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encodes the NF-κB regulatory protein A20. High-penetrance heterozygous mutations in cause a haploinsufficiency of A20 (HA20), inadequate inhibition of NF-κB pathway, and an early onset autoinflammatory disorder. However, the clinical phenotype of patients with HA20 varies greatly and clinical diagnoses prior to establishing the genetic cause, included both autoimmune and autoinflammatory conditions. Here, we present the first patient with HA20, who was previously diagnosed with AOSD but was later found to have a novel heterozygous variant in and who was successfully treated with anti-IL6 receptor biologic tocilizumab (RoActemra). We discovered a novel heterozygous mutation in c.1906C>T, not previously found in ExAC database. Further analysis shows that this single-nucleotide variant at the terminal residue of exon 7 produces an alternatively spliced mRNA resulting in p.His636fs 1. Additional genetic analysis of family members shows that this variant does segregate with the inflammatory clinical phenotypes. Subsequent functional test show that NF-κB activation, measured as intracellular phosphorylation of p65 in CD14 + monocytes, was more enhanced in the patient compared with healthy controls (HC) following stimulation with LPS. This was associated with higher production of inflammatory cytokines by the patients PBMC in response to LPS and ATP and enhanced activation of NLRP3 inflammasome complex. Furthermore, increased activation of NLRP3 inflammasome was evident systemically, since we detected higher levels of ASC specks in patients' sera compared with HC. Finally, we used population genetics data from GnomAD to construct a map of both genetic conservation and most probable disease-causing variants in which might be found in future cases of HA20.

The treatment of rheumatoid arthritis (RA) has been transformed with the introduction of biologic disease modifying anti-rheumatic drugs (bDMARD) and more recently, targeted synthetic DMARD (tsDMARD) therapies in the form of janus-kinase inhibitors. Nevertheless, response to these agents varies such that a trial and error approach is adopted; leading to poor patient quality of life, and long-term outcomes. There is thus an urgent need to identify effective biomarkers to guide treatment selection. A wealth of research has been invested in this field but with minimal progress. Increasingly recognized is the importance of evaluating synovial tissue, the primary site of RA, as opposed to peripheral blood-based investigation. In this mini-review, we summarize the literature supporting synovial tissue heterogeneity, the conceptual basis for stratified therapy. This includes recognition of distinct synovial pathobiological subtypes and associated molecular pathways. We also review synovial tissue studies that have been conducted to evaluate the effect of individual bDMARD and tsDMARD on the cellular and molecular characteristics, with a view to identifying tissue predictors of response. Initial observations are being brought into the clinical trial landscape with stratified biopsy trials to validate toward implementation. Furthermore, development of tissue based omics technology holds still more promise in advancing our understanding of disease processes and guiding future drug selection.

The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-κB, AP1 and C/EBPβ controls this CRE, which is strictly dependent on NF-κB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKα, known to be recruited by NF-κB to NF-κB-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-κB binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKKα detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.

BackgroundWe previously reported increased constitutive but unchanged induced CD4+ p-STAT3 in established RA versus healthy control (HC).1 This study aims to evaluate in early RA (ERA) (i) cell-subset constitutive and Cis (IL-6) and Trans (IL-6/sIL6R)-induced IL-6 signalling (ii) effect of tocilizumab (TCZ; +/-MTX) (iii) for an association between baseline phosphorylation intensities and TCZ (+/- MTX) response.Materials and methodsMultiparameter phosphoflow cytometry was performed on 20 treatment-naive, ERA patients randomised to TCZ monotherapy or TCZ-MTX to determine STAT3, Akt and Erk phosphorylation intensities. PBMCs were isolated and cryopreserved at baseline, and weeks 24 and 48 post-TCZ. HC samples (n=10) were also obtained. PBMCs were unmodulated, stimulated with IL-6 or soluble IL-6 receptor (sIL-6R) and median fluorescence intensities (MFI) were measured.Results Baseline data, HC and ERA Constitutive CD4+ p-STAT3 was numerically higher in ERA compared to HC, with non-significant increase following stimulation in both groups.There was no significant difference in constitutive monocyte p-STAT3 in HC vs ERA. Stimulation did not induce further increase in monocyte p-STAT3 in ERA.Constitutive CD4+ p-Akt was significantly higher in ERA versus HC (p=0.01), with no change post-stimulation. Pre/Post TCZ in ERA Constitutive CD4+ pSTAT3 expression did not alter following TCZ exposure.Statistically significant decreases in IL-6 stimulated CD4+ pSTAT3 expression from baseline to weeks 24 and 48 were observed (p=0.005 and 0.01 respectively); and similarly using IL-6/sIL-6R at week 48 (p=0.01).Constitutive monocyte p-STAT3 expression appeared to increase at week 24 compared to baseline (p=0.03); similar observation following stimulation with IL-6/sIL-6R at both weeks 24 and 48 was observed (p=0.01)TCZ led to non-significant decrease in constitutive and IL6-stimulated CD4+ p-Akt expression weeks 24 and 48. TCZ otherwise had no significant effect on p-Akt and p-Erk in other studied cell subsets.ConclusionsTCZ reduced induced IL-6 signalling via CD4+ STAT3. This preliminary analysis also suggests importance of monocyte subset and relevance of Akt in IL-6 signalling. Complete analysis of these data will clarify the relative roles and impact of IL-6R targeting further.ReferencesOuboussad L, Wong C, Hunt L, Emery P, McDermott MF, Buch MF. Investigating IL-6 pathway signalling kinetics in peripheral blood single cell subsets with tocilizumab therapy in patients with early rheumatoid arthritis. Ann Rheum Dis2016; 75(Suppl 1):A31.2-A31.

BackgroundWe previously reported increased constitutive but unchanged induced CD4+ p-STAT3 in established RA versus healthy control (HC).1 This study aims to evaluate in early RA (ERA) (i) cell-subset constitutive and Cis (IL-6) and Trans (IL-6/sIL6R)-induced IL-6 signalling (ii) effect of tocilizumab (TCZ; +/-MTX) (iii) for an association between baseline phosphorylation intensities and TCZ (+/- MTX) response.Materials and methodsMultiparameter phosphoflow cytometry was performed on 20 treatment-naïve, ERA patients randomised to TCZ monotherapy or TCZ-MTX to determine STAT3, Akt and Erk phosphorylation intensities. PBMCs were isolated and cryopreserved at baseline, and weeks 24 and 48 post-TCZ. HC samples (n=10) were also obtained. PBMCs were unmodulated, stimulated with IL-6 or soluble IL-6 receptor (sIL-6R) and median fluorescence intensities (MFI) were measured.Results Baseline data, HC and ERA Constitutive CD4+ p-STAT3 was numerically higher in ERA compared to HC, with non-significant increase following stimulation in both groups.There was no significant difference in constitutive monocyte p-STAT3 in HC vs ERA. Stimulation did not induce further increase in monocyte p-STAT3 in ERA.Constitutive CD4+ p-Akt was significantly higher in ERA versus HC (p=0.01), with no change post-stimulation. Pre/Post TCZ in ERA Constitutive CD4+ pSTAT3 expression did not alter following TCZ exposure.Statistically significant decreases in IL-6 stimulated CD4+ pSTAT3 expression from baseline to weeks 24 and 48 were observed (p=0.005 and 0.01 respectively); and similarly using IL-6/sIL-6R at week 48 (p=0.01).Constitutive monocyte p-STAT3 expression appeared to increase at week 24 compared to baseline (p=0.03); similar observation following stimulation with IL-6/sIL-6R at both weeks 24 and 48 was observed (p=0.01)TCZ led to non-significant decrease in constitutive and IL6-stimulated CD4+ p-Akt expression weeks 24 and 48. TCZ otherwise had no significant effect on p-Akt and p-Erk in other studied cell subsets.ConclusionsTCZ reduced induced IL-6 signalling via CD4+ STAT3. This preliminary analysis also suggests importance of monocyte subset and relevance of Akt in IL-6 signalling. Complete analysis of these data will clarify the relative roles and impact of IL-6R targeting further.ReferencesOuboussad L, Wong C, Hunt L, Emery P, McDermott MF, Buch MF. Investigating IL-6 pathway signalling kinetics in peripheral blood single cell subsets with tocilizumab therapy in patients with early rheumatoid arthritis. Ann Rheum Dis2016;75(Suppl 1):A31.2-A31.

The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-[kappa]B, AP1 and C/EBP[beta] controls this CRE, which is strictly dependent on NF-[kappa]B binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKK[alpha], known to be recruited by NF-[kappa]B to NF-[kappa]B-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-[kappa]B binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKK[alpha] detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.

Background and Objectives Rheumatoid arthritis (RA) is a polygenic disorder usually arising from combined genetic predisposition and environmental influences with associated dysfunctional immune responses. The X-box binding protein 1 (XBP1) transcription factor is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes including RA. XBP1 can also be activated in direct response to Toll-like Receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Our aim was to investigate the relevance of interactions between UPR and TLR signalling pathways in the serum and synovial fibroblasts (SFs) of patients with RA, using samples from healthy individuals and patients with osteoarthritis (OA) as controls Materials and Methods Peripheral blood mononuclear cells (PBMC) were obtained from control groups: healthy individuals (n = 24) and RA patients, comprising active disease (n = 47) and remission (n = 12). SFs from RA and OA patients were isolated by digestion of synovial biopsies. Gene expression profiling was performed using qPCR for the detection of sXBP1 and enzyme-linked immunosorbent assays (ELISA) to quantify levels of pro-inflammatory cytokines, IL-6 and TNF. siRNA targeting of XBP1 was used for knockdown experiments in SFs. Results We investigated the expression of ER stress response genes in patients with active RA and patients in remission. We show that TLR-dependent XBP1 activation is operative in the SFs of patients with active RA. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission (p = 0.01). Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SFs by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent IL-6 and TNF production. We also show that TNF itself induces sXBP1 in SFs, thus generating a potential feedback loop for sustained activation of these cells. Conclusions sXBP1 plays a central role in two different cellular processes that may first appear unconnected. However, linking inflammatory pathways with ER stress provides SFs with a timely, coordinated and protective response. XBP1 activation may therefore be a suitable target in the treatment of RA, since it forms a cornerstone of two different molecular processes implicated in the pathogenesis of RA.

The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF- Kappa B, AP1 and C/EBP beta controls this CRE, which is strictly dependent on NF- Kappa B binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKK alpha , known to be recruited by NF- Kappa B to NF- Kappa B-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF- Kappa B binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKK alpha detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.

Skin cancer is one of the most common forms of adult solid tumour. The incidence is increasing rapidly making skin cancer a major health problem in several countries. Cutaneous Malignant Melanoma (CMM) is the least common but the most life threatening type of skin cancers and is responsible for 90% of all skin malignancy associated deaths. The precise cellular and molecular etiology of malignant melanoma is quite complex and the molecular events directly related to melanoma progression are yet to be elucidated. However, recent advances in molecular biology have resulted in a clearer understanding of the cellular and molecular events of skin cancer development. The best-characterized locus associated with CMM development is the CDKN2A that maps to chromosome 9p21 and encodes for the cell cycle regulator p16 tumour suppressor gene (TSG), and is frequently inactivated in melanoma tumours. In addition to p16, other loci located in 9p21 appear to be important in CMM development and functional evidence for the presence of TSG(s) has been provided (Parris et al., 1999). The aim of our study is to contribute to the understanding of CMM development by isolating and characterising novel TSG(s) at this location. In order to pursue identifying potential TSG(s), we have developed several monochromosome hybrids using microcell mediated chromosome transfer, and evaluated the tumourigenicity of the constructed hybrids by anchorage independent growth in soft agar. For the molecular biology aspects, expression analysis of the genes in the 9p21 region was carried out by reverse transcription PCR. Potential candidate tumour suppressor genes were then carefully evaluated by generating expression profiles via conducting real time PCR. Experimental evidence is provided which supports the candidacy of interferon alpha 1 (IFNA1) as a tumour suppressor gene for melanoma development.