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1.
Clin Chim Acta ; 462: 60-64, 2016 Nov 01.
Article in English | MEDLINE | ID: mdl-27570064

ABSTRACT

3 Screen, a new ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8, has been developed and evaluated. In the assay serum samples were incubated (overnight; 2-8°C) in ELISA plate wells coated with GAD65, IA-2 and ZnT8 followed by a wash step and incubation with biotinylated GAD65, IA-2 and ZnT8 (1h; 2-8°C,). The assay was completed by addition of streptavidin-peroxidase and tetramethylbenzidine. Samples tested in the 3 Screen were also analysed in ELISAs and radiobinding assays for the three individual autoantibodies. 129/132 (98%) samples from newly diagnosed T1DM children and 1/100 non-diabetic children controls were positive in 3 Screen. There was good agreement between 3 Screen and the individual autoantibody assays. Dilution of positive samples showed good linearity characteristics. In the 2015 Islet Autoantibody Standardization Program 3 Screen achieved 94% sensitivity, 95.6% specificity and 0.948 area under curve by ROC analysis. 3 Screen provides a simple and sensitive method for combined measurement of three major diabetes associated autoantibodies in a single sample. The assay should be a useful tool for large scale population screening for individuals at risk of developing T1DM.


Subject(s)
Autoantibodies/blood , Cation Transport Proteins/blood , Diabetes Mellitus, Type 1/blood , Enzyme-Linked Immunosorbent Assay/methods , Glutamate Decarboxylase/blood , Islets of Langerhans/immunology , Receptor-Like Protein Tyrosine Phosphatases, Class 8/blood , Adolescent , Autoantibodies/immunology , Cation Transport Proteins/immunology , Child , Child, Preschool , Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 1/immunology , Enzyme-Linked Immunosorbent Assay/standards , Female , Glutamate Decarboxylase/immunology , Glutamate Decarboxylase/metabolism , Humans , Infant , Male , Receptor-Like Protein Tyrosine Phosphatases, Class 8/immunology , Receptor-Like Protein Tyrosine Phosphatases, Class 8/metabolism , Zinc Transporter 8
2.
Diabetes Technol Ther ; 18(11): 687-693, 2016 11.
Article in English | MEDLINE | ID: mdl-27552135

ABSTRACT

BACKGROUND: Testing for beta cell autoantibodies is used for wide-scale identification of early stages of type 1 diabetes. This requires suitable screening assays. We aimed to establish screening that utilized a first step assay (3 Screen) able to detect autoantibodies to the target antigens glutamic acid decarboxylase-65 (GAD), insulinoma-associated antigen 2 (IA-2), and zinc transporter 8 (ZnT8) to identify children positive for multiple beta cell autoantibodies. METHODS: An ELISA format was used where plates were coated with a mixture of recombinant GAD, IA-2, and ZnT8325W/R-dimer molecules. The performance was determined in venous blood from 686 first-degree relatives of patients with type 1 diabetes, and 200 patients at onset of type 1 diabetes, and applied as a screening assay in capillary blood from 33,639 general population children. RESULTS: The 3 Screen assay sensitivity for detecting autoantibody-positive patients at onset of type 1 diabetes was similar to that achieved by separate radiobinding assays (RBAs) for antibodies to GAD, IA-2, and ZnT8. Results in venous and capillary serum were correlated (R = 0.987). At a threshold corresponding to the 98th centile (29.1 U/mL) of all 33,639 capillary samples, the 3 Screen was positive in 123 samples with two or more RBA-positive antibodies to insulin, GAD, IA-2, or ZnT8, 146 with one antibody, and 479 that were RBA negative for beta cell autoantibodies. CONCLUSION: A 3 Screen ELISA was developed that was suitable for first step screening of multiple beta cell autoantibodies in capillary blood.


Subject(s)
Autoantibodies/blood , Capillaries , Diabetes Mellitus, Type 1/immunology , Enzyme-Linked Immunosorbent Assay/methods , Insulin-Secreting Cells/immunology , Adolescent , Cation Transport Proteins/immunology , Child , Female , Glutamate Decarboxylase/immunology , Humans , Male , Receptor-Like Protein Tyrosine Phosphatases, Class 8/immunology , Sensitivity and Specificity , Zinc Transporter 8
3.
Clin Chim Acta ; 447: 90-5, 2015 Jul 20.
Article in English | MEDLINE | ID: mdl-26006309

ABSTRACT

AIMS: A bridging-type ELISA for measuring autoantibodies to zinc transporter 8 (ZnT8A) was assessed using samples from different forms of diabetes mellitus. METHODS: ZnT8A were measured using an ELISA in patients with type 1 diabetes mellitus (T1DM; n=94), latent autoimmune diabetes of adulthood (LADA; n=51), type 2 diabetes mellitus (T2DM; n=59) and healthy blood donors (HBD; n=200). ZnT8A in ELISA and immunoprecipitation assays (IPA) using ZnT8 dimer (W325/R325) and monomers (W325, R325 and Q325) were compared. RESULTS: Inter- and intra-assay coefficients of variation (CV) were 7.1% and 1.7%, respectively (medium ZnT8A) and 8.5% and 2.7%, respectively (high ZnT8A). In the ELISA 51/94 (54.3%) T1DM, 16/51 (31.4%) LADA and 1/59 (1.7%) T2DM sera were ZnT8A positive. ROC analysis of T1DM and HBD for the ELISA showed 54% sensitivity and 99% specificity (cutoff 15u/mL) and AUC 0.80 (95% CI, 0.74-0.86). ELISA and IPA measurements were in very good agreement (r=0.856, k=0.889, n=204). Measurement of ZnT8A in addition to autoantibodies for GAD, IA-2 and insulin increased antibody positivity in T1DM by 4.3%, from 80.9% to 85.1%. CONCLUSIONS: The bridging-type ELISA is a convenient and reproducible method for determination of ZnT8A in serum. Measurement of ZnT8A increased autoantibody positivity in adult T1DM.


Subject(s)
Autoantibodies/blood , Autoantibodies/immunology , Cation Transport Proteins/immunology , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 2/blood , Enzyme-Linked Immunosorbent Assay/methods , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Cation Transport Proteins/chemistry , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 2/immunology , Female , Glutamate Decarboxylase/immunology , Humans , Insulin/immunology , Male , Middle Aged , Protein Multimerization , Protein Structure, Quaternary , Young Adult , Zinc Transporter 8
4.
Biomaterials ; 34(38): 10267-77, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24090839

ABSTRACT

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.


Subject(s)
Cystic Fibrosis/genetics , Lipids/chemistry , Lung/metabolism , Aerosols , Animals , Epithelium/metabolism , Female , Gene Transfer Techniques , Male , Sheep
5.
Br J Psychol ; 102(3): 443-63, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21751999

ABSTRACT

Despite evidence of widespread belief in conspiracy theories, there remains a dearth of research on the individual difference correlates of conspiracist ideation. In two studies, we sought to overcome this limitation by examining correlations between conspiracist ideation and a range of individual psychological factors. In Study 1, 817 Britons indicated their agreement with conspiracist ideation concerning the July 7, 2005 (7/7), London bombings, and completed a battery of individual difference scales. Results showed that stronger belief in 7/7 conspiracy theories was predicted by stronger belief in other real-world conspiracy theories, greater exposure to conspiracist ideation, higher political cynicism, greater support for democratic principles, more negative attitudes to authority, lower self-esteem, and lower Agreeableness. In Study 2, 281 Austrians indicated their agreement with an entirely fictitious conspiracy theory and completed a battery of individual difference measures not examined in Study 1. Results showed that belief in the entirely fictitious conspiracy theory was significantly associated with stronger belief in other real-world conspiracy theories, stronger paranormal beliefs, and lower crystallized intelligence. These results are discussed in terms of the potential of identifying individual difference constellations among conspiracy theorists.


Subject(s)
Culture , Delusions/psychology , Individuality , Personality , Adolescent , Adult , Austria , Female , Humans , Male , Middle Aged , Politics , Self Concept , United Kingdom
6.
Biomaterials ; 32(10): 2614-24, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21239054

ABSTRACT

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.


Subject(s)
Gene Transfer Techniques , Genes, Reporter/genetics , Luciferases/genetics , Luciferases/metabolism , Lung/metabolism , Animals , Bronchoalveolar Lavage Fluid , Cells, Cultured , Electricity , Gene Expression Profiling , Gene Expression Regulation , HEK293 Cells , Humans , Lipids/chemistry , Luciferases/blood , Mice , Polyethyleneimine/chemistry , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sheep , Time Factors , Transfection , Viruses/genetics , Whole Body Imaging
7.
Br J Health Psychol ; 15(Pt 4): 921-39, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20392339

ABSTRACT

OBJECTIVE: Although a number of recent health promotion interventions targeted at men have recognized the plurality of masculinities and adopted multifaceted approaches, in the main there continues to be a reliance on stereotypes of gendered behaviour that focus on hegemonic masculinities and a 'one-size-fits-all' approach to health care. The present study sought to overcome this limitation. DESIGN: The present study used a qualitative design, in which data were analysed using framework analysis. METHOD: A total of 82 middle-aged and older men, in a socially deprived area of Britain, took part in focus groups about health promotion. RESULTS: Analysis of focus group transcripts revealed four key themes: (1) that the 'doing' of gender in relation to health must be seen as contingent and in constant flux; (2) that, despite stereotypes of typical behaviour, men were keen to engage with health care services; (3) that men felt there were a number of barriers to help seeking, but generally welcomed the opportunity to discuss their health care needs, and; (4) that they were keen to see the above themes translated into directed advertising and health information for men. CONCLUSION: These results have practical implications for the way in which health promotion interventions target men, which we discuss in conclusion.


Subject(s)
Health Promotion , Men's Health , Needs Assessment , Patient Acceptance of Health Care , Adult , Aged , England , Focus Groups , Gender Identity , Humans , Male , Marketing of Health Services , Middle Aged , Patient Preference , Poverty , Professional-Patient Relations
8.
J Gene Med ; 12(1): 55-63, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19937989

ABSTRACT

BACKGROUND: When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs). METHODS: We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model. RESULTS: Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19% (range = 13%) RECs (n = 6). The nasal brushing method was superior, yielding 92% (range = 15%) RECs (n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels. CONCLUSIONS: Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Gene Expression Regulation , Nasal Cavity/pathology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Transgenes/genetics , Animals , Cell Separation , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fatty Acid-Binding Proteins/metabolism , Gene Transfer Techniques , Genetic Vectors/genetics , Humans , Lasers , Mice , Mice, Inbred C57BL , Microdissection , Nasal Cavity/metabolism , Nasal Septum/metabolism , Nasal Septum/pathology , Pronase/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
9.
Am J Respir Cell Mol Biol ; 43(1): 46-54, 2010 Jul.
Article in English | MEDLINE | ID: mdl-19648474

ABSTRACT

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.


Subject(s)
Gene Transfer Techniques , Nose/pathology , Animals , Bacterial Adhesion , Cystic Fibrosis/genetics , Cytomegalovirus/genetics , Enhancer Elements, Genetic , Female , Genetic Therapy/methods , Liposomes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Plasmids/metabolism , Promoter Regions, Genetic
10.
Mol Cell Probes ; 23(6): 272-80, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19615439

ABSTRACT

BACKGROUND: To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery. METHODS: We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR. RESULTS: The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells. CONCLUSION: We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis , Epithelial Cells/metabolism , Immunohistochemistry/methods , Transgenes , Antibodies/immunology , Cells, Cultured , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis/pathology , Cystic Fibrosis/therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/immunology , Epithelial Cells/pathology , Genetic Therapy/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Microscopy, Fluorescence , Nasal Mucosa/metabolism , Nasal Mucosa/pathology , Reproducibility of Results , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Transfection
11.
Mol Ther ; 15(2): 348-54, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17235313

ABSTRACT

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.


Subject(s)
Gene Transfer Techniques/instrumentation , Genetic Vectors/genetics , Lung/metabolism , Nebulizers and Vaporizers , Animals , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Female , Gene Expression , Gene Transfer Techniques/adverse effects , Genes, Reporter/genetics , Lung Diseases/etiology , Lung Diseases/pathology , Male , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sheep
12.
Am J Respir Cell Mol Biol ; 35(1): 72-83, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16498081

ABSTRACT

A panel of 11 human cystic fibrosis transmembrane conductance regulator (hCFTR) antibodies were tested in ovine nasal, tracheal, and bronchial epithelial brushings. Two of these, G449 (polyclonal) and MATG1104 (monoclonal), recognized hCFTR but did not cross react with endogenous sheep CFTR. This specificity allows immunologic detection of hCFTR expressed in gene transfer studies in sheep against the background of endogenous ovine CFTR, thus enhancing the value of the sheep as a model animal in which to study CFTR gene transfer. Studies on mixed populations of human and sheep nasal epithelial cells showed that detection of hCFTR by these two antibodies was possible even at the lowest proportion of human cells (1:100). The hCFTR gene was delivered in vivo by local instillation using polyethylenimine-mediated gene transfer to the ventral surface of the ovine trachea and hCFTR mRNA and protein levels scored in a blinded fashion. Despite abundant hCFTR mRNA expression, the number of cells expressing hCFTR protein detectable by G449 was low (approximately 0.006-0.05%). Immunohistochemistry for hCFTR in animals treated by whole-lung aerosol demonstrated positive cells in sections of tracheal epithelium and in distal conducting airways. The strategic use of hCFTR-specific antibodies supports the utility of the normal sheep as a model for hCFTR gene transfer studies.


Subject(s)
Antibodies/immunology , Cystic Fibrosis Transmembrane Conductance Regulator/immunology , Gene Transfer Techniques , Respiratory System/metabolism , Sheep/metabolism , Amino Acid Sequence , Animals , Antibody Specificity , Cell Count , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Epithelial Cells/cytology , Female , Gene Expression , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Species Specificity
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