Direct measurement of BDP and 17-BMP in bronchial and peripheral lung tissue after inhalation of HFA- vs CFC-driven aerosols.

Indirect assessments have shown a superior lung deposition of HFA-BDP (Ventolair/Qvar) compared to CFC-BDP (Aerobec). The aim of this study was to assess the concentrations of BDP and its metabolite 17-BMP in airways and peripheral tissue from resected lung specimens after inhalation of these BDP formulations. Immediately prior to surgery for lung cancer, 10 patients inhaled 1000 microg of either CFC-BDP (n = 5) or HFA-BDP (n = 5) Mouthwash was collected after inhalation, and serum before, during, and after surgery. There was no significant difference between CFC and HFA in the concentration of 17-BMP in bronchi (median, 4365 vs 4121 pg/g tissue). After CFC, concentrations of 17-BMP were lower in peripheral tissue (1424 vs 2089 pg/g; ANCOVA, p = 0.001) and in serum taken immediately after inhalation (688 vs 1219 pg/ml, p < 0.01). Furthermore, the CFC group showed a higher concentration of BDP in the mouthwash (17,660 vs 1320 ng/ml, p < 0.05), but the concentration of 17-BMP was lower (452 vs 1028 ng/ml, n.s.). These findings indicate a predominantly peripheral deposition of HFA-BDP, in line with previous data. They also provide evidence for a faster uptake and metabolism of HFA-BDP, probably because BDP is dissolved in HFA and has a smaller particle size distribution than the CFC suspensions.

Human alveolar epithelial cells induce nitric oxide synthase-2 expression in alveolar macrophages.

It was hypothesized that cell-to-cell interaction between human alveolar macrophages (AM) and alveolar epithelium, might be an important factor leading to nitric oxide synthase-2 (NOS2) messenger ribonucleic acid (mRNA) and protein expression by constituent cells of the alveolar wall and/or AM. NOS2 mRNA and the protein expression patterns of human AM and alveolar epithelial cells type II (AEC-II) isolated from normal parts of lung resections of patients with pulmonary malignancies were determined. In addition, NOS2 mRNA expression in human AM co-cultured with autologous AEC-II in the presence of pro-inflammatory cytokines interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma or lipopolysaccharide (LPS) was investigated. The effect of human surfactant protein-A (SP-A) on IFN-gamma-mediated NOS2 mRNA expression in human AM was also studied. Neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated AEC-II. In contrast, freshly isolated AM from bronchoalveolar lavage or lung tissue samples expressed immunoreactivity for NOS2 protein, but no NOS2 mRNA could be detected by reverse transcriptase polymerase chain reaction. All stimuli tested failed to induce NOS2 mRNA expression in human AM in vitro. Only AM-AEC-II co-culture in the presence of IFN-gamma led to NOS2 mRNA and protein expression. In situ hybridization of NOS2 mRNA on lung tissue explants and immunohistochemical staining of cytospin preparations of AM-AEC-II co-cultures demonstrated that NOS2 is expressed in AM but not in AEC-II. This co-culture effect could not be reproduced by substitution of AEC-II with SP-A. These data give evidence of a regulatory network controlling human nitric oxide synthase-2 expression in the lower respiratory tract.

Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II.

The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS3, and produces nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of type II human alveolar epithelial cells (AEC II) in primary culture. We detected NOS3 mRNA in freshly isolated AEC II and after 24 h of culture. NOS3 mRNA levels were much higher in AEC II cultured for 24 h with or without interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha, compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC II compared with unstimulated cells. NOS3 protein expression was verified by Western blot, and measuring nitrate/nitrite revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC II in 24- or 72-h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC II and A549 cells in NOS mRNA expression pattern.

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture.

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.

Guidelines for the clinical use of transdermal fentanyl.

Transdermal (TTS) fentanyl therapy has emerged as an effective alternative to the use of oral opioids for the control of pain in certain cancer patients. These patients are those with moderate to severe chronic pain, with a stable baseline pain pattern. Patients receiving this treatment should first be titrated to stable pain relief with oral opioids and should have recourse during therapy to fast-acting, short-duration analgesics for the control of incident pain. TTS fentanyl dosing schedules should be based upon the patient's requirement for rescue dosing and duration of effective pain control. The average requirement to change fentanyl patches is every 72 h, although 48-h dosing is necessary in a few patients. This novel route of fentanyl administration allows convenient outpatient treatment, the possibility of a lower incidence of side effects, and may thus aid compliance.

Determination of DNA single-strand breaks in lymphocytes of smokers and nonsmokers exposed to environmental tobacco smoke using the nick translation assay.

The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick translation assay is presented. This assay allows rapid and sensitive examination of SSB using only 5 ml heparinized blood for an eightfold determination. The assay was standardized by incubation of MBWC in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known genotoxic agent. In vitro incubation of MWBC with MNNG induced a dose-dependent increase in DNA-SSB at doses between 5 and 500 microM MNNG. The detection limit for the assay was 5 microM MNNG. To assess the suitability of this assay to detect SSB in vivo a controlled study was performed in which volunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental tobacco smoke (ETS), and nonsmokers controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On control days (days 1 and 3) smokers and nonsmokers sat in an unventilated 45 m3 room for 8 h. On the exposure days (days 2 and 4) each of the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High exposure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (CO-Hb), plasma nicotine and cotinine levels. Blood was drawn before and after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel technique for the detection of DNA single-strand breaks in human white blood cells and its combination with the unscheduled DNA synthesis assay.

A modified assay for the detection of DNA single-strand breaks (SSBs) in human mononucleated white blood cells (MWBCs) based on the nick translation (NT) reaction was developed and combined with the test for unscheduled DNA synthesis (UDS). Both assays were performed on disposable 96-well filtration plates and therefore allowed rapid and sensitive examination of SSBs and UDS. Only 5-8 ml of heparinized blood is required for an eightfold determination in both assays. The uptake of radioactive nucleotide precursors was demonstrated to depend linearly upon the NT reaction time and in both assay systems on the number of investigated cells. The best results and the lowest signal to noise ratio were obtained when the NT assay was performed at 25 degrees C for 20 min. The test was standardized for 150,000 MWBCs/well and a polymerase I concentration of 20 U/ml. The same number of cells were used to measure UDS during a 4-h incubation at 37 degrees C. We observed a dose-dependent increase in SSBs after in vitro incubation with N-methyl-N-nitrosoguanidine (MNNG), with a detection limit of 50 microM when MNNG was present for 1 h and of 5 microM after 20-h incubation period. UDS in MWBCs was increased after treatment for 1 h with MNNG (200 microM) only if poly(ADP)ribose synthesis was inhibited by 3-aminobenzamide. UDS was induced by 320 microM methyl methanesulfonate, but SSBs could only be detected after inhibition of UDS by 100 microM hydroxyurea.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of DNA single-strand breaks in lymphocytes of smokers.

In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five nonsmokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37 degrees C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0 degree C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some interindividual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)