Carnosine, oxidative and carbonyl stress, antioxidants, and muscle fiber characteristics of quadriceps muscle of patients with COPD.

Oxidative/carbonyl stress is elevated in lower-limb muscles of patients with chronic obstructive pulmonary disease (COPD). Carnosine is a skeletal muscle antioxidant particularly present in fast-twitch fibers. The aims of the present study were to compare muscle carnosine, oxidative/carbonyl stress, antioxidants, and fiber characteristics between patients with COPD and healthy controls (HCs) and between patients after stratification for airflow limitation (mild/moderate vs. severe/very severe), as well as to investigate correlates of carnosine in patients with COPD. A vastus lateralis muscle biopsy was obtained from 40 patients with stable COPD and 20 age- and sex-matched HCs. Carnosine, oxidative/carbonyl stress, antioxidants, fiber characteristics, quadriceps strength and endurance (QE), V̇o2peak (incremental cycle test), and physical activity (PA) were determined. Patients with COPD had a similar carnosine concentration [4.16 mmol/kg wet weight (WW; SD = 1.93)] to HCs [4.64 mmol/kg WW (SD = 1.71)] and significantly higher percentage of fast-twitch fibers and lower QE, V̇o2peak, and PA versus HCs. Patients with severe/very severe COPD had a 31% lower carnosine concentration [3.24 mmol/kg WW (SD = 1.79); n = 15] versus patients with mild/moderate COPD [4.71 mmol/kg WW (SD = 1.83); n = 25; P = 0.02] and significantly lower V̇o2peak and PA versus patients with mild/moderate COPD. Carnosine correlated significantly with QE (rs = 0.427), V̇o2peak (rs = 0.334), PA (rs = 0.379), and lung function parameters in patients with COPD. In conclusion, despite having the highest proportion of fast-twitch fibers, patients with severe/very severe COPD displayed a 31% lower muscle carnosine concentration compared with patients with mild/moderate COPD. As no other markers of oxidative/carbonyl stress or antioxidants were affected, the observed carnosine deficiency is thought to be a possible first sign of muscle redox balance abnormalities.NEW & NOTEWORTHY Carnosine, particularly present in fast-twitch fibers, was investigated in the quadriceps of patients with chronic obstructive pulmonary disease (COPD). Carnosine concentration was similar between patients with COPD and healthy controls but was 31% lower in patients with severe/very severe COPD, despite their high proportion of fast-twitch fibers, versus patients with mild/moderate COPD. As no other markers of oxidative/carbonyl stress or antioxidants were affected, the observed carnosine deficiency is thought to be a possible first sign of muscle redox balance abnormalities.

Automatic path proposal computation for CT-guided percutaneous liver biopsy.

PURPOSE: To evaluate feasibility of automatic software-based path proposals for CT-guided percutaneous biopsies. METHODS: Thirty-three patients (60 [Formula: see text] 12 years) referred for CT-guided biopsy of focal liver lesions were consecutively included. Pre-interventional CT and dedicated software (FraunhoferMeVis Pathfinder) were used for (semi)automatic segmentation of relevant structures. The software subsequently generated three path proposals in downward quality for CT-guided biopsy. Proposed needle paths were compared with consensus proposal of two experts (comparable, less suitable, not feasible). In case of comparable results, equivalent approach to software-based path proposal was used. Quality of segmentation process was evaluated (Likert scale, 1 [Formula: see text] best, 6 [Formula: see text] worst), and time for processing was registered. RESULTS: All biopsies were performed successfully without complications. In 91 % one of the three automatic path proposals was rated comparable to experts' proposal. None of the first proposals was rated not feasible, and 76 % were rated comparable to the experts' proposal. 7 % automatic path proposals were rated not feasible, all being second choice ([Formula: see text]) or third choice ([Formula: see text]). In 79 %, segmentation at least was good. Average total time for establishing automatic path proposal was 42 [Formula: see text] 9 s. CONCLUSION: Automatic software-based path proposal for CT-guided liver biopsies in the majority provides path proposals that are easy to establish and comparable to experts' insertion trajectories.

An unusual cause of difficult weaning in a patient with newly diagnosed small cell lung cancer.

We describe a patient with acute respiratory insufficiency and difficult ventilator weaning in the ICU ward, leading to diagnosis of small cell lung cancer with superior vena cava superior syndrome. Bilateral vocal cord paralysis caused his respiratory distress and weaning difficulties. Thyroidectomy and neurological problems (such as Parkinson disease and Guillain Barré syndrome) are more common causes of bilateral vocal cord paralysis. Lung cancer patients are also at risk due to mediastinal invasion. The left recurrent laryngeal nerve is more prone to paralysis because of the typical anatomy. In contrary, bilateral vocal cord paralysis is rare and doesn't result in speech problems but rather breathing difficulties. Tracheostomy is the classic therapy, but laser cordectomy and Botulinum toxin injection in the laryngeal muscles are alternatives.

Endobronchial lipoma, an extremely rare benign tumour of the lung, mimicking asthma bronchiale.

Endobronchial lipomas are extremely rare benign tumours of the lung. Their clinical presentation mimics that of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), leading to a delay in diagnosis and errors in treatment. Therefore, making precise diagnosis may be challenging. We report a case of a 63-year-old man with paroxysmal attacks of dyspnea, non-productive cough, and wheezing, initially suspect for adult onset asthma, but with a final diagnosis of endobronchial lipoma.

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors.

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

Combining tiotropium and salmeterol in COPD: Effects on airflow obstruction and symptoms.

BACKGROUND: Clinical information on 24-h spirometric efficacy of combining tiotropium and salmeterol compared to single-agent therapy is lacking in patients with COPD. METHODS: A randomized, double-blind, four-way crossover study of 6-week treatment periods comparing combination therapy of tiotropium 18 microg plus qd or bid salmeterol 50 microg versus single-agent therapy. Serial 24-h spirometry (FEV(1), FVC), effects on dyspnea (TDI focal score) and rescue salbutamol use were evaluated in 95 patients. RESULTS: Tiotropium plus qd salmeterol was superior to tiotropium or salmeterol alone in average FEV(1) (0-24h) by 72 mL and 97 mL (p<0.0001), respectively. Compared to this qd regimen, combination therapy including bid salmeterol provided comparable daytime (0-12h: 12 mL, p=0.38) bronchodilator effects, but significantly more bronchodilation during the night-time (12-24h: 73 mL, p<0.0001). Clinically relevant improvements in TDI focal score were achieved with bronchodilator combinations including salmeterol qd or bid (2.56 and 2.71; p<0.005 versus components). Symptom benefit of combination therapies was also reflected in less need for reliever medication. All treatments were well tolerated. CONCLUSION: Compared to single-agent therapy, combination therapy of tiotropium plus salmeterol in COPD provided clinically meaningful improvements in airflow obstruction and dyspnea as well as a reduction in reliever medication.

The efficacy of tiotropium administered via Respimat Soft Mist Inhaler or HandiHaler in COPD patients.

BACKGROUND: Tiotropium, a once daily inhaled anticholinergic delivered via HandiHaler, provides bronchodilation for >24h and improves patient-centred outcomes. The Respimat Soft Mist Inhaler (SMI), a novel, propellant-free inhaler, has been developed and proposed as an alternative delivery device for use with tiotropium. METHODS: In a pre-specified, pooled analysis of two 30-week, double-blind, double-dummy, crossover studies, 207 patients with Chronic Obstructive Pulmonary Disease (COPD) were randomised to receive once daily tiotropium 5 microg or 10 microg (aqueous solution delivered via Respimat SMI), tiotropium 18 microg (inhalation powder via HandiHaler) or placebo. The primary endpoint was trough forced expiratory volume in 1s (FEV(1)) response. Forced vital capacity (FVC), peak expiratory flow rate (PEFR), rescue medication use, safety and pharmacokinetics (in a subgroup of patients) were also assessed. RESULTS: Both tiotropium doses delivered by Respimat SMI were significantly superior to placebo and non-inferior to tiotropium 18 microg HandiHaler on the primary endpoint (all p<0.0001). All active treatments were significantly superior to placebo (all p<0.0001) and both doses of tiotropium Respimat SMI were non-inferior to tiotropium 18 microg HandiHaler on the secondary spirometry variables and rescue medication use. The systemic exposure was similar between tiotropium 5 microg Respimat SMI and tiotropium 18 microg HandiHaler but was higher for tiotropium 10 microg Respimat SMI. All active treatments were well tolerated. CONCLUSIONS: Tiotropium 5 microg Respimat SMI is comparable with tiotropium 18 microg HandiHaler in terms of efficacy, pharmacokinetics and safety. Respimat SMI is an effective alternative, multi-dose delivery device for tiotropium.

Comparison of tiotropium once daily, formoterol twice daily and both combined once daily in patients with COPD.

This study compared the bronchodilator effects of tiotropium, formoterol and both combined in chronic obstructive pulmonary disease (COPD). A total of 71 COPD patients (mean forced expiratory volume in one second (FEV1) 37% predicted) participated in a randomised, double-blind, three-way, crossover study and received tiotropium 18 microg q.d., formoterol 12 microg b.i.d. or both combined q.d. for three 6-week periods. The end-points were 24-h spirometry (FEV1, forced vital capacity (FVC)) at the end of each treatment, rescue salbutamol and safety. Compared with baseline (FEV1 prior to the first dose in the first period), tiotropium produced a significantly greater improvement in average daytime FEV1 (0-12 h) than formoterol (127 versus 86 mL), while average night-time FEV1 (12-24 h) was not different (tiotropium 43 mL, formoterol 38 mL). The most pronounced effects were provided by combination therapy (daytime 234 mL, night-time 86 mL); both differed significantly from single-agent therapies. Changes in FVC mirrored the FEV1 results. Compared with both single agents, daytime salbutamol use was significantly lower during combination therapy (tiotropium plus formoterol 1.81 puffs.day(-1), tiotropium 2.41 puffs x day(-1), formoterol 2.37 puffs x day(-1)). All treatments were well tolerated. In conclusion, in chronic obstructive pulmonary disease patients, tiotropium q.d. achieved a greater improvement in daytime and comparable improvement in night-time lung function compared with formoterol b.i.d. A combination of both drugs q.d. was most effective and provided an additive effect throughout the 24-h dosing interval.

Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems.

The sensitive detection of human cells in immunodeficient rodents is a prerequisite for the monitoring of micrometastasis of solid tumours, dissemination of leukaemic cells, or engraftment of haematological cells. We developed a universally applicable polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17. The method allows the detection of one human cell in 10(6) murine cells and could be established as both, a conventional DNA polymerase chain reaction-assay for routine screening, and a quantitative real-time polymerase chain reaction-assay using TaqMan-methodology. It was applied to the following xenotransplantation systems in SCID and NOD/SCID mice: (1) In a limiting dilution assay, cells of the MDA-MB 435 breast carcinoma were injected into the mammary fat pad of NOD/SCID mice. It could be shown that 10 cells mouse(-1) were sufficient to induce a positive polymerase chain reaction signal in liver and lung tissue 30 days after transplantation as an indicator for micrometastasis. At this time a palpable tumour was not yet detectable in the mammary fat pad region. (2) Cells of a newly established human acute lymphatic leukaemia were administered intraperitoneally to SCID mice. These cells apparently disseminated and were detectable as early as day 50 in the peripheral blood of living mice, while the leukaemia manifestation was delayed by day 140. (3) In a transplantation experiment using mature human lymphocytes we wanted to standardise conditions for a successful survival of these cells in NOD/SCID mice. It was established that at least 5 x 10(7) cells given intravenously were necessary and that the mice had to be conditioned by 2 Gy body irradiation to get positive polymerase chain reaction bands in several organs. (4) Engraftment studies with blood stem cells originating from cytapheresis samples of tumour patients or from cord blood were undertaken in NOD/SCID mice in order to define conditions of successful engraftment and to use this model for further optimisation strategies. The polymerase chain reaction method presented allowed a reliable prediction of positive engraftment and agreed well with the results of immunohistochemical or FACS analysis. All together, the polymerase chain reaction method developed allows a sensitive and reliable detection of low numbers of human cells in immunodeficient hosts. In combination with real-time (TaqMan) technique it allows an exact quantification of human cells. As this method can be performed with accessible material of living animals, follow up studies for the monitoring of therapeutic interventions are possible in which the survival time of mice as evaluation criteria can be omitted.

Treatment of asthma by the inhaled corticosteroid ciclesonide given either in the morning or evening.

The study addressed the question whether the novel inhaled prodrug corticosteroid ciclesonide is equally effective when inhaled in the morning compared to the evening. For this purpose a double-blind, randomized, parallel group study was initiated in which 209 asthmatic patients (forced expiratory volume in one second = 50-90% predicted) inhaled either 200 microg ciclesonide in the morning or in the evening, for 8 weeks. Efficacy was assessed by means of spirometry as well as daily recordings of morning and evening peak expiratory flow (PEF), symptoms and use of rescue medication. The 24-h urinary cortisol excretion was measured to evaluate any effect on hypothalamic-pituitary-adrenol axis. Ciclesonide significantly improved asthma control. Morning and evening administration was shown to be equally effective for the different spirometry variables, evening PEF, symptoms, use of rescue medication and number of asthma exacerbations. Regarding morning PEF, the improvements after evening dosing were more prominent and equivalence of morning and evening administration could not be demonstrated. No relevant influence on cortisol excretion was found. Overall, the study indicates that ciclesonide can be given either in the morning or in the evening to meet the patients' preference and individual medical needs, although evening administration may lead to a more pronounced improvement in morning peak expiratory flow.

Quantification of human cells in NOD/SCID mice by duplex real-time polymerase-chain reaction.

BACKGROUND AND OBJECTIVES: The aim of this study was the development of a fast and reliable polymerase chain reaction (PCR) assay which quantifies the proportion of human cells in immunodeficient chimeric mice, for example transplanted with human hematopoietic stem cells. DESIGN AND METHODS: We developed a TaqMan chemistry-based, real-time duplex PCR assay to quantify human and murine DNA in a single-tube reaction in parallel (HUmu PCR). Two independent sets of primers and exonuclease probes, located in the tumor necrosis factor-a gene of both species, were selected to amplify specifically human and murine genomic DNA. Serial dilutions of defined numbers of human cells in mouse cells served to construct calibration curves. The test was applied to NOD/SCID mice transplanted with CD34(+) cells isolated from human cord blood and compared to FACS analysis. RESULTS: Analysis of DNA from human cells diluted stepwise into a fixed number of murine cells - and vice versa - led to calibration curves with good correlation for human and murine cells (r(2)>0.99) with a detection limit of 2% human cells. Results obtained with the HUmu PCR paralleled those of FACS analysis. However, in contrast to FACS analysis, which requires fresh single cell suspensions, the HUmu PCR can be carried out on already stored samples, even from solid organs and, moreover, the quantity of material required for analysis is very low. INTERPRETATION AND CONCLUSIONS: The HUmu PCR presented here is the first real-time PCR assay for simultaneous quantification of human and murine cells. It is extremely fast, accurate and is an interesting alternative method for quantifying the proportion of human DNA in organs of chimeric mice.

Donor stromal cells from human blood engraft in NOD/SCID mice.

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice. (Blood. 2000;96:3971-3978)

Physico-chemical properties of the female sex pheromone of Heterodera schachtii (Nematoda: Heteroderidae).

Several physico-chemical properties of the female sex pheromone of the beet cyst nematode Heterodera schachtii were elucidated. At least one component of the pheromone can be extracted from aqueous solutions with diethyl ether. However, the pheromone has a higher solubility in water, as most of the pheromone activity remained in the water fraction. Ion exchange chromatography revealed that the pheromone or at least one of its components is positively charged, whereas another pheromone component may be negatively charged. Fractional distillation of pheromone extracts showed that it is, indeed, composed of at least two components. These components may interact additively rather than synergistically.

Second primary lung cancer in Flanders: frequency, clinical presentation, treatment and prognosis.

Patient and tumour characteristics of 23 patients presenting with a second primary lung cancer were analysed and compared with 534 patients with radically resected stage 1 non-small cell lung cancer (NSCLC). None of these characteristics is associated with a higher occurrence rate for second primary lung cancer. Prognosis in the latter patients is significantly worse than after resection of a 'solitary' NSCLC: the median survival time (MST) after resection of the first tumour is 50 months; after diagnosis of the second tumour only 14 months. Surgically retreated patients have a prognosis that is similar to that after resection of a 'solitary' NSCLC. No separate independent prognostic factors responsible for this survival difference could be isolated. Squamous histology and central location are associated with a longer recurrence free survival time. We conclude that the occurrence of a second primary lung cancer can not be predicted based on patient or tumour characteristics and that only surgical retreatment offers a chance of long survival in these patients.

Relationship between lung tissue and blood plasma concentrations of inhaled budesonide.

In 11 patients, in whom a lung lobe or whole lung was to be resected, a single dose of 1.6 mg inhaled budesonide was given pre-operatively. In 9 of them, concentrations of the drug in both lung tissue and blood plasma were measured. Budesonide concentrations in lung tissue, at least 90 min after dosage, were 2.1-8.9 nmol kg-1. Concentrations in blood plasma (0.27-1.1 nmol kg-1) were 1/8th of those in lung tissue.

Panagrellus redivivus: failure to find evidence for the occurrence and biosynthesis of sialic acids.

The occurrence of sialic acids in the free-living nematode Panagrellus redivivus was studied by periodate oxidation/[3H]sodium borohydride reduction of about 10(7) nematodes. In parallel, the capability of sialic acid biosynthesis was examined by metabolic labeling of the same number of nematodes with N-[3H]acetylmannosamine. In both experiments, radioactivity was incorporated into the nematodes. Mild acid hydrolysis, however, did not release radioactively labeled sialic acids or derivatives as tested by radio thin-layer chromatography, suggesting that P. redivivus does not contain or synthesize sialic acids.

Influence of lectins on female sex pheromone reception byHeterodera schachtii (Nematoda: Heteroderidae) males.

A bioassay was developed in which the chemotactic behavior of males of the sugarbeet cyst nematodeHeterodera schachtii towards the female sex pheromone can be observed and documented at any time. The standardized test, performed under sterile conditions, gives reproducible results within 1-2 hr. Incubation of males with the lectins fromCanavalia ensiformis, Triticum vulgare, Arachis hypogaea, Helix pomatia, andLimax flavus did not affect chemotactic attraction to the sex pheromone. Supplementing the bioassay medium with the lectin fromC. ensiformis also had no effect.C. ensiformis lectin binding to the surface of the exudate produced by the secretory gland cell of the amphids, the main chemoreceptors, did not inhibit the passage of fluorochrome fluorescein isothiocyanate into the amphidial canals, where the receptor dendrites are localized. Amphidial exudate synthesis was enhanced during pheromone reception.

Early emphysema. Ten years' evolution.

In this study, functional evolution over ten years was evaluated in 13 patients with early emphysema. The diagnosis was made on the basis of a decrease in single-breath DCO (55 +/- 14 percent predicted, mean +/- 1 SD), a loss of elastic recoil (CL,st = 0.76 +/- 0.25 L/cm H2O), and only minor airway obstruction (FEV1 = 87 +/- 13 percent predicted, Sgaw = 0.09 +/- 0.04 cm H2O-1.s-1), and compatible chest radiographs. During the ten years, there was a decrease in FEV1 of 0.89 +/- 0.40 L p less than 0.001), with a range of 0.20 to 1.55 L (which could not clearly be related to smoking habits or to initial lung function), a decrease in elastic recoil (p less than 0.05, with a decrease of Ptp, TLC by 6 +/- 7 cm H2O; p approximately equal to 0.05), an increase in TLC of 0.46 +/- 0.80 1 (p approximately equal to 0.05), and in RV/TLC of 9 +/- 3 percent (p less than 0.001). The resistance of the upstream segment (ratio Ptp/Vmax) increased slightly but generally remained within normal limits. In conclusion, patients with early emphysema resemble those with classic COPD, with a mean yearly decline in FEV1 similar to that in COPD.