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Toll-like receptors (TLRs) are innate immune receptors that respond to both exogenous and endogenous stimuli and are suggested to contribute to the perpetuation of chronic inflammation associated with rheumatoid arthritis (RA). In particular, the endosomal TLRs 3, 7, 8, and 9 have more recently been postulated to be of importance in RA pathogenesis. In this study, pan inhibition of the endosomal TLRs by a phosphorothioate-modified inhibitory oligodeoxynucleotide (ODN) is demonstrated in primary human B cells, macrophages, and RA fibroblasts. Inhibition of TLR8 was of particular interest as TLR8 has been associated with RA pathogenesis in both human and murine arthritis models. ODN1411 competitively inhibited TLR8 signaling and was observed to directly bind to a purified TLR8 ectodomain, suggesting inhibition was through a direct interaction with the receptor. Addition of ODN1411 to human RA synovial membrane cultures significantly inhibited spontaneous cytokine production from these cultures, suggesting a potential role for one or more of the endosomal TLRs in inflammatory cytokine production in RA and the potential for inhibitory ODNs as novel therapies.

This project examined methodologies used to establish primary cell cultures from non-small cell lung carcinoma (NSCLC) tumours. The objective was to enhance the in vitro proliferative potential of these clinically important tumours, particularly the squamous cell carcinoma (SCC) subtype. Existing techniques were successfully employed and refined here, permitting short-term maintenance in vitro and resulting in the establishment of a novel lung SCC cell line, DLRP, which was charaterised herein. The ability to generate long-term NSCLC cultures and cell lines on a routine basis however, remained elusive. The apparent defecit of relevant growth factors in currently available media for NSCLC was addressed. To this end, two likely sources of growth stimulators for malignant epithelial cells were explored. The first approach pursued literature reports of growth-promoting activités in the malignant effusions (MEs) and preliminary evidence in this project detected the presence in these fluids, of both stimulatory and inhibitory activity for NSCLC growth (including DLRP) in vitro. However, the active stimulatory species did not appear to be tumour-type specific and was conceivably a serum-derived systemic species. The second option evolved from the first, with evidence for the production of ‘self-stimulating’ autocrine growth factors by the human epidermoid carcinoma cell line, HEp-2. A bioassay was developed using HEp-2, which detected the presence of growth stimulators in MEs and HEp-2 conditioned medium (CM), based on their ability to stimulate the colony growth in monolayer of low density HEp-2 cells. Two separate fractions of HEp-2 CM, prepared by ultrafiltration (UF), i.e a 10-30 kDa and a >30 kDa fraction, both exhibited autocrine stimulatory activity (ASA). These fractions were distinguished by differential sensitivities to proteinase treatment. Production of several growth factors by HEp-2 is indicated by ASA elution profiles from gel-filtration, hydrophobic interaction, and heparin affinity chromatographic separations. Both heparin-binding and non-heparin-binding ASA are present in HEp-2 CM, but the heterologus elution profile of the heparin-binding ASA indicates that more than one high affinity heparin-binding mitogen is involved. Neutralising antibodies suggest that at least 60% of the ASA in the 10-30 MW fraction is attributable to a bFGF-like factor, and a similar species accounts for 40% of the > 30 kDa activity. Antisense studies designed to inhibit bFGF gene expression in HEp-2 cells did not support these findings. The possibility remains that one or more of the less well characterised heparin-binding growth factors are involved in HEp-2 autocrine growth control.

International audience; QuestionA better understanding of the response of Sphagnum mosses and associated vascular plants to climate warming is relevant for predicting the carbon balance of peatlands in a warmer world. Open-top chambers (OTCs) have been used to investigate the effect on soil biogeochemical processes in peatlands, but little information is available on the effects of OTCs on microclimate conditions and the associated response of the plant community. We aimed to understand how simulated warming and differences in soil moisture affect plant species cover.LocationA Sphagnum-dominated peatlands in French Jura.MethodsWe used OTCs to measure the effect of a near-ground temperature increase (+1.5 °C on average) on vegetation dynamics over five growing seasons (2008–2012) in a Sphagnum-dominated peatland, in two adjacent microhabitats with different hydrological conditions – wet and dry. Microclimatic conditions and plant species abundance were monitored at peak biomass in years 1, 2, 3 and 5 and monthly during the plant growing season in year 5.ResultsThe response to warming differed between vascular plants and bryophytes, as well as among species within these groups, and also varied in relation to soil moisture. Andromeda polifolia abundance responded positively to warming, while Vaccinium oxycoccus responded negatively, and Eriophorum vaginatum showed a high resistance.ConclusionDepth of rooting of vascular plants appeared to control the response in plant abundance, while moss abundance depended on various other interacting factors, such as shading by the vascular plant community, precipitation and soil moisture.

Regulatory T cells control many aspects of the immune system by enforcing dominant negative responses on other immune cells. Defective regulatory T cell function has been linked to many autoimmune diseases, including rheumatoid arthritis. Cytotoxic T lymphocyte antigen 4 (CTLA-4) plays a critical role in the development and function of regulatory T cells. Moreover, CTLA-4 function has been shown to be defective in rheumatoid arthritis; however the exact mechanism by which reduced expression of CTLA-4 leads to an inability of regulatory T cells to suppress effector T cells is still not clear. In this thesis I show that regulatory T cells isolated from peripheral blood of patients with rheumatoid arthritis are unable to suppress the proliferation and IFN-γ cytokine secretion of effector T cells in vitro. This lack of suppression was associated with reduced expression of total and surface CTLA-4. The mechanism underlying this reduction was found to be through methylation of a key CpG in an NFAT binding site within the CTLA-4 promoter. Methylation of this site resulted in reduced NFAT binding, and hence a reduction in CTLA-4 transcription. The observation that epigenetic changes could be responsible, in part, for defective Treg function raises the possibility for therapeutic intervention using methylation inhibitors. CTLA-4 is a known inducer of the immunoregulatory enzyme, indolamine 2,3- dioxygenase (IDO), and in this study it was shown that regulatory T cells from patients with rheumatoid arthritis were unable to induce the expression and enzymatic activity of IDO-1 in co-cultures with antigen presenting cells. CTLA-4 blockade was found to impair the ability of Tregs in co-culture to induce the expression of IDO-1, thereby mimicking the phenotype of a RA Treg. These findings suggest that one of the consequences of the reduced CTLA-4 expression displayed by regulatory T cells in rheumatoid arthritis is a failure to induce IDO expression in antigen presenting cells. This could explain, at least in part, the reduced suppressive capacity of regulatory T cells in rheumatoid arthritis. In addition to CTLA-4, a number of other molecules associated with regulatory T cell suppression such as CREM, CREB, IL-10 and CXCL10 were also found to be dysregulated in regulatory T cells isolated from patients with rheumatoid arthritis. These results suggest that other molecules associated with regulatory T cell suppression may also be epigenetically controlled and provide an insight into the inability of regulatory T cells to maintain immunological homeostasis in patients with RA. Open Access