Avelumab as neoadjuvant therapy in patients with urothelial non-metastatic muscle invasive bladder cancer: a multicenter, randomized, non-comparative, phase II study (Oncodistinct 004 - AURA trial).

INTRODUCTION: Cisplatin-based neoadjuvant chemotherapy (NAC) followed by surgery is the standard treatment for patients with non-metastatic muscle invasive bladder cancer (MIBC). Unfortunately, many patients are not candidates to receive cisplatin due to renal impairment. Additionally, no predictive biomarkers for pathological complete response (pCR) are currently validated in clinical practice. Studies evaluating immune checkpoint inhibitors in the peri-operative setting are emerging with promising results. Clinical trials are clearly required in the neoadjuvant setting in order to improve therapeutic strategies. METHODS AND ANALYSIS: Oncodistinct 004 - AURA is an ongoing multicenter phase II randomized trial assessing the efficacy and safety of avelumab single-agent or combined to different NAC regimens in patients with non-metastatic MIBC. Patients are enrolled in two distinct cohorts according to their eligibility to receive cisplatin-based NAC. In the cisplatin eligible cohort, patients are randomized in a 1:1 fashion to receive avelumab combined with cisplatin-gemcitabine or with dose-dense methotrexate-vinblastine-doxorubicin-cisplatin. In the cisplatin ineligible cohort, patients are randomized at a 1:1 ratio to paclitaxel-gemcitabine associated to avelumab or avelumab alone. Primary endpoint is pCR. Secondary endpoints are pathological response and safety. ETHICS AND DISSEMINATION: The study is approved by ethics committee from all participating centers. All participants provide informed consent prior inclusion to the study. Once completed, results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03674424).

Efficacy of dashboard driven dosing of infliximab in inflammatory bowel disease patients; a randomized controlled trial.

OBJECTIVES: Loss of response (LOR) to infliximab (IFX) remains a challenge in the management of inflammatory bowel diseases (IBD). Proactive dosing strategies to achieve and maintain predefined IFX trough levels (TL) may prevent LOR. We aimed to investigate the efficacy of dashboard driven IFX dosing compared to standard dosing in a prospective trial in IBD patients. METHODS: In this multicentre 1:1 'PRECISION' trial, we randomized IBD patients in clinical remission (Harvey Bradshaw Index ≤4 for Crohn's disease (CD) or a partial Mayo score ≤2 for ulcerative colitis (UC)) receiving IFX maintenance treatment. The precision group (PG) received IFX dosing guided by a Bayesian pharmacokinetic model, aiming to achieve and maintain a TL of 3 µg/ml by treatment (de)escalation as indicated by the dashboard. Patients in the control group (CG) continued treatment without dose adaptations. The primary endpoint was the proportion of patients in sustained clinical remission after 1 year. RESULTS: Eighty patients were enrolled (66 CD, 14 UC), and the median [interquartile range] age was 37 years [27-51]). After one year, 28/32 (88%) of patients in the PG were in sustained clinical remission versus 25/39 (64%) in the CG (p = .017). PG patients had lower median faecal calprotectin levels after 1 year (p = .031), whereas no significant differences in median CRP levels were found. CONCLUSION: We demonstrated that the use of a Bayesian dashboard for IFX dosing in maintenance treatment for IBD reduced the incidence of LOR compared to standard dosing. Precision dosing also resulted in lower FCP levels. CLINICALTRIALS.GOV NUMBER: NCT02453776.

Incorporating anti-VEGF pathway therapy as a continuum of care in metastatic colorectal cancer.

Metastatic cancer was previously treated with distinctive lines of chemotherapy regimens upon disease progression or toxicity, yet the choices of therapy are actually interrelated, with the selection of a first-line regimen in part determining the choices available for subsequent treatment. Lately the therapeutic approach based on separate lines of treatment, tends to be replaced from a perspective strategical approach, that of the "continuum of care". This strategy targets to an improved overall survival, improved of quality of life and minimization of toxicity through upfront design of treatment selection and sequencing, exposure to all available drugs and minimization of unnecessary treatment. Anti-VEGF treatment has a well-documented role in this approach. Bevacizumab should be included in upfront treatment regimens for all mCRC patients independently of RAS status, unless contraindicated. Upfront bevacizumab could be combined with all available regimens since the optimal choice of backbone chemotherapy is yet to be defined. In RAS wild-type population, when metastasectomy is the target, an anti-EGFR combination is also a valid approach. Maintenance with bevacizumab and fluoropyrimidines should be considered upon intolerance of induction treatment and/or disease stabilization; maintenance with bevacizumab monotherapy should be avoided. In highly selected patients, complete treatment cessation could be also an option. Continuation with bevacizumab upon first progression and switch of the "backbone" chemotherapy is a validated approach. Patients progressing after first-line oxaliplatin regimen including bevacizumab combinations could be treated with an aflibercept-irinotecan combination. When no more options are available, regorafenib monotherapy should be the following choice. Combinations of anti-VEGF and anti-EGFR treatment have no place in this approach and are not indicated.

Safety, tolerability and anti-tumour activity of the androgen biosynthesis inhibitor ASP9521 in patients with metastatic castration-resistant prostate cancer: multi-centre phase I/II study.

BACKGROUND: ASP9521 is a first-in-class orally available inhibitor of the enzyme 17 ß-hydroxysteroid dehydrogenase type 5 (17 ßHSD5; AKR1C3), catalysing the conversion of dehydroepiandrosterone and androstenedione into 5-androstenediol and testosterone. It has demonstrated anti-tumour activity in in vitro and in vivo preclinical models. MATERIAL AND METHODS: This first-in-man phase I/II study utilised a 3 + 3 dose escalation design starting at 30 mg ASP9521/day, with the aim of defining a maximum tolerated dose, as defined by the incidence of dose-limiting toxicities. Eligible patients received ASP9521 orally for 12 weeks. Safety, tolerability, pharmacokinetics (PK), pharmacodynamics and anti-tumour activity were assessed. RESULTS: Thirteen patients (median age: 68 years; range 52-76) with metastatic castration-resistant prostate cancer (mCRPC) progressing after chemotherapy were included; 12 patients discontinued treatment at or before week 13, mainly due to disease progression. The most common adverse events were grade 1/2 and included asthenia (N = 5), constipation (N = 4), diarrhoea (N = 3), back pain (N = 3) and cancer pain (N = 3). PK demonstrated a half-life (t1/2) ranging from 16 to 35 h, rapid absorption and dose proportionality. No biochemical or radiological responses were identified; neither endocrine biomarker levels nor circulating tumour cell counts were altered by ASP9521. Given the lack of observable clinical activity, the study was terminated without implementing a planned 12-week dose expansion part at selected doses or a planned food-effect study part. CONCLUSIONS: In patients with mCRPC, ASP9521 demonstrated dose-proportional increase in exposure over the doses evaluated, with an acceptable safety and tolerability profile. However, the novel androgen biosynthesis inhibitor showed no relevant evidence of clinical activity.

Endoscopic trimodal imaging detects colonic neoplasia as well as standard video endoscopy.

BACKGROUND & AIMS: Endoscopic trimodal imaging (ETMI) is a novel endoscopic technique that combines high-resolution endoscopy (HRE), autofluorescence imaging (AFI), and narrow-band imaging (NBI) that has only been studied in academic settings. We performed a randomized, controlled trial in a nonacademic setting to compare ETMI with standard video endoscopy (SVE) in the detection and differentiation of colorectal lesions. METHODS: The study included 234 patients scheduled to receive colonoscopy who were randomly assigned to undergo a colonoscopy in tandem with either ETMI or SVE. In the ETMI group (n=118), first examination was performed using HRE, followed by AFI. In the other group, both examinations were performed using SVE (n=116). In the ETMI group, detected lesions were differentiated using AFI and NBI. RESULTS: In the ETMI group, 87 adenomas were detected in the first examination (with HRE), and then 34 adenomas were detected during second inspection (with AFI). In the SVE group, 79 adenomas were detected during the first inspection, and then 33 adenomas were detected during the second inspection. Adenoma detection rates did not differ significantly between the 2 groups (ETMI: 1.03 vs SVE: 0.97, P=.360). The adenoma miss-rate was 29% for HRE and 28% for SVE. The sensitivity, specificity, and accuracy of NBI in differentiating adenomas from nonadenomatous lesions were 87%, 63%, and 75%, respectively; corresponding values for AFI were 90%, 37%, and 62%, respectively. CONCLUSIONS: In a nonacademic setting, ETMI did not improve the detection rate for adenomas compared with SVE. NBI and AFI each differentiated colonic lesions with high levels of sensitivity but low levels of specificity.

Docetaxel, ifosfamide and cisplatin in solid tumour treatment: a phase I study.

Docetaxel, ifosfamide and cisplatin have proven activity in a broad range of solid tumours and interfere with different phases of the cell cycle. We performed a phase I study with the aim to determine the maximum tolerated dose (MTD) of docetaxel, ifosfamide and cisplatin in patients with solid tumours and to define the safety, dose-limiting toxicity (DLT) and the recommended dose and administration schedule of docetaxel, ifosfamide and cisplatin for further phase II testing. Docetaxel was given by 1-h infusion on day 1, followed by ifosfamide 1000 mg/m(2)/day as a continuous infusion for 5 days. Mesna was added at the same doses to the same infusion bag and was continued for 12 h after the end of ifosfamide. Cisplatin was administered as a 24-h infusion concomitantly with ifosfamide, but in separate infusion bags, either on day 5 (schedule A) or on day 1 (schedule B). Escalation steps were planned only for docetaxel (60, 75, 85 mg/m(2)) and cisplatin (50, 75, 100 mg/m(2)). No intrapatient dose escalation was permitted. Prophylactic ciprofloxacin was used after a protocol amendment was implemented. No prophylactic haematopoietic growth factors were used. Cycles of docetaxel, ifosfamide and cisplatin were given at 3-week intervals. Toxicity was scored according to National Cancer Institute Canada-Common Toxicity Criteria 2. The MTD was defined as the dose at which a DLT was observed in fewer than two of six patients during the first treatment cycle. In total, 85 patients received 309 cycles. Only three escalation steps could be explored and DLTs were observed at each dose level. In total, 32 patients and 49 cycles showed DLTs. Febrile neutropenia occurred in 20 patients (24%). Only two DLTs were nonhaematological (one cerebral infarction and one encephalopathy grade 4). Neutropenia grade 4 lasted for greater than 7 days and/or thrombocytopenia grade 4 was dose limiting in 10 patients. Febrile neutropenia occurred in five of 41 patients (12%) who received prophylactic ciprofloxacin and in 15 of 44 patients (34%) who did not. MTD was reached at level 3 (docetaxel, 75 mg/m(2) and cisplatin, 75 mg/m(2)). With a lower dose of docetaxel (60 mg/m(2)) both schedules A and B were feasible, although, overall, schedule A seemed to be better tolerated. On the basis of this phase I study, the recommended docetaxel, ifosfamide and cisplatin regimen is docetaxel (60 mg/m(2)) on day 1, ifosfamide (1000 mg/m(2)/day) on days 1-5 and cisplatin (75 mg/m(2)) given on day 5. It is associated with substantial haematological toxicity, but this is feasible provided prophylactic antibiotics are used.

A non-randomized comparison of gemcitabine-based chemoradiation with or without induction chemotherapy for locally advanced squamous cell carcinoma of the head and neck.

BACKGROUND: Concomitant chemotherapy and radiotherapy (chemoradiation; CRT) is the standard treatment for locoregionally advanced squamous cell carcinoma of the head and neck (LA-SCCHN). CRT improves local control and overall survival (OS) when compared to radiotherapy (RT) alone. Induction chemotherapy (IC) reduces the risk of distant metastases (DM) and improves OS by 5% with the use of cisplatin/infusional 5 fluorouracil (PF) in meta-analysis. Adding a taxane to PF in the IC regimen confers a better outcome. Sequential treatment (ST) of IC followed by CRT is therefore under active investigation in multiple phase III trials. METHODS: We compared the outcome of two cohorts of patients (pts) with LA-SCCHN treated at our institution with CRT (n = 27) or ST (n = 31), respectively. CRT consisted of GEM 100 mg/m2 weekly + conventional RT (70 Gy); ST consisted of the same CRT preceded by platinum-based IC. RESULTS: Response to IC: complete 8 (26%), partial 20 (65%), stable 1, progressive 1, not evaluable 1. Median follow up of the surviving pts: for CRT 73 months, for ST 51 months. Median time to distant metastasis (TDM) was for CRT 23.6 months, for ST not reached. Median OS was for CRT 20.2 months, for ST 40.2 months. Cox regression analysis, taking into account age, T and N stage and tumor site, showed a hazard ratio with ST of 1.190 for time to locoregional failure (p = 0.712), 0.162 for TDM (p = 0.002), and 0.441 for overall survival (OS) (p = 0.026). CONCLUSION: TDM and OS were found significantly longer in the ST cohort without a reduced locoregional control. Notwithstanding the limitations of a non-randomized single-center comparison, the results are in line with very preliminary data of randomized comparisons suggesting an improved outcome with ST.

Laparoscopic ileocolic resection versus infliximab treatment of distal ileitis in Crohn's disease: a randomized multicenter trial (LIR!C-trial).

BACKGROUND: With the availability of infliximab, nowadays recurrent Crohn's disease, defined as disease refractory to immunomodulatory agents that has been treated with steroids, is generally treated with infliximab. Infliximab is an effective but expensive treatment and once started it is unclear when therapy can be discontinued. Surgical resection has been the golden standard in recurrent Crohn's disease. Laparoscopic ileocolic resection proved to be safe and is characterized by a quick symptom reduction. The objective of this study is to compare infliximab treatment with laparoscopic ileocolic resection in patients with recurrent Crohn's disease of the distal ileum with respect to quality of life and costs. METHODS/DESIGN: The study is designed as a multicenter randomized clinical trial including patients with Crohn's disease located in the terminal ileum that require infliximab treatment following recent consensus statements on inflammatory bowel disease treatment: moderate to severe disease activity in patients that fail to respond to steroid therapy or immunomodulatory therapy. Patients will be randomized to receive either infliximab or undergo a laparoscopic ileocolic resection. Primary outcomes are quality of life and costs. Secondary outcomes are hospital stay, early and late morbidity, sick leave and surgical recurrence. In order to detect an effect size of 0.5 on the Inflammatory Bowel Disease Questionnaire at a 5% two sided significance level with a power of 80%, a sample size of 65 patients per treatment group can be calculated. An economic evaluation will be performed by assessing the marginal direct medical, non-medical and time costs and the costs per Quality Adjusted Life Year (QALY) will be calculated. For both treatment strategies a cost-utility ratio will be calculated. Patients will be included from December 2007. DISCUSSION: The LIR!C-trial is a randomized multicenter trial that will provide evidence whether infliximab treatment or surgery is the best treatment for recurrent distal ileitis in Crohn's disease. TRIAL REGISTRATION: Nederlands Trial Register NTR1150.

Burden of disease and increasing prevalence of inflammatory bowel disease in a population-based cohort in the Netherlands.

BACKGROUND: Reported epidemiology and phenotype distributions vary widely and disease burden of inflammatory bowel disease (IBD) is poorly described. Our aim was to establish these features in a population-based cohort covering 319 976 inhabitants. Furthermore, differences between tertiary referral and peripheral hospital patients were quantified. METHODS: IBD patients in the adherence area of three peripheral hospitals (2004-2012) were included. Medical and surgical treatment data were obtained. Quality of life and disease activity were evaluated. An outpatient cohort from a tertiary referral centre was accrued. RESULTS: A total of 1461 patients were included: 761 (52.1%) with ulcerative colitis (UC), 579 (39.5%) with Crohn's disease (CD) and 121 (8.3%) with IBD-unspecified. Point prevalence of IBD was 432.1 per 100 000 inhabitants in 2010, which increased significantly over time, P-value of less than 0.0001. The mean annual incidence was 17.2 for UC, 10.5 for CD and 2.2 for IBD-unspecified. Tertiary referral Crohn's patients used thiopurines and biological therapy and underwent surgery significantly more often than patients in peripheral hospitals (P<0.0001). Disease activity correlated negatively with quality of life (P<0.0001) in UC and CD. CONCLUSION: The prevalence of IBD is still increasing. Burden of disease was significantly more severe, mainly in Crohn's patients, in the referral centre, highlighting the importance of population-based studies to accurately describe phenotype distribution and disease burden.

Diverse effects of infliximab and etanercept on T lymphocytes.

The tumor necrosis factor (TNF) antagonists infliximab and etanercept have proven to be useful additions to the armamentarium of agents used to manage patients with inflammatory disorders. However, as discussed in detail elsewhere in this supplement, these agents have different mechanisms of action and distinct safety and efficacy profiles in the clinical setting. Of particular interest are differing effects on T lymphocytes, thymocyte-derived cells that are responsible for cell-mediated immunity. Recent studies in 2 disease states, ankylosing spondylitis and Crohn's disease, have assessed the effects of TNF antagonists on T lymphocytes and reported differences that could partially explain some of the clinical disparities that have been reported.

Dichotomy between Lactobacillus rhamnosus and Klebsiella pneumoniae on dendritic cell phenotype and function.

The reaction of the intestinal immune system to intestinal bacteria shows striking differences between various bacterial strains. Whereas Klebsiella pneumoniae induces a fierce proinflammatory reaction, the probiotic strain Lactobacillus rhamnosus has clear anti-inflammatory effect in gastrointestinal disease and allergy. The molecular basis for this dichotomy is poorly understood but is likely to involve different modulation of antigen-presenting dendritic cells (DC) by L. rhamnosus and K. pneumoniae. Hence we evaluated phenotypic and functional characteristics of DC matured in the presence of L. rhamnosus and K. pneumoniae. Monocyte-derived immature DC were cultured in the presence of live bacteria to obtain mature DC. Both micro-organisms induced maturation of immature DC as shown by CD83 and CD86 expression, but receptors involved in activation of Th1 cells were expressed predominantly on DC exposed to K. pneumoniae. In contrast to K. pneumoniae, maturation with L. rhamnosus resulted in lower TNF-alpha, IL-6, and IL-8 production by immature DC and lower IL-12 and IL-18 production by mature DC. Moreover, L. rhamnosus led to the development of T cells without a typical Th phenotype whereas K. pneumoniae induced a Th1 immune response, dependent mainly on IL-12 production. Thus our results strongly support the concept that differential modulation of DC explains the differences in the immune response to various bacterial strains and indicates that K. pneumoniae induces Th1 immune responses via DC.

Infliximab induced T lymphocyte apoptosis in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract of unknown origin. Therapies include immune modulating agents, biological therapies, and surgery. The activity and efficacy of the anti-tumor necrosis factor (TNF) therapies infliximab and etanercept have proved to be different: infliximab is effective to induce and maintain remission in refractory CD, while etanercept is not. This brief review considers the question of whether this disparity can be explained by the different structure of the proteins, their different binding affinities, or the subsequent effects on T lymphocytes.

Lactobacillus rhamnosus induces peripheral hyporesponsiveness in stimulated CD4+ T cells via modulation of dendritic cell function.

BACKGROUND: Although it is widely recognized that the intake of so-called probiotic microorganisms is beneficial in chronic mucosal inflammation and topical allergic disease, the immunologic details explaining how such bacteria can exert these effects remain obscure. OBJECTIVE: We determined whether Lactobacillus rhamnosus can modulate T cell responses in vitro and in vivo. DESIGN: In vitro, human monocyte-derived dendritic cells (DCs) matured in the presence of L. rhamnosus were used to instruct naive CD4+ T cells; subsequently, the T cell response was assessed with the use of CD3/CD28 and interleukin (IL) 2. Cytokine production by ex vivo-stimulated naive cells and memory T cells was measured before and after oral supplementation with L. rhamnosus in 6 healthy volunteers and 6 patients with Crohn disease. RESULTS: A decreased T cell proliferation and cytokine production, especially of IL-2, IL-4, and IL-10, was observed in CD3/CD28-stimulated T cells derived from L. rhamnosus-matured DCs. This T cell hyporesponsiveness was associated with enhanced DC-T cell interaction and normal responsiveness of T cells for IL-2. In vivo oral supplementation of L. rhamnosus for 2 wk induced a similar T cell hyporesponsiveness, including impaired ex vivo T helper subsets 1 and 2 responses without up-regulation of immunoregulatory cytokines in cohorts of both healthy volunteers and patients with Crohn disease. CONCLUSIONS: We propose that L. rhamnosus modulates DC function to induce a novel form of T cell hyporesponsiveness; this mechanism might be an explanation for the observed beneficial effects of probiotic treatment in clinical disease.

Treating Crohn's disease by inducing T lymphocyte apoptosis.

Apoptosis is one of the most important regulatory mechanisms in immunological homeostasis. Disturbances in the apoptotic pathways lead to autoimmune disease. Crohn's disease is a chronic inflammatory bowel disease of unknown origin, which seems to be mediated by excessive T cell-mediated immunity. Recently, disturbances in apoptotic pathways of lamina propria T lymphocytes of patients with Crohn's disease have been identified. In the uninflamed, normal intestinal mucosa, lamina propria (LP) T cells are susceptible to activation-induced cell death, but these cells show a resistance to apoptosis based on several disturbances compared to controls. Recently, intriguing data were published using cytokine-targeted therapy (anti-IL12, anti-IL6 receptor, anti-TNF). Actually, these medications restored mucosal immunological imbalance by inducing apoptosis of the LP T cells and seemed to be beneficial in models of Crohn's disease. In this review, mechanisms of immunological homeostasis will be discussed. We will also discuss the fascinating new results of cytokine-targeted therapy in animal models of Crohn's disease and the effects of these drugs in patients with Crohn's disease.

Use of antiemetics in the prevention of chemotherapy-induced nausea and vomiting: review and focus on the Belgian situation.

Chemotherapy-induced nausea and vomiting (CINV) is a common, distressing, debilitating and costly side effect, experienced by up to 90% of patients receiving highly emetogenic drugs. During the last 20 years great advances have been made in the prevention and treatment of CINV. Aprepitant (a neurokinin-1 antagonist) and palonosetron (a 5-HT3 antagonist) are the most recent additions to the available armamentarium. The aim of this paper is to review the most recent findings concerning the pathophysiology and prevention of CINV, and the international guidelines currently in place for its prevention and treatment. Among the treatments available, 5-HT3 antagonists and NK-1 antagonists are compared. In a large meta-analysis (8 studies in 3 592 patients) statistically significant differences in favour of palonosetron compared with first-generation 5-HT3 antagonists have been demonstrated in the prevention of acute, delayed and overall CINV. A recent, large phase III randomized, gender-stratified, double-blind trial in 848 patients receiving a broad range of moderately emetogenic chemotherapy regimens with a variety of tumour types showed superiority of an aprepitant triple regimen compared to a control regimen of ondansetron and dexamethasone. A new combined 5-HT3/ NK-1 treatment, called NEPA (palonosetron/netupitant), has provided very promising preliminary data and is awaited with great anticipation by clinicians. The specific Belgian situation in terms of Health Authorities recommendations and reimbursement policies is also presented. It is concluded that further improvements are still desirable, particularly in the prevention and treatment of delayed emesis.

Development of a real-time PCR for identification of brachyspira species in human colonic biopsies.

BACKGROUND: Brachyspira species are fastidious anaerobic microorganisms, that infect the colon of various animals. The genus contains both important pathogens of livestock as well as commensals. Two species are known to infect humans: B. aalborgi and B. pilosicoli. There is some evidence suggesting that the veterinary pathogenic B. pilosicoli is a potential zoonotic agent, however, since diagnosis in humans is based on histopathology of colon biopsies, species identification is not routinely performed in human materials. METHODS: The study population comprised 57 patients with microscopic evidence of Brachyspira infection and 26 patients with no histopathological evidence of Brachyspira infection. Concomitant faecal samples were available from three infected patients. Based on publically available 16S rDNA gene sequences of all Brachyspira species, species-specific primer sets were designed. DNA was extracted and tested by real-time PCR and 16S rDNA was sequenced. RESULTS: Sensitivity and specificity for identification of Brachyspira species in colon biopsies was 100% and 87.7% respectively. Sequencing revealed B. pilosicoli in 15.4% of patients, B. aalborgi in 76.9% and a third species, tentatively named "Brachyspira hominis", in 26.2%. Ten patients (12.3%) had a double and two (3.1%) a triple infection. The presence of Brachyspira pilosicoli was significantly associated with inflammatory changes in the colon-biopsy (p=0.028). CONCLUSIONS: This newly designed PCR allows for sub-differentiation of Brachyspira species in patient material and thus allows large-scaled surveillance studies to elucidate the pathogenicity of human Brachyspira infections. One-third of affected patients appeared to be infected with a novel species.