinical signs of bacterial endophthalmitis. Ocular samples were also collected from 38 eyes that underwent routine surgery and from 15 eyes with intraocular inflammation due to nonbacterial causes. Panbacterial PCR was performed with a nested pair of 16S rRNA gene primers. Subsequent bacterial identification was completed for 18 paired samples (nine eyes) using restriction fragment length polymorphism (RFLP) and DNA sequencing. RESULTS: A 100% concordance was obtained between PCR and culture-positive samples. A PCR product was amplified from all 37 intraocular samples from eyes with suspected infection, whereas only 15 of 22 vitreous samples and 5 of 15 aqueous samples were culture positive. Culture-negative PCR-positive samples contained a preponderance of gram-negative bacterial sequences. Cloning and DNA analysis revealed 30 DNA sequences and included eight bacterial 16S rDNA, which currently remain unidentifiable. The presence of bacterial DNA was associated with an inflammatory response suggestive of infection and not colonization. All 15 samples from inflamed eyes with diverse uveitis diagnoses were PCR negative. The false-positive rate, due to contamination during sampling, was 5%. CONCLUSIONS: Bacterial DNA was detected in all patients with typical clinical signs of endophthalmitis. Gram-negative organisms seem to play a much more important role in the pathogenesis of this disease than previously thought. PCR-based techniques have great value in the confirmation of the diagnosis of bacterial endophthalmitis especially in culture-negative eyes.

Adult↗
PubMed2000

Use of PCR in endophthalmitis.

In eyes with suspected endophthalmitis, early diagnosis and appropriate treatment have been noted to be associated with a better visual outcome. Currently, however, confirmation of the diagnosis of endophthalmitis (bacterial and/or fungal) is dependent on conventional techniques of microbiological isolation of organisms which require between one and twelve days. Furthermore, many samples prove to be culture-negative. In order to improve the rate of microbiological diagnosis, PCR technology has been successfully applied to the detection of bacteria and fungi in ocular samples. Specific oligonucleotide primers have been used to detect the presence of pathogens, which have been subsequently identified using RFLP analysis, DNA sequencing, and/or cloning techniques. Results demonstrated that PCR-based methods are rapidly able to confirm the presence of pathogens with high specificity and sensitivity. PCR-based techniques have also been used to rule out with confidence the presence of pathogens, a unique advantage of this methodology. The use of molecular methods has significantly increased the number of intraocular samples from which a confirmed diagnosis is made and reduced the time to laboratory diagnosis. PCR-based methods promise to be useful diagnostic tools in the management of these patients, especially those from whom ocular samples prove to be culture-negative.

Aged↗
PubMed1999

Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway.

Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins.

ADP Ribose Transferases↗
PubMed1999

Cytokine production and mRNA expression by conjunctival T-cell lines in chronic allergic eye disease.

BACKGROUND: Activated CD4+ T cells, mast cells and eosinophils are the main cytokine-producing cell-types infiltrating the conjunctiva during chronic allergic eye diseases. Interactions between these cells are thought to play an important immunopathogenic role in these disorders (giant papillary conjunctivitis; vernal keratoconjunctivitis; atopic keratoconjunctivitis). OBJECTIVE: The objective was to compare the cytokine profiles of conjunctival T-cell lines from patients with different forms of chronic allergic eye disease. METHODS: T cells were isolated from conjunctival biopsies and non-specifically expanded into lines. The lines were immunophenotyped by flow cytometry. Cytokine production was quantified by immunoassays and more sensitive molecular techniques were used to investigate cytokine mRNA expression to identify the presence of interleukin (IL) -2, IL-4 and interferon (IFN) -gamma transcripts. RESULTS: Following four to six rounds of stimulation, the conjunctival T-cell populations were CD3+ (> 93%), with variable levels of CD4 and CD8 expression. All were HLA-DR+ (> 80%) with some HLA-DQ expression. Conjunctival T-cell lines from atopic keratoconjunctivitis produced selective increases in IFN-gamma, IL-10 and IL-13 (P<0.01), those from vernal keratoconjunctivitis produced increased IL-5 (P<0.01) whereas T-cell lines from giant papillary conjunctivitis produced only low levels of cytokines. IL-4 was only detected at the mRNA level and was expressed in four out of five T-cell lines in the vernal keratoconjunctivitis group. In contrast there was moderate to strong expression of IFN-gamma in five out of six T-cell lines in atopic keratoconjunctivitis. CONCLUSION: Different patterns of T-cell cytokine profiles were observed for each disease, with low-level, non-polarized cytokine production in giant papillary conjunctivitis, a TH2-like profile in vernal keratoconjunctivitis and a shift towards a TH1-like profile in atopic keratoconjunctivitis.

Adult↗