Online measurement of oxygen consumption by goat bone marrow stromal cells in a combined cell-seeding and proliferation perfusion bioreactor.

In an effort to produce clinically useful volumes of tissue engineered bone products, a direct perfusion bioreactor system was developed. Perfusion flow rate, flow direction, and the position of the bioreactor are factors that influenced the amounts and homogeneity of the cells seeded on the scaffold surface. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cm(3)) of small-sized macroporous biphasic calcium phosphate (BCP) scaffolds (2-6 mm). Cell load and cell distribution were shown using Methylene Blue block staining, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate the viability of the cells. Although cells were not distributed homogenously after cell seeding, the scaffolds were covered with a viable, homogeneous cell layer after 25 days of cultivation. The hybrid structures became interconnected, and a dense layer of extracellular matrix formed on and in the scaffolds. Online oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption could possibly be used to estimate GBMSC population doubling times during growth in this bioreactor system. On the basis of our results, we conclude that a direct perfusion bioreactor system is capable of seeding and proliferating GBMSCs on BCP ceramic scaffolds that can be monitored online during cultivation.

Parallel high-resolution confocal Raman SEM analysis of inorganic and organic bone matrix constituents.

In many multi-disciplinary fields of science, such as tissue engineering, where material and biological sciences are combined, there is a need for a tool that combines ultrastructural and chemical data analysis in a non-destructive manner at high resolution. We show that a combination of confocal Raman spectroscopy (CRS) and scanning electron microscopy (SEM) can be used for such analysis. Studies of atomic composition can be done by X-ray microanalysis in SEM, but this is only possible for atomic numbers greater than five and does not reveal molecular identity. Raman spectroscopy, however, can provide information on molecular composition and identity by detection of wavelength shifts caused by molecular vibrations. In this study, CRS-SEM revealed that early in vitro-formed bone extracellular matrix (ECM) produced by rat osteoprogenitor cells resembles mature bone chemically. We gained insight into the structure and chemical composition of the ECM, which was composed of mainly mineralized collagen type I fibres and areas of dense carbonated calcium phosphate related to the collagen fibre density, as revealed by Raman imaging of SEM samples. We found that CRS-SEM allows the study of specimens in a non-destructive manner and provides high-resolution structural and chemical information about inorganic and organic constituents by parallel measurements on the same sample.

Studying the effect of different macrostructures on in vitro cell behaviour and in vivo bone formation using a tissue engineering approach.

In the present study, we tested the in vitro process of differentiation and mineralization as well as the process of in vivo bone formation on substrates with different macrostructures. We used carbonated apatite-coated titanium discs that were respectively smooth, plasma spayed with titanium or had a porous structure. Subcultured rat bone marrow cells were seeded on the substrates and after 7 days of culture, the tissue-coated substrates were subcutaneously implanted in nude mice for 4 weeks. After 1 week of culture in the presence of the osteogenic differentiation promoter dexamethasone, the cells had formed a continuous layer of mineralized tissue on the smooth and titanium plasma-sprayed discs. In the case of the porous titanium discs, the bone-like tissue coverage was restricted to the outer surface and the peripheral pores. The influence of the macrostructure on the process of differentiation of the cultured cells depended on the presence of dexamethasone. When dexamethasone was present, the highest ALP/DNA ratios were obtained with the smooth surfaces. In the absence of dexamethasone, the highest ALP/DNA values were obtained with the rough macrostructured discs. We postulate that these different patterns were due to the shielding of cells in pits or pores of rough structured substrates by dense overlying cell layers. These cell layers are suggested to increase the exposure of excreted osteoinductive proteins and decrease the exposure of dexamethasone to underlying cells. Four weeks post-implantation, abundant bone formation could be observed on all in vitro tissue-coated substrates. The percentage of direct bone contact on the porous discs (42.3 +/- 22.3) was significantly lower compared to the non-porous discs. This was related to the process of bone infiltration into the central oriented pores that predominantly occurred in a centrifugal manner. The percentage of direct bone contact on the smooth discs (96.3 +/- 2.3) was significantly higher compared to the titanium plasma-sprayed discs (81.5 +/- 10.7). This was not due to fibrous tissue infiltration, but due to the extensive formation of bone marrow. Nevertheless, for practical reasons regarding protection of the layer of cultured cells during the implantation procedure, the use of rough or porous surface structures is suspected to be advantageous in revision surgery.

Rapid fluorescence ratio assay for detecting changes in radiosensitivity.

To test modifications in sensitization to radiation or drugs in preclinical studies of cancer therapy, the colony-forming assay is regarded as the gold standard. Because this assay is time consuming, somewhat laborious, and unsuitable for rapid screening, development of other assays is desirable. We describe here an assay based on the detection of enhanced green fluorescence protein (EGFP) with flow cytometry that is particularly suitable for genetic manipulation studies in which the gene of interest is introduced together with EGFP as reporter. It is easily adaptable to other reporters, however, whether naturally fluorescent or requiring immunochemical staining. Cells are irradiated as mixed populations of a known standard cell line (nonfluorescent) together with the genetically manipulated cell line expressing EGFP. Ratios of fluorescent and nonfluorescent cells are measured before treatment and several days after treatment. If the cell populations have equal radiosensitivities, the ratio remains unchanged. Changes in the ratio indicate changes in radiosensitivity. The assay was validated for two situations in which dominant negative peptides inhibiting DNA repair were expressed in A549 human lung cells and affected radiosensitivity.

Is the increase in bronchial responsiveness or FEV1 shortly after cessation of beta2-agonists reflecting a real deterioration of the disease in allergic asthmatic patients? A comparison between short-acting and long-acting beta2-agonists.

Regular use of beta2-agonists might result in increased bronchial hyper-responsiveness (BHR) and decreased forced expiratory volume in 1 sec (FEV1). It has been suggested that these possible detrimental effects are not a real deterioration of the disease, but that it might be only a transient (rebound) effect shortly after discontinuing this regular use. Moreover, these effects are thought to occur especially during short-acting and not during long-acting beta2-agonists use. The aim of this study was to invest gate whether a rebound effect (a pharmacological deterioration effect diminishing after several hours) in FEV1 and PC20 (concentration of histamine causing a 20% fall in FEV1 with regard to baseline) occurred after cessation of regular use of beta2-agonists, and whether this occurred both after short-acting and long-acting beta2-agonists. Allergic asthmatic patients (n = 134) were randomly allocated to the use of a short-acting (salbutamol), a long-acting beta2-agonist (formoterol) or placebo for 12 weeks (double-blind, double-dummy). No other asthma medication was allowed, including inhaled corticosteroids. At the start and every 4 weeks later FEV and PC20 were measured, each time at least 12 h after the last doses of study medication, which is in the possible rebound period. To investigate whether a (transient) rebound effect occurred, parameters were additionally measured at least 72 h later after discontinuation of the study medication. After 12 weeks of short-acting beta2-agonist use, a drop was seen in FEV1 from 85.6 (+/- 2.21)% predicted to 78.8 (+/- 2.9)% predicted, measured 15 h (median) after the last doses of medication. This was significantly different compared to placebo. When measured 168 h (median) later FEV1 recovered to 85.5 (+/- 2.4)% predicted, comparable to baseline. PC20 decreased with -1.17 (+/- 0.44) doubling dose after 12 weeks of short-acting beta2-agonist use, measured 15 h after the last doses of medication, which was significantly different compared to placebo. However, 168 h later PC20 recovered slightly with +0.55 (+/- 0.34) doubling dose, but this value was still lower compared to placebo. In contrast, during long-acting beta2-agonist and placebo use no significant changes were seen. In conclusion, the use of short-acting beta2-agonists resulted in a transient (rebound) effect in FEV while the effects on PC20 may point to a real deterioration of the disease. Long-acting beta2-agonist and placebo use showed no changes. We conclude that a mono-therapy of short-acting and not of long-acting beta2-agonists might have deleterious effects in asthma.

Potential masking effect on dyspnoea perception by short- and long-acting beta2-agonists in asthma.

Asthma patients evaluate the effect of medication treatment through the degree of their asthma symptoms, which might be affected by their ability to perceive these symptoms. It has been suggested that beta2-agonists may mask the effects of an increase in airway inflammation. This study compared the perception of histamine-induced bronchoconstriction during monotherapy with short- or long-acting beta2-agonists. Asthmatic patients (68 male and 60 female, mean age 35+/-11 yrs, forced expiratory volume in one second (FEV1) 86+/-15% of the reference value, provocative concentration causing a 20% fall in FEV1 (PC20) geometric mean 0.97 mg x mL(-1) (95% confidence interval (CI): 0.73-1.30)) were selected and randomly allocated to use either a short-acting (salbutamol, n=41) or long-acting beta2-agonist (formoterol, n=46) or placebo (n=41) for 12 weeks. Perception of dyspnoea provoked by histamine-induced bronchoconstriction was measured at the start and every 4 weeks thereafter. Subjects quantified their sensation of breathlessness during the challenge tests on a modified Borg scale at the start of the study and every 4 weeks thereafter. The sensitivity to changes in FEV1 was analysed by the linear regression slope (alpha) Borg versus % fall in FEV1. The absolute perceptual magnitude (PS20) was determined by the perception score at the 20% fall in FEV1. Although the geometric mean PC20 decreased significantly within the group using short-acting beta2-agonists (in the group with initial PC20 > or = 2 mg x mL(-1) there was a drop from 5.26-1.94 mg x mL(-1); p=0.013), repeated measurement analysis showed no difference in the course of time of perception (both slope alpha and PS20) between the three medication groups. This study showed that chronic use of short- or long-acting beta2-agonists in asthmatics for a period of 12 weeks, did not significantly change the perception of histamine-induced bronchoconstriction compared with placebo. Further investigation is required to establish whether this suggests that these drugs do not mask a deterioration of asthma.

Inhaled corticosteroids, combined with long-acting beta(2)-agonists, improve the perception of bronchoconstriction in asthma.

The relationship between asthma medication and the perception of asthma symptoms is of interest for daily practice. Poor perception of asthma symptoms might influence patients' health care behavior and subsequently might lead to undertreatment and deterioration of their disease. This study investigated the influence of the chronic use of short-acting and long-acting beta(2)-agonists, compared with the additional use of inhaled corticosteroids on the perception of histamine-induced bronchoconstriction. Patients with asthma (33 male and 31 female, mean age 35 +/- 11 yr, FEV(1) 87 +/- 14% of the reference value, PC(20) geometric mean 1.08 mg/ml (95% CI: 0.76-1.52) were selected and randomly allocated to the use of either a short-acting beta(2)-agonists (salbutamol, n = 22) or a long-acting beta(2)-agonists (formoterol, n = 22) or placebo (n = 20), which has been used for 12 wk. This medication treatment was repeated exactly 1 yr later, with patients receiving the same medication plus an inhaled corticosteroid. Perception of histamine-induced bronchoconstriction was measured at the start of each treatment period and every 4 wk thereafter. Subjects quantified their sensation of respiratory discomfort during the challenge tests on a modified Borg scale. The perceptive "sensitivity" for changes in FEV(1) was analyzed by the linear regression slope (alpha) "Borg versus percentage fall in FEV(1)." The "absolute perceptual magnitude" was determined by the perception score at the 20% fall in FEV(1) (PS(20)). The additional use of inhaled corticosteroids during the second year resulted in an improved perception of histamine-induced bronchoconstriction (slope alpha) compared with the first year for only the long-acting beta(2)-agonists group (p value 0.036). This improvement was not observed for the "absolute perceptual magnitude" (PS(20)). The additional use of inhaled corticosteroids during chronic use of long-acting beta(2)-agonists improves the perceptive "sensitivity" for changes in FEV(1) during histamine-induced bronchoconstriction, which was not observed for short-acting bronchodilators. This result might indicate that the positive effects on perception of airway obstruction might be another reason (besides the beneficial effects on the clinical condition) for prescribing a combination of long-acting beta(2)-agonists and inhaled steroids.

A placebo-controlled clinical trial of regular monotherapy with short-acting and long-acting beta(2)-agonists in allergic asthmatic patients.

BACKGROUND: Some recent studies suggest that regular beta(2)-agonist use may result in inadequate control of asthma. It has been hypothesized that this occurs particularly in allergic asthmatic patients exposed to relevant allergens. Moreover, it is still unclear whether this occurs during the use of both short-acting and long-acting beta(2)-agonists. METHODS: Asthmatic patients (n = 145) allergic to house dust mite (HDM) were randomly allocated to monotherapy with a short-acting beta(2)-agonist (SA; n = 48), a long-acting beta(2)-agonist (LA; n = 50), or placebo (n = 47), double blind, double dummy. The study covered three periods: (1) a 4-week run-in period, in which no changes took place; followed by (2) cessation of treatment with asthma medication including inhaled corticosteroids, introduction of allergen avoidance measures (active/placebo treatment) to lower HDM exposure in the active group, and an 8-week washout period to adjust patients to these changes; followed by (3) a 12-week study medication period. At the start of the 12-week medication period, and every 4 weeks thereafter, spirometric measurements (FEV(1) and provocative concentration of histamine causing a 20% fall in FEV(1) [PC(20)]) were performed. Peak flow and asthma symptoms were recorded daily. Additionally, at the start and every 6 weeks thereafter, dust samples were collected from mattresses and living room and bedroom floors to assess HDM (der p 1) concentrations. Effects on FEV(1), PC(20), peak flow, and asthma symptoms were analyzed with repeated-measurement analysis and corrected for the exposure to HDM allergens. RESULTS: There were no significant differences among the three medication groups after 12 weeks for FEV(1). However, a significant decrease in mean FEV(1) percent predicted (95% confidence interval [CI]) was observed within the SA group: - 6.6 (- 10.4 to - 2.8) (p = 0.0002). A decrease in geometric mean PC(20) (95% CI) of - 1.2 (- 1.96 to - 0.44) doubling concentration was observed within the SA group (p = 0.05). No significant changes in FEV(1) and PC(20) were observed > 12 weeks within the LA group or the placebo group. There were neither changes in peak flow and asthma symptom scores among the three medication groups nor within the groups. Moreover, none of the parameters showed interactive effects with allergen exposure. CONCLUSION: There were no significant differences among the three medication groups for FEV(1) and PC(20). The within-treatment group comparison showed a significant small decline in FEV(1) for the SA group (but not for the LA group), which could indicate that monotherapy with SAs might have negative effects on FEV(1). This was not seen during regular use of LAS: No clear pathophysiologic mechanism can explain these findings at the moment. Relatively high or low exposure to allergens did not alter these findings.

Hypoxia and perfusion measurements in human tumors--initial experience with pimonidazole and IUdR.

We describe our preliminary studies on the development of methods to measure hypoxia in standard paraffin sections of human tumors. Three parameters were investigated. First, image analysis of tumor vascularity yielded the parameter diffusion limited fraction (DLF), which is the amount of tumor tissue greater than a fixed distance from the nearest blood vessel. Secondly, the amount of tumor tissue stained with antibodies against bound reduced products of the bioreductive marker pimonidazole was assessed. Finally, the fraction of blood vessels showing no surrounding tumor tissue labeled with lUdR, a cell kinetic marker, was measured. DLF and pimonidazole monitor primarily chronic hypoxia, while it is hypothesized that the IUdR-negative fraction monitors acute hypoxia. Feasibility was demonstrated in a series of 10 esophageal and 10 rectal tumors (no drug administration), 10 cervix tumors (pimonidazole) and 14 head and neck tumors (pimonidazole and lUdR). Significant differences between tumors were found for all parameters. DLF correlated significantly with the pimonidazole fraction when all images of all tumors were included, although mean values per tumor showed no correlation. The IUdR-negative fraction did not correlate with either of the other two parameters. We conclude that it is feasible to measure hypoxia-related, and possibly perfusion-related, parameters on paraffin sections for predictive purposes, although each method needs further validation. Each parameter will be correlated with outcome in a larger study on head and neck tumors treated with surgery with or without postoperative radiotherapy.

Use of thymidine analogues to indicate vascular perfusion in tumours.

Temporary reduction in blood-flow within tumour blood vessels can reduce oxygen supply leading to transient perfusion-limited hypoxia. Consequent selection of cells with mutations and reduced radiosensitivity can lead to disease progression and treatment-resistance. In the present study, we investigated whether heterogeneity of labelling after thymidine analogue administration is related to perfusion variations, and if so, could it be quantified and used as a perfusion indicator. Perfusion in murine RIF1 tumours was reduced by hydralazine or increased by nicotinamide and the mice subsequently injected with IdUrd. Tumours were halved for analysis by both flow cytometry and immunohistochemistry. Tumour sections were stained for vasculature and IdUrd. Each blood vessel was scored for the density of IdUrd-labelled cells surrounding it, using a semi-quantitative scoring system. Flow cytometry showed that the IdUrd labelling index and intensity decreased by approximately 50% after hydralazine. In tumour sections of control animals, 2.9% of vessels showed no IdUrd label. In contrast, after hydralazine almost 50% of vessels had no surrounding IdUrd labelling, whereas after nicotinamide there were fewer vessels with low labelling and a higher median score. In conclusion, changes of tumour perfusion by pharmacological agents is reflected in changes in tumour-cell labelling by the thymidine analogue IdUrd, suggesting that IdUrd labelling could be used to indicate perfusion in individual vessels in human tumours.

Diffusion limited hypoxia estimated by vascular image analysis: comparison with pimonidazole staining in human tumors.

PURPOSE: To assess diffusion limited hypoxia in human tumors using image analysis of vasculature and to compare it with the bioreductive marker pimonidazole as an independent method. MATERIALS AND METHODS: To set up the method, nine rectal adenocarcinomas and ten squamous cell carcinomas were analyzed. To validate the method, ten squamous cell carcinomas of the cervix were analyzed from patients who were injected with pimonidazole and biopsied approximately 24 h later. Sections of the rectal and esophageal tumors were stained for vasculature, while cervix tumor sections were double stained for vasculature and pimonidazole. Tumor areas were delineated on digitized images, and the proportion of tumor tissue greater than a fixed distance from the nearest blood vessel (called diffusion limited fraction, DLF) was then calculated. The proportion of tumor area stained for pimonidazole was also measured. RESULTS: There was a wide variation between tumors in both the vascular-derived DLF and in the pimonidazole-stained fraction. Average DLFs varied between 1.5 and 92% for different tumors, with significant differences between them. The area stained by pimonidazole was significantly smaller than DLF for all tumors. The correlation between pimonidazole area and DLF was significant in three of seven tumors containing > or = 3 images. When images from all tumors (n=123) were analyzed together, the correlation was highly significant (r=0.47, P<0.0001). CONCLUSION: The vascular derived DLF correlates significantly with pimonidazole staining, but there was large scatter. Both methods may underestimate perfusion limited hypoxia.

Perception of respiratory sensation assessed by means of histamine challenge and threshold loading tests.

BACKGROUND: Some asthmatic patients perceive the severity of their disease rather poorly. These patients may not receive optimal therapy because of underpresentation of their respiratory symptoms. It is therefore important to identify these patients. The present study evaluates a new threshold loading device for measuring the perception of respiratory sensation. This method for measuring the perception of respiratory sensation may be a viable alternative to the bronchial provocation test. The aim of the present study was to investigate whether the assessment of the perception of respiratory sensation based on a threshold loading test (inspiratory and expiratory) identifies the same subjects as poor perceivers as compared to assessment by histamine bronchial provocation test. METHOD: In 36 subjects, the perception of respiratory sensation through a threshold loading device was compared to the perception of respiratory sensation during a histamine provocation test. Each test was performed with scoring of the magnitude of the respiratory sensation on a visual analog scale (VAS). The magnitude of the stimulus intensity was indicated by the percentage of decrease in FEV(1) during the histamine challenge test and by the percentage of the subject's maximum mouth pressure (percent maximal inspiratory pressure and percent maximal expiratory pressure) during the threshold loading test. The relationship between VAS values and the stimulus intensity was analyzed by determining the linear regression coefficient between the two parameters. RESULTS: No relationship was found between the perception of the sensation induced by the histamine challenge and that during breathing through a threshold loading device for both inspiration (Rs = 0.15; p = 0.40) and expiration (Rs = 0.13; p = 0.47). We did find a significant relation between the perception of respiratory sensation during the inspiratory and expiratory threshold loading test (Rs = 0.67; p = 0. 0001). Furthermore, we defined a subgroup of patients of special interest: those with low symptoms of breathlessness and high bronchial responsiveness. Compared to the inspiratory and expiratory threshold loading test, the histamine challenge test identified more patients in the special interest group as poorest perceivers. CONCLUSION: The measurement of perception by means of a threshold loading device and a histamine provocation test did not identify the same subjects as poor perceivers, probably because we did not measure the patient's perceptiveness for exactly the same stimuli. In the population with relatively more severe asthma and very low symptoms of breathlessness, the histamine-induced bronchoconstriction test identified more patients as poorest perceivers compared to the threshold loading test.

Perception of bronchoconstriction in asthma patients measured during histamine challenge test.

This study investigated two aspects of the perception of bronchoconstriction ("sensitivity" and "absolute perceptual magnitude") in asthmatic patients and identified which clinical characteristics are related to these two aspects of perception of bronchoconstriction. The perception of histamine induced bronchoconstriction was measured in 128 asthmatic patients. Subjects quantified their breathlessness on a Visual Analogue Scale (VAS) before forced expiratory volume in one second (FEV1 was measured after each inhalation of histamine. The perceptive "sensitivity" for changes in FEV1 was analysed by the "VAS percentage fall in FEV1" slope. The "absolute perceptual magnitude" was determined by the VAS value at a 20% fall in FEV1. Spearman correlations were used for analysis between the two aspects of perception and asthma symptoms, peak flow variability, bronchial responsiveness and FEV1 % predicted. Patients with a low "sensitivity" for changes in FEV1 were more likely to show a frequent peak flow variability (Rs=-0.21; p<0.05), a high bronchial responsiveness (Rs= 0.37; p<0.001) and a low baseline FEV1 % pred (Rs=0.22; p<0.05). Patient's "absolute perceptual magnitude" correlated positively with symptoms during daily life (significant correlations varied 0.21-0.32) but not with the lung function parameters. The severity of asthma reflected by a low lung function and a high bronchial responsiveness, is associated with a low "sensitivity" for changes in forced expiratory volume in one second. A patient's "absolute perceptual magnitude" is positively related with asthma symptoms during daily life.

Effects of house dust mite avoidance measures on Der p 1 concentrations and clinical condition of mild adult house dust mite-allergic asthmatic patients, using no inhaled steroids.

BACKGROUND: Exposure to house dust mite (HDM) allergens often results in worsening of asthma. Therefore, avoidance of exposure to HDM allergens is often proposed. Unfortunately, the most effective and feasible avoidance strategy is still not completely assessed. Consequently, we investigated the effects of a combined HDM avoidance strategy on HDM allergen concentrations and clinical condition of allergic, mild asthmatic, patients using no inhaled steroids. METHODS: Asthmatic patients, allergic to HDM, using no inhaled corticosteroids, were randomly allocated to an active (n = 76) or a placebo allergen-avoidance group (n = 81). Avoidance measures consisted of applying Acarosan(R) (placebo: water) to the living room and bedroom floors, and the use of HDM-impermeable covers for mattresses and bedding (placebo: cotton covers for mattresses only). Effects on allergen concentrations (Der p 1), FEV1, bronchial hyperresponsiveness, peak flow parameters and asthma symptom scores were studied during 20 weeks and controlled for the allergic status of the patients. RESULTS: The active covers reduced Der p 1 concentrations to 9.4% (P = 0.0001), and were always significant lower than in the placebo group (P = 0.0002). Acarosan(R) resulted in slight but significant decreases (twofold, P = 0.0001), both on living room and bedroom floors, but concentrations were never significantly lower than the placebo group. Although the combined avoidance strategy resulted in a considerable reduction in allergen load in the active group, no differences were seen between the two groups in any of the clinical parameters during the follow-up period in this group of allergic asthmatics, using no inhaled corticosteroids. Corrections for the allergic status did not alter these results. CONCLUSIONS: The combined avoidance strategy was effective in reducing HDM allergen concentration. This was especially achieved by the allergen-impermeable covers, while the effects of Acarosan(R) were only marginal. However, this allergen reduction was not reflected in a convincing improvement in clinical condition in this group of mild allergic asthmatics, using no inhaled steroids. Perhaps, a longer follow-up period would have resulted in more pronounced effects.

Measuring breathlessness during histamine challenge: a simple standardized procedure in asthmatic patients.

One of the problems in research on symptom perception during histamine challenge has been the difficulty in finding both a valid and practical parameter of the "perceptiveness" for bronchoconstriction in a subject. The purpose of this study was to validate whether the slope in the linear regression model between stimulus and sensation during histamine challenge is an appropriate index for the "perceptiveness" for bronchoconstriction by comparing it with the classical Stevens' law. One hundred and thirty-four asthmatic patients were included in the study and underwent a bronchial challenge with histamine. The relationship between the change in visual analogue scale (VAS) values and the change in forced expiratory volume in one second (FEV1) as percentage of baseline value was analysed by determining both the exponent n in deltaVAS=k x (%deltaFEV1)n and the slope alpha in deltaVAS=k + alpha(%deltaFEV1). The best-fitting line of both the exponential and the linear regression model were determined by the least-squares method in which the percentage explained variation (R2) was compared. The median value of R2 of the exponential regression line and the linear regression line was 0.76 and 0.83, respectively, and significantly different. The Spearman rank correlation coefficient between exponent n in the exponential model deltaVAS=k x (%deltaFEV1)n and the slope alpha in the linear regression model deltaVAS=k + alpha(%deltaFEV1) was 0.87 (95% confidence interval 0.83-0.91). On the basis of the results, it was concluded that the linear regression coefficient can be used as a valid expression to describe the "perceptiveness" of an asthmatic subject instead of Stevens' power function during histamine challenge.

Relation of the perception of airway obstruction to the severity of asthma.

BACKGROUND: Patients with a poor perception of their symptoms of asthma seem to have an increased risk of an asthma attack. The influence of factors such as airway calibre, bronchial hyperresponsiveness, age and sex on the "perceptiveness" of a patient are poorly understood. It is of clinical importance to identify patients who are likely to have a poor perception of their symptoms. We have studied the perception of bronchoconstriction by asthmatic patients during a histamine provocation test and analysed the influence of bronchial obstruction, hyperresponsiveness, sex, and age. We were particularly interested to establish whether there was any difference in perception between subjects with a greater or lesser severity of asthma (expressed as bronchial obstruction, hyperresponsiveness). METHODS: One hundred and thirty four patients with allergic asthma underwent a histamine provocation test. The FEV1 was measured after each inhalation of histamine. Subjects were asked to rate subjective quantification of the sensation of breathlessness on a visual analogue scale (VAS). The relationship between changes in VAS values and the reduction in FEV1 as a percentage of the baseline value was analysed by determining the linear regression slope (alpha) between the two parameters and indicates the perception of airway obstruction. Multiple regression analysis was performed to investigate the effect of baseline FEV1, bronchial hyperresponsiveness, sex and age on the "perceptiveness" for bronchoconstriction. RESULTS: The median value of the slope alpha (indicating the perception of airway obstruction) was 0.91 (25-75th percentile: 0.48-1.45). Age and sex had no influence on the perception of bronchoconstriction. Both initial bronchial tone (baseline FEV1) and bronchial hyperresponsiveness (PC20) showed a significant correlation with the perception of bronchoconstriction. The regression coefficients for FEV1 and 2log PC20 in the multiple regression model were 0.20 and 0.10. Patients who had a low baseline FEV1 and/or a high bronchial responsiveness to histamine were more likely to show a low perceptiveness for bronchoconstriction during the challenge test. CONCLUSIONS: Low baseline FEV1 and high bronchial responsiveness are associated with a low degree of "perceptiveness" for bronchoconstriction. This suggests that patients with a more severe degree of asthma either show adaptation of "perceptiveness" for airway obstruction or that low perceptiveness leads to more severe asthma.

Exertional dyspnoea in patients with airway obstruction, with and without CO2 retention.

BACKGROUND: Dyspnoea is a common and disabling symptom in patients with cardiopulmonary disease. Unfortunately the mechanisms that produce dyspnoea are still poorly understood. The relationship between dyspnoea and the load on the ventilatory muscles, chemical drive, and ventilatory indices was therefore assessed in patients with obstructive pulmonary disease during an incremental exercise test. METHODS: Fifty patients with a wide range of obstructive pulmonary disease (mean forced expiratory volume in one second (FEV1) 66.1 (28.8)% predicted) performed an incremental cycle ergometer test. A subdivision was made between subjects with CO2 retention (delta PaCO2 > or = 0, n = 22) and subjects without CO2 retention (delta PaCO2 < 0, n = 28) during exercise. During the test dyspnoea (Borg score), oesophageal pressures (mechanical load on the ventilatory muscles (time tension index (TTI), blood gas tensions, and minute ventilation were measured. Correlations for changes in mechanical and chemical factors with changes in dyspnoea score were calculated to assess relevant factors. An analysis of covariance was used to examine whether there was a relationship between dyspnoea score and each of these factors and whether this relationship was different between the subgroups with and without CO2 retention. Multiple regression analysis was used to assess the independent effect of each parameter on dyspnoea sensation. Furthermore, the amplitude of pleural pressure swing ((Pi + Pe)act) generated at maximal work load (Ptot, an indication of the load on all respiratory muscles) was calculated. Analysis of covariance was used to assess whether there was a relationship between tidal volume (VT) and Ptot and whether this relationship was different between the groups (slopes are an expression of the length-tension inappropriateness, LTI). RESULTS: In the total group and the group without CO2 retention a significant correlation between dyspnoea and the increase in the inspiratory time tension index (TTIi) was present. In the group with CO2 retention a significant correlation was seen between dyspnoea and delta PaCO2. The factors delta PaO2, delta VE%MVV and delta (VT/Ti) showed a correlation with a p value of < or = 0.10 both in the total group and in those without CO2 retention. In an analysis of covariance the relationship between dyspnoea score and delta PaCO2 appeared to be significantly different between the two subgroups, being more pronounced in the group with CO2 retention. No other relationships with change in dyspnoea score were found. There was no significant relationship between VT and Ptot in the total group nor in the two subgroups, indicating some length-tension inappropriateness in both groups. CONCLUSIONS: In patients with distinctive pulmonary disease who are normocapnic or hypocapnic the mechanical load (delta TTIi) and length-tension inappropriateness (LTI) on ventilatory muscles seem to be the main determinant of exertional dyspnoea. As soon as hypercapnia occurs, this seems to override all other inputs for dyspnoea.

House dust mite avoidance measures improve peak flow and symptoms in patients with allergy but without asthma: a possible delay in the manifestation of clinical asthma?

BACKGROUND: Asthma caused by allergy to house dust mite is a growing problem. Patients with allergy who do not have asthma (yet) might develop asthma depending on exposure to precipitating factors. OBJECTIVE: We sought to determine whether house dust mite avoidance measures have an effect on the development of asthma. METHODS: Patients with allergy (n = 29) who had no diagnosis of asthma (FEV1 of 99.1% +/- 10.6% of predicted, peak flow variability of 5.21% +/- 3.41%, reversibility of FEV1 after 400 microg salbutamol of 3.92% +/- 3.75% according to the reference values) were randomly allocated (subjects blinded) to a treatment (n = 16) and a placebo group (n = 13). House dust mite avoidance treatment consisted of applying Acarosan (Allergopharma, J. Ganzer KG, Hamburg, Germany) (the placebo group used water) to the floors (living room, bedroom), and the use of covers for mattresses and bedding that were impermeable to house dust mite (the placebo group used cotton covers for mattresses only). We tested whether the intervention had an effect on peak flow parameters and asthma symptom scores during 6 weeks of treatment. RESULTS: Significant improvements were seen in the treatment group in symptom scores (Borg score) for disturbed sleep, breathlessness, wheeze, and overall symptom score. Slight but statistically significant improvements in peak flow (morning, evening, and variability) were seen in the treatment group also. No significant changes were seen in the placebo group. CONCLUSIONS: Although this study is not long enough to study the development of asthma, the results indicates that house dust mite avoidance measures had an effect on peak flow parameters and asthma symptoms in patients with allergy but without asthma. These findings might implicate that a shift in developing clinically manifest asthma could be achieved with house dust mite avoidance measures. To give a better answer to whether preventing the development of asthma is possible, larger studies with a longer follow-up period are necessary.

Cell kinetic measurements in prostate cancer.

PURPOSE: Two approaches have been suggested for escalating the total dose in radiotherapy treatment of prostate cancer. One is conformal radiotherapy; the other is hyperfractionation using many small fractions. Both imply some possible prolongation in overall treatment time. To judge whether prolonged treatment schedules would be detrimental, it is necessary to know the proliferation rates in human prostate tumors, specifically, the potential doubling time (Tpot). There is a lack of data on this parameter in the literature. METHODS AND MATERIALS: Seven patients with adenocarcinoma of the prostate were studied. A tracer dose of 100 mg/m2 of IUdR was infused intravenously 4-12 h before biopies were taken. Biopsies were fixed in 70% ethanol, stored at 4 degrees C, and later prepared and stained by standard methods for flow cytometry, using the red fluorescence signal for DNA and the green fluorescence signal (fluorescein isothiocyanate) for 5-iodo-2'-deoxyuridine. The duration of DNA synthesis (Ts) was determined by the relative movement (RM) method, knowing the interval between tracer administration and biopsy. Tpot was calculated as the quotient of Ts by labeling index (LI). RESULTS: In two of the seven tumors the LI was too low (<0.6%) for a reliable estimate of RM to be made, so no determination of Tpot was possible for these tumors. The mean LI values in the other five tumors were 2.4%, 1.4%, 1.0%, 3.0%, and 0.9%. The durations of Ts were 13.2, 9.5, 10.0, 11.7, and 12.7 h, respectively. The resulting values of Tpot were 23, 28, 42, 16, and 61 days, respectively. CONCLUSION: The low labeling indices in prostate tumors, also reported by others, made estimation of Ts by RM impossible in about a third of these tumors. However, five tumors yielded long estimates for Tpot, implying that prolongation from 6 to about 8 weeks should not be detrimental.

Oral platinum analogue JM216, a radiosensitizer in oxic murine cells.

This study was designed to compare radiosensitization by the oral platinum compound JM216 with cisplatin. RIF1 mouse tumour cells were treated at various doses and at various exposure times with JM216 and irradiated 15 min before the end of drug exposure. The fraction of cells surviving treatment was assessed by colony formation. Results were compared with those for equivalent treatments with cisplatin. JM216 alone showed exponential killing of RIF1 cells, being approximately three times less efficient than cisplatin on a molar basis. For radiosensitization studies, drug doses used gave approximately 50 or 90% cell killing alone. No radiosensitization was seen after 2-h drug exposures, but significant radiosensitization occurred after 1- and 0.5-h exposures (shorter times required proportionally higher drug doses, giving equivalent drug kill). The enhancement ratio and time dependence were similar for the two platinum compounds, reaching 1.5 at the highest concentrations tested. Drug DNA adduct formation was assessed using immunocytochemistry with the NKI-A59 antiserum raised to cisplatin-DNA adducts. The antiserum was shown to recognize JM216-DNA adducts in a dose-dependent manner and maximum nuclear staining was found to be correlated with cell kill for both drugs. However, neither the level of staining at the time of irradiation nor at the time of maximum adducts correlated with radiosensitization, indicating that the number of DNA adducts did not determine radiosensitization. Intracellular glutathione levels were shown to be decreased by the drug, but only by approximately 50%, implying that this was not the cause of the increased radiosensitivity. In summary, JM216 was shown capable of radiosensitizing a platinum-sensitive tumour line to an extent similar to cisplatin. Radiosensitization was exposure-time and drug-concentration dependent, but was not dependent on DNA adduct levels nor glutathione depletion. In contrast, cell kill after drug alone was well correlated with adduct levels. These data suggest that JM216 could replace cisplatin in combined radiotherapy-chemotherapy studies, and also indicate that the NKI-A59 antibody could be used to monitor exposure levels in vivo.