AAT Research

Am J Respir Crit Care Med 2002 Aug 15;166(4):490-5

4-Hydroxy-2-nonenal, a specific lipid peroxidation product, is elevated in lungs
of patients with chronic obstructive pulmonary disease.

Rahman I, van Schadewijk AA, Crowther AJ, Hiemstra PS, Stolk J, MacNee W, De
Boer WI.

Respiratory Medicine Unit, ELEGI Laboratory, University of Edinburgh Medical
School, Wilkie Building, Teviot Place, Edinburgh EH8 9AG, Scotland, UK.
irfan.rahman@ed.ac.uk

Cigarette smoking results in oxidative stress and inflammation in the lungs,
which are involved in the pathogenesis of chronic obstructive pulmonary disease
(COPD). 4-Hydroxy-2-nonenal (4-HNE), a highly reactive diffusible product of
lipid peroxidation, is a key mediator of oxidant-induced cell signaling and
apoptosis. 4-HNE has a high affinity toward cysteine, histidine, and lysine
groups and forms direct protein adducts. We investigated the presence of
4-HNE-modified proteins in lung tissue obtained from subjects with and without
COPD. We studied 23 current or ex-smokers with similar smoking histories with
COPD (n = 11; FEV(1) < 70% predicted) or without COPD (n = 12; FEV(1) > 84%
predicted) who had undergone lung resection. As 4-HNE and transforming growth
factor-beta(1) (TGF-beta(1)) can modulate gamma-glutamylcysteine synthetase
(gamma-GCS) mRNA levels in lung cells, we assessed the relations between
4-HNE-modified protein levels, FEV(1), gamma-GCS, and TGF-beta(1).
4-HNE-modified protein levels were elevated in airway and alveolar epithelial
cells, endothelial cells, and neutrophils in subjects with COPD, compared with
the levels in subjects without COPD (p < 0.01). We also observed a significant
inverse correlation between the levels of 4-HNE adducts in alveolar epithelium,
airway endothelium, and neutrophils and FEV(1) (p < 0.05) and a positive
correlation between 4-HNE adducts and TGF-beta(1) protein and mRNA as well as
gamma-GCS mRNA levels in airway and alveolar epithelium (p < 0.01). The elevated
levels of 4-HNE may play a role in the signaling events in lung inflammation
leading to the imbalance of the expression of both proinflammatory mediators and
protective antioxidant genes in COPD.

Free Radic Biol Med 2001 Dec 1;31(11):1442-7

Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine
in asthmatics.

Peters EA, Hiltermann JT, Stolk J.

Department of Pulmonology, Leiden University Medical Center, The, Leiden,
Netherlands.

Apocynin is an inhibitor of NADPH oxidase present in inflammatory cells such as
eosinophils and neutrophils. We investigated the effect of inhaled apocynin on
ozone-induced bronchial hyperresponsiveness in vivo. Seven mild atopic
asthmatics participated in a placebo-controlled, cross-over study with two
exposures to O(3) at 2-week intervals. Apocynin (3 ml of 0.5 mg/ml) was inhaled
2 times before and 6 times after O(3) exposure at hourly intervals. At 36 h
before and 16 h after O(3) exposure, methacholine inhalation challenge tests
(Mch) were performed, and PC(20) and maximal % fall from baseline (MFEV(1)) were
calculated from dose-response curves. O(3)-induced change in PC(20) (Delta
PC(20)) after placebo treatment was -1.94 +/- 0.39 DD (mean +/- SEM doubling
dose Mch) (p =.001) and apocynin was -0.6 +/- 0.33 DD (p =.17). The difference
between apocynin and placebo treatment was 1.3 DD +/- 0.42 (p =.02).
O(3)-induced Delta MFEV(1) was 11.9 +/- 1.5% (p =.008) during placebo inhalation
and 3.85 +/- 1.8% during apocynin (p =.47). Apocynin reduced the Delta MFEV(1)
by 8.05% compared to placebo (p =.025). We conclude that apocynin markedly
reduced O(3)-induced hyperreactivity for Mch as well as maximal airway
narrowing. The results suggest that apocynin may have a role in preventing
ozone-induced exacerbations of asthma.

Invest Radiol 2001 Nov;36(11):648-51

Repeatability of lung density measurements with low-dose computed tomography in
subjects with alpha-1-antitrypsin deficiency-associated emphysema.

Stolk J, Dirksen A, van der Lugt AA, Hutsebaut J, Mathieu J, de Ree J, Reiber
JH, Stoel BC.

Department of Pulmonology (C3-P), Leiden University Medical Center, Leiden, the
Netherlands. j.stolk.long@lumc.nl

RATIONALE AND OBJECTIVES: Multislice computed tomography (MSCT) of the lungs
provides a new opportunity for longitudinal assessment of lung densities because
of shorter scan duration. The aim of the present study was to assess the
intraindividual variation of lung densities measured by MSCT of patients with
emphysema. METHODS: Ten patients with emphysema participated in a study in which
MSCT was obtained on two occasions, approximately 2 weeks apart. Scanning
parameters were 140 kV, 20 mAs, 4 x 2.5-mm collimation, and effective slice
thickness of 2.5 mm. Lung density was measured as the 15th percentile point and
the relative area below -910 Hounsfield units (HU) by using Pulmo-LKEB software.
RESULTS: The mean difference of the 15th percentile point was -1.29 +/- 3.2 HU,
and that for the relative area below the -910-HU parameter was -1.02% +/- 3.09%.
Intraclass coefficients of variation were 0.96 (0.86-0.99) and 0.94 (0.8-0.98),
respectively (95% confidence interval). CONCLUSIONS: Lung density parameters of
emphysema derived by MSCT provide an opportunity for analysis of the treatment
effects of new drugs on the progression of emphysema.

Free Radic Biol Med 2000 Mar 15;28(6):920-5

Localization of gamma-glutamylcysteine synthetase messenger rna expression in
lungs of smokers and patients with chronic obstructive pulmonary disease.

Rahman I, van Schadewijk AA, Hiemstra PS, Stolk J, van Krieken JH, MacNee W, de
Boer WI.

Respiratory Medicine Unit, ELEGI Laboratories, University of Edinburgh, Medical
School, Edinburgh, Scotland, UK. ir@srv1.med.ed.ac.uk

Cigarette smoking results in an oxidant/antioxidant imbalance in the lungs and
inflammation, which are considered to be key factors in the pathogenesis of
chronic obstructive pulmonary disease (COPD). Glutathione (GSH) is an important
protective antioxidant in lung epithelial cells and epithelial lining fluid. De
novo GSH synthesis in cells occurs by a two-enzyme process. The rate-limiting
enzyme is gamma-glutamylcysteine synthetase (gamma-GCS), in which the heavy
subunit (HS) constitutes most of its catalytic activity. The localization and
expression of gamma-GCS-HS in specific lung cells as well as possible
differences in its expression between smokers with and without COPD have not yet
been studied. The purpose of this study was to investigate gamma-GCS-HS
expression using messenger RNA in situ hybridization in peripheral lung tissue.
We studied 23 current or ex-smokers with similar smoking histories with (n = 11;
forced expiratory volume in 1 s [FEV(1)] < 75% predicted) or without COPD (n =
12; FEV(1) < 84% predicted). We assessed the relations between pulmonary
gamma-GCS-HS expression, FEV(1) and transforming growth factor-beta1
(TGFbeta(1)), because TGFbeta(1) can modulate gamma-GCS-HS expression in lung
epithelial cells. Gamma-GCS-HS is predominantly expressed by airway and alveolar
epithelial cells, alveolar CD68+ cells (macrophages), and endothelial cells of
both arteries and veins. In subjects with COPD, semiquantitative analysis
revealed higher levels of gamma-GCS-HS messenger RNA in alveolar epithelium (1.5
times, p <.04) and a trend for a higher expression in bronchiolar epithelium
(1.3 times, p =.075) compared with subjects without COPD. We did not observe a
significant correlation between airway and alveolar epithelial gamma-GCS-HS
expression and TGFbeta(1) expression (r =.20), FEV(1) percentage predicted (r
=.18), or FEV(1)/forced vital capacity ratio (r =.14; p.05). Our results show
that gamma-GCS-HS is localized, particularly in lung epithelium, and shows
higher expression in smokers with COPD. This suggests a specific role for
enhanced GSH synthesis as a mechanism to provide an adaptive response against
oxidative stress in patients with COPD.

J Pathol 2000 Apr;190(5):619-26

Monocyte chemoattractant protein 1, interleukin 8, and chronic airways
inflammation in COPD.

de Boer WI, Sont JK, van Schadewijk A, Stolk J, van Krieken JH, Hiemstra PS.

Department of Pulmonology, Leiden University Medical Center, Leiden, The
Netherlands. pdeboer@pulmonology.azl.nl

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of
death, with cigarette smoking among the main risk factors. Hallmarks of COPD
include chronic airflow obstruction and chronic inflammation in the airway walls
or alveolar septa. An earlier study reported elevated numbers of macrophages and
mast cells within the bronchiolar epithelium in smokers with COPD, compared with
smokers without. Since specific chemokines may be involved in this influx, the
in situ protein and mRNA expression of monocyte chemoattractant protein 1
(MCP-1) and of interleukin 8 (IL-8) were studied in tumour-free peripheral lung
tissue resected for lung cancer of current or ex-smokers with COPD (FEV(1)<75%;
n=14) and without COPD (FEV(1)>84; n=14). MCP-1 was expressed by macrophages, T
cells, and endothelial and epithelial cells. Its receptor, CCR2, is expressed by
macrophages, mast cells, and epithelial cells. IL-8 was found in neutrophils,
epithelial cells, and macrophages. In subjects with COPD, semi-quantitative
analysis revealed 1.5-fold higher levels of MCP-1 mRNA and IL-8 mRNA and protein
in bronchiolar epithelium (p<0.01) and 1.4-fold higher levels of CCR2 in
macrophages (p=0.014) than in subjects without COPD. The bronchiolar epithelial
MCP-1 mRNA expression correlated with both CCR2 expression on macrophages and
mast cells (p<0.05) and the numbers of intra-epithelial macrophages and mast
cells (p<0.04). The epithelial IL-8 expression did not correlate with the
numbers of neutrophils, macrophages, CD45RO+, CD8+, or mast cells. These data
suggest that MCP-1 and CCR2 are involved in the recruitment of macrophages and
mast cells into the airway epithelium in COPD. Copyright 2000 John Wiley & Sons,
Ltd.

Chest 2000 Mar;117(3):786-9

Long-term effect of bilateral plication of the diaphragm.

Stolk J, Versteegh MI.

Departments of Pulmonology, Leiden University Medical Center, Leiden, The
Netherlands. jstolk@pulmonology.azl.nl

STUDY OBJECTIVES: To assess the feasibility and clinical outcome of bilateral
plication of the diaphragm in patients with bilateral diaphragmatic paralysis
(BDP) caused by neuralgic amyotrophy (NA), a mononeuritis of the phrenic nerves.
DESIGN: Prospective, case-control study over a 1-year period. SETTING: A
university hospital in The Netherlands. PATIENTS: Six patients who presented
with BDP caused by NA. METHODS: The diagnosis of BDP was based on the absence of
muscle response after cervical magnetic stimulation of both phrenic nerves.
Three patients did not undergo surgery but were observed for a period of 2
years, and the other three patients underwent a limited lateral thoracotomy at
the eighth intercostal space. Plication was performed by U-stitches until the
diaphragm was as tight as possible. Vital capacity (VC) and arterial blood gas
was measured during follow-up. RESULTS: One month postoperatively, mean VC
measured in the supine position was significantly improved by 17%, and this
effect was sustained for 12 months. Arterial PO(2) increased by 45%. VC and
blood gas levels did not improve in the three patients that were only observed
during the 2-year period. All three surgical patients could sleep in the supine
position after the operation. CONCLUSION: Bilateral plication of the diaphragm
for NA-induced paralysis results in improvement of ventilation and blood gas
exchange, allowing patients to sleep in the supine position without dyspnea.

Thorax 2000 Mar;55(3):189-93

Is there any relationship between plasma antioxidant capacity and lung function
in smokers and in patients with chronic obstructive pulmonary disease?

Rahman I, Swarska E, Henry M, Stolk J, MacNee W.

Respiratory Medicine Unit, Royal Infirmary, Edinburgh, UK.

BACKGROUND: It has been suggested that oxidative stress is an important factor
in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have
shown that an oxidant/antioxidant imbalance occurs in the distal air spaces of
smokers and in patients with COPD which is reflected systemically in the plasma.
A study was undertaken to determine whether plasma antioxidant status correlated
with lung function as assessed by forced expiratory volume in one second
(FEV(1)) and forced vital capacity (FVC) in smokers and patients with COPD.
METHODS: Plasma antioxidant capacity, assessed by the Trolox equivalent
antioxidant capacity (TEAC) as an index of overall systemic oxidative stress,
and protein thiol levels were measured in 95 patients with stable COPD, in 82
healthy smokers, and in 37 healthy non-smokers. RESULTS: Mean (SE) plasma TEAC
levels were significantly decreased in patients with COPD (0.81 (0.03) mmol/l,
p<0.001) and in healthy smokers (0.87 (0.04) mmol/l, p<0. 001) compared with
healthy non-smokers (1.31 (0.11) mmol/l). The mean differences in plasma
antioxidant capacity (mM) were (0.81, 95% confidence interval (CI) 0.22 to
1.48), (0.87, 95% CI 0.2 to 1.46), and (1.31, 95% CI 1.09 to 1.58) in patients
with COPD, healthy smokers, and healthy non-smokers, respectively. This
reduction was associated with a 29% (95% CI 18 to 38) and a 30% (95% CI 19 to
40) decrease in plasma protein thiol levels in COPD patients and smokers,
respectively. Current smoking was not the main contributor to the reduction in
antioxidant capacity in patients with COPD as those patients who were still
smokers had similar TEAC levels (mean (SE) 0. 78 (0.05); n = 25) to those who
had stopped smoking (0.84 (0.02); n = 70). No significant correlations were
found between spirometric data measured as FEV(1) % predicted or FEV(1)/FVC %
predicted and the plasma levels of TEAC in patients with COPD, healthy smokers,
or healthy non-smokers. Similarly, there was no significant correlation between
FEV(1) % predicted or FEV(1)/FVC % predicted and the levels of plasma protein
thiols in the three groups. CONCLUSIONS: These data confirm decreased
antioxidant capacity in smokers and patients with COPD, indicating the presence
of systemic oxidative stress. However, no relationship was found between protein
thiols or TEAC levels and measurements of airflow limitation in either smokers
or in patients with COPD.

Free Radic Biol Med 1999 Dec;27(11-12):1448-54

Ozone-induced inflammation assessed in sputum and bronchial lavage fluid from
asthmatics: a new noninvasive tool in epidemiologic studies on air pollution and
asthma.

Hiltermann JT, Lapperre TS, van Bree L, Steerenberg PA, Brahim JJ, Sont JK,
Sterk PJ, Hiemstra PS, Stolk J.

Department of Pulmonology, Leiden University Medical Centre, The Netherlands.

We investigated correlations between ozone-induced increases in inflammatory
markers in induced sputum and in bronchial lavage fluid. Sixteen volunteers with
intermittent asthma participated in a placebo-controlled parallel study with two
exposures. Six days before and 16 h after the first exposure to ozone (0.4 ppm
during 2 h) sputum was induced with hypertonic saline. This resulted in a
significant increase in the sputum levels of eosinophil cationic protein (ECP;
1.8-fold; p = .03), neutrophil elastase (5.0-fold; p = .005) and the total cell
number (1.6-fold; p = .02). After 4 weeks, a second exposure was randomized for
air or ozone. Six days before and 16 h after the second exposure a bronchial
lavage was performed. ECP values in sputum and in bronchial lavage fluid
obtained after ozone correlated significantly (Rs = .79; p = .04), as did
interleukin-8 (IL-8) values (Rs = .86; p = .01), and the percentage eosinophils
(Rs = .89; p = .007). Moreover, the ozone-induced changes in percentage
eosinophils observed in sputum and lavage fluid were highly correlated (Rs =
.93; p = .003). In conclusion, changes in eosinophils, IL-8, and ECP markers
induced by ozone and measured in sputum reflect the inflammatory responses in
the lower airways of asthmatics, and may provide a noninvasive tool in
epidemiologic studies on air pollution and asthma.

Am J Respir Crit Care Med 1999 Nov;160(5 Pt 1):1468-72

A randomized clinical trial of alpha(1)-antitrypsin augmentation therapy.

Dirksen A, Dijkman JH, Madsen F, Stoel B, Hutchison DC, Ulrik CS, Skovgaard LT,
Kok-Jensen A, Rudolphus A, Seersholm N, Vrooman HA, Reiber JH, Hansen NC,
Heckscher T, Viskum K, Stolk J.

Department of Respiratory Medicine, The Rigshospital, Copenhagen, Denmark.
adi@dadlnet.dk

We have investigated whether restoration of the balance between neutrophil
elastase and its inhibitor, alpha(1)-antitrypsin, can prevent the progression of
pulmonary emphysema in patients with alpha(1)-antitrypsin deficiency. Twenty-six
Danish and 30 Dutch ex-smokers with alpha(1)-antitrypsin deficiency of PI*ZZ
phenotype and moderate emphysema (FEV(1) between 30% and 80% of predicted)
participated in a double-blind trial of alpha(1)-antitrypsin augmentation
therapy. The patients were randomized to either alpha(1)-antitrypsin (250 mg/kg)
or albumin (625 mg/kg) infusions at 4-wk intervals for at least 3 yr.
Self-administered spirometry performed every morning and evening at home showed
no significant difference in decline of FEV(1) between treatment and placebo.
Each year, the degree of emphysema was quantified by the 15th percentile point
of the lung density histogram derived from computed tomography (CT). The loss of
lung tissue measured by CT (mean +/- SEM) was 2.6 +/- 0.41 g/L/yr for placebo as
compared with 1.5 +/- 0.41 g/L/yr for alpha(1)-antitrypsin infusion (p = 0.07).
Power analysis showed that this protective effect would be significant in a
similar trial with 130 patients. This is in contrast to calculations based on
annual decline of FEV(1) showing that 550 patients would be needed to show a 50%
reduction of annual decline. We conclude that lung density measurements by CT
may facilitate future randomized clinical trials of investigational drugs for a
disease in which little progress in therapy has been made in the past 30 yr.

Invest Radiol 1999 Apr;34(4):303-9

Sources of error in lung densitometry with CT.

Stoel BC, Vrooman HA, Stolk J, Reiber JH.

Department of Radiology, Leiden University Medical Center, The Netherlands.

RATIONALE AND OBJECTIVES: To determine and analyze the most important error
sources in lung CT densitometry in vivo. METHODS: The authors examined the
influences of CT acquisition errors, physiologic changes, and image segmentation
errors on lung densitometry. Among others, spatial dependency and long-term
reproducibility of the density measurements of blood and air were examined over
a period of 4 years in a group of 28 patients with pulmonary emphysema. These
results were related to the measured lung densities in this group. RESULTS: The
density measurement of blood and air is strongly dependent on the position in
the thorax. Despite full-scanner calibrations, x-ray tube replacement can induce
a significant increase in measured blood density. CONCLUSIONS: A change in a
lung density parameter over time can actually be the result of tube replacement
or changing blood density. A simple postprocessing technique can correct for
these changes.

FEBS Lett 1999 Jan 25;443(2):235-9

Apocynin increases glutathione synthesis and activates AP-1 in alveolar
epithelial cells.

Lapperre TS, Jimenez LA, Antonicelli F, Drost EM, Hiemstra PS, Stolk J, MacNee
W, Rahman I.

Department of Pulmonology, Leiden University Medical Centre, The Netherlands.

Apocynin (4-hydroxy-3-methoxy-acetophenone) is a potent intracellular inhibitor
of superoxide anion production in neutrophils. In this study, we studied the
effect of apocynin on the regulation of the antioxidant glutathione (GSH) and
activation of the transcription factor AP-I in human alveolar epithelial cells
(A549). Apocynin enhanced intracellular GSH by increasing gamma-glutamylcysteine
synthetase activity in A549 cells. Apocynin also increased the expression of
gamma-GCS heavy subunit mRNA. This was associated with increased AP-1 DNA
binding as measured by the electrophoretic mobility shift assay. These data
indicate that apocynin displays antioxidant properties, in part, by increasing
glutathione synthesis through activation of AP-1.

Eur Respir J 1998 Nov;12(5):1200-8

Neutrophil serine proteinases and defensins in chronic obstructive pulmonary
disease: effects on pulmonary epithelium.

Hiemstra PS, van Wetering S, Stolk J.

Dept of Pulmonology, Leiden University Medical Center, The Netherlands.

Neutrophils have the capacity to accumulate in high numbers in the lung during
infection and inflammation. Because they play an important role in host defence
against infection, but may also cause tissue injury, these cells are thought to
be involved in the pathogenesis of various inflammatory lung disorders,
including chronic bronchitis and chronic obstructive pulmonary disease.
Neutrophil products that may mediate tissue injury at sites of
neutrophil-dominated inflammation include the neutrophil serine proteinases
elastase, cathepsin G and proteinase 3, and the nonenzymatic defensins. One of
the targets of the neutrophil is the lung epithelium, and in vitro studies have
revealed that both the serine proteinases and neutrophil defensins markedly
affect the integrity of the epithelial layer, decrease the frequency of ciliary
beat, increase the secretion of mucus, and induce the synthesis of
epithelium-derived mediators that may influence the amplification and resolution
of neutrophil-dominated inflammation. Both neutrophil elastase and defensins
induce the release of the neutrophil chemoattractant chemokine interleukin-8
from respiratory epithelial cells. The alpha1-proteinase inhibitor (alpha1-PI)
is a well-characterized inhibitor of neutrophil elastase, that also blocks the
cytotoxic and stimulatory activity of defensins towards epithelial cells. The
elastase inhibitory activity of alpha1-PI is also abrogated by the binding of
defensins to this inhibitor. Incubation of epithelial cells with neutrophil
defensins in combination with either elastase or cathepsin G resulted in
decreased effects on the epithelial cells compared with those observed when the
cells were incubated with defensins, elastase or cathepsin G separately. These
results suggest that neutrophil defensins and serine proteinases cause injury
and stimulate epithelial cells to produce chemokines that attract more
neutrophils to the site of inflammation. The effects of neutrophil defensins and
serine proteinases on epithelial cells appear to be restricted by proteinase
inhibitors and by inhibitory interactions between these sets of neutrophil
granule proteins.

Am J Respir Crit Care Med 1998 Dec;158(6):1951-7

Transforming growth factor beta1 and recruitment of macrophages and mast cells
in airways in chronic obstructive pulmonary disease.

de Boer WI, van Schadewijk A, Sont JK, Sharma HS, Stolk J, Hiemstra PS, van
Krieken JH.

Departments of Pulmonology and Pathology, Leiden University Medical Center,
Leiden, The Netherlands.

Chronic airways inflammation is one of the features of chronic obstructive
pulmonary disease (COPD). We demonstrated previously that bronchiolar epithelium
in COPD contains increased numbers of macrophages and mast cells. Transforming
growth factor beta1 (TGF-beta1) may be involved in this influx because it has
chemotactic activity for macrophages and mast cells. In this study, we examined
expression patterns of TGF-beta1, TGF-beta receptors type I and II (TGF-betaRI
and TGF-betaRII) by immunohistochemistry and mRNA in situ hybridization in
peripheral lung tissue of 14 current or ex-smokers with COPD (FEV1 < 75%) and 14
without COPD (FEV1 > 84%). In both groups, TGF-beta1 and its receptors are
present in airway and alveolar epithelial cells, airway and vascular smooth
muscle cells, and tissue and alveolar CD68(+) cells (considered herein to be
macrophages). In subjects with COPD, a semiquantitative analysis revealed
approximately twofold higher levels of TGF-beta1 mRNA and protein in bronchiolar
and alveolar epithelium (p < 0.02) as compared with subjects without COPD. With
regard to bronchiolar epithelial cells, we found a significant correlation
between TGF-beta1 mRNA and protein expression (r = 0.62; p < 0.002), and between
the FEV1 of all subjects together and TGF-beta1 protein (r = -0.60; p < 0.0002)
and mRNA (r = -0.67; p < 0. 002) levels. The epithelial expression of TGF-beta1
mRNA and TGF-beta1 protein correlates with the number of intraepithelial
macrophages (both: r = 0.44; p < 0.03) whereas intraepithelial mast cell numbers
correlate with epithelial TGF-beta1 mRNA expression. These data suggest a role
for TGF-beta1 in recruiting macrophages into the airway epithelium in COPD.

Eur Respir J 1998 May;11(5):1116-20

Prospective evaluation of World Health Organization criteria to assist diagnosis
of tuberculosis in children.

Houwert KA, Borggreven PA, Schaaf HS, Nel E, Donald PR, Stolk J.

Dept of Pulmonology, Leiden University Medical Center, The Netherlands.

Because of the difficulty in confirming childhood tuberculosis (TB), the World
Health Organization (WHO) proposes a hierarchical approach to diagnosis using
history and certain clinical features to help to improve the control of TB in
communities. The objective of this study was to evaluate prospectively in
children the diagnostic value of recent weight loss or failure to gain weight
adequately, cough or wheezing for >2 weeks and recent household contact with an
adult case of pulmonary TB. These evaluations were performed in 627 children
presenting to the paediatric outpatient department of a tertiary hospital
situated in the Western Cape Province of South Africa and serving a community
with a very high incidence of TB (>1,000 per 100,000). If at least one of the
criteria was present, the diagnosis of TB was investigated further by clinical
investigation, Mantoux test, chest radiography and TB culture from gastric
aspirate. One or more of the proposed criteria for diagnosing TB in childhood
were present in 206 children (33%). TB confirmed by culture of Mycobacterium
tuberculosis from gastric aspirate was found in 10 children (5%). After
diagnostic work-up, 23 children (11%) were considered to have probable TB,
whereas 173 (84%) were, after follow-up of 8 weeks, thought not to have TB. In
this study the simultaneous presence of the three WHO criteria for suspecting TB
had a positive predictive value of 63%. These results should assist in the more
precise delineation of the predictive value of the proposed World Health
Organization approach to the diagnosis of tuberculosis in childhood.

Free Radic Biol Med 1998 Apr;24(6):952-8

Ozone-induced airway hyperresponsiveness in patients with asthma: role of
neutrophil-derived serine proteinases.

Hiltermann TJ, Peters EA, Alberts B, Kwikkers K, Borggreven PA, Hiemstra PS,
Dijkman JH, van Bree LA, Stolk J.

Department of Pulmonology, Leiden University Hospital, The Netherlands.

Proteinase inhibitors may be of potential therapeutic value in the treatment of
respiratory diseases such as chronic obstructive pulmonary disease (COPD) or
asthma. Our aim was to study the role of neutrophils, and neutrophil-derived
serine proteinases in an acute model in patients with asthma. Exposure to ozone
induces an acute neutrophilic inflammatory reaction accompanied by an increase
in airway hyperresponsiveness. It is thought that these two effects of ozone are
linked, and that neutrophil-derived serine proteinases (i.e. elastase) may play
a role in the ozone-induced airway hyperresponsiveness. Therefore, we examined
the effect of recombinant antileukoprotease (rALP), one of the major serine
proteinase inhibitors in the lung, on ozone-induced changes in airway
hyperresponsiveness in this model. We observed that 16 h after exposure to
ozone, airway hyperresponsiveness to methacholine was increased both following
placebo and rALP treatment. There was no significant difference between placebo
and rALP treatment (change in area under the dose-response curve to
methacholine: 117.3+/-59.0 vs 193.6+/-59.6 % fall x DD; p=.12). Moreover, the
immediate decrease in FEV1 after ozone exposure was not significantly different
between the two groups (placebo: -29.6+/-6.7%; rALP: -20.9+/-3.8%; p=.11). In
addition, no significant differences were observed in plasma levels of
fibrinogen degradation products generated by neutrophil serine proteinases
before and after exposure to ozone. We conclude that neutrophil-derived serine
proteinases are not important mediators for ozone-induced hyperresponsiveness.

Eur Respir J 1998 Mar;11(3):686-93

Asthma severity and susceptibility to air pollution.

Hiltermann TJ, Stolk J, van der Zee SC, Brunekreef B, de Bruijne CR, Fischer PH,
Ameling CB, Sterk PJ, Hiemstra PS, van Bree L.

Dept of Pulmonology, Leiden University Medical Centre, The Netherlands.

Exacerbations of asthma have been associated with exposure to ozone or particles
with a 50% cut-off aerodynamic diameter of 10 microm (PM10). We postulated in
this study that the association of summertime air pollution (i.e. ozone and
PM10) with acute respiratory symptoms, medication use and peak expiratory flow
differs among patients grouped according to asthma severity. During the summer
of 1995, effects of ambient air pollution on these parameters were studied in a
panel of 60 nonsmoking patients with intermittent to severe persistent asthma.
These patients were recruited from our Pulmonary Out-patient Clinic. Subgroup
analysis was performed on the degree of hyperresponsiveness and lung steroid use
before the start of the study, as indictors for the severity of asthma.
Associations of the parameters studied with ozone, PM10, nitrogen dioxide (NO2),
sulphur dioxide (SO2) and black smoke were evaluated using time series analysis.
Several episodes with increased summertime air pollution occurred during the 96
day study period. Eight hour average ozone concentrations exceeded the World
Health Organization (WHO) Air Quality Guidelines (120 microg x m(-3)) on 16
occasions. Daily mean levels of PM10 were moderately elevated (range 16-98
microg x m(-3)). Levels of the other measured pollutants were low. There was a
consistent, positive association of the prevalence of shortness of breath
(maximal relative risk (RRmax) 1.18) with ozone, PM10, black smoke and NO2. In
addition, bronchodilator use was associated with both ozone and PM10 levels
(RRmax 1.16). Stratification by airway hyperresponsiveness and steroid use did
not affect the magnitude of the observed associations. No associations with peak
expiratory flow measurements were found. We conclude that the severity of asthma
is not an indicator for the sensitivity to air pollution.

Ned Tijdschr Geneeskd 1997 Jul 5;141(27):1327-30

A new surgical treatment with reduction of lung volume
for severe pulmonary emphysema]

Stolk J, Versteegh MI, van der Peijl ID, Jaddoe VW.

Afd. Longziekten, Universitair Medisch Centrum, Leiden.

Current drug treatment of pulmonary emphysema has little effect on quality of
life and duration of survival. Surgical treatment for patients with severe
pulmonary emphysema was recently introduced; it consists of resection of lung
tissue with poor ventilation and perfusion. Surgical reduction of lung volume
improves the forced expiratory pressure per second by 80-100% from 0.8 to 1.51.
This increases the exercise tolerance and improves the patient's functioning in
everyday life. The postoperative mortality and morbidity are acceptable.

Am J Respir Crit Care Med 1997 Dec;156(6):1765-72

Effects of photochemical air pollution and allergen exposure on upper
respiratory tract inflammation in asthmatics.

Hiltermann TJ, de Bruijne CR, Stolk J, Zwinderman AH, Spieksma FT, Roemer W,
Steerenberg PA, Fischer PH, van Bree L, Hiemstra PS.

Department of Pulmonology, Leiden University Medical Centre, The Netherlands.

Asthma is an inflammatory disease of the airways, and exacerbations of this
disease have been associated with high levels of air pollution. The objective of
this study was to examine whether ambient air pollution and/or allergen exposure
induces inflammatory changes in the upper airways of asthmatics. Sixty patients
with intermittent to severe persistent asthma visited the Hospital's Out Patient
Clinic every 2 wk for a period of 3 mo, and on each visit a nasal lavage was
obtained. Associations between nasal inflammatory parameters and seasonal
allergens and/or air pollution exposures were analyzed using linear regression
analysis. The study ran from July 3 to October 6, 1995, during which period
ozone (8-h mean: 80 micrograms/m3) and PM10 (24-h mean: 40 micrograms/m3) were
the major air pollutants; the major aeroallergen was mugwort pollen (24-h mean:
27 pollen grains/m3). Effects on both cellular and soluble markers in nasal
lavage were demonstrated for both ozone and mugwort pollen, but not for PM10.
Ambient ozone exposure was associated with an increase in neutrophils (112% per
100 micrograms/m3 increase in 8-h average ozone concentration), eosinophils
(176%), epithelial cells (55%), IL-8 (22%), and eosinophil cationic protein
(ECP) (19%). Increases in environmental mugwort pollen counts were associated
with an increase in nasal eosinophils (107% per 100 pollen/m3) and ECP (23%),
but not with neutrophils, epithelial cells, or lL-8. This study demonstrated
that both ambient ozone and allergen exposure are associated with inflammatory
responses in the upper airways of subjects with asthma, although the type of
inflammation is qualitatively different.

Am J Pathol 1997 Dec;151(6):1785-90

Chronic obstructive pulmonary disease: role of bronchiolar mast cells and
macrophages.

Grashoff WF, Sont JK, Sterk PJ, Hiemstra PS, de Boer WI, Stolk J, Han J, van
Krieken JM.

Department of Pulmonology, Leiden University Medical Center, The Netherlands.

Chronic obstructive pulmonary disease (COPD) is considered to be caused in part
by smoking-induced inflammation, but it is unknown which inflammatory cells
within the small airways are associated with the obstruction. We investigated
the inflammatory infiltrate in the small airways of 16 current or ex-smokers
with COPD (FEV1 < or = 75% predicted) and 15 without COPD (FEV1 > or = 85%
predicted) in pneumectomy specimens that were removed for lung cancer. Mast
cells, macrophages, neutrophils, eosinophils, T cells, and B cells were
identified using immunohistochemistry on formalin-fixed, paraffin-embedded
specimens. These cells were quantified in the epithelium and the remainder of
the airway wall. The number of mast cells and macrophages in the epithelium, but
not in the remainder of the airway wall, was significantly increased in patients
with COPD. Neutrophil and T cell numbers did not differ between the groups. Only
few B cells and eosinophils were present in both groups. Smoking history,
perioperative steroid usage, tumor localization, or reversibility in the FEV1 to
salbutamol could not account for the observed differences. We conclude that the
number of epithelial mast cells and macrophages is increased in the bronchioli
in smokers with airflow limitation, suggesting a role in development of COPD.

J Clin Epidemiol 1997 Aug;50(8):881-90

Antimicrobial treatment in acute maxillary sinusitis: a meta-analysis.

de Bock GH, Dekker FW, Stolk J, Springer MP, Kievit J, van Houwelingen JC.

Department of General Practice, University of Leiden, The Netherlands.

OBJECTIVE: The aims of this study were to assess which antibiotic is most
effective in the treatment of acute maxillary sinusitis in otherwise healthy
adults and adolescents, and which has the fewest side effects. DESIGN: To assess
the short-term effects of antimicrobial treatments, a meta-analysis was
performed using Mantel-Haenszel procedures on 16 comparative, randomized studies
with a total number of 3358 patients. No placebo-controlled studies were
available. Antimicrobial treatments were categorized according to type,
spectrum, beta-lactamase inhibition, and bactericidal effect. Outcomes were
clinical cure, clinical success, and adverse events. RESULTS: When studies were
analyzed separately, we found significant differences between cefpodoxim and
cefaclor in relation to clinical cure, and between loracarbef and doxycycline in
relation to clinical success. When data was pooled, sulphonamides were
significantly more effective than penicillins in relation to clinical cure, and
macrolids were more effective than penicillins in relation to clinical success,
whereas cephalosporins caused significantly less adverse events than
penicillins. When studies were stratified (standard classic meta-analysis),
antibiotics with beta-lactamase inhibition offered significantly more clinical
cures than antibiotics without beta-lactamase inhibition. However, this
significant effect was only due to one study from Southern Europe, published
before 1991. CONCLUSION: Differences in outcome between antimicrobial treatments
of acute sinusitis in otherwise healthy adults and adolescents appear to be
small. Therefore, the cheapest antimicrobial treatment can be selected.

Respir Med 1997 Apr;91(4):241-4

Bronchiolitis obliterans organizing pneumonia after adjuvant radiotherapy for
breast carcinoma.

van Laar JM, Holscher HC, van Krieken JH, Stolk J.

Department of General Internal Medicine, Leiden University Hospital, The
Netherlands.

Eur J Clin Invest 1997 Feb;27(2):148-56

An enzyme immunoassay for polymorphonuclear leucocyte-mediated fibrinogenolysis.

Bos R, van Leuven CJ, Stolk J, Hiemstra PS, Ronday HK, Nieuwenhuizen W.

TNO-Prevention and Health, Leiden, The Netherlands.

Upon stimulation, polymorphonuclear leucocytes (PMNs) release potent serine
proteases, i.e. elastase, cathepsin G and proteinase 3, which contribute to the
degradation of tissue and plasma components. Here, we describe the development
of a plasma test to assess PMN-mediated fibrinogenolysis as a biochemical marker
for actual PMN-derived proteolysis in vivo, useful for monitoring therapeutic
efficacy, i.e. of elastase inhibitors. We generated a monoclonal antibody (MAb),
designated 1-1/B3, with a high affinity for elastase-degraded fibrinogen (EDF).
The epitope for 1-1/B3 becomes exposed in a time-dependent manner during
digestion of fibrinogen with purified PMN-derived serine proteases and with
isolated PMNs in vitro. However, 1-1/B3 does not react with plasma fibrinogen or
with fibrin(ogen) degradation products generated by plasmin or by other active
proteases that may occur locally, i.e. metalloproteases and lysosomal
cathepsins. On the basis of MAb 1-1/B3, we developed a plasma test for the
assessment of PMN-mediated fibrin(ogen) degradation products (PMN-FDP). In a
panel of control plasmas, we observed concentrations of PMN-FDP of 8.2 +/- 0.9
ng mL-1 (n = 18). These values were increased twofold in patients with alpha
1-proteinase inhibitor deficiency (18.6 +/- 3.3 ng mL-1; n = 12; P < 0.0001) and
even more in patients with sepsis (365.7 +/- 97.7 ng mL-1; n = 16; P < 0.0001).
Furthermore, synovial tissue extracts from patients with rheumatoid arthritis
contained increased levels of PMN-FDP, compared with synovial tissue extracts (P
< 0.005) from patients with osteoarthritis.

Invest Radiol 1996 Dec;31(12):761-7

Assessment of the progression of emphysema by quantitative analysis of
spirometrically gated computed tomography images.

Zagers H, Vrooman HA, Aarts NJ, Stolk J, Schultze Kool LJ, Dijkman JH, Van
Voorthuisen AE, Reiber JH.

Department of Diagnostic Radiology and Nuclear Medicine, Leiden University
Hospital, The Netherlands.

RATIONALE AND OBJECTIVES: The authors assessed the progression of pulmonary
emphysema by means of quantitative analysis of computed tomography images.
METHODS: Twenty-three patients suffering from emphysema due to an alpha
1-antitrypsin deficiency, aged 45 +/- 7 years and exsmokers, were scanned twice
with a 1-year time interval. At 90% of the vital lung capacity, slices with a
thickness of 1.5 mm were acquired at the level of the carina and 5 cm above the
carina; slices with a thickness of 1 cm were acquired 5 cm below the carina. The
entire lung was scanned spirally at a respiratory status, corresponding with 75%
of the total lung capacity at baseline. The mean lung densities (MLD) were
calculated in an objective manner with new analytic software featuring automated
detection of the lung contours. RESULTS: Mean lung densities decreased by 14.2
+/- 12.0 Hounsfield units (HU; P < 0.001) above the carina, by 18.1 +/- 14.4 HU
(P < 0.001) at the carina level, by 23.6 +/- 15.0 HU (P < 0.001) below the
carina, and by 12.8 +/- 22.2 HU (P < 0.01) for the entire lung. The decrease in
MLD was most obvious in the lower lung lobes. For the same patient group, the
annual decrease in the forced expiratory volume (FEV1) and the carbon
monoxide-diffusion were 120 +/- 190 mL (P < 0.01) and 10 +/- 70 mmol/kg/minute (
P < 0.2), respectively. No significant correlation was found between the
decrease in MLD and the decrease in FEV1. CONCLUSIONS: Progression of emphysema
can be assessed in an objective manner based on the mean lung density (MLD),
measured from computed tomography volume scans as well as from single-slice
scans. Mean lung density has proved to be more sensitive than FEV1 and carbon
monoxide-diffusion.

Infect Immun 1996 Nov;64(11):4520-4

Antibacterial activity of antileukoprotease.

Hiemstra PS, Maassen RJ, Stolk J, Heinzel-Wieland R, Steffens GJ, Dijkman JH.

Department of Pulmonology, Leiden University Hospital, The Netherlands.

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an
endogenous inhibitor of serine proteinases that is present in various external
secretions. ALP, one of the major inhibitors of serine proteinases present in
the human lung, is a potent reversible inhibitor of elastase and, to a lesser
extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that
has some of the characteristics of ALP has been identified (M. A. Couto, S. S.
L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun.
60:5042-5047, 1992). This report, together with the cationic nature of ALP, led
us to investigate the antimicrobial activity of ALP. ALP was shown to display
marked in vitro antibacterial activity against Escherichia coli and
Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that
of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP
comprises two homologous domains: its proteinase-inhibitory activities are known
to be located in the second COOH-terminal domain, and the function of its first
NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated
first domain with E. coli or S. aureus resulted in killing of these bacteria,
whereas its second domain displayed very little antibacterial activity. Together
these data suggest a putative antimicrobial role for the first domain of ALP and
indicate that its antimicrobial activity may equip ALP to contribute to host
defense against infection.

Purine enzyme activities in recent onset rheumatoid arthritis: are there
differences between patients and healthy controls?

Stolk JN, Boerbooms AM, De Abreu RA, Kerstens PJ, de Koning DG, de Graaf R,
Mulder J, van de Putte LB.

Department of Rheumatology, University Hospital Nijmegen, Netherlands.

OBJECTIVE: Purine enzyme activities may predict the effectiveness of
azathioprine treatment and be associated with increased deaths from infectious
diseases. In rheumatoid arthritis, patients show variable responses to
azathioprine and a higher percentage of death is caused by infections. The aim
of the study was to investigate possible rheumatoid arthritis associated
abnormalities of purine enzyme activities by measuring several of these enzymes
in patients with recent onset rheumatoid arthritis before treatment with disease
modifying antirheumatic drugs or prednisone. METHODS: 23 patients with recent
onset rheumatoid arthritis and 28 healthy controls were studied. Activities of
the enzymes 5'-nucleotidase, purine nucleoside phosphorylase (PNP), hypoxanthine
guanine phosphoribosyltransferase (HGPRT), and thiopurine methyltransferase
(TPMT) were measured. Assessment of disease activity and blood sampling for
routine measurements and HLA typing were done simultaneously. RESULTS: Purine
enzyme activities did not differ between patients and healthy controls. Enzyme
activities had no significant relations with indices of disease activity or
rheumatoid factor titre or with the rheumatoid arthritis associated HLA types.
Activity of 5'nucleotidase decreased with age (P < or = 0.05) and was lower by
about 27% (P = 0.007) in males than in females. CONCLUSIONS: In rheumatoid
arthritis patients, neither the variability in azathioprine effectiveness nor
the increased death rate from infections can be explained by pre-existing
abnormalities in the activities of the purine enzymes 5'-nucleotidase, PNP,
HGPRT, or TPMT at an early stage of the disease, before disease modifying
antirheumatic drugs or prednisone treatment. Besides adjustment for age, results
of studies involving purine 5' nucleotidase activity should also be adjusted for
sex.

J Exp Med 1996 Oct 1;184(4):1305-12

Colony growth of human hematopoietic progenitor cells in the absence of serum is
supported by a proteinase inhibitor identified as antileukoproteinase.

Goselink HM, van Damme J, Hiemstra PS, Wuyts A, Stolk J, Fibbe WE, Willemze R,
Falkenburg JH.

Department of Hematology, Leiden University Hospital, The Netherlands.

Serum contains many growth factors and nutrients that stimulate colony formation
of hematopoietic progenitor cells (HPC) in semisolid cultures. In the absence of
serum, no proliferation of HPCs could be obtained in semisolid medium cultures
of partially purified bone marrow cells in the presence of multiple
hematopoietic growth factors, insulin, cholesterol, and purified clinical-grade
human albumin. This appeared to be due to a suppressive activity induced by
monocyte- and T lymphocyte-depleted accessory cells on CD34+ HPCs. Serum-free
conditioned medium from the bladder carcinoma cellline 5637 could replace serum
to support the growth of HPCs in these cultures. After gel filtration and
reverse-phase high-performance liquid chromatography of 5637 supernatants, this
activity could be attributed to a 15-kD protein that was further identified by
NH2-terminal sequence analysis as the serine proteinase inhibitor
antileukoproteinase (ALP). The growth-supportive activity from the 5637
conditioned medium and the (partially) purified fractions could be completely
neutralized by a polyclonal rabbit IgG antibody against human ALP (huALP).
Similar supportive effects on the growth of HPC could be obtained in the
presence of recombinant huALP. We demonstrated that the COOH-terminal domain of
ALP containing the proteinase inhibitory activity was responsible for this
effect. alpha-1 proteinase inhibitor was capable of similar support of in vitro
HPC growth. These results illustrate that proteinase inhibitors play an
important role in the in vitro growth of hematopoietic cells by the
neutralization of proteinases produced by bone marrow accessory cells. This may
be of particular relevance for in vitro expansion of human hematopoietic stem
cells in serum-free media.

Complications in the use of the subcutaneous tunnelled intratracheal oxygen
catheter.

in't Veen JC, Stolk J, Dijkman JH.

Department of Pulmonology, University Hospital, Leiden, Netherlands.

Transtracheal oxygen delivery seems to be a safe procedure in the treatment of
chronic obstructive pulmonary disease (COPD) with chronic hypoxaemia. Even so,
serious complications do occur. Three patients in whom we used a subcutaneous
tunnelled intratracheal oxygen catheter (ITO2C) are described. Surgical
intervention was required in all because of complications from the procedure.
One of the complications--tracheal and catheter obstruction with stridor and
subcutaneous emphysema by granulomatous tissue--has to our knowledge not been
reported before.

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