18 research outputs found

    Oral Abstracts 7: RA ClinicalO37. Long-Term Outcomes of Early RA Patients Initiated with Adalimumab Plus Methotrexate Compared with Methotrexate Alone Following a Targeted Treatment Approach

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    Background: This analysis assessed, on a group level, whether there is a long-term advantage for early RA patients treated with adalimumab (ADA) + MTX vs those initially treated with placebo (PBO) + MTX who either responded to therapy or added ADA following inadequate response (IR). Methods: OPTIMA was a 78- week, randomized, controlled trial of ADA + MTX vs PBO + MTX in MTX-naïve early (<1 year) RA patients. Therapy was adjusted at week 26: ADA + MTX-responders (R) who achieved DAS28 (CRP) <3.2 at weeks 22 and 26 (Period 1, P1) were re-randomized to withdraw or continue ADA and PBO + MTX-R continued randomized therapy for 52 weeks (P2); IR-patients received open-label (OL) ADA + MTX during P2. This post hoc analysis evaluated the proportion of patients at week 78 with DAS28 (CRP) <3.2, HAQ-DI <0.5, and/or ΔmTSS ≤0.5 by initial treatment. To account for patients who withdrew ADA during P2, an equivalent proportion of R was imputed from ADA + MTX-R patients. Results: At week 26, significantly more patients had low disease activity, normal function, and/or no radiographic progression with ADA + MTX vs PBO + MTX (Table 1). Differences in clinical and functional outcomes disappeared following additional treatment, when PBO + MTX-IR (n = 348/460) switched to OL ADA + MTX. Addition of OL ADA slowed radiographic progression, but more patients who received ADA + MTX from baseline had no radiographic progression at week 78 than patients who received initial PBO + MTX. Conclusions: Early RA patients treated with PBO + MTX achieved comparable long-term clinical and functional outcomes on a group level as those who began ADA + MTX, but only when therapy was optimized by the addition of ADA in PBO + MTX-IR. Still, ADA + MTX therapy conferred a radiographic benefit although the difference did not appear to translate to an additional functional benefit. Disclosures: P.E., AbbVie, Merck, Pfizer, UCB, Roche, BMS—Provided Expert Advice, Undertaken Trials, AbbVie—AbbVie sponsored the study, contributed to its design, and participated in the collection, analysis, and interpretation of the data, and in the writing, reviewing, and approval of the final version. R.F., AbbVie, Pfizer, Merck, Roche, UCB, Celgene, Amgen, AstraZeneca, BMS, Janssen, Lilly, Novartis—Research Grants, Consultation Fees. S.F., AbbVie—Employee, Stocks. A.K., AbbVie, Amgen, AstraZeneca, BMS, Celgene, Centocor-Janssen, Pfizer, Roche, UCB—Research Grants, Consultation Fees. H.K., AbbVie—Employee, Stocks. S.R., AbbVie—Employee, Stocks. J.S., AbbVie, Amgen, AstraZeneca, BMS, Celgene, Centocor-Janssen, GlaxoSmithKline, Lilly, Pfizer (Wyeth), MSD (Schering-Plough), Novo-Nordisk, Roche, Sandoz, UCB—Research Grants, Consultation Fees. R.V., AbbVie, BMS, GlaxoSmithKline, Human Genome Sciences, Merck, Pfizer, Roche, UCB Pharma—Consultation Fees, Research Support. Table 1.Week 78 clinical, functional, and radiographic outcomes in patients who received continued ADA + MTX vs those who continued PBO + MTX or added open-label ADA following an inadequate response ADA + MTX, n/N (%)a PBO + MTX, n/N (%)b Outcome Week 26 Week 52 Week 78 Week 26 Week 52 Week 78 DAS28 (CRP) <3.2 246/466 (53) 304/465 (65) 303/465 (65) 139/460 (30)*** 284/460 (62) 300/460 (65) HAQ-DI <0.5 211/466 (45) 220/466 (47) 224/466 (48) 150/460 (33)*** 203/460 (44) 208/460 (45) ΔmTSS ≤0.5 402/462 (87) 379/445 (86) 382/443 (86) 330/459 (72)*** 318/440 (72)*** 318/440 (72)*** DAS28 (CRP) <3.2 + ΔmTSS ≤0.5 216/462 (47) 260/443 (59) 266/443 (60) 112/459 (24)*** 196/440 (45) 211/440 (48)*** DAS28 (CRP) <3.2 + HAQ-DI <0.5 + ΔmTSS ≤0.5 146/462 (32) 168/443 (38) 174/443 (39) 82/459 (18)*** 120/440 (27)*** 135/440 (31)** aIncludes patients from the ADA Continuation (n = 105) and OL ADA Carry On (n = 259) arms, as well as the proportional equivalent number of responders from the ADA Withdrawal arm (n = 102). bIncludes patients from the MTX Continuation (n = 112) and Rescue ADA (n = 348) arms. Last observation carried forward: DAS28 (CRP) and HAQ-DI; Multiple imputations: ΔmTSS. ***P < 0.001 and **iP < 0.01, respectively, for differences between initial treatments from chi-squar

    Quantification of global transcription patterns in M.tuberculosis

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    A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

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    <div><p>The integrity of the epithelium is maintained by a complex but regulated interplay of processes that allow conversion of a proliferative state into a stably differentiated state. In this study, using human embryonic stem cell (hESC) derived Retinal Pigment Epithelium (RPE) cells as a model; we have investigated the molecular mechanisms that affect attainment of the epithelial phenotype. We demonstrate that RPE undergo a Mesenchymal–Epithelial Transition in culture before acquiring an epithelial phenotype in a FOXM1 dependent manner. We show that FOXM1 directly regulates proliferation of RPE through transcriptional control of cell cycle associated genes. Additionally, FOXM1 modulates expression of the signaling ligands BMP7 and Wnt5B which act reciprocally to enable epithelialization. This data uncovers a novel effect of FOXM1 dependent activities in contributing towards epithelial fate acquisition and furthers our understanding of the molecular regulators of a cell type that is currently being evaluated as a cell therapy.</p></div

    Epithelial fate acquisition is density dependent.

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    <p>A. Quantification of change in cell density (number of DAPI positive nuclei per cm<sup>2</sup> imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm<sup>2</sup>) or low (8000 cells/cm<sup>2</sup>) density. C. Plot showing differential expression of <i>BMP7</i> and <i>Wnt5B</i> transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.</p

    BMP/Wnt signalling is required for MET.

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    <p>A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm<sup>2</sup>) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm<sup>2</sup>) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of <i>BEST1</i> under the same conditions (top and bottom right). <i>ACTB</i> and <i>B2M</i> are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of <i>BMP7</i> and <i>Wnt5B</i> transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). <i>GAPDH</i>, <i>HPRT1</i> and <i>IPO8</i> were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).</p

    A Mesenchymal-Epithelial Transition with temporal FOXM1 expression during RPE culture.

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    <p>A. Immunocytochemistry was performed for CRALBP, ZO1, αSMA and Ki67 at Day 0 (D0), Day 3 (D3) and Day 35 (D35). Arrowheads point towards Ki67 positive nuclei. B. Microarray heatmap of the expression profiles of the top 250 genes, ranked by the significance of their expression changes, over time in culture. Raw expression data are mean centred and scaled to unit variance prior to clustering. A schematic of the scaled expression is shown on the right where individual gene profiles are in light grey and the mean expression profile is shown in black. C. Microarray heatmap showing transcript expression for a panel of representative markers over a timecourse of RPE culture. D. Immunocytochemistry for FOXM1 at Day 2 and Day 14 of RPE culture. Arrowheads point towards FOXM1 positive nuclei. E. Quantification of immunocytochemistry showing percentage of nuclei staining positive for FOXM1 over time. Bars represent Mean ± SD (n = 3). F. Expression of <i>FOXM1</i> transcript measured using qPCR (relative to housekeeping genes <i>ACTB</i> and <i>GAPDH</i>) in iPSC derived RPE, human foetal RPE and ARPE19 cells over time. Bars represent Mean ± SD (n = 3)</p

    FOXM1 regulates RPE proliferation.

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    <p>A. Graph showing quantification of immunocytochemistry where % Ki67 (n = 3) or % EdU (n = 6) is plotted on the left Y axis and relative expression of <i>FOXM1</i> transcript (n = 3; <i>ACTB</i> used as housekeeping gene) on the right Y axis over days in culture (x axis). B. Quantification of change in <i>FOXM1</i> transcript upon transient overexpression (pFOXM1) or knockdown (siFOXM1), 48h post transfection, measured by qPCR. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 3). C. Quantification of change in EdU incorporation upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). D. Quantification of immunocytochemistry for Ki67 upon siRNA mediated knockdown of non-targeting control, GAPDH, SNAI1, SNAI2 and FOXM1, at Day 6 post transfection. Bars represent Mean + SD (n = 3). n.s non-significant, * p<0.05 Student’s t-test. E. Effect of Thiostrepton on EdU incorporation [left Y axis, red] and <i>FOXM1</i> transcript expression measured by qPCR [right Y axis, blue]. Bars represent Mean ± SD (n = 6). F. Bright-field microscopy showing a scratch introduced in a RPE monolayer at 0 hrs and 19hrs in the presence of DMSO or 10μM Thiostrepton. Edge of the scratch is marked with a white line. Scale bar = 200 μm. G. Quantification of F (above). Bars represent Mean + SD (n = 7). P<0.0001 (Student’s t-test)</p

    FOXM1 promotes RPE epithelial fate.

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    <p>A. qPCR based measurement of transcript expression of a panel of epithelial (red) and mesenchymal (green) markers at Day 10 post siFOXM1 transfection (except levels of <i>FOXM1</i> itself which are measured at Day 2 post knockdown). Data is normalized to transfection with non-targeting siRNA used as a control. <i>ACTB</i>, <i>GAPDH</i>, <i>IPO8</i> and <i>HPRT1</i> are used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test). B. Immunocytochemistry for PMEL17 upon FOXM1 knockdown (siFOXM1) or overexpression (pFOXM1) at Day 10 post transfection. C. Level of knockdown obtained upon transient transfection of siRNA against SNAI2, SNAI1, ZEB1, TWIST1 and GSC. Knockdown was measured by qPCR at Day 6 post transfection and is expressed relative to non-targeting siRNA used as control. <i>CYC1</i> and <i>GAPDH</i> were used as housekeeping genes. Bars represent Mean ± SD (n = 6–9). Knockdown of EMT-TF expression was significant, P<0.05 (Student’s t-test). D. No significant effect on <i>PMEL</i>, <i>MITF</i> or <i>BEST1</i> expression was observed under the same conditions described above for Fig 2C.</p

    Model showing proposed roles of FOXM1 in epithelial fate acquisition.

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    <p>RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.</p

    Genes contributing to pain sensitivity in the normal population:an exome sequencing study

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    Sensitivity to pain varies considerably between individuals and is known to be heritable. Increased sensitivity to experimental pain is a risk factor for developing chronic pain, a common and debilitating but poorly understood symptom. To understand mechanisms underlying pain sensitivity and to search for rare gene variants (MAF<5%) influencing pain sensitivity, we explored the genetic variation in individuals' responses to experimental pain. Quantitative sensory testing to heat pain was performed in 2,500 volunteers from TwinsUK (TUK): exome sequencing to a depth of 70× was carried out on DNA from singletons at the high and low ends of the heat pain sensitivity distribution in two separate subsamples. Thus in TUK1, 101 pain-sensitive and 102 pain-insensitive were examined, while in TUK2 there were 114 and 96 individuals respectively. A combination of methods was used to test the association between rare variants and pain sensitivity, and the function of the genes identified was explored using network analysis. Using causal reasoning analysis on the genes with different patterns of SNVs by pain sensitivity status, we observed a significant enrichment of variants in genes of the angiotensin pathway (Bonferroni corrected p = 3.8×10(-4)). This pathway is already implicated in animal models and human studies of pain, supporting the notion that it may provide fruitful new targets in pain management. The approach of sequencing extreme exome variation in normal individuals has provided important insights into gene networks mediating pain sensitivity in humans and will be applicable to other common complex traits
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