A systematic survey of randomised trials that stopped early for reasons of futility.

BACKGROUND: Randomised trial protocols may incorporate interim analyses, with the potential to stop the study for futility if early data show insufficient promise of a treatment benefit. Previously, we have shown that this approach will theoretically lead to mis-estimation of the treatment effect. We now wished to ascertain the importance of this phenomenon in practice. METHODS: We reviewed the methods and results in a set of trials that had stopped for futility, identified through an extensive literature search. We recorded clinical areas, interventions, study design, outcomes, trial setting, sponsorship, planned and actual treatment effects, sample sizes; power; and if there was a data safety monitoring board, or a published protocol. We identified: if interim analyses were pre-specified, and how many analyses actually occurred; what pre-specified criteria might define futility; if a futility analysis formed the basis for stopping; who made the decision to stop; and the conditional power of each study, i.e. the probability of statistically significant results if the study were to continue to its complete sample size. RESULTS: We identified 52 eligible trials, covering many clinical areas. Most trials had multiple centres, tested drugs, and 40% were industry sponsored. There were 75% where at least one interim analysis was planned a priori; a majority had only one interim analysis, typically with about half the target total sample size. A majority of trials did not pre-define a stopping rule, and a variety of reasons were given for stopping. Few studies calculated and reported low conditional power to justify the early stop. When conditional power could be calculated, it was typically low, especially under the current trend hypothesis. However, under the original design hypothesis, a few studies had relatively high conditional power. Data collection often continued after the interim analysis. CONCLUSIONS: Although other factors will typically be involved, we conclude that, from the perspective of conditional power, stopping early for futility was probably reasonable in most cases, but documentation of the basis for stopping was often missing or vague. Interpretation of truncated trials would be enhanced by improved reporting of stopping protocols, and of their actual execution.

Randomised trials with provision for early stopping for benefit (or harm): The impact on the estimated treatment effect.

Stopping rules for clinical trials are primarily intended to control Type I error rates if interim analyses are planned, but less is known about the impact that potential stopping has on estimating treatment benefit. In this paper, we derive analytic expressions for (1) the over-estimation of benefit in studies that stop early, (2) the under-estimation of benefit in completed studies, and (3) the overall bias in studies with a stopping rule. We also examine the probability of stopping early and the situation in meta-analyses. Numerical evaluations show that the greatest concern is with over-estimation of benefit in stopped studies, especially if the probability of stopping early is small. The overall bias is usually less than 10% of the true benefit, and under-estimation in completed studies is also typically small. The probability of stopping depends on the true treatment effect and sample size. The magnitude of these effects depends on the particular rule adopted, but we show that the maximum overall bias is the same for all stopping rules. We also show that an essentially unbiased meta-analysis estimate of benefit can be recovered, even if some component studies have stopping rules. We illustrate these methods using data from three clinical trials. The results confirm our earlier empirical work on clinical trials. Investigators may consult our numerical results for guidance on potential mis-estimation and bias in the treatment effect if a stopping rule is adopted. Particular concern is warranted in studies that actually stop early, where interim results may be quite misleading.

Quantifying the bias in the estimated treatment effect in randomized trials having interim analyses and a rule for early stopping for futility.

In this paper, we consider the potential bias in the estimated treatment effect obtained from clinical trials, the protocols of which include the possibility of interim analyses and an early termination of the study for reasons of futility. In particular, by considering the conditional power at an interim analysis, we derive analytic expressions for various parameters of interest: (i) the underestimation or overestimation of the treatment effect in studies that stop for futility; (ii) the impact of the interim analyses on the estimation of treatment effect in studies that are completed, i.e. that do not stop for futility; (iii) the overall estimation bias in the estimated treatment effect in a single study with such a stopping rule; and (iv) the probability of stopping at an interim analysis. We evaluate these general expressions numerically for typical trial scenarios. Results show that the parameters of interest depend on a number of factors, including the true underlying treatment effect, the difference that the trial is designed to detect, the study power, the number of planned interim analyses and what assumption is made about future data to be observed after an interim analysis. Because the probability of stopping early is small for many practical situations, the overall bias is often small, but a more serious issue is the potential for substantial underestimation of the treatment effect in studies that actually stop for futility. We also consider these ideas using data from an illustrative trial that did stop for futility at an interim analysis. Copyright © 2017 John Wiley & Sons, Ltd.

Planning and reporting of quality-of-life outcomes in cancer trials.

BACKGROUND: Information about the impact of cancer treatments on patients' quality of life (QoL) is of paramount importance to patients and treating oncologists. Cancer trials that do not specify QoL as an outcome or fail to report collected QoL data, omit crucial information for decision making. To estimate the magnitude of these problems, we investigated how frequently QoL outcomes were specified in protocols of cancer trials and subsequently reported. DESIGN: Retrospective cohort study of RCT protocols approved by six research ethics committees in Switzerland, Germany, and Canada between 2000 and 2003. We compared protocols to corresponding publications, which were identified through literature searches and investigator surveys. RESULTS: Of the 173 cancer trials, 90 (52%) specified QoL outcomes in their protocol, 2 (1%) as primary and 88 (51%) as secondary outcome. Of the 173 trials, 35 (20%) reported QoL outcomes in a corresponding publication (4 modified from the protocol), 18 (10%) were published but failed to report QoL outcomes in the primary or a secondary publication, and 37 (21%) were not published at all. Of the 83 (48%) trials that did not specify QoL outcomes in their protocol, none subsequently reported QoL outcomes. Failure to report pre-specified QoL outcomes was not associated with industry sponsorship (versus non-industry), sample size, and multicentre (versus single centre) status but possibly with trial discontinuation. CONCLUSIONS: About half of cancer trials specified QoL outcomes in their protocols. However, only 20% reported any QoL data in associated publications. Highly relevant information for decision making is often unavailable to patients, oncologists, and health policymakers.

First-line treatment and outcome of elderly patients with primary central nervous system lymphoma (PCNSL)--a systematic review and individual patient data meta-analysis.

BACKGROUND: To investigate prognosis and effects of first-line therapy in elderly primary central nervous system lymphoma (PCNSL) patients. PATIENTS AND METHODS: A systematic review of studies about first-line therapy in immunocompetent patients ≥60 years with PCNSL until 2014 and a meta-analysis of individual patient data from eligible studies and international collaborators were carried out. RESULTS: We identified 20 eligible studies; from 13 studies, we obtained individual data of 405 patients, which were pooled with data of 378 additional patients (N = 783). Median age and Karnofsky Performance Score (KPS) was 68 years (range: 60-90 years) and 60% (range: 10%-100%), respectively. Treatments varied greatly, 573 (73%) patients received high-dose methotrexate (HD-MTX)-based therapy. A total of 276 patients received whole-brain radiotherapy (median 36 Gy, range 28.5-70 Gy). KPS ≥ 70% was the strongest prognostic factor for mortality [hazard ratio (HR) 0.50, 95% confidence interval (CI) 0.41-0.62]. After a median follow-up of 40 months, HD-MTX-based therapy was associated with improved survival (HR 0.70, 95% CI 0.53-0.93). There was no difference between HD-MTX plus oral chemotherapy and more aggressive HD-MTX-based therapies (HR 1.39, 95% CI 0.90-2.15). Radiotherapy was associated with an improved survival, but correlated with an increased risk for neurological side-effects (odds ratio 5.23, 95% CI 2.33-11.74). CONCLUSIONS: Elderly PCNSL patients benefit from HD-MTX-based therapy, especially if combined with oral alkylating agents. More aggressive HD-MTX protocols do not seem to improve outcome. WBRT may improve outcome, but is associated with increased risk for neurological side-effects. Prospective trials for elderly PCNSL patients are warranted.

Somatostatin-based radiopeptide therapy with [177Lu-DOTA]-TOC versus [90Y-DOTA]-TOC in neuroendocrine tumours.

PURPOSE: Somatostatin-based radiopeptide treatment is generally performed using the ß-emitting radionuclides (90)Y or (177)Lu. The present study aimed at comparing benefits and harms of both therapeutic approaches. METHODS: In a comparative cohort study, patients with advanced neuroendocrine tumours underwent repeated cycles of [(90)Y-DOTA]-TOC or [(177)Lu-DOTA]-TOC until progression of disease or permanent adverse events. Multivariable Cox regression and competing risks regression were employed to examine predictors of survival and adverse events for both treatment groups. RESULTS: Overall, 910 patients underwent 1,804 cycles of [(90)Y-DOTA]-TOC and 141 patients underwent 259 cycles of [(177)Lu-DOTA]-TOC. The median survival after [(177)Lu-DOTA]-TOC and after [(90)Y-DOTA]-TOC was comparable (45.5 months versus 35.9 months, hazard ratio 0.91, 95% confidence interval 0.63-1.30, p = 0.49). Subgroup analyses revealed a significantly longer survival for [(177)Lu-DOTA]-TOC over [(90)Y-DOTA]-TOC in patients with low tumour uptake, solitary lesions and extra-hepatic lesions. The rate of severe transient haematotoxicities was lower after [(177)Lu-DOTA]-TOC treatment (1.4 vs 10.1%, p = 0.001), while the rate of severe permanent renal toxicities was similar in both treatment groups (9.2 vs 7.8%, p = 0.32). CONCLUSION: The present results revealed no difference in median overall survival after [(177)Lu-DOTA]-TOC and [(90)Y-DOTA]-TOC. Furthermore, [(177)Lu-DOTA]-TOC was less haematotoxic than [(90)Y-DOTA]-TOC.

Mould-active compared with fluconazole prophylaxis to prevent invasive fungal diseases in cancer patients receiving chemotherapy or haematopoietic stem-cell transplantation: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND: Objectives were to compare systemic mould-active vs fluconazole prophylaxis in cancer patients receiving chemotherapy or haematopoietic stem cell transplantation (HSCT). METHODS: We searched OVID MEDLINE and the Cochrane Central Register of Controlled Trials (1948-August 2011) and EMBASE (1980-August 2011). Randomised controlled trials of mould-active vs fluconazole prophylaxis in cancer or HSCT patients were included. Primary outcome was proven/probable invasive fungal infections (IFI). Analysis was completed by computing relative risks (RRs) using a random-effects model and Mantel-Haenszel method. RESULTS: From 984 reviewed articles, 20 were included in this review. Mould-active compared with fluconazole prophylaxis significantly reduced the number of proven/probable IFI (RR 0.71, 95% CI 0.52 to 0.98; P=0.03). Mould-active prophylaxis also decreased the risk of invasive aspergillosis (IA; RR 0.53, 95% confidence interval (CI) 0.37-0.75; P=0.0004) and IFI-related mortality (RR 0.67, 95% CI 0.47-0.96; P=0.03) but is also associated with an increased risk of adverse events (AEs) leading to antifungal discontinuation (RR 1.95, 95% CI 1.24-3.07; P=0.004). There was no decrease in overall mortality (RR 1.0; 95% CI 0.88-1.13; P=0.96). CONCLUSION: Mould-active compared with fluconazole prophylaxis significantly reduces proven/probable IFI, IA, and IFI-related mortality in cancer patients receiving chemotherapy or HSCT, but increases AE and does not affect overall mortality. (PROSPERO Registration: CRD420111174).

Adverse events associated with individual statin treatments for cardiovascular disease: an indirect comparison meta-analysis.

BACKGROUND: Statins are the most widely prescribed drug available. Due to this reason, it is important to understand the risks involved with the drug class and individual statins. AIM: We conducted a meta-analysis and employed indirect comparisons to identify differing risk effects across statins. DESIGN: We included any randomized clinical trial (RCT) of atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin used for cardiovascular disease event prevention. The main outcome was adverse events [all-cause mortality, cancers, rhabdomylosis, diabetes, aspartate and alanine aminotransferase (AST/ALT), and creatinine kinase (CK) increases beyond the upper limit of normal]. In order to evaluate the relative effects of each drug on adverse events, we calculated adjusted indirect comparisons of the adverse-event outcomes. RESULTS: Seventy-two trials involving 159,458 patients met our inclusion criteria. Overall, statin treatments significantly increased the rate of diabetes when compared to controls (OR: 1.09; 95% CI: 1.02-1.16) and elevated AST (OR: 1.31; 95% CI: 1.04-1.66) and ALT (OR: 1.28; 95% CI: 1.11-1.48) levels when compared to controls. Using indirect comparisons, we also found that atorvastatin significantly elevated AST levels compared to pravastatin (OR: 2.21; 95% CI: 1.13-4.29) and simvastatin significantly increased CK levels when compared to rosuvastatin (OR: 4.39; 95% CI: 1.01-19.07). Higher dose studies had increased risk of AST elevations. DISCUSSION: Although statins are generally well tolerated, there are risks associated with almost all drugs. With few exceptions, statins appear to exert a similar risk across individual drugs.

Efficacy and safety of statin treatment for cardiovascular disease: a network meta-analysis of 170,255 patients from 76 randomized trials.

BACKGROUND: Statins represent the largest selling class of cardiovascular drug in the world. Previous randomized trials (RCTs) have demonstrated important clinical benefits with statin therapy. AIM: We combined evidence from all RCTs comparing a statin with placebo or usual care among patients with and without prior coronary heart disease (CHD) to determine clinical outcomes. DESIGN: We searched independently, in duplicate, 12 electronic databases (from inception to August 2010), including full text journal content databases, to identify all statin versus inert control RCTs. We included RCTs of any statin versus any non-drug control in any populations. We abstracted data in duplicate on reported major clinical events and adverse events. We performed a random-effects meta-analysis and meta-regression. We performed a mixed treatment comparison using Bayesian methods. RESULTS: We included a total of 76 RCTs involving 170,255 participants. There were a total of 14,878 deaths. Statin therapy reduced all-cause mortality, Relative Risk (RR) 0.90 [95% confidence interval (CI) 0.86-0.94, P ≤ 0.0001, I(2)=17%]; cardiovascular disease (CVD) mortality (RR 0.80, 95% CI 0.74-0.87, P<0.0001, I(2)=27%); fatal myocardial infarction (MI) (RR 0.82, 95% CI 0.75-0.91, P<0.0001, I(2)=21%); non-fatal MI (RR 0.74, 95% CI 0.67-0.81, P ≤ 0.001, I(2)=45%); revascularization (RR 0.76, 95% CI 0.70-0.81, P ≤ 0.0001); and a composite of fatal and non-fatal strokes (0.86, 95% CI 0.78-0.95, P=0.004, I(2)=41%). Adverse events were generally mild, but 17 RCTs reported on increased risk of development of incident diabetes [Odds Ratio (OR) 1.09; 95% CI 1.02-1.17, P=0.001, I(2)=11%]. Studies did not yield important differences across populations. We did not find any differing treatment effects between statins. DISCUSSION: Statin therapies offer clear benefits across broad populations. As generic formulations become more available efforts to expand access should be a priority.

Perceived competence in children and adolescents with haemophilia: an explorative study.

With the introduction of prophylaxis, restricting children with haemophilia to participate in physical activities was no longer necessary. Subsequently, many studies report on improved physical functioning in children and adolescents with haemophilia. However, little is known about psychological aspects such as perceived competence and impact of disease. Therefore, the aims of this study were to explore: (i) perceived competence, (ii) perceived impact of illness, and (iii) analyse associations between perceived competence and demographic factors, disease-related factors and joint status in young haemophiliacs in the Netherlands. Fifty-four children (age 8-12 years) and 72 adolescents (12-18 years) with haemophilia participated in this cross-sectional, multi-centre, explorative study. Measurements included perceived competence (Self Perception Profile for Children/Adolescents; range 6-24/5-20), impact of disease (Revised Perception Illness Experience; range 1-5), demographic factors, disease-related factors, joint status and functional status. Mean (SD) scores for perceived competence in the children ranged from 17.3 (±4.0) to 19.6 (±4.0), and for adolescents from 13.3 (±2.4) to 15.7 (±2.8) points. In general, scores were comparable with those of healthy peers, but children with haemophilia had a lower global self-worth score and competence in close friendship was lower for adolescents when compared with those of healthy peers. Mean (SD) scores for impact of disease ranged from 1.2 (±0.4) to 2.3 (±0.8) in children and from 1.3 (±0.4) to 2.0 (±0.8) in adolescents. Severe haemophilia, prophylactic medication, high impact of disease and a shorter walking distance showed a weak to moderate association with perceived competence. Children and adolescents with haemophilia in general have a perceived competence that is nearly comparable with that of healthy peers, with the exception of a lower global self-worth in children and a lower competence for close friendship in adolescents. Haemophiliacs seem to perceive their disease as having relatively low impact on their life. Severe disease, prophylactic treatment and low functional status seemed to be associated with lower perceived competence.

Prescribed medications and pharmacy interventions for acute respiratory tract infections in Swiss primary care.

BACKGROUND AND OBJECTIVES: Symptomatic medications are often not considered in clinical studies assessing interventions to reduce prescribing of antibiotics for acute respiratory tract infections (ARTI). Our study objectives were to examine prescribing patterns of antibiotics and symptomatic medications for ARTI in Swiss primary care and to monitor pharmacists' interventions during the prescription-dispensing process. METHODS: Medical records of 695 patients participating in a clinical trial which was designed to reduce use of antibiotics for ARTI in primary care, were linked to their prescriptions. Matching of prescribed and dispensed medications enabled the assessment of interventions by community pharmacists. RESULTS: On average, 2.4 different drugs were prescribed per patient (in total 142 antibiotics, 1599 symptomatic medications, and 56 non-ARTI-medication). Most patients (80%) were treated only with symptomatic medications. Most frequently prescribed symptomatic ARTI-medications were nasal decongestants (39%), cough suppressants (36%), and mucolytics (31%). Patients with prescribed antibiotics received significantly fewer symptomatic medications (odds ratio, 0.24; 95% confidence interval 0.16-0.37). Over 20% of prescriptions prompted at least one intervention by a pharmacist in the dispensing process. A discrepancy between prescribed and dispensed medications was seen in 19% of patients. CONCLUSIONS: Prescription rates of antibiotics for ARTI in this trial were low and patients were treated mainly with non-antibiotic symptomatic medications. Efforts to reduce antibiotic prescribing may induce higher rates of use of medications for intensive symptomatic treatment. Considerable differences between prescribed and dispensed medications were noted.

Adjunctive corticosteroids for Pneumocystis jiroveci pneumonia in patients with HIV-infection.

BACKGROUND: Pneumocystis jiroveci pneumonia (PCP) remains the most common opportunistic infection in patients infected with the human immunodeficiency virus (HIV). Among patients with HIV infection and PCP the mortality rate is 10 to 20% during the initial infection and increases substantially with the need for mechanical ventilation. It was suggested that in these patients corticosteroids adjunctive to standard treatment for PCP could prevent the need for mechanical ventilation and decrease mortality. OBJECTIVES: To assess the effects of adjunctive corticosteroids on overall mortality and the need for mechanical ventilation in HIV-infected patients with PCP and substantial hypoxemia (arterial oxygen partial pressure <70 mmHg or alveolar-arterial gradient >35 mmHg on room air). SEARCH STRATEGY: We searched Medline (January 1980-December 2004), EMBASE (January 1985-December 2004) and The Cochrane Library (Issue 4, 2004) without language restrictions to identify randomised controlled trials that compared adjunctive corticosteroids to control in HIV-infected patients with PCP. We further reviewed the reference lists from previously published overviews, we searched UptoDate version 2005 and Clinical Evidence Concise (Issue 12, 2004), contacted experts of the field, and searched reference lists of identified publications for citations of additional relevant articles. SELECTION CRITERIA: Trials were considered eligible for this review if they compared corticosteroids to placebo or usual care in HIV-infected patients with PCP in addition to baseline treatment with trimethoprim-sulfamethoxazole, pentamidine or dapsone-trimethoprim, used random allocation, and reported mortality data. We excluded trials in patients with no or mild hypoxemia (arterial oxygen partial pressure >70 mmHg or an alveolar-arterial gradient <35 mmHg on room air) and trials with a follow-up of less than 30 days. DATA COLLECTION AND ANALYSIS: Two teams of reviewers independently evaluated the methodology and extracted data from each primary study. We pooled treatment effects across studies and calculated a weighted average risk ratio of overall mortality in the treatment and control groups by using a random effects model. MAIN RESULTS: Six studies were included in the review and meta-analysis. Risk ratios for overall mortality for adjunctive corticosteroids were 0.56 (95% confidence interval [CI], 0.32-0.98) at 1 month and 0.68 (95% CI, 0.50-0.94) at 3-4 months of follow-up. To prevent 1 death, numbers needed to treat are 9 patients in a setting without highly active antiretroviral therapy (HAART) available, and 23 patients with HAART available. Only the 3 largest trials provided data on the need for mechanical ventilation with a risk ratio of 0.38 (95% CI, 0.20-0.73) in favour of adjunctive corticosteroids. AUTHORS' CONCLUSIONS: The number and size of trials investigating adjunctive corticosteroids for HIV-infected patients with PCP is small, but evidence from this review suggests a beneficial effect for patients with substantial hypoxemia.

Cystic fibrosis and CFTR.

Cystic fibrosis (CF) is a complex disease affecting epithelial ion transport. There are not many diseases like CF that have triggered such intense research activities. The complexity of the disease is due to mutations in the CFTR protein, now known to be a Cl(-) channel and a regulator of other transport proteins. The various interactions and the large number of disease-causing CFTR mutations is the reason for a variable genotype-phenotype correlation and sometimes unpredictable clinical manifestation. Nevertheless, the research of the past 10 years has resulted in a tremendous increase in knowledge, not only in regard to CFTR but also in regard to molecular interactions and completely new means of ion channel and gene therapy.

Motor performance of children during treatment for acute lymphoblastic leukemia.

BACKGROUND: Daily life motor skills of children with acute lymphoblastic leukemia (ALL) were studied during treatment using the Movement Assessment Battery for Children (Movement ABC). In addition, the possible relation with vincristine treatment was investigated. PROCEDURE: Seventeen children treated for ALL, aged 4-12 years, were compared to an age- and sex-matched control group. RESULTS: The leukemia group performed more poorly than the control group on both fine and gross motor skills. In looking at the number of children with ALL who scored in the clinical range of the different subtests, problems in balance skills were found to be most pronounced at the end of induction therapy. Remarkably, half a year after reinduction therapy, problems with balance had decreased, whereas the number of children with fine motor problems had increased. CONCLUSIONS: A relation between the gross motor problems and vincristine neurotoxicity seems plausible based on a descriptive analysis of the data, but this was not supported statistically.

No evidence for direct activation of the cystic fibrosis transmembrane conductance regulator by 8-cyclopentyl-1,3-dipropylxanthine.

8-Cyclopentyl-1,3-dipropylxanthine (CPX) is a selective A1-adenosine receptor antagonist which has been reported to activate Cl- efflux at very low concentrations in cells carrying the cystic fibrosis (CF) defect, but not in cells expressing the wild-type form of the CF transmembrane conductance regulator (CFTR). CPX was suggested as a new therapeutic drug for the treatment of CF. In the present study, we examined the effects of CPX on various types of recombinant cells (Xenopus oocytes, Chinese hamster ovary cells, CF tracheal cells) and native non-CF and CF respiratory epithelial cells. CPX did not activate a whole-cell conductance when applied at concentrations ranging from 1 nmol/l to 100 micromol/l in oocytes injected with water or expressing either wild-type CFTR or mutant deltaF508-CFTR. Correspondingly, CPX (10 micromol/l) did not activate whole-cell conductance in non-CF or CF respiratory epithelial cells and Chinese hamster ovary cells expressing either wild-type CFTR or deltaF508-CFTR. Instead, CPX depolarized Vm by inhibition of a K+ conductance in CF respiratory epithelial cells. At 10 micromol/l CPX marginally decreased intracellular pH in respiratory epithelial cells, independent of expression of wild-type CFTR or mutant CFTR. According to these data, CPX-induced 36Cl efflux reported in previous studies cannot be attributed to direct activation of deltaF508-CFTR Cl- conductance and is probably related to the CPX-induced changes in intracellular pH.

Cl- transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co-expressing CFTR and ENaC.

1. Epithelial Na+ channels (ENaCs) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is activated by protein kinase A. Since cAMP-dependent activation of CFTR Cl- conductance is defective in cystic fibrosis (CF), ENaC currents are not inhibited by CFTR. This could explain the enhanced Na+ conductance found in CF. In the present study, we examined possible mechanisms of interaction between CFTR and ENaC co-expressed in Xenopus oocytes. 2. The magnitude of CFTR Cl- currents activated by 3-isobutyl-1-methylxanthine (IBMX) in oocytes co-expressing either wild-type or mutant CFTR and ENaC determined the degree of downregulation of ENaC currents. 3. The ability of CFTR to inhibit ENaC currents was significantly reduced either when extracellular Cl- was replaced by poorly conductive anions, e.g. SCN- or gluconate, or when CFTR was inhibited by diphenylamine-carboxylate (DPC, 1 mmol l-1). 4. Downregulation of ENaC was more pronounced at positive when compared with negative clamp voltages. This suggests that outward currents, i.e. influx of Cl- through activated CFTR most effectively downregulated ENaC. 5. Activation of endogenous Ca2+-activated Cl- currents by 1 micromol l-1 ionomycin did not inhibit ENaC current. This suggests that inhibition of ENaC mediated by Cl- currents may be specific to CFTR. 6. The present findings indicate that downregulation of ENaC by CFTR is correlated to the ability of CFTR to conduct Cl-. The data have implications for how epithelia switch from NaCl absorption to NaCl secretion when CFTR is activated by secretagogues.

KVLQT channels are inhibited by the K+ channel blocker 293B.

Previous data have indicated that the chromanol 293B blocks a cAMP activated K+ conductance in the colonic crypt, a K+ conductance in pig cardiac myocytes and the K+ conductance induced by IsK protein expression in Xenopus oocytes. We have also shown that cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) up-regulates, apart from the typical Cl- current, a 293B- inhibitable K+ current. Very recently it has been shown that the IsK protein interacts with KVLQT subunits to produce a K+ channel. These data have prompted us to ask the following questions: Is the 293B-inhibitable current in oocytes expressing CFTR and activated by cAMP caused by an endogenous Xenopus KVLQT (XKVLQT), and is mouse KVLQT (mKVLQT) expressed in oocytes inhibited by 293B? Antisense and sense probes for XKVLQT were coinjected with CFTR cRNA into oocytes. After 3-4 days the oocytes were examined by two electrode voltage clamp. It was found that in control oocytes expressing CFTR and stimulated by isobutylmethylxanthine (IBMX, 1 mmol/l) 293B (10 micromol/l) reduced the conductance (Gm). In oocytes coinjected with the sense probe for XKVLQT and pretreated with IBMX 293B still reduced Gm, whilst the 293B-inhibitable Gm was almost completely absent in oocytes coinjected with XKVLQT antisense. In another series a full length clone for mKVLQT was generated by PCR techniques and the cRNA was injected into oocytes. After several days these oocytes, unlike water injected ones, were found to be strongly hyperpolarized and their Gm was increased significantly. The oocytes were depolarized significantly and their Gm was reduced reversibly by 10 micromol/l 293B. These data indicate that CFTR activation by IBMX indeed co-activates an endogenous oocyte XKVLQT channel and that this channel is inhibited by a new class of channel blockers, of which 293B is the prototype.

The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl- conductance of Xenopus oocytes.

Oocytes from Xenopus laevis activate a Ca2+ dependent Cl- conductance when exposed to the Ca2+ ionophore ionomycin. This Ca2+ activated Cl- conductance (CaCC) is strongly outwardly rectifying and has a halide conductivity ratio (GI- / GCl-) of about 4.4. This is in contrast to the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- conductance, which produces more linear I/V curves with a GI- / GCl- ratio of about 0.52. Ionomycin enhanced CaCC (DeltaG) in water injected and CFTR expressing ooyctes in the absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/l) by (microS) 23 +/- 1.9 (n=9) and 23.6 +/- 2.3 (n=11). Stimulation by IBMX did not change CaCC in water injected oocytes. CaCC was inhibited in CFTR-expressing ooyctes after stimulation with IBMX or a membrane permeable form of cAMP and was only 5.1 +/- 0.48 microS (n=18) and 6. 9 +/- 0.6 (n=3), respectively. Inhibition of CaCC was correlated to the amount of CFTR-current activated by IBMX. DeltaF508-CFTR which demonstrates only a small residual function in activating a cAMP dependent Cl- channel in oocytes inhibited CaCC to a lesser degree (DeltaG=12.1 +/- 1.1 microS; n=7). Changes of CFTR and CaCC-Cl- whole cell conductances were also measured when extracellular Cl- was replaced by I-. The results confirmed the reduced activation of CaCC in the presence of activated CFTR. No evidence was found for inhibition of CFTR-currents by increase of intracellular Ca2+. Moreover, intracellular cAMP was not changed by ionomycin and stimulation by IBMX did not change the ionomycin induced Ca2+ increase in Xenopus oocytes. Taken together, these results suggest that activation of CFTR-Cl- currents is paralleled by an inhibition of Ca2+ activated Cl- currents in ooyctes of Xenopus laevis. These results provide another example for CFTR-dependent regulation of membrane conductances other than cAMP-dependent Cl- conductance. They might explain previous findings in epithelial tissues of CF-knockout mice.

Fine motor and handwriting problems after treatment for childhood acute lymphoblastic leukemia.

Motor skills were investigated in 18 children 2 years after treatment for acute lymphoblastic leukemia (ALL). Gross and fine motor functioning were examined with the Movement Assessment Battery for Children. Handwriting as a specific fine motor skill was studied with a computerized writing task. We conclude that 2 years after cessation of treatment motor problems in ALL survivors were still present. Dysfunctions were mainly pronounced in handwriting and fine motor skills.