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1.
Cleft Palate Craniofac J ; : 10556656241236580, 2024 Mar 18.
Article in English | MEDLINE | ID: mdl-38500357

ABSTRACT

OBJECTIVE: Despite growing recognition that congenital craniofacial conditions have lifelong implications, psychological support for adults is currently lacking. The aim of this project was to produce a series of short films about living with craniosynostosis in adulthood, alongside a psychoeducational booklet. DESIGN: The resources were developed using multiple focus groups and meetings attended by researchers, patient representatives, a leading charitable organisation, an award-winning film production company, clinicians, and other experts in the field. RESULTS: An online mixed-methods survey was developed based on prior work to request feedback on the acceptability and utility of the resources from the craniosynostosis community. While data collection to evaluate the resources is ongoing, preliminary results (n = 36) highlight an acceptability rating of 100%. CONCLUSIONS: The resources developed represent a step forward in addressing the unmet information and support needs of adults with craniosynostosis and highlight the benefits of co-production in research.

2.
J Immunol ; 207(2): 555-568, 2021 07 15.
Article in English | MEDLINE | ID: mdl-34233910

ABSTRACT

As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ Mϕ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ Mϕ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ Mϕ and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-γ can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics.


Subject(s)
Cell Differentiation/physiology , Inflammation/metabolism , Interferon-gamma/metabolism , Macrophages/metabolism , Monocytes/metabolism , Psoriasis/metabolism , Biomarkers/metabolism , Cells, Cultured , Humans , Macrophage Colony-Stimulating Factor/metabolism , Phenotype , Proteomics/methods , Skin/metabolism
3.
Sci Rep ; 12(1): 3114, 2022 02 24.
Article in English | MEDLINE | ID: mdl-35210470

ABSTRACT

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.


Subject(s)
SARS-CoV-2
4.
J Biomol Screen ; 17(8): 1041-9, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22772056

ABSTRACT

The development of assays in single-addition mode is of great interest for screening purposes given the multiple advantages of minimizing the number of intervention steps. Binding assays seem to be more prone to this attractive format because no functional biological activity is taking place but instead a biophysical process, whose dynamics seem easier to control without introducing significant alterations, is happening. Therefore, single-addition assays based on the displacement of prebound labeled ligands can be conceived, but careful kinetic considerations must still be taken to maximize the sensitivity of the assay and to avoid jeopardizing the identification of compounds with slow-binding kinetics. This article shows the development of a single-addition, displacement-based binding assay intended to identify modulators that act by binding to the gabapentin site of the ion channel regulatory protein α2δ1. After studying the kinetics of gabapentin binding and the influence they might have on the assay sensitivity, the best conditions were identified, and the sensitivity was compared with that of the more classical two-additions competition-based assay. Although the present study focuses on α2δ1 and its interaction with gabapentin, the rationale and the methodology followed are of broad purpose and can be applied to virtually every binding assay.


Subject(s)
Amines/pharmacology , Calcium Channels, N-Type/metabolism , Cyclohexanecarboxylic Acids/pharmacology , High-Throughput Screening Assays , Radioligand Assay , gamma-Aminobutyric Acid/pharmacology , Amines/pharmacokinetics , Binding, Competitive , Cyclohexanecarboxylic Acids/pharmacokinetics , Gabapentin , HEK293 Cells , Humans , Kinetics , Ligands , Models, Chemical , Protein Binding , Scintillation Counting , gamma-Aminobutyric Acid/pharmacokinetics
5.
J Immunol ; 178(9): 5533-42, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17442935

ABSTRACT

Different signals in addition to the antigenic signal are required to initiate an immunological reaction. In the context of sulfamethoxazole allergy, the Ag is thought to be derived from its toxic nitroso metabolite, but little is known about the costimulatory signals, including those associated with dendritic cell maturation. In this study, we demonstrate increased CD40 expression, but not CD80, CD83, or CD86, with dendritic cell surfaces exposed to sulfamethoxazole (250-500 microM) and the protein-reactive metabolite nitroso sulfamethoxazole (1-10 microM). Increased CD40 expression was not associated with apoptosis or necrosis, or glutathione depletion. Covalently modified intracellular proteins were detected when sulfamethoxazole was incubated with dendritic cells. Importantly, the enzyme inhibitor 1-aminobenzotriazole prevented the increase in CD40 expression with sulfamethoxazole, but not with nitroso sulfamethoxazole or LPS. The enzymes CYP2C9, CYP2C8, and myeloperoxidase catalyzed the conversion of sulfamethoxazole to sulfamethoxazole hydroxylamine. Myeloperoxidase was expressed at high levels in dendritic cells. Nitroso sulfamethoxazole immunogenicity was inhibited in mice with a blocking anti-CD40L Ab. In addition, when a primary nitroso sulfamethoxazole-specific T cell response using drug-naive human cells was generated, the magnitude of the response was enhanced when cultures were exposed to a stimulatory anti-CD40 Ab. Finally, increased CD40 expression was 5-fold higher on nitroso sulfamethoxazole-treated dendritic cells from an HIV-positive allergic patient compared with volunteers. These data provide evidence of a link between localized metabolism, dendritic cell activation, and drug immunogenicity.


Subject(s)
Anti-Infective Agents/pharmacology , Dendritic Cells/drug effects , Drug Hypersensitivity/immunology , Sulfamethoxazole/analogs & derivatives , Sulfamethoxazole/pharmacology , Animals , Anti-Infective Agents/immunology , Anti-Infective Agents/metabolism , Aryl Hydrocarbon Hydroxylases/metabolism , CD40 Antigens/analysis , CD40 Antigens/metabolism , CD40 Ligand/antagonists & inhibitors , Cytochrome P-450 CYP1B1 , Cytochrome P-450 CYP2C8 , Cytochrome P-450 CYP2C9 , Dendritic Cells/immunology , Granulocyte Colony-Stimulating Factor/metabolism , HIV Infections/immunology , Humans , Interleukin-3/metabolism , Mice , Mice, Inbred BALB C , Recombinant Fusion Proteins/metabolism , Recombinant Proteins , Sulfamethoxazole/immunology , Sulfamethoxazole/metabolism , Up-Regulation
6.
Drug Metab Dispos ; 34(6): 1063-9, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16531474

ABSTRACT

Drug-induced changes in expression of cytochrome P450 (P450) genes are a significant issue in the preclinical development of pharmaceuticals. For example, preclinically, P450 induction can affect safety studies by reducing the systemic exposure of a compound undergoing toxicological evaluation, thus limiting the exposure that can be safely investigated in patients. Therefore, the induction potential of candidate drugs has been studied as part of the drug development process, typically using protein and/or catalytic end points. However, measuring changes in the levels of mRNA using TaqMan technology offers the opportunity to investigate this issue with the advantages of better dynamic range and specific enzyme identification. Here, we describe the TaqMan application to study ex vivo the P450 gene induction in the rat. Initially, livers from rats dosed with the prototypic P450 inducers beta-napthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX), and clofibric acid (CLO) were analyzed for mRNA levels of CYP1A1, 1A2, 2B1, 2B2, 2E1, 3A2, 3A23, and 4A1 and compared with control animals. The maximum fold induction of mRNA varied: 2500-fold for CYP1A1 with BNF, 680-fold for CYP2B1 with PB, 59-fold for CYP3A23 with DEX, and 16-fold for CYP4A1 with CLO. This method was then applied to estimate the inductive potential of putative drug candidates undergoing rodent toxicological evaluation. We present a summary of these data that demonstrates the sensitivity and specificity of the TaqMan assay to distinguish between inducers and noninducers and that offers a highly specific alternative to the quantification of drug effects on P450 expression using immunodetection and substrate metabolism.


Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Liver/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Catalysis , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 CYP2B1/genetics , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Dexamethasone/pharmacology , Drug Evaluation, Preclinical , Enzyme Induction , Liver/drug effects , Liver/enzymology , Male , Phenobarbital/pharmacology , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Taq Polymerase , beta-Naphthoflavone/pharmacology
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