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Background: rituximab is often used as rescue therapy in interstitial lung disease (ILD) associated with connective tissue disease (CTD), but has not been studied in clinical trials. This study aimed to assess whether rituximab is superior to cyclophosphamide as a treatment for severe or progressive CTD associated ILD.Methods: we conducted a randomised, double-blind, double-dummy, phase 2b trial to assess the superiority of rituximab compared with cyclophosphamide. Patients aged 18-80 years with severe or progressive ILD related to scleroderma, idiopathic inflammatory myositis, or mixed CTD, recruited across 11 specialist ILD or rheumatology centres in the UK, were randomly assigned (1:1) to receive rituximab (1000 mg at weeks 0 and 2 intravenously) or cyclophosphamide (600 mg/m2 body surface area every 4 weeks intravenously for six doses). The primary endpoint was rate of change in forced vital capacity (FVC) at 24 weeks compared with baseline, analysed using a mixed-effects model with random intercepts, adjusted for baseline FVC and CTD type. Prespecified secondary endpoints reported in this Article were change in FVC at 48 weeks versus baseline; changes from baseline in 6 min walk distance, diffusing capacity of the lung for carbon monoxide (DLCO), physician-assessed global disease activity (GDA) score, and quality-of-life scores on the St George's Respiratory Questionnaire (SGRQ), King's Brief Interstitial Lung Disease (KBILD) questionnaire, and European Quality of Life Five-Dimension (EQ-5D) questionnaire at 24 and 48 weeks; overall survival, progression-free survival, and time to treatment failure; and corticosteroid use. All endpoints were analysed in the modified intention-to-treat population, which comprised all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov (NCT01862926).Findings: between Dec 1, 2014, and March 31, 2020, we screened 145 participants, of whom 101 participants were randomly allocated: 50 (50%) to receive cyclophosphamide and 51 (50%) to receive rituximab. 48 (96%) participants in the cyclophosphamide group and 49 (96%) in the rituximab group received at least one dose of treatment and were included in analyses; 43 (86%) participants in the cyclophosphamide group and 42 (82%) participants in the rituximab group completed 24 weeks of treatment and follow-up. At 24 weeks, FVC was improved from baseline in both the cyclophosphamide group (unadjusted mean increase 99 mL [SD 329]) and the rituximab group (97 mL [234]); in the adjusted mixed-effects model, the difference in the primary endpoint at 24 weeks was -40 mL (95% CI -153 to 74; p=0·49) between the rituximab group and the cyclophosphamide group. KBILD quality-of-life scores were improved at 24 weeks by a mean 9·4 points (SD 20·8) in the cyclophosphamide group and 8·8 points (17·0) in the rituximab group. No significant differences in secondary endpoints were identified between the treatment groups, with the exception of change in GDA score at week 48, which favoured cyclophosphamide (difference 0·90 [95% CI 0·11 to 1·68]). Improvements in lung function and respiratory-related quality-of-life measures were observed in both treatment groups. Lower corticosteroid exposure over 48 weeks of follow-up was recorded in the rituximab group. Two (4%) of 48 participants who received cyclophosphamide and three (6%) of 49 who received rituximab died during the study, all due to complications of CTD or ILD. Overall survival, progression-free survival, and time to treatment failure did not significantly differ between the two groups. All participants reported at least one adverse event during the study. Numerically fewer adverse events were reported by participants receiving rituximab (445 events) than those receiving cyclophosphamide (646 events). Gastrointestinal and respiratory disorders were the most commonly reported adverse events in both groups. There were 62 serious adverse events of which 33 occurred in the cyclophosphamide group and 29 in the rituximab group.Interpretation: rituximab was not superior to cyclophosphamide to treat patients with CTD-ILD, although participants in both treatment groups had increased FVC at 24 weeks, in addition to clinically important improvements in patient-reported quality of life. Rituximab was associated with fewer adverse events. Rituximab should be considered as a therapeutic alternative to cyclophosphamide in individuals with CTD-ILD requiring intravenous therapy.Funding: efficacy and Mechanism Evaluation Programme (Medical Research Council and National Institute for Health Research, UK).

In patients with fibrotic interstitial lung disease (ILD), hypoxemia on exertion is frequent, and contributes to exercise intolerance, exertional dyspnoea and reduced quality of life [1–3]. Clinically significant exertional hypoxemia is typically defined as a drop in transcutaneous arterial oxygen saturation (SpO2) to ≤88% on a six-minute walk test (6MWT) [4], and is associated with reduced survival in ILD patients [5]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Alfieri has nothing to disclose. Conflict of interest: Dr. Crisafulli has nothing to disclose. Conflict of interest: Dr. Visca has nothing to disclose. Conflict of interest: Dr. Chong has nothing to disclose. Conflict of interest: Dr. Stock has nothing to disclose. Conflict of interest: Dr. Mori has nothing to disclose. Conflict of interest: Dr. De Lauretis has nothing to disclose. Conflict of interest: Dr. Tsipouri has nothing to disclose. Conflict of interest: Dr. Chua reports lecture fees and advisory board fees from Boeringher Ingelheim and from Roche, outside the submitted work. Conflict of interest: Dr. Kouranos has nothing to disclose. Conflict of interest: Dr. Kokosi has nothing to disclose. Conflict of interest: Dr. Hogben has nothing to disclose. Conflict of interest: PLM has, via his institution, received industry-academic funding from Roche, Boehringer Ingelheim and Galapagos and has received speakers fees from Roche. Conflict of interest: Dr. George reports grants, personal fees and non-financial support from Boehringer Ingelheim, personal fees and non-financial support from Roche, personal fees from Teva, outside the submitted work. Conflict of interest: TMM has, via his institution, received industry-academic funding from GlaxoSmithKline R&D and UCB and has received consultancy or speakers fees from Apellis, Astra Zeneca, Bayer, Blade Therapeutics, Boehringer Ingelheim, Bristol-Myers Squibb, Galapagos, GlaxoSmithKline R&D, Indalo, Novartis, Pliant, ProMetic, Respivnat, Roche, Samumed and UCB. Conflict of interest: Dr. Chetta has nothing to disclose. Conflict of interest: Dr. Sestini has nothing to disclose. Conflict of interest: Dr. Wells reports lecture fees and advisory board fees from Boeringher Ingelheim, Roche and Bayer, outside the submitted work. Conflict of interest: Dr. Renzoni reports lecture fees and advisory board fees from Boeringher Ingelheim and from Roche, and lecture fees from Mundipharma, outside the submitted work.

BACKGROUND: In fibrotic interstitial lung diseases, exertional breathlessness is strongly linked to health-related quality of life (HRQOL). Breathlessness is often associated with oxygen desaturation, but few data about the use of ambulatory oxygen in patients with fibrotic interstitial lung disease are available. We aimed to assess the effects of ambulatory oxygen on HRQOL in patients with interstitial lung disease with isolated exertional hypoxia. METHODS: AmbOx was a prospective, open-label, mixed-method, crossover randomised controlled clinical trial done at three centres for interstitial lung disease in the UK. Eligible patients were aged 18 years or older, had fibrotic interstitial lung disease, were not hypoxic at rest but had a fall in transcutaneous arterial oxygen saturation to 88% or less on a screening visit 6-min walk test (6MWT), and had self-reported stable respiratory symptoms in the previous 2 weeks. Participants were randomly assigned (1:1) to either oxygen treatment or no oxygen treatment for 2 weeks, followed by crossover for another 2 weeks. Randomisation was by a computer-generated sequence of treatments randomly permuted in blocks of constant size (fixed size of ten). The primary outcome, which was assessed by intention to treat, was the change in total score on the King's Brief Interstitial Lung Disease questionnaire (K-BILD) after 2 weeks on oxygen compared with 2 weeks of no treatment. General linear models with treatment sequence as a fixed effect were used for analysis. Patient views were explored through semi-structured topic-guided interviews in a subgroup of participants. This study was registered with ClinicalTrials.gov, number NCT02286063, and is closed to new participants with all follow-up completed. FINDINGS: Between Sept 10, 2014, and Oct 5, 2016, 84 patients were randomly assigned, 41 randomised to ambulatory oxygen first and 43 to no oxygen. 76 participants completed the trial. Compared with no oxygen, ambulatory oxygen was associated with significant improvements in total K-BILD scores (mean 55·5 [SD 13·8] on oxygen vs 51·8 [13·6] on no oxygen, mean difference adjusted for order of treatment 3·7 [95% CI 1·8 to 5·6]; p

Background There are no ILD specific guidelines on the use of ambulatory oxygen. The AmbOx trial is a multicenter, randomised, cross-over controlled trial (NCT02286063), to assess quality of life during two weeks on ambulatory oxygen compared to two weeks off oxygen, in patients with fibrotic ILD. Methods Individuals with fibrotic ILD whose oxygen saturation was normal at rest, but dropped to ≤88% on a 6MWT, with stable symptoms during a two week run-in period, were recruited and randomised. Primary outcome: health status assessed by King’s Brief ILD questionnaire (KBILD). A simple question on whether breathlessness had changed (better, same, worse) over the previous two weeks was a key secondary outcome. Patients‘ experiences with portable oxygen were explored through interviews in a subgroup. At the end of the four week trial period, patients were asked if they wished to continue with the ambulatory oxygen. Results Out of 84 randomised patients, 76 completed the trial. Mean age 64.5±1.1 years, 58 males, 53 ever smokers, FVC 73.3%±19.1%, DLCO 38.7%±12.8%. 43 patients had possible/definite IPF. Ambulatory oxygen, compared to no oxygen, was associated with improvements in total KBILD score (p

Background Interstitial lung disease (ILD) frequently complicates systemic autoimmune disorders resulting in considerable morbidity and mortality. The connective tissue diseases (CTDs) most frequently resulting in ILD include: systemic sclerosis, idiopathic inflammatory myositis (including dermatomyositis, polymyositis and anti-synthetase syndrome) and mixed connective tissue disease. Despite the development, over the last two decades, of a range of biological therapies which have resulted in significant improvements in the treatment of the systemic manifestations of CTD, the management of CTD-associated ILD has changed little. At present there are no approved therapies for CTD-ILD. Following trials in scleroderma-ILD, cyclophosphamide is the accepted standard of care for individuals with severe or progressive CTD-related ILD. Observational studies have suggested that the anti-CD20 monoclonal antibody, rituximab, is an effective rescue therapy in the treatment of refractory CTD-ILD. However, before now, there have been no randomised controlled trials assessing the efficacy of rituximab in this treatment population. Methods/design RECITAL is a UK, multicentre, prospective, randomised, double-blind, double-dummy, controlled trial funded by the Efficacy and Mechanism Evaluation Programme of the Medical Research Council and National Institute for Health Research. The trial will compare rituximab 1 g given intravenously, twice at an interval of 2 weeks, with intravenously administered cyclophosphamide given monthly at a dose of 600 mg/m2 body surface area in individuals with ILD due to systemic sclerosis, idiopathic inflammatory myositis (including anti-synthetase syndrome) or mixed connective tissue disease. A total of 116 individuals will be randomised 1:1 to each of the two treatment arms, with stratification based on underlying CTD, and will be followed for a total of 48 weeks from first dose. The primary endpoint for the study will be change in forced vital capacity (FVC) at 24 weeks. Key secondary endpoints include: safety, change in FVC at 48 weeks as well as survival, change in oxygen requirements, total 48-week corticosteroid exposure and utilisation of health care resources. Discussion This is the first randomised control trial to study the efficacy of rituximab as first-line treatment in CTD-associated ILD. The results generated should provide important information on the treatment of a life-threatening complication affecting a rare group of CTDs. Trial registration ClinicalTrials.gov, NCT01862926. Registered on 22 May 2013. Electronic supplementary material The online version of this article (doi:10.1186/s13063-017-2016-2) contains supplementary material, which is available to authorized users.

Background Fibrotic interstitial lung diseases (ILDs) are chronic and often progressive conditions resulting in substantial morbidity and mortality. Shortness of breath, a symptom often linked to oxygen desaturation on exertion, is tightly linked to worsening quality of life in these patients. Although ambulatory oxygen is used empirically in their treatment, there are no ILD-specific guidelines on its use. To our knowledge, no studies are available on the effects of ambulatory oxygen on day-to-day life in patients with ILD. Methods/design Ambulatory oxygen in fibrotic lung disease (AmbOx) is a multicentre, randomised controlled crossover trial (RCT) funded by the Research for Patient Benefit Programme of the National Institute for Health Research. The trial will compare ambulatory oxygen used during daily activities with no ambulatory oxygen in patients with fibrotic lung disease whose oxygen saturation (SaO2) is ≥94% at rest, but drops to ≤88% on a 6-min Walk Test. The randomised controlled trial (RCT) will evaluate the effects on health status (measured by the King’s Brief ILD Questionnaire: K-BILD) of ambulatory oxygen used at home, at an optimal flow rate determined by titration at screening visit, and administered for a 2-week period, compared to 2 weeks off oxygen. Key secondary outcomes will include breathlessness on activity scores, as measured by the University of California San Diego Shortness of Breath Questionnaire, global patient assessment of change scores, as well as quality of life scores (St George’s Respiratory Questionnaire), anxiety and depression scores (Hospital Anxiety and Depression Scale), activity markers measured by SenseWear Armbands, pulse oximetry measurements, patient-reported daily activities, patient- and oxygen company-reported oxygen cylinder use. The study also includes a qualitative component and will explore in interviews patients’ experiences of the use of a portable oxygen supply and trial participation in a subgroup of 20 patients and carers. Discussion This is the first RCT of the effects of ambulatory oxygen during daily life on health status and breathlessness in fibrotic lung disease. The results generated should provide the basis for setting up ILD-specific guidelines for the use of ambulatory oxygen. Trial registration National Clinical Trials Registry, identifier: NCT02286063. Registered on 8 October 2014 (retrospectively registered). Electronic supplementary material The online version of this article (doi:10.1186/s13063-017-1912-9) contains supplementary material, which is available to authorized users.

Three mutant mice with pigmentation phenotypes were recovered from a genomewide random mouse chemical mutagenesis study. White toes (Whto; MGI:1861986), Belly spot and white toes (Bswt; MGI:2152776) and Dark footpads 2 (Dfp2; MGI:1861991) were identified following visual inspection of progeny from a male exposed to the point mutagen ethylnitrosourea (ENU). In order to rapidly localize the causative mutations, genome-wide linkage scans were performed on pooled DNA samples from backcross animals for each mutant line. Whto was mapped to proximal mouse chromosome (Mmu) 7 between Cen (the centromere) and D7Mit112 (8.0 cM from the centromere), Bswt was mapped to centric Mmul between D1Mit214 (32.1 cM) and D1Mit480 (32.8 cM) and Dfp2 was mapped to proximalMmu4 between Cen and D4Mit18 (5.2 cM). Whto, Bswt and Dfp2 may provide novel starting points in furthering the elucidation of genetic and biochemical pathways relevant to pigmentation and associated biological processes.

We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA[2, 3]. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8–12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.

Comparative mapping and sequencing was used to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome., Background Indian muntjac (Muntiacus muntjak vaginalis) has an extreme mammalian karyotype, with only six and seven chromosomes in the female and male, respectively. Chinese muntjac (Muntiacus reevesi) has a more typical mammalian karyotype, with 46 chromosomes in both sexes. Despite this disparity, the two muntjac species are morphologically similar and can even interbreed to produce viable (albeit sterile) offspring. Previous studies have suggested that a series of telocentric chromosome fusion events involving telomeric and/or satellite repeats led to the extant Indian muntjac karyotype. Results We used a comparative mapping and sequencing approach to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome. Specifically, we screened an Indian muntjac bacterial artificial-chromosome library with a telomere repeat-specific probe. Isolated clones found by fluorescence in situ hybridization to map to interstitial regions on Indian muntjac chromosomes were further characterized, with a subset then subjected to shotgun sequencing. Subsequently, we isolated and sequenced overlapping clones extending from the ends of some of these initial clones; we also generated orthologous sequence from isolated Chinese muntjac clones. The generated Indian muntjac sequence has been analyzed for the juxtaposition of telomeric and satellite repeats and for synteny relationships relative to other mammalian genomes, including the Chinese muntjac. Conclusions The generated sequence data and comparative analyses provide a detailed genomic context for seven ancestral chromosome fusion sites in the Indian muntjac genome, which further supports the telocentric fusion model for the events leading to the unusual karyotypic differences among muntjac species.