Association of FCGR3A and FCGR3B haplotypes with rheumatoid arthritis and primary Sjögren's syndrome [POSTER PRESENTATION]

Background Rheumatoid arthritis (RA) is an autoimmune disease that is thought to arise from a complex interaction between multiple genetic factors and environmental triggers. We have previously demonstrated an association between a Fc gamma receptor (FcγR) haplotype and RA in a cross-sectional cohort of RA patients. We have sought to confirm this association in an inception cohort of RA patients and matched controls. We also extended our study to investigate a second autoanti-body associated rheumatic disease, primary Sjögren's syndrome (PSS). Methods The FCGR3A-158F/V and FCGR3B-NA1/NA2 functional polymorphisms were examined for association in an inception cohort of RA patients (n = 448), and a well-characterised PSS cohort (n = 83) from the United Kingdom. Pairwise disequilibrium coefficients (D') were calculated in 267 Blood Service healthy controls. The EHPlus program was used to estimate haplotype frequencies for patients and controls and to determine whether significant linkage disequilibrium was present. A likelihood ratio test is performed to test for differences between the haplotype frequencies in cases and controls. A permutation procedure implemented in this program enabled 1000 permutations to be performed on all haplotype associations to assess significance. Results There was significant linkage disequilibrium between FCGR3A and FCGR3B (D' = -0.445, P = 0.001). There was no significant difference in the FCGR3A or FCGR3B allele or genotype frequencies in the RA or PSS patients compared with controls. However, there was a significant difference in the FCGR3A-FCGR3B haplotype distributions with increased homozygosity for the FCGR3A-FCGR3B 158V-NA2 haplotype in both our inception RA cohort (odds ratio = 2.15, 95% confidence interval = 1.1–4.2 P = 0.027) and PSS (odds ratio = 2.83, 95% confidence interval = 1.0–8.2, P = 0.047) compared with controls. The reference group for these analyses comprised individuals who did not possess a copy of the FCGR3A-FCGR3B 158V-NA2 haplotype. Conclusions We have confirmed our original findings of association between the FCGR3A-FCGR3B 158V-NA2 haplotype and RA in a new inception cohort of RA patients. This suggests that there may be an RA-susceptibility gene at this locus. The significant increased frequency of an identical haplotype in PSS suggests the FcγR genetic locus may contribute to the pathogenesis of diverse autoantibody-mediated rheumatic diseases

Automated Correlative Light and Electron Microscopy using FIB-SEM as a tool to screen for ultrastructural phenotypes

In Correlative Light and Electron Microscopy (CLEM), two imaging modalities are combined to take advantage of the localization capabilities of light microscopy (LM) to guide the capture of high-resolution details in the electron microscope (EM). However, traditional approaches have proven to be very laborious, thus yielding a too low throughput for quantitative or exploratory studies of populations. Recently, in the electron microscopy field, FIB-SEM (Focused Ion Beam -Scanning Electron Microscope) tomography has emerged as a flexible method that enables semi-automated 3D volume acquisitions. During my thesis, I developed CLEMSite, a tool that takes advantage of the semi-automation and scanning capabilities of the FIB-SEM to automatically acquire volumes of adherent cultured cells. CLEMSite is a combination of computer vision and machine learning applications with a library for controlling the microscope ( product from a collaboration with Carl Zeiss GmbH and Fibics Inc.). Thanks to this, the microscope was able to automatically track, find and acquire cell regions previously identified in the light microscope. More specifically, two main modules were implemented. First, a correlation module was designed to detect and record reference points from a grid pattern present on the culture substrate in both modalities (LM and EM). Second, I designed a module that retrieves the regions of interest in the FIB-SEM and that drives the acquisition of image stacks between different targets in an unattended fashion. The automated CLEM approach is demonstrated on a project where 3D EM volumes are examined upon multiple siRNA treatments for knocking down genes involved in the morphogenesis of the Golgi apparatus. Additionally, the power of CLEM approaches using FIB-SEM is demonstrated with the detailed structural analysis of two events: the breakage of the nuclear envelope within constricted cells and an intriguing catastrophic DNA Damage Response in binucleated cells. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible and that EM can provide incredible insights to guide new biological discoveries

A systems approach to determine how Toxoplasma gondii Infection causes neuropsychiatric disease

This thesis was previously held under moratorium from 16/03/2020 to 16/03/2022T. gondii infection acquired during life has been associated with psychoneurological disease in humans and behavioural changes in mice. However, less is known about the potential of congenitally acquired T. gondii infection, or for maternal T. gondii infection induced immune activation, to cause psychoneurological disease. The studies described herein, using LCMS (Liquid chromatography–mass spectrometry) demonstrate that adult acquired infection alters the neurochemistry and transcriptome of the brains of BALB/c mice. Notable changes to tryptophan, purine, arginine and carnitine metabolism were observed in infected mice. Congenitally infected and mice exposed to the maternal immune response to T. gondii, but not congenitally infected were found to have decreased mobility compared with control mice. Congenital T. gondii infection resulted in similar alterations in the neurochemistry of mice as seen in adult acquired infections. Some of these changes were observed, including tryptophan metabolism in mice exposed to the maternal immune response to T. gondii, but not congenitally infected. Both adult acquired T. gondii and congenital infection altered the brain transcriptome of mice relative to control uninfected mice with notable changes seen to transcripts of many immunologically important genes and enzymes in some of the metabolic pathways identified by LCMS. In addition, both adult acquired T. gondii infection, congenital infection and maternal exposure to different degrees were found to induce changes in a number of additional transcripts previously associated with psychoneurological diseases. These results demonstrate that maternal exposure to T. gondii infection during pregnancy induces a subset of neurochemical and transcriptomic changes found in mice with adult acquired and congenital T. gondii infection. The results therefore reinforce the potential of maternal immune activation to affect psychoneurological diseases and implicate T. gondii as a potential aetiological agent of this process.T. gondii infection acquired during life has been associated with psychoneurological disease in humans and behavioural changes in mice. However, less is known about the potential of congenitally acquired T. gondii infection, or for maternal T. gondii infection induced immune activation, to cause psychoneurological disease. The studies described herein, using LCMS (Liquid chromatography–mass spectrometry) demonstrate that adult acquired infection alters the neurochemistry and transcriptome of the brains of BALB/c mice. Notable changes to tryptophan, purine, arginine and carnitine metabolism were observed in infected mice. Congenitally infected and mice exposed to the maternal immune response to T. gondii, but not congenitally infected were found to have decreased mobility compared with control mice. Congenital T. gondii infection resulted in similar alterations in the neurochemistry of mice as seen in adult acquired infections. Some of these changes were observed, including tryptophan metabolism in mice exposed to the maternal immune response to T. gondii, but not congenitally infected. Both adult acquired T. gondii and congenital infection altered the brain transcriptome of mice relative to control uninfected mice with notable changes seen to transcripts of many immunologically important genes and enzymes in some of the metabolic pathways identified by LCMS. In addition, both adult acquired T. gondii infection, congenital infection and maternal exposure to different degrees were found to induce changes in a number of additional transcripts previously associated with psychoneurological diseases. These results demonstrate that maternal exposure to T. gondii infection during pregnancy induces a subset of neurochemical and transcriptomic changes found in mice with adult acquired and congenital T. gondii infection. The results therefore reinforce the potential of maternal immune activation to affect psychoneurological diseases and implicate T. gondii as a potential aetiological agent of this process

Advances in Image Processing, Analysis and Recognition Technology

For many decades, researchers have been trying to make computers’ analysis of images as effective as the system of human vision is. For this purpose, many algorithms and systems have previously been created. The whole process covers various stages, including image processing, representation and recognition. The results of this work can be applied to many computer-assisted areas of everyday life. They improve particular activities and provide handy tools, which are sometimes only for entertainment, but quite often, they significantly increase our safety. In fact, the practical implementation of image processing algorithms is particularly wide. Moreover, the rapid growth of computational complexity and computer efficiency has allowed for the development of more sophisticated and effective algorithms and tools. Although significant progress has been made so far, many issues still remain, resulting in the need for the development of novel approaches

O-linked beta N-acetylglucosamine (O-GlcNAc) post-translational modifications govern axon regeneration

Axonal regeneration within the mammalian central nervous system following traumatic damage is limited and interventions to enable regrowth is a crucial goal in regenerative medicine. The nematode Caenorhabditis elegans is an excellent model to identify the intrinsic genetic programs that govern axonal regrowth. Here we demonstrate that alterations in O-linked N- beta-acetylglucosamine (O-GlcNAc) post-translational modifications of proteins can increase the regenerative potential of individual neurons. O-GlcNAc are single monosaccharide protein modifications that occur on serines/threonines in nucleocytoplasmic compartments. Changes in O-GlcNAc levels serve as a sensor of cellular nutrients and acts in part through the insulin-signaling pathway. Loss of O-GlcNAc via mutation of the O-GlcNAc Transferase (OGT), the enzyme that adds O-GlcNAc onto target proteins, enhances regeneration by 70%. Remarkably, hyper-O-GlcNAcyation via mutation of the O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from target proteins, also enhances regeneration by 40%. Our results shed light on this apparent contradiction by demonstrating that O-GlcNAc enzyme mutants differentially modulate the insulin-signaling pathway. OGT mutants act through AKT1 to modulate glycolysis. In contrast, OGA mutants act through the FOXO/DAF-16 transcription factor to improve the mitochondrial stress response. These findings reveal for the first time the importance of O-GlcNAc post-translational modifications in axon regeneration and provide evidence that regulation of metabolic programs can dictate the regenerative capacity of a neuron.2021-02-20T00:00:00

Genetic, epigenetic and functional analysis of tumorigenesis in neurofibromatosis type 1 (NF1).

Neurofibromatosis Type 1 (NF1) is an autosomal dominant disorder caused by constitutional inactivation of the NF1 gene. NF1 is associated with extreme phenotypic variability and results in an increased risk of developing benign and malignant peripheral nerve sheath tumours (MPNSTs). The molecular mechanisms that underlie NF1 tumorigenesis are poorly understood although inactivation of additional loci in conjunction with NF1 mutations is postulated to be involved. A combinative approach encompassing genetic, epigenetic and functional analysis was employed to dissect the molecular mechanisms responsible for tumorigenesis and progression to malignancy in NF1. Genetic analysis of benign cutaneous neurofibromas from NF1 patients with a high tumour burden was undertaken involving PCR, sequencing, Loss of Heterozygosity (LOH) and Multiplex Ligation-dependent Probe Amplification (MLPA) at the NF1 locus. In addition, Microsatellite Instability (MSI) analysis and molecular analysis of the TP53, RB1, CDKN2A and MMR genes was performed in a subset of these tumours. It was revealed that a small proportion of tumours from such patients harbour LOH of the TP53 and RB1 genes, a novel finding which indicates that additional modifying loci may underlie the development of multiple cutaneous neurofibromas in patients with a high tumour burden. Furthermore, Schwann cell culture and Laser Capture Microdissection were used to enhance the somatic mutation detection rate in the NF1 gene. Pyrosequencing methylation analysis was employed to determine whether NF1 gene methylation was associated with NF1 tumorigenesis. Promoter methylation of potential modifying genes RASSF1A and MGMT was also assessed. Results of this study demonstrate that methylation of the NF1 and MGMT genes is unlikely to contribute to NF1 tumorigenesis. RASSF1A promoter methylation was significantly higher in tumour samples in comparison to controls. RASSF1A promoter methylation was correlated with a reduction in RASSF1A gene expression, as evidenced by 5aza2dc treatment. Non-synonymous missense mutations account for 15% of lesions in the NF1 germlihe and somatic mutational spectrum although without a suitable functional test, their pathogenicity cannot be defined with certainty. Site directed mutagenesis, a Ras ELISA and bioinformatic analysis were utilised to develop a novel functional assay to assess the potential pathogenicity of NF1 gap related domain (GRD) missense variants. Of the 16 mutations functionally analysed, 11 were found to be pathogenic. A panel of 14 candidate genes were found to be aberrantly expressed in 14 tumours and 5 MPNST tumour-derived cell lines by relative quantification. A number of genes in the Rho-GTPase pathway including RAC1 and ROCK2 are over-expressed in these tumours, suggesting that these genes might be critical for survival of MPNST cells. ShRNA (short hairpin RNA) for RAC1 and ROCK2 were used in adhesion, migration, invasion and wound healing assays on control an d MPNST derived cell lines. RAC1 and ROCK2 knockdown in MPNST cell lines resulted in a significant increase in cell adhesion and a reduction in wound healing, migratory and invasive activity. The cMET gene was also over-expressed in MPNSTs. Treatment of MPNST cell lines with cMET inhibitors also resulted in a reduction of cell growth and migration. This study has identified several novel therapeutic targets for NF1-associated malignancies

The inhibitor of differentiation-2 promotes synovial fibroblast-dependent osteoclastogenesis in rheumatoid arthritis

Objectives: Despite indirect evidence suggesting that low oxygen levels might occur in the rheumatoid arthritis (RA) synovium, direct proof of the presence of hypoxia in the arthritic synovium as well as the relevance of low oxygen levels for joint destruction is lacking. The aim of this study was to analyse the distribution of hypoxia in arthritic joints and to evaluate the molecular effects of the hypoxic environment on the phenotype of RA synovial fibroblasts (SF).<p></p> Methods: The hypoxia marker EF-5 was applied in mice with the collagen-induced arthritis (CIA). Expression profile analysis with hypoxic and normoxic SF was performed using subtractive hybridization and microarray. The expression of the inhibitor of differentiation-2 (Id-2), CD68 (macrophage marker) and prolyl hydroxylase (fibroblast marker) was evaluated by immunohistochemistry on synovial tissues from RA, osteoarthritis patients and CIA mice. To evaluate the function of Id-2 in SF, cells were transfected with the pcDNA3.1 containing cDNA for Id-2 or Id-2-specific siRNA or mock controls. The expression of Id-2 and genes regulated by Id-2 in transfected SF was evaluated by SYBR Green real-time PCR and western blot. SF stably transfected with Id-2 were cocultured with bone marrow cells in a transwell system. The expression of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin were measured by real-time PCR. The development of osteoclasts was evaluated by visualization of the activity of tartrate-resistant acid phosphatase.<p></p> Results: Using the hypoxia marker EF-5 we found that in mice with CIA, synovial cells invading bone and cartilage are exposed to reduced oxygen levels. Expression profile studies identified Id-2 as being upregulated under low oxygen conditions. In addition, IL-1beta stimulation increased the expression of Id-2 in these cells. Histological studies of RA synovium and CIA synovium showed strong expression of Id-2 in SF at sites of synovial invasion into bone. Overproduction of Id-2 in SF by stable transfection triggered the expression of several genes promoting osteoclastogenesis, including BMP-2, PTHrP, Wnt5a and vascular endothelial growth factor. Conversely, the suppression of endogenous Id-2 led to the downregulation of the expression of these molecules. Consistent with these findings coculture of Id-2 transfected SF with bone marrow cells increased the expression of the osteoclast differentiation factor RANKL, and decreased the expression of the osteoclast inhibitory factor osteoprotegerin in bone marrow stromal cells, which was followed by an increase in the number of osteoclasts.<p></p> Conclusion: Taken together, our data provide evidence that hypoxia is present at sites of synovial invasion in RA and that Id-2 induced by hypoxia contributes at these sites to joint destruction by promoting SF-dependent osteoclastogenesis