Benefit of Filgotinib, a JAK1 Preferential Inhibitor, in Rheumatoid Arthritis Patients with Previous Rapid Radiographic Progression: Post Hoc Analysis of Two Trials.

INTRODUCTION: We conducted a post hoc analysis of efficacy and safety of filgotinib stratified by estimated radiographic progression rate before baseline (BL) in patients with rheumatoid arthritis (RA) who had inadequate response to methotrexate (MTX; FINCH 1; NCT02889796) or were naïve to it (FINCH 3; NCT02886728). METHODS: Radiographic progression rate was BL-Modified Total Sharp Score (mTSS) divided by RA duration (BL mTSS/year); estimated rapid radiographic progression (e-RRP) was BL change in mTSS/year ≥ 5; and estimated nonrapid radiographic progression (e-NRRP) was BL mTSS/year < 5. Efficacy and safety were compared between subgroups. All p-values are nominal. RESULTS: In FINCH 1 and FINCH 3, 558/1755 (31.8%) and 787/1249 (63.0%) patients, respectively, had BL e-RRP. BL characteristics were generally similar between subgroups within each trial. At week (W) 24, in FINCH 1, proportions achieving a Disease Activity Score 28 for rheumatoid arthritis with C-reactive protein < 2.6 were significantly greater with filgotinib 200 (FIL200) and 100 mg (FIL100) versus placebo among e-RRP and e-NRRP subgroups. In each study, proportions of FIL-treated patients achieving Clinical Disease Activity Index ≤ 2.8 and Simple Disease Activity Index ≤ 3.3 were similar between subgroups. In FINCH 3, disease activity measures were at least numerically improved among patients receiving FIL versus MTX monotherapy. At W24, mTSS changes from BL (CFB) were greater among patients with e-RRP in FINCH 1 and FINCH 3 versus e-NRRP (0.81 versus 0.19, p = 0.001; 0.67 versus 0.25, p = 0.31, respectively). At W52, in FINCH 1, mTSS CFBs were smaller among e-RRP patients treated with FIL200 (0.40; p < 0.001) and FIL100 (0.77; p = 0.024) versus adalimumab (ADA; 1.46). In FINCH 3 at W52, mTSS CFBs were significantly smaller with FIL200 versus MTX among e-RRP patients. Rates of treatment-emergent adverse events (AEs) were comparable between subgroups and across treatment arms. CONCLUSIONS: Patients with previous e-RRP who received standard care tended to progress radiographically. FIL200 demonstrated persistent, consistent benefit for disease activity control among e-RRP and e-NRRP subgroups, and AE profiles were similar between subgroups. Although filgotinib efficacy was somewhat reduced among patients with e-RRP, filgotinib treatment slowed radiographic progression in both subgroups. TRIAL REGISTRATION: Clinicaltrials.gov NCT02889796, NCT02886728.

Efficacy and Safety of Filgotinib in Patients with High Risk of Poor Prognosis Who Showed Inadequate Response to MTX: A Post Hoc Analysis of the FINCH 1 Study.

INTRODUCTION: This exploratory analysis of FINCH 1 (NCT02889796) examined filgotinib (FIL) efficacy and safety in a subgroup of patients with rheumatoid arthritis (RA) and inadequate response to methotrexate (MTX; MTX-IR) who had four poor prognostic factors (PPFs). METHODS: Patients with MTX-IR received placebo up to week (W)24 or FIL200 mg, FIL100 mg, or adalimumab up to W52; all received MTX. Efficacy and safety data were stratified by four PPFs versus fewer than four PPFs: seropositivity, high-sensitivity C-reactive protein (CRP) ≥ 6 mg/L, Disease Activity Score in 28 joints with CRP > 5.1, and erosions on X-rays. RESULTS: At baseline, 687/1755 patients had four PPFs. At W12, whether with four PPFs or fewer than four PPFs, response rates on all American College of Rheumatology (ACR) measures were significantly greater with FIL200 and FIL100 versus placebo. At W52, FIL200 ACR20/50/70 response rates remained at least numerically higher versus adalimumab in both subgroups. At W52, FIL200 reduced modified total Sharp score (mTSS) change versus adalimumab in patients with four or fewer than four PPFs. CONCLUSIONS: In high-risk (four PPFs) patients with MTX-IR RA, FIL200 and FIL100 showed similar reductions in disease activity versus placebo at W12 as in patients with fewer than four PPFs. mTSS in patients receiving FIL200 changed little from W24 to W52, while that in patients receiving FIL100 progressed comparably to patients who received adalimumab. Tolerability was comparable across treatment arms and subgroups.

Primary genetic linkage maps of the ascidian, Ciona intestinalis.

For whole-genome analysis in a basal chordate (protochordate), we used F1 pseudo-testcross mapping strategy and amplified fragment length polymorphism (AFLP) markers to construct primary linkage maps of the ascidian tunicate Ciona intestinalis. Two genetic maps consisted of 14 linkage groups, in agreement with the haploid chromosome number, and contained 276 and 125 AFLP loci derived from crosses between British and Neapolitan individuals. The two maps covered 4218.9 and 2086.9 cM, respectively, with an average marker interval of 16.1 and 18.9 cM. We observed a high recombinant ratio, ranging from 25 to 49 kb/cM, which can explain the high degree of polymorphism in this species. Some AFLP markers were converted to sequence tagged sites (STSs) by sequence determination, in order to create anchor markers for the fragmental physical map. Our recombination tools provide basic knowledge of genetic status and whole genome organization, and genetic markers to assist positional cloning in C. intestinalis.

Application of Minos, one of the Tc1/mariner superfamily transposable elements, to ascidian embryos as a tool for insertional mutagenesis.

As it has a simple genome structure, Ciona intestinalis is a good chordate species for studying the function of genes. To this end, it is a key requirement to introduce insertional mutagenesis using a transposable element to the ascidian system. The present study focuses on Minos, one of the Tc1/mariner superfamily transposons that is already used in a human cell line. By extrachromosomal excision and transposition assays, we found that Minos activity is very high in C. intestinalis. We also demonstrated the nuclear localization activity of Minos transposase in Ciona embryos. From these tests, we concluded that Minos transposase has complete activity when it is expressed in C. intestinalis, suggesting that Minos has the potential to be used for genome-wide insertional mutagenesis of C. intestinalis.

Genomics and developmental approaches to an ascidian adenohypophysis primordium.

Ascidians, which are the closest phylogenetic relatives to vertebrates, lack a distinct pituitary gland, which is the major endocrine gland in vertebrates. Nevertheless, for the past 130 years, it has been debated that the ascidian neural complex (NC) is homologous to the pituitary. Of the three major components of the NC, the neural gland (NG) has mainly been thought to be the ascidian counterpart of the pituitary. Recently, however, the ciliated funnel, and not the NG, was postulated to be the adenohypophysis (AH) primordium because it is likely derived from oral ectoderm, and because the expression of several placodal genes is comparable to their expression in vertebrates. An extensive in silico survey of the Ciona intestinalis genome sequence revealed that genes encoding pituitary hormones are absent in ascidians. Under the circumstances, this thesis attempts to find a path that shows that the AH primordium is recognizable in the ascidian by revisiting molecular and developmental data from recent public resources on C. intestinalis, and through the use of advanced bio-imaging techniques. A putative Ciona genetic pathway, which was constructed by referring to data from mammals, shows that only a patchwork of the genetic network exists to achieve terminal differentiation of the AH endocrine cells in the Ciona genome. Re-annotation on glycoprotein hormone related proteins, a GPA2/ARP and two GPB5/BRP ones previously reported, reveals that the GPA2 locus contains two splicing variants, and one variant likely formed a three-dimensional conformation similar to that of human GPA2. No clone of the GPB5/BRP1 locus has been isolated, and another candidate, BRP2, is unlikely to be a GPB5. Next, I argued a possibility that endocrine activities of Ciona species could be specialized in association with its short generation time, and I suggest that not only Ciona species but also other ascidians should be studied in order to understand ascidian endocrinology. Confocal images of the stages of tailbud development reconfirmed the presence of an oral ectoderm placode, and I propose to update the stomodeum development by adding descriptions of a folded structure of the stomodeum and deeply positioned opening of the sensory vesicle. Finally, YFP expression driven by Ci-Six3 promoter demonstrated a boundary between the pharyngeal endoderm and other ectodermal and neuroectodermal tissues around the ciliated funnel. These updates on the ascidian model, which complement other lower chordates and vertebrates, shed light on the evolutionary origin of the pituitary primordium.

Midline signaling and evolution of the forebrain in chordates: a focus on the lamprey Hedgehog case.

Lampreys are agnathans (vertebrates without jaws). They occupy a key phylogenetic position in the emergence of novelties and in the diversification of morphology at the dawn of vertebrates. We have used lampreys to investigate the possibility that embryonic midline signaling systems have been a driving force for the evolution of the forebrain in vertebrates. We have focused on Sonic Hedgehog/Hedgehog (Shh/Hh) signaling. In this article, we first review and summarize our recent work on the comparative analysis of embryonic expression patterns for Shh/Hh, together with Fgf8 (fibroblast growth factor 8) and Wnt (wingless-Int) pathway components, in the embryonic lamprey forebrain. Comparison with nonvertebrate chordates on one hand, and jawed vertebrates on the other hand, shows that these morphogens/growth factors acquired new expression domains in the most rostral part of the neural tube in lampreys compared to nonvertebrate chordates, and in jawed vertebrates compared to lampreys. These data are consistent with the idea that changes in Shh, Fgf8 or Wnt signaling in the course of evolution have been instrumental for the emergence and diversification of the telencephalon, a part of the forebrain that is unique to vertebrates. We have then used comparative genomics on Shh/Hh loci to identify commonalities and differences in noncoding regulatory sequences across species and phyla. Conserved noncoding elements (CNEs) can be detected in lamprey Hh introns, even though they display unique structural features and need adjustments of parameters used for in silico alignments to be detected, because of lamprey-specific properties of the genome. The data also show conservation of a ventral midline enhancer located in Shh/Hh intron 2 of all chordates, the very species which possess a notochord and a floor plate, but not in earlier emerged deuterostomes or protostomes. These findings exemplify how the Shh/Hh locus is one of the best loci to study genome evolution with regards to developmental events.

Two lamprey Hedgehog genes share non-coding regulatory sequences and expression patterns with gnathostome Hedgehogs.

Hedgehog (Hh) genes play major roles in animal development and studies of their evolution, expression and function point to major differences among chordates. Here we focused on Hh genes in lampreys in order to characterize the evolution of Hh signalling at the emergence of vertebrates. Screening of a cosmid library of the river lamprey Lampetra fluviatilis and searching the preliminary genome assembly of the sea lamprey Petromyzon marinus indicate that lampreys have two Hh genes, named Hha and Hhb. Phylogenetic analyses suggest that Hha and Hhb are lamprey-specific paralogs closely related to Sonic/Indian Hh genes. Expression analysis indicates that Hha and Hhb are expressed in a Sonic Hh-like pattern. The two transcripts are expressed in largely overlapping but not identical domains in the lamprey embryonic brain, including a newly-described expression domain in the nasohypophyseal placode. Global alignments of genomic sequences and local alignment with known gnathostome regulatory motifs show that lamprey Hhs share conserved non-coding elements (CNE) with gnathostome Hhs albeit with sequences that have significantly diverged and dispersed. Functional assays using zebrafish embryos demonstrate gnathostome-like midline enhancer activity for CNEs contained in intron2. We conclude that lamprey Hh genes are gnathostome Shh-like in terms of expression and regulation. In addition, they show some lamprey-specific features, including duplication and structural (but not functional) changes in the intronic/regulatory sequences.

Initial stage of genetic mapping in Ciona intestinalis.

The use of classic genetics is emerging in the ascidian Ciona intestinalis; recent advances in genomics and high-quality developmental and evolutionary studies have made this animal an attractive model for research purposes. Genetic mapping in Ciona will likely make a major contribution to ascidian genomics and developmental biology by providing support for genome assembly and annotation and for the isolation of genes with particular mutations, while construction of genetic maps advances classic genetics in this species. Two major issues must be overcome before fine genetic maps can be constructed: the choice of proper genetic backgrounds and the establishment of laboratory strains. A high degree of polymorphism is useful for genetic mapping if we consider particular combinations of genetic backgrounds and techniques, although it is necessary to pay attention to the confused classification of C. intestinalis. Thus, it is preferred to establish laboratory strains instead of using samples with various genetic backgrounds. As these issues are unresolved, only amplified fragment length polymorphism-based maps have been created, while bulk segregant analysis is expected to isolate markers flanking mutant loci. However, rich genomic resources should facilitate the next stage of genetic map construction based on type I markers using coding sequences. The meiotic events that occur in crossing experiments for mapping purposes should shed light on population genetics and speciation issues. The results of such investigations may provide feedback for comparative genomics and developmental genetics in the near future.

Evolutionary modification of mouth position in deuterostomes.

In chordates, the oral ectoderm is positioned at the anterior neural boundary and is characterized by pituitary homeobox (Pitx) and overlapping Dlx and Six3 expressions. Recent studies have shown that the ectoderm molecular map is also conserved in hemichordates and echinoderms. However, the mouth develops in a more posterior position in these animals, in a domain characterized by Nkx2.1 and Goosecoid expression, in a manner similar to that observed in protostomes. Furthermore, BMP signaling antagonizes mouth development in echinoderms and hemichordates, but seems to promote oral ectoderm specification in chordates. Conversely, Nodal signaling appears to be required for oral ectoderm specification in sea urchins but not in chordates. The Nodal/BMP antagonism at work during ectoderm patterning thus seems to constitute a conserved feature in deuterostomes, and mouth relocation may have been accompanied by a change in the influence of BMP/Nodal signals on oral ectoderm specification. We suggest that the mouth primordium was located at the anterior neural boundary, in early chordate evolution. In extant chordate embryos, subsequent mouth positioning differ between urochordates and vertebrates, presumably as a consequence of surrounding tissues remodelling. We illustrate these morphogenetic movements by means of morphological data obtained by the confocal imaging of ascidian tailbud embryos, and provide a table for determining the tailbud stages of this model organism.

Windows of the brain: towards a developmental biology of circumventricular and other neurohemal organs.

We review the anatomical and functional features of circumventricular organs in vertebrates and their homologous neurohemal organs in invertebrates. Focusing on cyclostomes (lamprey) and urochordates (ascidians), we discuss the evolutionary origin of these organs as a function of their cell type specification and morphogenesis.

Culture of Ciona intestinalis in closed systems.

Improvements in closed-system culturing methods for marine invertebrates are important prerequisites for the generalized use of transgenic lines. We discuss here the effects of several closed-system conditions on the growth and survival of the solitary ascidian, Ciona intestinalis. In Shimoda, close to the sea, a small-tank system was used to ensure that tanks and systems were reasonably equipped, water exchange was rapid, and animals separated to minimize the risk of infection. In Gif-sur-Yvette, an inland site, we tried to determine the optimal conditions to limit handling operations, and to save artificial seawater by avoiding water pollution. A mixture of at least two types of live algae was better than any single-organism diet. With these maintenance protocols, we were able to obtain several generations of Ciona intestinalis, including several transgenic lines. Because these systems make it easier to rear Ciona intestinalis in laboratories, they increase the potentialities of this model organism for research.

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.