Daily CBCT-based dose calculations for enhancing the safety of dose-escalation in lung cancer radiotherapy.

PURPOSE: Dose-escalation in lung cancer comes with a high risk of severe toxicity. This study aimed to calculate the delivered dose in a Scandinavian phase-III dose-escalation trial. METHODS: The delivered dose was evaluated for 21 locally-advanced non-small cell lung cancer (LA-NSCLC) patients treated as part of the NARLAL2 dose-escalation trial. The patients were randomized between standard and escalated heterogeneous dose-delivery. Both treatment plans were created and approved before randomization. Daily cone-beam CT (CBCT) for patient positioning, and adaptive radiotherapy were mandatory. Standard and escalated plans, including adaptive re-plans, were recalculated on each daily CBCT and accumulated on the planning CT for each patient. Dose to the clinical target volume (CTV), organs at risk (OAR), and the effects of plan adaptions were evaluated for the accumulated dose and on each treated fraction scaled to full treatment. RESULTS: For the standard treatment, plan adaptations reduced the number of patients with CTV-T underdosage from six to one, and the total number of fractions with CTV-T underdosage from 161 to 56; while for the escalated treatment, the number of patients was reduced from five to zero and number of fractions from 81 to 11. For dose-escalation, three patients had fractions exceeding trial constraints for heart, bronchi, or esophagus, and one had an accumulated heart dose above the constraints. CONCLUSION: Dose-escalation for LA-NSCLC patients, using daily image guidance and adaptive radiotherapy, is dosimetrically safe for the majority of patients. Dose calculation on daily CBCTs is an efficient tool to monitor target coverage and OAR doses.

Outcome of conventional radiotherapy in small centrally located tumours or lymph nodes: minimal toxicity, remarkable survival but challenging loco-regional control.

BACKGROUND: In peripheral lung tumours, stereotactic body radiotherapy (SBRT) is superior to conventional RT. SBRT has also shown high loco-regional control (LC) in centrally located tumours, but there is a high risk of severe toxicity. The STRICTSTARLung trial (NCT05354596) examines if risk-adapted SBRT for central tumours is feasible. In this study, we examined overall survival (OS), Disease-free survival (DSF), LC, and toxicity in patients with central tumours that could have been candidates for SBRT but received conventional RT. MATERIAL AND METHODS: Retrospectively, we evaluated 49 lung cancer patients that between 2008 and 2021 received RT (60-70Gy in 2 Gy fractions) for a solitary tumour or lymph node with a diameter <5cm located <2cm from the bronchial tree, oesophagus, aorta or heart. All tumours were pathologically verified; 30 were primary lung tumours (T1b-T4) and 19 were solitary lymph nodes (T0N1-N2). Chemotherapy was administered as concomitant (29) or sequential (4). OS and LC were analysed using Kaplan Meier. Cox proportional hazards model for OS and disease-free survival (DFS) was performed including tumour volume, histology, sex, T- vs N-site and chemotherapy. Toxicity was scored. RESULTS: In 42 patients, the tumour was located <1 cm to mediastinum. Median follow-up time was 44 months (range: 7-123). The median OS was 51 months. OS at 1-, 3- and 5-year was 88% (SE:5), 59% (SE:7) and 50% (SE:8). Loco-regional recurrences occurred in 16 patients resulting in 1-, and 3-year LC rates of 77% (SE:6) and 64% (SE:8). The majority occurred within 3 years after RT. Only stage showed significant impact on OS and DFS. No patients experienced grade 4-5 toxicity. Seven patients developed grade 3 toxicity (5 oesophageal stenosis, 2 pneumonitis). CONCLUSION: Conventional RT for patients with small central lung tumours or solitary lymph nodes is feasible. Median OS was 51 months, and toxicity was low with no grade 4-5 events.

A phase IIb, open-label, randomized controlled dose ranging multi-centre trial to evaluate the safety, tolerability, pharmacokinetics and exposure-response relationship of different doses of delpazolid in combination with bedaquiline delamanid moxifloxacin in adult subjects with newly diagnosed, uncomplicated, smear-positive, drug-sensitive pulmonary tuberculosis.

BACKGROUND: Linezolid is an effective, but toxic anti-tuberculosis drug that is currently recommended for the treatment of drug-resistant tuberculosis. Improved oxazolidinones should have a better safety profile, while preserving efficacy. Delpazolid is a novel oxazolidinone developed by LegoChem Biosciences Inc. that has been evaluated up to phase 2a clinical trials. Since oxazolidinone toxicity can occur late in treatment, LegoChem Biosciences Inc. and the PanACEA Consortium designed DECODE to be an innovative dose-ranging study with long-term follow-up for determining the exposure-response and exposure-toxicity relationship of delpazolid to support dose selection for later studies. Delpazolid is administered in combination with bedaquiline, delamanid and moxifloxacin. METHODS: Seventy-five participants with drug-sensitive, pulmonary tuberculosis will receive bedaquiline, delamanid and moxifloxacin, and will be randomized to delpazolid dosages of 0 mg, 400 mg, 800 mg, 1200 mg once daily, or 800 mg twice daily, for 16 weeks. The primary efficacy endpoint will be the rate of decline of bacterial load on treatment, measured by MGIT liquid culture time to detection from weekly sputum cultures. The primary safety endpoint will be the proportion of oxazolidinone class toxicities; neuropathy, myelosuppression, or tyramine pressor response. Participants who convert to negative liquid media culture by week 8 will stop treatment after the end of their 16-week course and will be observed for relapse until week 52. Participants who do not convert to negative culture will receive continuation phase treatment with rifampicin and isoniazid to complete a six-month treatment course. DISCUSSION: DECODE is an innovative dose-finding trial, designed to support exposure-response modelling for safe and effective dose selection. The trial design allows assessment of occurrence of late toxicities as observed with linezolid, which is necessary in clinical evaluation of novel oxazolidinones. The primary efficacy endpoint is the change in bacterial load, an endpoint conventionally used in shorter dose-finding trials. Long-term follow-up after shortened treatment is possible through a safety rule excluding slow-and non-responders from potentially poorly performing dosages. TRIAL REGISTRATION: DECODE was registered in ClinicalTrials.gov before recruitment start on 22 October 2021 (NCT04550832).

Thorough design and pre-trial quality assurance (QA) decrease dosimetric impact of delineation and dose planning variability in the STRICTLUNG and STARLUNG trials for stereotactic body radiotherapy (SBRT) of central and ultra-central lung tumours.

INTRODUCTION: SBRT of central lung tumours implies significant risk of toxicity. We are initiating two phase II trials prescribing 56 Gy/eight fractions to PTV, allowing for dose escalation of GTV. We prioritize organs at risk (OAR) constraints over target coverage, making the treatment plans very sensitive to OAR delineation variations. The aim of this study is to quantify the dosimetric impact of contouring variations and to provide a thorough description of pre-trial quality assurance to be used in upcoming trials to provide consistent clinical care. MATERIALS AND METHODS: Delineation: Seven physicians delineated OAR in three rounds, with evaluations in-between. For each patient case, seven treatment plans, repeatedly using each of the OAR structure sets from the seven physicians, were made and compared to evaluate the dosimetric effect of delineation variability. Treatment planning: Treatment plans for seven cases were made at six departments in two rounds, with discussion in-between. RESULTS: OAR delineation variation between centres resulted in high variabilities in OAR dose for simulated plans and led to potential overdosage of the lobar bronchus (constraint: D0.03cc < 45 Gy), with maximum doses ranging between 58 Gy (first round), and 50 Gy (third round). For mediastinal tissue, the constraint (D0.03cc < 45 Gy) was violated for the majority of the delineations in all three rounds, with maximum doses of 84 Gy (first round), and 72 Gy (third round).For the treatment planning study, the range of the standard deviation for GTV mean dose was 12.8-18.5 Gy (first round) and 2.8-3.5 Gy (second round). CONCLUSIONS: Even small variations in OAR delineation led to high OAR overdosage. The study demonstrates the importance of having extensive QA procedures in place before initiating clinical trials on dose escalation in SBRT.

A comparison of two methods for segmentation of functional volumes in radiotherapy planning of lung cancer patients.

BACKGROUND: In radiotherapy (RT) of lung cancer, dose to functional lung (FL) volumes segmented with two different methods (perfusion SPECT (Q-SPECT) and 4D-CT (4D) ventilation (V)) have been shown to correlate with the incidence of radiation pneumonitis (RP). This study aims to compare the FL volumes identified by both methods. MATERIAL AND METHODS: Thirty lung cancer patients had a 4D and Q-SPECT prior to treatment. Seventeen of these patients also had a ventilation SPECT (V-SPECT). FL sub-volumes were segmented automatically, using cut-off values. The volumes were compared in terms of overlap fraction (OF) relative to the minimal volume, and intersection fraction (IF) of the FL volume relative to the total lung volume (VLung). RESULTS: Cut-off values suggested in literature for Q-SPECT and 4D-V resulted in volumes differing in size by a median 18% [6%;31%], and a median OF and IF of 0.48 [0.23;0.70] and 0.09 [0.02;0.25], respectively. Segmenting volumes of comparable size of about 1/3 of VLung (FL-m(1/3), m = method) resulted in a median OF and IF of 0.43 [0.23;0.58] and 0.12 [0.06;0.19], respectively. Twenty-five patients (83%) had a reasonable overlap between FL-Q(1/3) and FL-4D-V(1/3) volumes, with OF values above 0.33. IF increased significantly (p = .036) compared to using fixed cut-off values. Similarly, volumes of comparable size of about 1/3 VLung were produced for V-SPECT, and FL-Q(1/3), FL-V(1/3), and FL-4D-V(1/3) were compared. The overlaps and intersections of FL-V(1/3) with FL-Q(1/3) volumes were significantly (p<.001) larger than the corresponding overlaps and intersections of FL-Q(1/3) with FL-4D(1/3) and FL-V(1/3) with FL-4D(1/3). CONCLUSION: The Q-SPECT and 4D-V methods do not segment entirely the same FL volumes. A reasonable overlap of the volumes along with the findings of other studies that both correlate to RP incidence, suggests that a combination of both volumes, e.g. using the IF, may be useful in RT treatment planning.

Optimal beam angle selection and knowledge-based planning significantly reduces radiotherapy dose to organs at risk for lung cancer patients.

BACKGROUND: Lung cancer patients struggle with high toxicity rates. This study investigates if IMRT plans with individually set beam angles or uni-lateral VMAT plans results in dose reduction to OARs. We investigate if introduction of a RapidPlan model leads to reduced dose to OARs. Finally, the model is validated prospectively. MATERIAL AND METHODS: Seventy-four consecutive lung cancer patients treated with IMRT were included. For all patients, new IMRT plans were made by an experienced dose planner re-tuning beam angles aiming for minimized dose to the lungs and heart. Additionally, VMAT plans were made. The IMRT plans were selected as input for a RapidPlan model, which was used to generate 74 new IMRT plans. The new IMRT plans were used as input for a second RapidPlan model. This model was clinically implemented and used for generation of clinical treatment plans. Dosimetric parameters were compared using a Wilcoxon signed rank test or a 1-sided student's t-test. p < .05 was considered significant. RESULTS: IMRT plans significantly reduced mean doses to lungs (MLD) and heart (MHD) by 1.6 Gy and 1.7 Gy in mean compared to VMAT plans. MLD was significantly (p < .001) reduced from 10.8 Gy to 9.4 Gy by using the second RapidPlan model. MHD was significantly (p < .001) reduced from 4.9 Gy to 3.9 Gy. The model was validated in prospectively collected treatment plans showing significantly lower MLD after the implementation of the second RapidPlan model. CONCLUSION: Introduction of RapidPlan and beam angles selected based on the target and OARs position reduces dose to OARs.

Circulating microRNAs associated with liver fibrosis in chronic hepatitis C patients.

A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.

Energy layer optimization strategies for intensity-modulated proton therapy of lung cancer patients.

PURPOSE: When treating lung cancer patients with intensity-modulated proton therapy (IMPT), target coverage can only be guaranteed when utilizing motion mitigation. The three motion mitigation techniques, gating, breath-hold, and dose repainting, all benefit from a more rapid application of the treatment plan. A lower limit for the ungated treatment time is defined by the number of energy layers in the IMPT plan. By limiting this number during treatment planning, IMPT could become more viable for lung cancer patients. We investigate to what extend the number of layers can be reduced in single-field optimization (SFO) and multifield optimization (MFO) plans and which implications it has on the plan quality and robustness. METHODS: We have implemented three distinct layer-reducing strategies in the treatment planning system Hyperion; constant energy steps, exponential energy steps, and an adaptive strategy, where the spot weights are exposed to a group sparsity penalty in combination with layer exclusion during optimization. Four levels of increasing layer removal are planned for each strategy. SFO and MFO plans with three treatment fields are created for eleven locally advanced NSCLC patients on the midventilation 4DCT phase to simulate a breath-hold. A minimum dose to the target is ensured for each degree of layer reduction, reflecting the plan quality in the homogeneity index (HI). Plan quality was also assessed by a robustness evaluation, where the patient setup was shifted 2 mm or 4 mm in six directions. RESULTS: The three strategies result in very similar target coverages and robustness levels as a function of removed layers. The HI increases unacceptably for all the SFO plans after 50% layer removal as compared to the reference plan, while all the MFO plans are clinically acceptable with up to a highest removed percentage of 75%. The robustness level is constant as a function of removed layers. The SFO plans are significantly more robust than the MFO plans with all P-values below 0.001 (Wilcoxon signed-rank). The overall mean D98% CTV dose difference is at 2-mm setup error amplitude: 0.7 Gy (SFO) and 1.9 Gy (MFO), and at 4 mm: 3.2 Gy (SFO) and 5.4 Gy (MFO), respectively. CONCLUSIONS: The number of layers in MFO plans can be reduced substantially more than in SFO plans without compromising plan quality. Furthermore, as the robustness is independent of the number of layers, it follows that if the level of robustness is acceptable or enforced via robust optimization, MFO plans could be candidates for treatment time reductions via energy layer reductions.

Local failure after radical radiotherapy of non-small cell lung cancer in relation to the planning FDG-PET/CT.

OBJECTIVES: Local recurrence (rec) in lung cancer is associated with poor survival. This study examined whether the pattern of failure is associated with the most PET avid volume in the planning-FDG-PET/CT scan (p-PET/CT). METHODS: 162 consecutive inoperable NSCLC patients (pts) receiving radiotherapy between January 2012 and April 2014 were reviewed. Radiotherapy was delivered in 2 Gy/fraction (5f/week) to a total dose of 60-66 Gy. Pts were followed with CT scans every third month. Patients with local rec as first event were analyzed. For the primary tumor (T) the overlap-fraction (OF) between 50% of SUVpeak on p-PET/CT and the volume of T-rec was calculated: OF = (SUVp50∩T-rec)/min(SUVp50, T-rec). Similarly for the GTV on the p-CT: OF = (GTV∩T-rec)/min(GTV, T-rec). OF was based on a rigid registration between p-PET/CT and rec-CT with PET guided delineation of T- rec. For lymph nodes (LN), the correlation between the location of treated-LN and the location of recurrence-LN was evaluated. RESULTS: 67 patients developed local rec. 51 pts had rec in T-site, 45 pts in LN-site. Due to anatomical changes, reliable registration between p-CT and rec-CT was only obtained in 26 pts with T-rec. The median OFSUVp50 was 52, 8% [range 26; 100%] and the median OFGTV was 80.5% [19.7; 100%]. Eleven pts had higher OFSUVp50 than OFGTV. LN-rec predominantly occurred in the station 2R (32%), 4R (46%), 7 (46%) and right hilum (36%). Pts with malignant LNs in station 4R or 7 on p-CT had a high risk of rec in these stations; 4R (55%) and 7 (83%). CONCLUSIONS: This study indicates that the most PET active volume on p-PET-CT is a driver for rec at T-site. LN-recurrences predominantly appear in station 2R, 4R, 7 and right hilum. Additional confirmatory studies regarding lymph node mapping and selective lymph node irradiation is needed.

Reliability of dose volume constraint inference from clinical data.

Dose volume histogram points (DVHPs) frequently serve as dose constraints in radiotherapy treatment planning. An experiment was designed to investigate the reliability of DVHP inference from clinical data for multiple cohort sizes and complication incidence rates. The experimental background was radiation pneumonitis in non-small cell lung cancer and the DVHP inference method was based on logistic regression. From 102 NSCLC real-life dose distributions and a postulated DVHP model, an 'ideal' cohort was generated where the most predictive model was equal to the postulated model. A bootstrap and a Cohort Replication Monte Carlo (CoRepMC) approach were applied to create 1000 equally sized populations each. The cohorts were then analyzed to establish inference frequency distributions. This was applied to nine scenarios for cohort sizes of 102 (1), 500 (2) to 2000 (3) patients (by sampling with replacement) and three postulated DVHP models. The Bootstrap was repeated for a 'non-ideal' cohort, where the most predictive model did not coincide with the postulated model. The Bootstrap produced chaotic results for all models of cohort size 1 for both the ideal and non-ideal cohorts. For cohort size 2 and 3, the distributions for all populations were more concentrated around the postulated DVHP. For the CoRepMC, the inference frequency increased with cohort size and incidence rate. Correct inference rates >[Formula: see text] were only achieved by cohorts with more than 500 patients. Both Bootstrap and CoRepMC indicate that inference of the correct or approximate DVHP for typical cohort sizes is highly uncertain. CoRepMC results were less spurious than Bootstrap results, demonstrating the large influence that randomness in dose-response has on the statistical analysis.

Plasma total bilirubin levels predict amputation events in type 2 diabetes mellitus: the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study.

AIMS/HYPOTHESIS: Bilirubin has antioxidant and anti-inflammatory activities. Previous studies demonstrated that higher bilirubin levels were associated with reduced prevalence of peripheral arterial disease (PAD). However, the relationship between bilirubin and lower-limb amputation, a consequence of PAD, is currently unknown. We hypothesised that, in patients with type 2 diabetes, bilirubin concentrations may inversely associate with lower-limb amputation. METHODS: The relationship between baseline plasma total bilirubin levels and amputation events was analysed in 9,795 type 2 diabetic patients from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study. The analysis plan was pre-specified. Lower-limb amputation was adjudicated blinded to treatment allocation. Relevant clinical and biochemical data were available for analyses. Amputation was a pre-specified tertiary endpoint. RESULTS: Bilirubin concentrations were significantly inversely associated with lower-limb amputation, with a greater than threefold risk gradient across levels. Individuals with lower bilirubin concentrations had a higher risk for first amputation (HR 1.38 per 5 µmol/l decrease in bilirubin concentration, 95% CI 1.07, 1.79, p = 0.013). The same association persisted after adjustment for baseline variables, including age, height, smoking status, γ-glutamyltransferase level, HbA1c, trial treatment allocation (placebo vs fenofibrate), as well as previous PAD, non-PAD cardiovascular disease, amputation or diabetic skin ulcer, neuropathy, nephropathy and diabetic retinopathy (HR 1.38 per 5 µmol/l decrease in bilirubin concentration, 95% CI 1.05, 1.81, p = 0.019). CONCLUSIONS/INTERPRETATION: Our results identify a significant inverse relationship between bilirubin levels and total lower-limb amputation, driven by major amputation. Our data raise the hypothesis that bilirubin may protect against amputation in type 2 diabetes.

Determination of atrazine and degradation products in Luxembourgish drinking water: origin and fate of potential endocrine-disrupting pesticides.

Several pesticides have been hypothesized to act as endocrine-disrupting compounds, exhibiting hormonal activity and perturbing normal physiological functions. Among these, especially s-triazine herbicides have received increased attention. Despite being banned in many countries, including the European Union, atrazine is still the world's most widely used herbicide. Despite its discontinued use, considerable concentrations of atrazine and its degradation products, mainly desethylatrazine (DEA) and deisopropylatrazine (DIA), are still found in the environment, including drinking water sources. The aim of this investigation was to study concentrations of especially s-triazine herbicides and major degradation products in drinking water, including spring water, tap water and bottled water in Luxembourg. Spring water (2007/2008/2009, n = 69/69/69), tap water (2008/2009, n = 19/26), and bottled water (2007/2008/2009, n = 5/13/7) were sampled at locations in Luxembourg and investigated for pesticides by LC-ESI-MS/MS. Atrazine was the predominant triazine, detectable in many spring water locations, tap and bottled water, ranging (mean) from 0-57 (9), 0-44 (4), and 0-4 (1) ng l(-1), respectively. DEA and DIA in spring water ranged (mean) from 0-120 (19) and 0-27 (3) ng l(-1), with higher concentrations from agricultural areas and low molar ratios of DEA:atrazine <0.5 and high ratios of atrazine:nitrate suggesting point-source contamination. Levels (mean) of DEA and DIA in tap water were 0-62 (14) and 0-6 (<1) ng l(-1) and in bottled water 0-11 (2) and 0-7 (2) ng l(-1). Simazine and other triazines were detected in traces (<5 ng l(-1)). Thus, the conducted monitoring suggested the presence of low concentrations of s-triazines in raw and finished water, presumably partly due to non-agricultural contamination, with concentrations being below thresholds advocated by the European Union Directive 98/83/EC.

Tolerance levels of EPID-based quality control for volumetric modulated arc therapy.

PURPOSE: Volumetric modulated arc therapy (VMAT) includes features such as a variable dose rate and gantry speed in addition to the beam modulation achieved with multileaf collimator (MLC) motion patterns employed in intensity modulated radiotherapy. Three tests have previously been proposed for the evaluation of the performance of VMAT delivery. In order to enable a convenient and accurate routine machine quality control (QC) program, the present study proposes tolerance levels for these tests based on a department-wide implementation of an electronic portal imaging device (EPID)-based QC. METHODS: Three different VMAT tests--a picket fence (PF) test, a dose rate versus gantry speed (DRGS) test, and a dose rate versus MLC leaf speed (DRMLC) test--were performed on nine accelerators using two different EPIDs (aS1000 and aS500, Varian Medical Systems). All tests were repeated six times for each accelerator. The images were analyzed using an in-house-developed software. For the PF test, the positions and widths of individual MLC leaf gaps were compared to the mean value. In the DRGS and DRMLC tests, different combinations of dose rate, gantry speed, and MLC leaf speed were used to deliver identical doses to separate parts of the EPID. The tests were evaluated by looking for deviations in the constancy of the measured dose for the preset combinations of dose rate, gantry speed, and MLC leaf speed. RESULTS: For the PF test, a 0.3 mm tolerance level was suggested for the positioning of the MLC leaves. The tolerance level for the gap width was 0.5 mm. For the DRGS and DRMLC tests, a 3% tolerance level was proposed. CONCLUSIONS: With the adapted levels of tolerance for an EPID-based approach, the PF, the DRGS, and the DRMLC tests offer a convenient and accurate machine QC program for linear accelerators used for VMAT.

Solid phase extraction coupled to liquid chromatography-tandem mass spectrometry analysis of sulfonamides, tetracyclines, analgesics and hormones in surface water and wastewater in Luxembourg.

In the early 1990s different studies highlighted the relationship between pharmaceuticals, human health and the environment. Among the emerging contaminants, antibiotics are obviously of high concern, because of their potential for inducing antibiotic resistance. In addition, natural and synthetic hormones are relevant because of their potential endocrine-disrupting effects on wildlife. This investigation focuses on the analysis of four classes of veterinary and human pharmaceuticals (sulfonamides, tetracyclines, analgesics and hormones) in surface water and wastewater in Luxembourg. The selected eleven pharmaceuticals include four sulfonamides (sulfathiazole, sulfamethoxazole, sulfadimethoxine and sulfamethazine), two tetracyclines (tetracycline and oxytetracycline), two analgesics (ibuprofen and diclofenac), and three hormones (2 naturals, estrone and beta-estradiol, and a synthetic one, 17-alpha-ethinyl estradiol). The most innovative parts of this study are the simultaneous extraction of the above-mentioned pharmaceuticals as well as tracking their behaviour during flood events in a small river catchment. The method includes pre-concentration by solid phase extraction using Oasis HLB (Hydrophilic Lipophilic Balance) which gave superior results compared to Chromabond C-18EC, Chromabond(R) EASY and Bond Elut PLEXA cartridges, also evaluated in this investigation. The analysis of the investigated pharmaceutical compounds is carried out by high performance liquid chromatography coupled to a triple quadrupole mass spectrometer. The limits of quantification were 1 ng L(-1), except for beta-estradiol (2 ng L(-1)) and 17-alpha-ethinyl estradiol (6 ng L(-1)). Recovery rates range from 70 to 94%, with relative standard deviations between 4 and 19%. Application of this method to river concentration and flood events revealed high concentrations of ibuprofen (10-4000 ng L(-1)), with highest levels during flood events, while concentrations of estrogens (1-240 ng L(-1)) and sulfonamides (1-20 ng L(-1)) were comparatively low.

Distinct molecular requirements for activation or stabilization of soluble guanylyl cyclase upon haem oxidation-induced degradation.

BACKGROUND AND PURPOSE: In endothelial dysfunction, signalling by nitric oxide (NO) is impaired because of the oxidation and subsequent loss of the soluble guanylyl cyclase (sGC) haem. The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid (BAY 58-2667) is a haem-mimetic able to bind with high affinity to sGC when the native haem (the NO binding site) is removed and it also protects sGC from ubiquitin-triggered degradation. Here we investigate whether this protection is a unique feature of BAY 58-2667 or a general characteristic of haem-site ligands such as the haem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt (HMR 1766), the haem-mimetic Zn-protoporphyrin IX (Zn-PPIX) or the haem-dependent sGC stimulator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272). EXPERIMENTAL APPROACH: The sGC inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to induce oxidation-induced degradation of sGC. Activity and protein levels of sGC were measured in a Chinese hamster ovary cell line as well as in primary porcine endothelial cells. Cells expressing mutant sGC were used to elucidate the molecular mechanism underlying the effects observed. KEY RESULTS: Oxidation-induced sGC degradation was prevented by BAY 58-2667 and Zn-PPIX in both cell types. In contrast, the structurally unrelated sGC activator, HMR 1766, and the sGC stimulator, BAY 41-2272, did not protect. Similarly, the constitutively haem-free sGC mutant beta(1)H105F was stabilized by BAY 58-2667 and Zn-PPIX. CONCLUSIONS: The ability of BAY 58-2667 not only to activate but also to stabilize oxidized/haem-free sGC represents a unique example of bimodal target interaction and distinguishes this structural class from non-stabilizing sGC activators and sGC stimulators such as HMR 1766 and BAY 41-2272, respectively.

Multiple sclerosis and the TNFRSF1A R92Q mutation: clinical characteristics of 21 cases.

OBJECTIVE: Tumor necrosis factor receptor 1-associated periodic syndrome (TRAPS) is an autosomal dominantly inherited autoinflammatory disorder resulting from mutations in the TNFRSF1A gene, which encodes the p55 receptor for tumor necrosis factor alpha. We recently identified the R92Q mutation encoded by exon 4 in six patients with multiple sclerosis (MS) who reported at least two symptoms suggestive of TRAPS. The current study presents the characteristics of a larger cohort of MS patients carrying this mutation. METHODS: Clinical and laboratory parameters, including human leukocyte antigen (HLA)-DR15 status, were evaluated, and genetic testing was performed. Whenever possible, family members were also invited for interview and mutation analysis. RESULTS: Twenty TNFRSF1A R92Q carriers had MS according to the McDonald criteria, and 1 had clinically isolated syndrome. The majority of patients had typical onset and features of MS. Nine patients carried an HLA-DR15 haplotype. All individuals showed TRAPS-compatible symptoms, which consisted mainly of myalgias, arthralgias, headache, severe fatigue, and skin rashes; were milder than usually described; and appeared mainly in adulthood. Most patients experienced severe side effects during immunomodulatory therapy for MS. Seventeen family members carried the identical mutation, and 15 of them reported symptoms suggestive of TRAPS. CONCLUSION: In most cases with multiple sclerosis (MS) and coexisting tumor necrosis factor receptor 1-associated periodic syndrome (TRAPS), features of MS were quite typical, whereas TRAPS presented mostly without the fever episodes observed in childhood. The penetrance of the R92Q mutation in affected family members was higher than reported. We recommend careful observation of MS patients with coexisting TRAPS with regard to unexpected side effects of immunomodulatory therapies.