Potential application of measuring serum infliximab levels in rheumatoid arthritis management: A retrospective study based on KURAMA cohort data

Infliximab (IFX) therapy has considerably improved the treatment of rheumatoid arthritis (RA). However, some patients still do not respond adequately to IFX therapy, or the efficacy of the treatment diminishes over time. Although previous studies have reported a relationship between serum IFX levels and therapeutic efficacy, the potential applications of IFX therapeutic drug monitoring (TDM) in clinical practice remain unclear. The purpose of this study was to investigate the potential applications of IFX TDM by analyzing a Japanese cohort database. Data were collected retrospectively from the Kyoto University Rheumatoid Arthritis Management Alliance cohort between January 1, 2011, and December 31, 2018. Serum IFX levels were measured using a liquid chromatography-tandem mass spectrometer. Out of the 311 RA patients that used IFX, 41 were eligible for the analysis. Serum IFX levels were significantly higher in responders than in non-responders. An optimal cut-off value was determined to be 0.32 μg/mL based on a receiver operating characteristic curve. At the IFX measurement point, a better therapeutic response was observed in the high IFX group (n = 32) than in the low IFX group (n = 9). Conversely, at the maximum effect point, when DAS28-ESR was the lowest between IFX introduction and measurement points, there were no differences in responder proportions between the low and high IFX groups. IFX primary ineffectiveness could be avoided with appropriate dose escalation without blood concentration measurement in clinical practice. In conclusion, IFX TDM could facilitate the identification of secondary non-responders and in turn, proper IFX use

Critical Role of IRF-3 in the Direct Regulation of dsRNA-Induced Retinoic Acid-Inducible Gene-I (RIG-I) Expression

<div><p>The cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I), which is also known as an IFN-stimulated gene (ISG), senses viral RNA to activate antiviral signaling. It is therefore thought that RIG-I is regulated in a STAT1-dependent manner. Although RIG-I-mediated antiviral signaling is indispensable for the induction of an appropriate adaptive immune response, the mechanism underlying the regulation of RIG-I expression remains elusive. Here, we examined the direct regulation of RIG-I expression by interferon regulatory factor 3 (IRF-3), which is an essential molecule for antiviral innate immunity. We initially found that RIG-I can be induced by dsRNA in both IFN-independent and IRF-3-dependent manners. A sequence analysis revealed that the RIG-I gene has putative IRF-3-binding sites in its promoter region. Using a combination of cellular, molecular biological, and mutational approaches, we first showed that IRF-3 can directly regulate the expression of RIG-I via a single IRF-element (IRF-E) site in the proximal promoter region of the RIG-I gene in response to dsRNA. IRF-3 is considered a master regulator in antiviral signaling for the generation of type I interferons (IFNs). Thus, our findings demonstrate that RIG-I expression induced by the IRF-3-mediated pathway may serve as a crucial antiviral factor for reinforcing a surveillance system against viral invasion through the regulation of the cytoplasmic viral sensor RIG-I.</p></div

Proposal model for the direct role of IRF-3 in both constitutive and induced RIG-I expression.

<p>Upon viral infection, a low level of constitutively expressed RIG-I recognizes viral RNA, inducing the cells to reach an antiviral state. In the antiviral state, IRF-3 is phosphorylated in response to RLR signaling and translocates to the nucleus to induce type I IFNs. Our findings indicate that activated IRF-3 is also able to directly enhance the expression of RIG-I to enhance antiviral signaling. Infected cells produce IFNs, which subsequently activate STAT1, leading to the robust expression of RIG-I in neighboring cells.</p

IRF-3 binds to IRF-E in the RIG-I promoter.

<p>(A) EMSA was performed using a DIG-labelled RIG-I IRF-E wild-type (WT) probe. Nuclear extracts were prepared from poly I:C-transfected HeLa cells. For the supershift assay, rabbit anti-IRF-3 antibody was pre-incubated with the reaction mixture. (B) The labeled RIG-I IRF-E-WT probe was combined with 500 nM (r)GST or (r)GST-IRF-3(5D) fusion protein at various concentrations (500 nM, 125 nM, and 50 nM; wedges). Arrows indicate the IRF-3-oligonucleotide probe complex.</p

IRF-3 binding to the RIG-I promoter <i>in vivo</i>.

<p>HeLa cells were transfected with poly I:C (100 ng) for 4 h and fixed; the DNA was then fragmented. Chromatin immunoprecipitation was performed using an anti-IRF-3 antibody or control IgG. The means (±SD) of three experiments are shown. †<i>P</i> < 0.05</p

Promoter analysis of the human RIG-I gene.

<p>(A) Putative consensus sequences of STAT1, ISRE, c-Rel and IRF-E on the RIG-I promoter are shown. (B) and (C) HeLa cells were co-transfected with pGL4.11 (empty) or serial human RIG-I luciferase reporter constructs and Renilla luciferase expression vector (pGL4.74) for 24 h. The cells were further transfected with poly I:C (100 ng) for 4 h. The reporter activities are shown as relative values, specifically ratios of the firefly luciferase activities driven by the RIG-I promoters to the Renilla luciferase activities. The means (±SD) of three experiments are shown; †<i>P</i> < 0.05, *<i>P</i> < 0.01.</p

＜Poster＞Alteration of peptides in rat brain treated with angiotensin-converting enzyme Inhibitor, captopril

Although animal studies suggest that centrally active angiotensin-converting enzyme （ACE） inhibitors may protect against dementia beyond HTN control, the mechanism（s） underlying these improvements in cognitive function remains unclear. We measured the brain peptide levels in rat treated with captopril（ 50 mg/kg） for 3 weeks by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry （SELDI TOF-MS）. Two protein chip arrays were used for peptide profi ling： one with a strong anion- exchanger and the other with a weak cation-exchanger. 　 Comparing with control group, 15 mass peaks were considered specifi c to experimental animals, and 6 peaks were signifi cantly up-regulated and 5 down-regulated

IRF-E on the RIG-I promoter regulates the transcriptional activity of RIG-I.

<p>(A) Putative consensus sequences of ISRE, GAS and IRF-E within the proximal region of the RIG-I promoter are shown. (B) A series of single (double or triple) deletion constructs on the RIG-I promoter is shown. (C) U3A and U5A cells were co-transfected with the RIG-I deletion constructs and a renilla luciferase expression vector as shown in (B); †<i>P</i> < 0.05, *<i>P</i> < 0.01.</p