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Biomedical subjects

F B Thunnissen

Publications and source records attributed to F B Thunnissen.

At least 19 recordsLinked to original sources
PubMed1999

High-frequency oscillatory ventilation, partial liquid ventilation, or conventional mechanical ventilation in newborn piglets with saline lavage-induced acute lung injury. A comparison of gas-exchange efficacy and lung histomorphology.

It has been reported that, in diseased lungs, either partial liquid ventilation (PLV) or high-frequency oscillatory ventilation (HFOV) can improve oxygenation better and with less lung injury than conventional mechanical ventilation (CMV). This study was intended as a preclinical comparison between the effects of HFOV, PLV and CMV on gas exchange, lung mechanics and histology. Fifteen anesthetized newborn piglets, with respiratory insufficiency due to repeated saline lung lavage, were allocated to either a PLV, HFOV or CMV (n = 5 each) strategy, and treated for 4 h. Within 30 min of commencing therapy, PLV, HFOV, and CMV improved arterial PO2 (Pa,O2), alveoloarterial oxygen gradient (P(A-a),O2), oxygenation index (OI), venous admixture (va), and arterial PCO2 (Pa,CO2). After 4 h, oxygenation parameters (Pa,O2, P(A-a),O2, OI and venous admixture) were significantly better in the HFOV group than in the PLV group; the CMV group showed a higher Pa,O2 and lower OI than the PLV group. Gas exchange at the end of the experiment was not different from baseline in the HFOV and CMV groups. Lung histology and morphometry were performed after perfusion-fixation at endotracheal deflation pressure corresponding to mean airway pressure at the end of the experiment. Lung injury score and mean linear intercept were not different between the three treatment groups. We conclude that in this model, gas exchange improved significantly in all three ventilation strategies. Indices of oxygenation improved less during PLV. The saline lavage-induced acute lung injury model used as in this study, is less stable than previously thought. The final lung injury is not influenced by the ventilation strategy. We speculate that the impaired gas exchange during PLV is an expression of diffusion limitation and ventilation-perfusion mismatch in a recovering lung.

Animals↗
PubMed1999

Number and proliferation of clara cells in normal human airway epithelium.

Experimental pathologic studies suggest that Clara cells are one of the types of airway stem cells but the proliferation of Clara cells in human lungs has not yet been examined. The purpose of this study was to assess in conducting airways of normal human lungs: (1) the number of Clara cells; and (2) the contribution of Clara cells to the proliferation compartment. Samples of histologic normal tissue were taken from seven lungs obtained by autopsy. A triple sequential (immuno)histochemical staining was performed, using MIB-1 as a proliferation marker and anti-CC10 for the identification of Clara cells; subsequently, a PAS stain was carried out as a marker for goblet cells, as these cells were reported to be CC10-immunoreactive in an unknown proportion. Clara cells were virtually absent in the proximal airway epithelium. The number of Clara cells in the terminal bronchioles was 11 +/- 3% (mean +/- SD) and in respiratory bronchioles 22 +/- 5%. The overall proliferation compartment of the conducting airway epithelium was 0.83 +/- 0.47%; the contribution of Clara cells was 9%. In the terminal bronchioles 15% of proliferating airway epithelial cells were Clara cells, and in the respiratory bronchioles this number increased to 44%. The contribution of Clara cells to the proliferation compartment of normal human tracheobronchial epithelium is substantial, demonstrating a role of the Clara cell in the maintenance of the normal epithelium of the distal conducting airways in humans.

Adult↗
PubMed1998

Interaction between glucocorticoids and beta2-agonists: alpha and beta glucocorticoid-receptor mRNA expression in human bronchial epithelial cells.

Recent studies have suggested that regular use of beta2-agonists has adverse effects on asthma control, due to the cross-talk between cAMP responsive element binding proteins (CREB) and glucocorticoid receptors (GR). The aim of this study was to investigate the interaction between GR and CREB on cytoplasmic protein level with a gel mobility shift assay and to determine the effect of this interaction on mRNA levels by Northern blot analysis. After exposing human bronchial epithelial cells for 1 hr to either 1 microM terbutaline or budesonide, more binding of CREB and GR, respectively, was observed to their responsive elements in DNA. Simultaneous exposure to terbutaline and budesonide also increased the binding of CREB and GR to DNA. After 4 hr, both alpha and beta GR mRNAs were down-regulated by 1 microM budesonide. Simultaneous addition of 1 microM terbutaline prevented this down-regulation. Adding 100 times more budesonide compared to terbutaline again down-regulated both GR forms, although significantly less compared to the down-regulation induced by 1 microM budesonide alone. Addition of terbutaline to cells already exposed to budesonide did not reverse the GR mRNA expression within 44 hr. Similar results were obtained with metallothionein-2 (MT2) mRNA levels. In conclusion, beta2-agonists interfere with the GR function in human bronchial epithelial cells when given simultaneously, with this being overcome by sequential exposure of the cells to first glucocorticoids and later beta2-agonists.

Activating Transcription Factor 2↗
PubMed1998

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection.

Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.

DNA Mutational Analysis↗
PubMed1998

Increased proliferative activity and p53 expression in normal glandular breast tissue after radiation therapy.

Radiation used in breast-conserving therapy (BCT) for early breast cancer, to eradicate residual malignant cells after tumour resection, induces DNA damage and cell death. Little is known about the effect of the commonly used doses of radiation therapy on normal breast tissue. Under physiological conditions, p53 plays a role in maintaining genomic stability and regulating progression through the cell cycle. In normal glandular breast tissue, p53 expression is very low, as is proliferative activity. The purpose of this study was to investigate the levels of p53 expression and proliferative activity in non-malignant glandular epithelium of the breast after BCT. The immunohistochemical expression of p53 and Ki-67 was semiquantitatively correlated in non-malignant glandular epithelium in biopsies before and after BCT in 24 women with breast cancer. In 18 cases, a recurrence was diagnosed and in the remaining cases, the clinical suspicion was not histologically confirmed. In addition, in six cases with contralateral breast cancer, the same immunohistochemical evaluation was performed in tissue from both breasts. The mean interval between the two surgical interventions was 50 months. The percentage of p53 immunoreactive epithelial cells in normal breast tissue was significantly (P < 0.01) higher after radiation therapy than before in the ipsilateral side (0.2 per cent +/- 0.3 and 4.6 per cent +/- 4.5, respectively). Ki-67 immunoreactivity was also significantly increased (P < 0.001) after radiation therapy, from 0.6 per cent to an average of 4.8 per cent in epithelial cells. In contrast, in the patients with contralateral breast cancer, the levels of p53 and Ki-67 immunoreactivity in the normal glandular breast tissue were comparable to the ipsilateral side (P = 0.7 and P = 0.1, respectively). In conclusion, increased expression of p53 and Ki-67 is present in normal glandular breast tissue, even 2-5 years after radiation therapy.

Adult↗
PubMed1998

Gut permeability, intestinal morphology, and nutritional depletion.

Nutritional depletion increases the risk for postoperative complications. The intestinal barrier may be important in the underlying pathophysiologic mechanism. In this study, 26 patients were evaluated to determine whether nutritional depletion was related to gut integrity and intestinal morphology. Nutritional depletion was estimated by calculating percentage ideal body weight (PIB) or percentage ideal fat free mass (PIFFM). To assess gut integrity, a lactulose/mannitol (L/M) test was performed. Duodenal biopsies were taken, and villous height, crypt depth, number of IgA-producing plasma cells, intraepithelial lymphocytes (IELs), and proliferating index were determined. The L/M ratio was increased, and villous height was decreased in depleted patients. Depletion was not associated with differences in the number of immune cells or proliferating index. The number of IgA-producing plasma cells was positively correlated with the L/M ratio. This study shows that nutritional depletion is associated with increased intestinal permeability and a decrease in villous height.

Adult↗
PubMed1998

Glucocorticoid receptor mRNA levels in bronchial epithelial cells of patients with COPD: influence of glucocorticoids.

Glucocorticoids (gcs) are known to be effective in the treatment of asthma. In chronic obstructive pulmonary disease (COPD), however, no beneficial effects are demonstrated in most patients. Hypothetically, this may be explained by an overexpressed beta-glucocorticoid receptor (GR) compared to the alpha-GR. The aim of this study was to investigate alpha- and beta-GR mRNA levels and ratios in patients with COPD with or without glucocorticoid treatment. GR and, as a control, metallothionein (MT) 2 mRNA levels were compared between patients with COPD receiving glucocorticoids (COPD + gcs), glucocorticoid naive COPD-patients (COPD - gcs) and non-COPD control patients not using gcs. Bronchoscopy was performed and bronchial epithelial cells were sampled with brushing. Smoking did not influence alpha- and beta-GR levels and ratios, nor the MT2 mRNA expression level. The alpha-GR mRNA expression was lower in the COPD - gcs group than in controls. Both GR forms were higher in the COPD + gcs patients than in the COPD - gcs patients, but not different from the levels measured in the controls. alpha 1/beta-GR mRNA ratios did not differ between the groups and averaged 1.7, suggesting no inhibitory effect of the beta-GR on the alpha 1 form. MT2 levels were upregulated in the COPD + gcs patients as compared to the COPD - gcs group, indicating a pharmacological glucocorticoid effect. In the present study it is demonstrated that basal GR mRNA levels are lower in patients with COPD. Although this needs to be investigated further, this might explain, in part, the non-responsiveness of patients with COPD to gcs.

Adult↗
PubMed1998

Alpha and beta glucocorticoid receptor mRNA expression in skeletal muscle.

The aim of the present study was to investigate the occurrence and autoregulation of both glucocorticoid receptor mRNAs in rat gastrocnemius muscle. The expression of both receptor forms was studied 1, 4 or 12 hours after intra-tracheal instillation of a high dose (100 micrograms) of budesonide; muscular expression was compared with glucocorticoid receptor expression in lung tissue. After Northern blot analysis, hybridization was performed with glucocorticoid receptor, glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase probes. In the gastrocnemius muscle, both the alpha and beta glucocorticoid receptor mRNA forms were detected and found to be downregulated four hours after the budesonide instillation. alpha/beta glucocorticoid receptor ratios were lower in the gastrocnemius (1.1 +/- 0.2) than in the lungs (2.6 +/- 0.6). In the lungs, at all time points, the average alpha glucocorticoid receptor mRNA levels did not differ from controls, although glutamine synthetase mRNA levels were upregulated. The beta glucocorticoid receptor mRNA was slightly reduced at 1 and 4 hours. In conclusion, after intra-tracheal instillation of budesonide, both alpha and beta glucocorticoid receptor forms were downregulated in muscle tissue. The difference in alpha/beta glucocorticoid receptor mRNA ratios and concentrations between lung and gastrocnemius muscle supports the hypothesis of differential gene regulation by glucocorticoids in different cell types.

Administration, Inhalation↗
PubMed1998

Number and proliferation of basal and parabasal cells in normal human airway epithelium.

Two roles have been suggested for basal cells on the basis of studies performed with laboratory animals: (1) anchoring of the tracheobronchial epithelium; and (2) being the epithelial stem cell. Parabasal cells located just above the basal cells have also been shown to contribute to cell renewal. However, a systematic study of the composition and proliferation of basal and parabasal cells in normal human lungs is lacking. The aims of this study were to determine in normal human conducting-airway epithelium: (1) the number of basal and parabasal cells; and (2) the contribution of basal and parabasal cells to the proliferation fraction. Samples of histologically normal tissue, free of pulmonary disease, were taken from seven lungs obtained by autopsy. Immunohistochemical staining was performed with the primary antibody MIB-1 as a proliferation marker and the antikeratin antibody 34betaE12 as a marker for basal and parabasal cells. In the largest conducting airways (diameter >= 4 mm), the percentages of basal and parabasal cells were 31% and 7%, respectively; the contribution to the proliferation compartment was 51% for basal and 33% for parabasal cells. In the smallest airways (diameter < 0.5 mm), 6% of epithelial cells were basal cells, with a 30% contribution to the proliferation compartment, whereas parabasal cells were absent. The high fraction of basal and parabasal cells contributing to the proliferation compartment of normal human conducting-airway epithelium supports the theory that cells at or near the basement membrane are likely to be progenitor cells.

Adult↗
PubMed1998

Detection of K-ras point mutations in sputum from patients with adenocarcinoma of the lung by point-EXACCT.

PURPOSE: Kirsten ras (K-ras) point mutations are found in 30% to 56% of pulmonary adenocarcinomas by means of highly sensitive techniques. Recently, the Point-EXACCT (point mutation detection using exonuclease amplification coupled capture technique) method was described, which detected one cell with a mutation in 15,000 normal cells. The aim of this study was to examine whether K-ras point mutations could be found with this rapid method in the sputum of patients with adenocarcinoma of the lung. PATIENTS AND METHODS: DNA from paraffin-embedded adenocarcinoma and corresponding sputum samples were analyzed for mutations of the K-ras gene. Twenty-eight biopsy specimens and 54 sputum samples of 22 patients were used for amplification and K-ras codon 12 point mutation detection. RESULTS: In 11 of 22 patients (50%), a mutation in K-ras codon 12 was shown in the tumor sample. In five of 11 patients (45%) with a K-ras mutation in the tumor, the same type of mutation was identified in at least one sputum sample. A mutation could not be detected in any of the sputum samples from patients with a K-ras-negative tumor. Time between K-ras point mutation detection in sputum and clinical diagnosis of lung cancer varied from 1 month to almost 4 years. In two of the five patients with K-ras-positive sputum specimens, malignant cells were found with cytologic examination. CONCLUSION: Point-EXACCT is suitable for the detection of K-ras point mutations in sputum samples of patients with adenocarcinoma of the lung. This approach may be an important adjunct to cytology in the early diagnosis of lung cancer.

Adenocarcinoma↗
PubMed1998

Validation of the Point-EXACCT method in non-small cell lung carcinomas.

K-ras point mutations are often detected in part of the lung carcinomas. For the validation of a highly sensitive and rapid assay for known point mutations, Point-EXACCT (Biochim Biophys Acta 1998; 1379:42-52), we analyzed 89 non-small cell lung carcinomas and compared the results with two sequencing methods. No point mutations were found with double-stranded sequencing. Single-stranded sequencing detected six patients positive for K-ras codon 12. When Point-EXACCT was used, K-ras codon 12 mutations were detected in 8 of 52 patients with squamous cell carcinomas, 10 of 29 patients with adenocarcinomas, and 3 of 8 patients with large cell carcinomas. The finding of K-ras mutations in squamous cell carcinomas is explained by the high sensitivity of the method. Therefore, Point-EXACCT may be applicable to detection of those alterations occurring at a low frequency among an excess of cells with wild-type DNA.

Carcinoma, Non-Small-Cell Lung↗
PubMed1997

Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators.

Human respiratory epithelium expresses inducible nitric oxide synthase (iNOS) continuously in vivo, however mechanisms responsible for maintenance of expression are not known. We show that IFNgamma is sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and IL-4 potentiates IFNgamma-induced iNOS expression in HAEC through stabilization of iNOS mRNA. IFNgamma/IL-4- induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 wk. Removal of overlying culture media resulted in loss of expression, while transfer of conditioned media induced iNOS mRNA in other HAEC. IFNgamma and IL-4 stimulation activated STAT1 and STAT6 in HAEC, but conditioned media transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines. Although cytokine induction of iNOS was dependent on new protein synthesis, conditioned media induction of iNOS in HAEC was not. Further, removal of overlying culture media from cells at different times after cytokine stimulation demonstrated that mediator synthesis and/or secretion important for induction and maintenance of iNOS occurs early after cytokine stimulation. In conclusion, a combination of IFNgamma/ IL-4, which occurs naturally in the lung epithelial lining fluid, leads to maintenance of iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA.

Adolescent↗
PubMed1997

Quality assurance in DNA image analysis on diploid cells.

DNA image analysis is presently performed in many laboratories. Before general extrapolation of results between different laboratories, validation has to be performed for interlaboratory studies on DNA image analysis. The aim of this study was to establish the performance of different DNA image analysis instruments when measuring different diploid cells. On three separate parts of the same object slide, human liver cells, white blood cells, and imprints of a breast fibroadenoma were sampled. In this quality assurance interlaboratory study, 13 laboratories participated voluntarily. Two slides were sent to each participating laboratory: one Feulgen and one unstained to be stained according the participating laboratory in-house procedure. The features integrated optical density (IOD) and object AREA were recorded for each nucleus. For calculation of the results, the average IOD value of liver diploid cells was set at 2c. A striking difference was observed between the different presumed diploid cell types, from almost 1c to 3c. This variation was not dependent on central or in-house staining. Although in-house calibration was performed for each image analysis system, a surprisingly large variation existed in the reported object AREA, irrespective of the cell type. These results clearly demonstrate that measurements performed in one laboratory cannot be extrapolated to the outcome of others and support the need for standardization. The use of external control cells works well for comparison of instruments. In conclusion, in DNA image analysis quality control is necessary, standardization is obligatory, and the use of an internal control for determination of the diploid peak in a histogram of patient samples is recommended.

Breast Neoplasms↗
PubMed1997

Which cytological criteria are the most discriminative to distinguish carcinoma, lymphoma, and soft-tissue sarcoma? A probabilistic approach.

The reliability of fine-needle aspiration cytology (FNA) for distinguishing between carcinoma, lymphoma, and sarcoma was established in a previous study (Thunnissen et al., Cytopathology 1993; 4:107-114). The purpose of this study was to investigate which criteria were useful for a probabilistic diagnosis. A total of 78 randomly chosen FNA smears (31 carcinomas, 24 lymphomas, and 23 sarcomas) was sent around and read "blindly" by six cytopathologists. Each pathologist completed a list of 16 criteria for every case. Histology was used as a reference standard. A statistical analysis led to the selection of three criteria: "lymphoglandular bodies," "well-defined clusters," and "spindle-cell nuclei," associated with lymphoma, carcinoma, and soft-tissue sarcoma, respectively. Given the scores on these criteria, the probabilities to be assigned to the three diagnostic categories can be read from a table. It turns out, as one might expect, that the classification of the most probable disease is pretty reliable if one cytologic criterion scores much higher than the other two criteria. On other cases, fuzziness appears and misclassifications are far from improbable. This study offers a general cytologic approach. The cytologic criteria "lymphoglandular bodies," "well-defined clusters," and "spindle-cell nuclei" can be used both in daily practice and in education to assign posterior probabilities to carcinoma, lymphoma, and soft-tissue sarcoma.

Biopsy, Needle↗
PubMed1997

Decreased Cu,Zn-SOD activity in asthmatic airway epithelium: correction by inhaled corticosteroid in vivo.

To investigate the antioxidant response of respiratory epithelium to the chronic airway inflammation in asthma, the major intracellular antioxidants [copper and zinc-containing superoxide dismutase (Cu,Zn-SOD) and manganese-containing SOD (Mn-SOD), catalase, and glutathione peroxidase] were quantitated in bronchial epithelial cells of healthy control and asthmatic individuals. Although catalase and glutathione peroxidase in bronchial epithelium of asthmatics were similar to control SOD activity in asthmatics not on inhaled corticosteroid (-CS) was lower than asthmatics on inhaled corticosteroid (+CS) and controls. Investigation of Mn-SOD and Cu,Zn-SOD activities revealed that the lower SOD activity in asthmatics -CS was because of decreased Cu,Zn-SOD activity. However, Mn-SOD and Cu,Zn-SOD mRNA and protein levels were similar among asthmatics -CS, asthmatics +CS, and controls. Importantly, Cu,Zn-SOD specific activity in asthmatics -CS was decreased in comparison with control and asthmatics +CS. Furthermore, in paired comparisons of asthmatics -CS and +CS, inhaled corticosteroids resulted in normalization of bronchial epithelial Cu,Zn-SOD specific activity. These findings suggest loss of Cu,Zn-SOD activity in asthma is related to inflammation, perhaps through oxidant inactivation of Cu,Zn-SOD protein.

Administration, Inhalation↗
PubMed1997

In vitro and in vivo modulation of alpha- and beta-glucocorticoid-receptor mRNA in human bronchial epithelium.

Despite the central role bronchial epithelial cells play in asthmatic reactions, and the widespread use of inhaled corticosteroids in asthma, no information is available on the effect of glucocorticoids on glucocorticoid- receptor (GR) gene expression. In this study, the effect of budesonide on alpha- and beta-GR gene expression in human bronchial epithelial cells was investigated in vitro and in vivo. A bronchial epithelial cell line was exposed in vitro to budesonide, and a dose- and time-dependent synchronous downregulation of alpha- and beta-GR mRNA was observed. A 1-h exposure resulted in a reversible and reduced downregulation as compared with continuous exposure. In healthy volunteers (n = 10), no difference on average was present in GR mRNA expression before or after 4 wk of budesonide inhalation in either bronchial epithelial cells or alveolar macrophages. The time between the last inhalation and sampling of cells ranged from 0.5 to 8 h. However, a significant downregulation of alpha-GR mRNA was observed when the time between the last inhalation and sampling of cells was more than 2 h. Normalization of the downregulation of GR mRNA expression in bronchial epithelial cells is explained by the pharmacokinetics of inhaled budesonide in the human lung.

Administration, Topical↗