Relationship Between Adalimumab Concentrations, Antidrug Antibodies, and Disease Activity in Rheumatoid Arthritis: A Cross-Sectional Observational Study.

OBJECTIVES: To determine the influence of patient characteristics and disease activity on adalimumab (ADA) concentrations; to assess the relationships between ADA concentrations, the presence of antidrug antibodies (ADAb), and disease activity in rheumatoid arthritis (RA); and to determine the association between cytokine concentrations and ADA concentrations. METHODS: A cross-sectional study of people with RA receiving ADA for at least 4 weeks was undertaken. Disease activity was assessed by the Disease Activity Score in 28 joints (DAS28), with responders defined as DAS28 ≤ 3.2. Serum and plasma were obtained for ADA concentrations and ADAb, and a panel of cytokines were obtained for a subgroup. ADA concentrations were compared between demographic and clinical subgroups using ANOVA. The independent associations between clinical and demographic features were analyzed using a general linear model. Variables significantly associated with ADA concentrations from the univariate analyses were entered into multivariate analyses. RESULTS: Of the 156 participants, 69.2% were female and the mean age was 57.4 (SD 12.7) years. Multivariate analysis revealed that higher C-reactive protein (P < 0.001) and higher weight (P < 0.004) were independently associated with lower ADA concentrations. ADA concentrations were higher in those with DAS28 ≤ 3.2 compared to those with DAS28 > 3.2 (median 10.8 [IQR 6.4-20.8] mg/L vs 7.1 [IQR 1.5-12.6] mg/L, P < 0.001). There was a significant negative correlation between interleukin 6 (IL-6) and ADA concentrations (r = -0.04, P < 0.01). CONCLUSION: ADA concentration correlates negatively with markers of inflammatory disease activity in RA, including IL-6. ADA concentration in the range 5 to 7 mg/L over the dose interval are associated with better disease control.

Activated CD90/Thy-1 fibroblasts co-express the Δ133p53ß isoform and are associated with highly inflamed rheumatoid arthritis.

BACKGROUND: The p53 isoform Δ133p53ß is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53ß is driving aggressive disease in RA. METHODS: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53ß. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. RESULTS: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53ß mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53ß. CONCLUSIONS: Induction of Δ133p53ß in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53ß could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53ß could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

Plasma interleukin-23 and circulating IL-17A+IFNγ+ ex-Th17 cells predict opposing outcomes of anti-TNF therapy in rheumatoid arthritis.

OBJECTIVES: TNF-α inhibitors are widely used in rheumatoid arthritis (RA) with varying success. Response to TNF-α inhibition may reflect the evolution of rheumatoid inflammation through fluctuating stages of TNF-α dependence. Our aim was to assess plasma concentrations of Th-17-related cytokines and the presence of circulating effector T-cells to identify predictors of response to TNF-α inhibitors. METHODS: Ninety-three people with RA were seen prior to and 4-6 months after commencing etanercept or adalimumab. Plasma concentrations of Th17-related cytokines, circulating effector T-cells, their production of relevant transcription factors and intracellular cytokines were measured at baseline. EULAR response criteria were used to define poor (ΔDAS28 ≤ 1.2 and/or DAS28 > 3.2) and good (ΔDAS28 > 1.2 and DAS28 ≤ 3.2) responders. Multivariate logistic regression was used to identify predictors of response. RESULTS: Participants with plasma IL-23 present at baseline were more likely to be poor responders [15/20 (75%) of IL-23+ versus 36/73 (49.3%) of IL-23-; p = 0.041]. While frequencies of Th1, Th17, ex-Th17 and Treg cell populations were similar between good and poor responders to anti-TNF therapy, IL-17A+IFNγ+ ex-Th17 cells were more prevalent in good responders (0.83% of ex-TH17 cells) compared to poor responders (0.24% of ex-Th17 cells), p = 0.023. Both plasma IL-23 cytokine status (OR = 0.17 (95% CI 0.04-0.73)) and IL-17A+IFNγ+ ex-Th17 cell frequency (OR = 1.64 (95% CI 1.06 to 2.54)) were independently associated with a good response to anti-TNF therapy. Receiver operator characteristic (ROC) analysis, including both parameters, demonstrated an area under the ROC curve (AUC) of 0.70 (95% CI 0.60-0.82; p = 0.001). CONCLUSIONS: Plasma IL-23 and circulating IL-17A+IFNγ+ ex-Th17 cells are independently associated with response to anti-TNF therapy. In combination, plasma IL-23 and circulating IL-17A+IFNγ+ ex-Th17 cells provide additive value to the prediction of response to anti-TNF therapy in RA.

The contribution from interleukin-27 towards rheumatoid inflammation: insights from gene expression.

We aimed to assess expression of genes encoding the heterodimeric IL-27 cytokine and constituent subunits of the Il-27 receptor in rheumatoid arthritis (RA), including in extra-articular, subcutaneous rheumatoid nodules. Comparing between nodules and joint synovia, significantly elevated expression of IL27A within nodules, and comparable IL27B expression, identified nodules as a significant source of IL-27 in RA. T-lymphocytes were the main source of IL27RA transcript, and IL27RA expression correlated with a number of plasma cytokines, as well as tissue TNF expression in both nodules and RA synovia. In synovia, correlations between IL27A, IL27RA IL17A and CD21L expression, and significantly elevated expression of the genes encoding IL-27, associated the presence of IL-27 with B cell-dominated synovial inflammation. Impact from nodule derived IL-27 on systemic or synovial inflammation in RA remains unknown and further study of these implications is required. Our study raises questions regarding the appropriate circumstances for the blockade or administration of IL-27 as a potential therapeutic adjunct in RA.

Co-expression of CD21L and IL17A defines a subset of rheumatoid synovia, characterised by large lymphoid aggregates and high inflammation.

OBJECTIVE: To determine whether the expression of IL17A and CD21L genes in inflamed rheumatoid synovia is associated with the neogenesis of ectopic lymphoid follicle-like structures (ELS), and if this aids the stratification of rheumatoid inflammation and thereby distinguishes patients with rheumatoid arthritis that might be responsive to specific targeted biologic therapies. METHODS: Expression of IL17A and CD21L genes was assessed by RT-PCR, qRT-PCR and dPCR in synovia from 54 patients with rheumatoid arthritis. A subset of synovia (n = 30) was assessed by immunohistology for the presence of CD20+ B-lymphocytes and size of CD20+ B-lymphocyte aggregates as indicated by maximum radial cell count. The molecular profiles of six IL17A+/CD21L+ and six IL17A-/CD21L- synovia were determined by complementary DNA microarray analysis. RESULTS: By RT-PCR, 26% of synovia expressed IL17A and 52% expressed CD21L. This provided the basis for distinguishing four subgroups of rheumatoid synovia: IL17A+/CD21L+ (18.5% of synovia), IL17A+/CD21L- (7.5%), IL17A-/CD21L+ (33.3%) and IL17A-/CD21L- (40.7%). While the subgroups did not predict clinical outcome measures, comparisons between the synovial subgroups revealed the IL17A+/CD21L+ subgroup had significantly larger CD20+ B-lymphocyte aggregates (P = 0.007) and a gene expression profile skewed toward B-cell- and antibody-mediated immunity. In contrast, genes associated with bone and cartilage remodelling were prominent in IL17A-/CD21L- synovia. CONCLUSIONS: Rheumatoid synovia can be subdivided on the basis of IL17A and CD21L gene expression. Ensuing molecular subgroups do not predict clinical outcome for patients but highlight high inflammation and the predominance of B-lymphocyte mediated mechanisms operating in IL17A+/CD21L+ synovia. This may provide a rationale for more refined therapeutic selection due to the distinct molecular profiles associated with IL17A+/CD21L+ and IL17A-/CD21L- rheumatoid synovia.

Digital-PCR for gene expression: impact from inherent tissue RNA degradation.

Subtle molecular differences indicate the heterogeneity present in a number of disease settings. Digital-PCR (dPCR) platforms achieve the necessary levels of sensitivity and accuracy over standard quantitative RT-PCR (qPCR) that promote their use for such situations, detecting low abundance transcript and subtle changes from gene expression. An underlying requisite is good quality RNA, principally dictated by appropriate tissue handling and RNA extraction. Here we consider the application of dPCR to measures of gene expression in pathological tissues with inherent necrosis, focusing on rheumatoid subcutaneous nodules. Variable RNA fragmentation is a feature of RNA from such tissues. Increased presence of transcript fragmentation is reflected in a proportionate decrease in Agilent DV200 metric and downstream, a reduction in endogenous control genes' expression, measured by RT-dPCR. We show that normalisation of target gene expression to that for endogenous control genes sufficiently corrects for the variable level of fragmented RNA. Recovery of target gene values was achieved in samples comprising as much as 50 percent fragmented RNA, indicating the suitability and appropriate limitation of such data treatment when applied to samples obtained from inherently necrotic tissues.

Expression of the genes facilitating methotrexate action within subcutaneous rheumatoid nodules.

OBJECTIVES: We sought further understanding of the association between methotrexate (MTX) therapy and accelerated development of subcutaneous rheumatoid nodules. The objective was to establish expression of genes involved in the transport, metabolism, and mechanism of action of MTX within nodule tissue. We also examined for differences in gene expression between nodules from patients actively receiving MTX compared to those not receiving MTX. METHODS: Subcutaneous nodule tissues (n=23) were obtained from 21 patients with RA, undergoing elective surgery. Expression of genes important to the transport (SLC19A1, ABCB1, ABCC1, ABCG2), metabolism (FPGS, GGH), and mechanism of action (TYMS, MTR, MTRR) of MTX, including for the adenosine receptors ADORA1, ADORA2A, ADORA2B, ADORA3 and ADORA3variant were quantitated by real-time PCR in each nodule sample. RESULTS: Transcripts for all genes were found in all nodules. Expression of MTR was significantly reduced in nodules from patients receiving MTX therapy. Patterns of gene expression differed, with those metabolising MTX more prominent in nodules from patients receiving MTX when compared to nodules from those not receiving MTX, where genes involved in MTX transport were more prominent. CONCLUSIONS: Genes involved in MTX handling are expressed in rheumatoid nodules, providing further evidence that metabolism of MTX within nodules could exert a local effect. Furthermore the profile of gene expression in nodules differed from that previously observed in rheumatoid synovial membrane. The significant reduction of MTR expression in nodules has implications for MTR- and MTRR-mediated re-methylation reactions. Our data suggest that in contrast to synovium, downstream methylation reactions involving methionine and the biosynthesis of S-adenosylmethionine (SAM) could be reduced in nodule tissue. This could help explain differing responses to MTX in rheumatoid nodules and synovium and warrants further investigation.

Characterization of primary cilia distribution and morphology during lactation, stasis, and involution in the bovine mammary gland.

Primary cilia are small, sensory organelles projecting from virtually all cells and are vital for cellular and tissue function. Their distribution in bovine mammary tissue has not previously been assessed, despite the potential for these organelles to provide specialized perceptive and regulatory functions to this acutely responsive and adaptive gland. The research objectives were to assess ciliary distribution and morphology during active lactation, milk stasis, and early involution using tissue samples obtained following the abrupt cessation of milk removal in nonpregnant, Friesian dairy cows at mid-lactation. Routinely processed tissue sections were obtained at intervals from 6 to 192 hr after the last milking (N = 3 animals per group) and assigned to active lactation (6-12 hr), milk stasis (18-36 hr), and early involution (72-192 hr). Primary cilia were observed in luminal secretory epithelial cells (SECs), myoepithelial cells, and stromal cells following fluorescent immunohistochemistry and confocal microscopy. In SECs, some primary cilia appeared deflected against the apical cell membrane. The proportion of those deflected was greater during milk stasis than active lactation. Data show that primary cilia were suitably placed in three important cell types to potentially coordinate various forms of signal transduction relying on both mechanosensation and chemosensation, according to the physical and physiological state of the gland. Their cell-type distribution and morphology provide new directions in the study of mammary regulation to enhance the understanding of how various mammary-specific cellular responses may be initiated by biochemical or local biophysical factors.

Expression of ovarian tumour suppressor OPCML in the female CD-1 mouse reproductive tract.

Opioid binding protein/cell adhesion molecule-like gene (OPCML) is frequently inactivated in epithelial ovarian cancer, but the role of this membrane protein in normal reproductive function is unclear. The ovarian surface epithelium (OSE) is thought to be the cell of origin of most epithelial ovarian cancers, some of which arise after transformation of OSE cells lining ovarian inclusion cysts, formed during ovulation. We used immunohistochemistry, immunoblotting and quantitative RT-PCR (qRT-PCR) to investigate OPCML expression in the uteri and ovaries of cycling 3-month CD-1 mice, as well as in ovaries from older mice containing inclusion cysts derived from rete ovarii tubules. Immunoblotting showed OPCML bands in uterine, but not whole ovarian or muscle extracts. Strong OPCML immunoreactivity was observed in oviduct, rete ovarii and uterus, whereas in ovary more immunoreactivity was seen in granulosa cells than OSE. No staining was observed in OSE around ovulation sites, where OSE cells divide to cover the site. OPCML immunoreactivity was also weaker in more dysplastic cells lining large ovarian inclusion cysts, compared with normal rete ovarii. No significant changes in Opcml mRNA expression were observed in whole ovarian and uterine extracts at different stages of the cycle. We conclude that murine OPCML is more consistently expressed in cells lining the uterus, oviduct and rete ovarii than in ovary and is not expressed in OSE associated with ovulation sites. This observation supports the hypothesis that a proportion of epithelial ovarian cancers arise from ductal cells and other epithelia of the secondary Mullerian system, rather than the OSE.

E-cadherin expression and bromodeoxyuridine incorporation during development of ovarian inclusion cysts in age-matched breeder and incessantly ovulated CD-1 mice.

BACKGROUND: Female CD-1/Swiss Webster mice subjected to incessant ovulation for 8 months and 12-month breeder mice both developed ovarian inclusion cysts similar to serous cystadenomas. The majority of cysts appeared to be dilated rete ovarii tubules, but high ovulation number resulted in more cortical inclusion cysts. We hypothesized that comparison of inclusion cyst pathology in animals of the same age, but with differences in total lifetime ovulation number, might allow us to determine distinguishing characteristics of the two types of cyst. METHODS: Ovaries from breeder mice (BR) or females subjected to incessant ovulation (IO) were compared at 6-, 9- and 12-months of age. Ovaries were serially sectioned and cysts characterized with regard to location and histology, E-cadherin immunoreactivity and rates of BrdU incorporation. RESULTS: Inclusion cysts developed with age in BR and IO ovaries. The majority of cysts were connected to the ovarian hilus. Two cortical inclusion cysts were observed in ten IO ovaries and one in ten BR ovaries. Low or no E-cadherin immuno-staining was seen in the OSE of all mice studied. Conversely, strong membrane immuno-staining was observed in rete ovarii epithelial cells. Variable E-cadherin immunoreactivity was seen in cells of hilar inclusion cysts, with strong staining observed in cuboidal ciliated cells and little or no staining in flat epithelial cells. Two of the three cortical cysts contained papillae, which showed E-cadherin immuno-staining at the edge of cells. However hilar and cortical cysts were not distinguishable by morphology, cell type or E-cadherin immunoreactivity. BrdU incorporation in cyst cells (1.4% [95% CI: 1.0 to 2.1]) was greater than in OSE (0.7% [95% CI: 0.4 to 1.2]) and very few BrdU-labeled cells were observed in rete ovarii at any age. Incessant ovulation significantly increased BrdU incorporation in OSE of older animals. CONCLUSION: These experiments confirm ovarian inclusion cysts develop with age in the CD-1 mouse strain, irrespective of total ovulation burden. We conclude longer periods of incessant ovulation do not lead to significant changes in inclusion cyst formation or steroidogenesis in CD-1 mice and inclusion cyst type can not be distinguished by morphology, cell proliferation rate or E-cadherin immunoreactivity.