A functional calcium-binding site in the metalloprotease domain of ADAMTS13.

ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca(2+) ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca(2+) concentrations, a high-affinity functional ADAMTS13 Ca(2+)-binding site was suggested with K(D(app)) of 80 muM (+/- 15 muM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca(2+)-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca(2+) dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca(2+)-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the K(D(app)) for Ca(2+) of ADAMTS13, and for D187A and E212A also reduced the V(max) to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca(2+) revealed an approximately 13-fold reduction in specificity constant, k(cat)/K(m), contributed by changes in both K(m) and k(cat). These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca(2+) upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.

ADAMTS13 and von Willebrand factor and the risk of myocardial infarction in men.

Von Willebrand factor (VWF) mediates the tethering/adhesion of platelets at sites of vascular injury. This function depends on its multimeric size, which is controlled by ADAMTS13. We measured plasma ADAMTS13 and VWF antigen levels by enzyme-linked immunosorbent assay (ELISA) in a large population-based case-control study (Study of Myocardial Infarctions Leiden [SMILE]), consisting of 560 men with a first myocardial infarction (MI) and 646 control subjects. Although ABO blood groups influenced VWF levels, they had no influence on ADAMTS13. Furthermore, there was no relationship between plasma ADAMTS13 and VWF levels. Similar to VWF, the estimated risk of MI was increased for every quartile of ADAMTS13 when compared to the lowest quartile (odds ratio, 1.5-1.6). If confirmed, the association of ADAMTS13 with MI may suggest an unexpected mechanistic action of ADAMTS13.

ADAMTS13 substrate recognition of von Willebrand factor A2 domain.

ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in theA2 domain. To examine substrate recognition, we expressed in bacteria and purified three A2 (VWF76-(1593-1668), VWF115-(1554-1668), VWFA2-(1473-1668)) and one A2-A3 (VWF115-A3-(1554-1874)) domain fragments. Using high pressure liquid chromatography analysis, the initial rates of VWF115 cleavage by ADAMTS13 at different substrate concentrations were determined, and from this the kinetic constants were derived (Km 1.61 microM; kcat 0.14 s(-1)), from which the specificity constant kcat/Km was calculated, 8.70 x 10(4) m(-1) s(-1). Similar values of the specificity constant were obtained for VWF76 and VWF115-A3. To identify residues important for recognition and proteolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved approximately 8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614 on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K1617A, E1638A, E1640A) revealed up to 13-fold reduction in kcat/Km for D1587A, D1614A, E1615A, and K1617A mutants. When introduced into the intact VWFA2 domain, proteolysis of the D1587A, D1614A, and E1615A mutants was also slowed, particularly in the presence of urea. Surface plasmon resonance demonstrated appreciable reduction in binding affinity between ADAMTS13 and VWF115 mutants (KD up to approximately 1.3 microM), compared with VWF115 (KD 20 nM). These results demonstrate an important role for Asp1614 and surrounding charged residues in the binding and cleavage of the VWFA2 domain by ADAMTS13.

Magnetic resonance screening of iron status in transfusion-dependent beta-thalassaemia patients.

The clinical utility of dual sequence (T1- and T2-weighted) magnetic resonance (MR) imaging in estimating liver iron concentration (LIC) in 32 transfusion-dependent beta-thalassaemia major (24 females; age 18.5+/-5.9 years) patients on desferrioxamine was evaluated. Signal intensity ratios (SIR) between liver, spleen and pancreas to psoas muscle were determined on both sequences. Relationships between clinical and MR parameters, and accuracy of SIR thresholds in determining adequacy of chelation from LIC were analysed. Liver T1- and T2-SIR were related to LIC (P < 0.001). T1-SIR < 0.60 predicted severe iron overload (LIC > 15 mg/g) with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100%, 87%, 33% and 100% respectively. T2-SIR < 0.1 yielded 100% sensitivity, 93% specificity, 50% PPV and 100% NPV. T1-SIR > or = 1.1 predicted LIC < 7 mg/g with 69% sensitivity, 88% specificity, 85% PPV and 74% NPV. T2-SIR > or = 0.20 yielded 56.5% sensitivity, 94% specificity, 90% PPV and 71% NPV. LIC correlated with liver T1-SIR, liver T2-SIR and serum ferritin (r = -0.76, -0.65, 0.47, respectively; P < 0.01). Serum ferritin was inversely related to liver T1-SIR, liver T2-SIR and spleen T2-SIR (r = -0.35, -0.43, -0.40, respectively; P < 0.05). Mean total transfusion burden was not related to any MR parameter. Although neither MR sequence was a highly accurate predictor of LIC, SIR thresholds are useful to determine presence of iron overload and adequacy of chelation treatment.

The effect of aging on nasal mucociliary clearance, beat frequency, and ultrastructure of respiratory cilia.

The increased susceptibility of the elderly to lower respiratory tract infection cannot be fully explained. Although mucociliary clearance, which is affected by ciliary beating and ultrastructure, plays a crucial role in the defense of the airways against inhaled microbes, little is known of the effects of aging on these parameters. We studied the nasal mucociliary clearance (NMCC) time, ciliary beat frequency, and ultrastructure of respiratory cilia in a cohort of healthy volunteers (age range 11 to 90 yr). Ciliary beat frequency of ciliated nasal epithelial cells was obtained via an established photometric method, and NMCC time was measured with the saccharine test. There was a correlation of ciliary beat frequency (r = -0.48, p = 0.0001) and NMCC time r = 0.64, p < 0.001) with increasing age. Transmission electron microscopy revealed an increase in the percent of subjects exhibiting microtubular disarrangement and single central microtubules with aging (p = 0.002 and p = 0.005, respectively). Subjects older than 40 yr of age had significantly slower ciliary beat frequency, higher percent of ciliary cross-sections displaying single tubules, and longer NMCC time than their younger counterparts (p < 0.05). These findings may help explain the frequent occurrence of respiratory infection in the elderly.

Serial HRCT lung changes after 3-field radiation treatment of breast cancer.

AIMS: To document serial high resolution computed tomography (HRCT) features of lung injury after 3-field radiotherapy for breast cancer. MATERIALS AND METHODS: Thirty women who received opposing tangential chest wall and supraclavicular field (SCF) irradiation after breast surgery were recruited. Thoracic HRCT was performed before and at 1, 3, 6 and 12 months after radiotherapy (RT). Lung injury at 3 months was quantified by applying a scoring system to each HRCT section. Findings were correlated with spirometric lung function tests. RESULTS: There was HRCT evidence of lung injury in 27 (90%) women at 1 month and in all 30 patients at 3 months. Spirometric lung function declined post-RT (P < 0.05), correlating with an increased SCF acute lung injury score at 3 months [r = -0.54 and -0.46, P = 0.01 and 0.03 for forced expiratory volume in 1st (FEV(1)) and forced vital capacity (FVC), respectively]. Lung injury on HRCT progressed from ground glass opacification at 1 month to nodular consolidation (3 months), increasing linear densities (6 months), and finally to residual subpleural linear and dense opacities (12 months). CONCLUSION: There is a high incidence of lung injury associated with 3-field radiotherapy for breast cancer, with concurrent SCF irradiation increasing the risk of lung damage and functional impairment. A characteristic sequence of HRCT changes is seen in most patients receiving this type of radiotherapy.Ooi, G. C. (2000). Clinical Radiology55, 817-824.

Diagnostic yield of bronchoscopic sampling methods in bronchial carcinoma.

OBJECTIVES: The aim was to study the yield of malignant or suspicious cells from bronchoalveolar lavage (BAL), endobronchial biopsy (BX) and endobronchial brushing (BR) specimens obtained at bronchoscopy. METHODOLOGY: We prospectively followed up all patients who had undergone bronchoscopy and studied those with subsequent diagnosis of bronchial carcinoma at a tertiary referral centre. Bronchoalveolar lavage, BX and BR were performed, sequentially whenever possible, by one pulmonologist. The parameters assessed were age, gender, macroscopic bronchoscopic findings, TNM staging, radiological findings, histological typing, and diagnostic yield from BAL, BX, and BR. RESULTS: One hundred patients (31 females, 69 males; mean age +/- SD, 61.8+/-12.7 years; range 32-81 years) were studied between 1995 and 1997. Of these, BAL, BX and BR were performed on 100, 64 and 37 cases which yielded diagnostic specimens for bronchial carcinoma in 69, 78.1, and 62.2% of cases, respectively (P > 0.05). Diagnostic specimens were therefore obtained in 69,50, and 23% of the entire patient cohort from BAL, BX, and BR, respectively (P < 0.001). The diagnostic yield of BAL was independent of patient age, gender, site of lesion, TNM staging, histological typing and macroscopic bronchoscopic findings. Addition of BX, BR and postbronchoscopic sputum sampling to BAL only increased the yield of diagnostic specimens for bronchial carcinoma by 7, 0, and 4%, respectively. CONCLUSION: Bronchoalveolar lavage is a highly effective sampling method to obtain cytological evidence for bronchial carcinoma. The diagnostic yield for BAL is independent of tumour or other clinical characteristics. Further studies should be performed to confirm these important and clinically relevant findings.

Helicobacter pylori sero-prevalence in asthma.

Asthma is a very common respiratory disease which is characterized by the presence of cytokine-mediated airway inflammation leading to smooth muscle contraction, oedema and progressive airway damage in some cases. In light of our recent finding of an increased sero-prevalence of Helicobacter pylori in bronchiectasis, we have determined serum levels of H. pylori-specific IgG in asthmatic and control subjects. Altogether 90 consecutive asthmatic [mean age +/- SD 42.6+/-16 years and 52 female (F)] and 97 healthy control subjects (mean age +/- SD 43.2+/-13.3 years and 51 female (F); P = 0.78 and 0.39 respectively) were recruited prospectively. H. pylori sero-prevalence was not significantly different between asthmatic and control subjects (P>0.05). Serum H. pylori IgG levels did not correlate with FEV1 % predicted, FVC % predicted or duration of asthma (P>0.05). Similar to the results of previously published sero-epidemiological studies, there was a weak correlation between serum H. pylori IgG with increasing age (r = 0.43, P = 0.004). Despite the sero-epidemiological association of H. pylori infection with many inflammatory conditions, our data showed no such association for middle age asthmatic patients with mild intermittent asthma in our locality.

Lung function reference values in Chinese children and adolescents in Hong Kong. II. Prediction equations for plethysmographic lung volumes.

As part of a comprehensive evaluation of lung function in Hong Kong Chinese children and adolescents, over a thousand healthy subjects aged 7-19 yr from seven schools were recruited for lung function testing that included spirometry and, in many cases, lung subdivision measurements. Lung function tests were performed using SensorMedics Automated Body Plethysmograph according to published standards. Of these, 551 subjects (219 males), aged 8-19 yr, had satisfactory lung subdivision indices recorded. Analysis for the values of lung subdivisions including total lung capacity (TLC), residual volume (RV), and functional residual capacity (FRC) demonstrated that standing height and sitting height were the best predictors of lung volumes. After allowing for standing height or sitting height in the regression models for lung volumes, age at examination was the second best parameter, although its inclusion into the equations contributed to less than 1% of explained variance for boys and 3% for girls. These are the first reported data in international literature on reference values for lung subdivisions in Chinese children and adolescents.

Previously unrecognized obstructive sleep apnea in Chinese subjects with essential hypertension.

Obstructive sleep apnea (OSA) is found to affect 2-4% of the middle-aged population in several Caucasian studies, whereas the prevalence among other ethnic groups have not been clearly documented. It has been reported that OSA and systemic hypertension are highly associated; we therefore conducted a study on Chinese subjects who were receiving treatment for essential hypertension to assess the prevalence of OSA among this group. Ninety-two consecutive patients being followed up at a hypertension clinic were recruited for a questionnaire survey. The entire study group was aged 54.7 +/- 11.7 years, with 40 men. One male subject had a diagnosis of obstructive sleep apnea on nasal continuous positive airway pressure (nCPAP) treatment and 46 subjects agreed to an overnight sleep study. Those who underwent sleep study showed selection bias with a higher body mass index and more symptoms associated with OSA. Of the 46 who underwent sleep study, 16 (34.8%) had an obstructive apnea-hypopnea (AHI) score of >/=5 and excessive daytime sleepiness, with a median score of 26.2 (range, 8.3-64.9). Patients in the group with obstructive sleep apnea syndrome (OSAS) thus defined compared with those without OSAS had more men (64.7 vs 17.20%, p = 0.001) and an excess of smokers (31.5 vs 3.3%. p = 0.01) and had significantly more symptoms of excessive daytime sleepiness (p = 0.001), daytime fatigue (p = 0.007), and witnessed apneas (p = 0.008). Seven patients accepted treatment with nCPAP and reported improvement in symptoms, but there was no detectable change in clinic blood pressure measurements after 3 months of nCPAP treatment. This study demonstrated a high prevalence of previously unidentified OSAS among Chinese patients with essential hypertension. Increased awareness of both doctors and patients toward this potentially treatable problem is warranted.

A pilot study of low-dose erythromycin in bronchiectasis.

Patients with bronchiectasis suffer from sputum production, recurrent exacerbations, and progressive airway destruction. Erythromycin is effective in diffuse panbronchiolitis, another suppurative airway disorder, although its efficacy is unknown in idiopathic bronchiectasis. A double-blind placebo-controlled study was therefore conducted to evaluate the effects of 8-week administration of low dose erythromycin (500 mg b.i.d.) in steady-state idiopathic bronchiectasis. Patients in the erythromycin group (n=11, 8 female, mean age 50+/-15 yrs), but not the placebo group (n=10, 8 female, mean age 59+/-16 yrs) had significantly improved forced expiratory volume in one second, forced vital capacity and 24-h sputum volume after 8 weeks (p<0.05). There was no parallel improvement in sputum pathogens, leukocytes, interleukin (IL)-1alpha and IL-8, tumour necrosis factor-alpha, or leukotriene B4. The results of this pilot study show that low-dose erythromycin improves lung function and sputum volume in bronchiectasis. Further studies are indicated to evaluate the efficacy of long-term erythromycin therapy in bronchiectasis.

Qualitative and quantitative magnetic resonance imaging in haemoglobin H disease: screening for iron overload.

OBJECTIVES: To evaluate the clinical utility of magnetic resonance imaging (MRI) in screening for iron overload in non-transfusion dependent Haemoglobin (Hb) H disease. PATIENTS AND METHODS: Thirty-six non-transfusion dependent HbH patients were evaluated with axial spin echo T1 and gradient echo T2 MRI of the abdomen and heart. The ratios of signal intensities (SIR) of the liver, spleen, pancreas and heart to paraspinous muscles were calculated. SIR <1 was taken as indicative of iron overload. Qualitative grading (0-4 scale) of iron overload was also performed. The relationship between T1 and T2 SIR and serum ferritin, and that between qualitative grading and serum ferritin were examined using standard statistical methods. Comparisons were also made between qualitative grading and quantitative T1 and T2 SIR data in diagnosing iron overload. Six patients underwent liver biopsies. RESULTS: T2 SIR was more sensitive in detecting iron overload than T1 SIR. Thirty-three livers, 13 spleens, six pancreas and one heart were diagnosed as having iron overload with T2 SIR, including three patients with normal serum ferritin. A positive diagnosis by T2 SIR was more closely related to that of qualitative grading than T1 SIR. Serum ferritin was negatively correlated with hepatic SIR (T1 and T2), and with T2 SIR of the spleen and pancreas, even after adjustment for age. Liver haemosiderosis was confirmed in all six patients who underwent liver biopsies. Liver iron concentration of only one and a half times the normal was found in one patient with positive MR findings. CONCLUSION: MR is a non-invasive, effective method for early detection of iron overload particularly in the liver and spleen. Qualitative grading and quantitative T2 SIR data are equivalent in diagnosing iron overload. Routine screening of non-transfusion dependent HbH patients will identify high risk patients in whom early therapeutic intervention may prevent further complications and morbidity.

Helicobacter pylori and upper gastrointestinal symptoms in bronchiectasis.

The recently reported increase in seroprevalence of Helicobacter pylori, the causative pathogen in peptic ulceration, in bronchiectasis is unexplained. Therefore, the association of antibodies directed against cytotoxin-associated gene A(CagA), whose expression indicates virulence of H. pylori, and upper gastrointestinal symptoms in patients with stable bronchiectasis and healthy volunteers evaluated. One hundred patients (mean +/- SD age 55.1+/-16.7 yrs) and 94 healthy asymptomatic subjects (54.6+/-7.6 yrs) underwent clinical and physiological assessment and serum levels of anti-H. pylori CagA were determined using standard clinical and enzyme-linked immunosorbent assay techniques. Samples were positive for anti-H. pylori CagA in 11.7% of controls and 24% of bronchiectatic subjects (p = 0.03). There was, however, no association between serum H. pylori CagA immunoglobulin G level and forced expiratory volume in one second (FEV1), forced vital capacity (FVC), sputum volume, respiratory symptoms or upper respiratory gastrointestinal symptoms (p>0.05). Patients who suffered from acid regurgitation or upper abdominal distension had significantly lower FEV1 and FVC (as a percentage of the predicted value) compared to their counterparts. The results of anticytotoxin-associated gene A measurements in this study contrasted with the previous finding that anti-Helicobacter pylori immunoglobulin G correlated with sputum volume. These findings, therefore, suggest that Helicobacter pylori, should it have a pathogenic role in bronchiectasis, could act via noncytotoxin-associated gene A-mediated mechanisms, and, in this context, gastro-oesophageal reflux might be of importance in bronchiectasis.

Inhaled fluticasone reduces sputum inflammatory indices in severe bronchiectasis.

Although corticosteroid therapy might be clinically beneficial for bronchiectasis, very little is known of its effects on the inflammatory and infective markers in bronchiectasis. We have therefore performed a double-blind, placebo-controlled study to evaluate the effects of a 4-wk administration of inhaled fluticasone in bronchiectasis. Twenty-four patients (12 female; mean age 51 yr) were randomized into receiving either inhaled fluticasone (500 microgram twice daily) via the Accuhaler device (n = 12) or placebo. At each visit, spirometry, 24-h sputum volume, sputum leukocyte density, bacterial densities, and concentrations of interleukin (IL)-1beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), and leukotriene B4 (LTB4) were determined. There was a significant (p < 0.05) decrease in sputum leukocyte density and IL-1beta, IL-8, and LTB4 after fluticasone treatment. The fluticasone group had one and the placebo group three episodes of exacerbation. There were no significant changes in spirometry (p > 0.05) or any reported adverse reactions in either group. The results of this study show that high-dose fluticasone is effective in reducing the sputum inflammatory indices in bronchiectasis. Large-scale and long-term studies are indicated to evaluate the effects of inhaled steroid therapy on the inflammatory components in bronchiectasis.

The effect of Pseudomonas aeruginosa infection on clinical parameters in steady-state bronchiectasis.

STUDY OBJECTIVE: To investigate the effect of Pseudomonas aeruginosa infection on clinical parameters in Chinese patients with noncystic fibrosis and steady-state bronchiectasis. DESIGN: Prospective, cross-sectional clinicomicrobiological study with informed consent. SETTING: Consecutive outpatient recruitment from a specialist bronchiectasis respiratory clinic. PATIENTS: Outpatients (n = 100; 62 women; 55.1+/-16.7 years old; FEV1/FVC 1.4+/-0.7/2.1+/-0.9 L), who had stable respiratory symptoms for more than 3 weeks. MEASUREMENTS AND RESULTS: Respiratory pathogens isolated from the sputum were: Pseudomonas aeruginosa (33), Haemophilus influenzae (10), Moraxella catarrhalis (2), other Gram-negative bacilli (5), Streptococcus pneumoniae (6), Staphylococcus aureus (5), mycobacteria (3), and yeast (1). Clinical parameters in patients with positive isolation of P aeruginosa were compared with those without the organism in the sputum culture (non-P aeruginosa). In the P aeruginosa group, the FEV1/FVC ratio and sputum volume were lower (p < 0.005) and higher (p < 0.0001), respectively, than those of the non-P aeruginosa group. The FEV1/FVC ratio (< 60%) and sputum volume (grading > 5) were independently associated with a positive sputum isolation of P aeruginosa with odds ratios of 3.1 (confidence interval [CI] 1.2 to 8.4; p < 0.01) and 4.7 (CI 1.6 to 13.3; p < 0.001), respectively. CONCLUSIONS: P aeruginosa is the predominant respiratory pathogen isolated in the sputum of Chinese patients with steady-state bronchiectasis, and its isolation is associated with high sputum output (> or = 75th quartile) and moderately severe airflow obstruction (FEV1/FVC < 60%).

Serum interleukin-2 receptor in infants and young children.

Soluble interleukin-2 receptor (IL-2R) concentrations were measured in 110 serum samples from 102 children undergoing routine small bowel biopsy for a wide range of gastrointestinal symptoms. Young children in the control group who had no gastrointestinal disease and a normal intestinal mucosa were found to have high concentrations of IL-2R. There was a significant inverse relationship between IL-2R concentration and age in the control group. Children with a gastrointestinal diagnosis appeared to have increased IL-2R concentrations, although some of the diagnostic groups were too small in size for statistical analysis. Children with cow's milk sensitive enteropathy had IL-2R concentrations equivalent to those of age-matched controls. High concentrations of IL-2R in young healthy children implies that serum IL-2R is of no clinical use as an index of inflammation in this age group.

Endomysial antibody screening in children.

It has been suggested that endomysial antibodies are specific markers for coeliac disease. In a 13-month study, we examined the usefulness of screening for these antibodies in the diagnosis of coeliac disease in children. Twenty-one of 223 (9.4%) serum samples [or 17 of 192 (9%) children undergoing investigation for GI disorders] were found to be positive for serum IgA class endomysial antibodies. These included eight strong positives, eight positives, and five weak positives. One-hundred-thirty-four children had small bowel biopsies performed. Endomysial antibodies were found in all children with severe villous atrophy on a gluten-containing diet who were diagnosed as having coeliac disease. Three children with positive and four with weak-positive results did not have coeliac disease. One had partial villous atrophy consistent with the diagnosis of cow-milk-sensitive enteropathy and the others had a normal small bowel mucosa. This study confirms a strong association between endomysial antibodies and coeliac disease; however, not all cases with positive antibodies had the disease. At the present time, small bowel biopsy remains essential for the diagnosis of coeliac disease.

Enteroaggregative Escherichia coli: another cause of acute and chronic diarrhoea in England?

Enteroaggregative Escherichia coli (EAggEC) has been found to be associated with acute and persistent diarrhoea in children in developing countries. Its clinical significance in developed countries has not been examined in much detail. In a survey of faecal samples from children with diarrhoea presenting to a children's hospital in East London between August and December 1988, EAggEC strains were isolated in 8 of 297 (2.7%) consecutive stool samples collected from 289 children and in 5 of 34 typed E. coli isolates during 1988. Of the 13 children found to be excreting EAggEC, 8 had acute diarrhoea and 5 had chronic diarrhoea of more than 14 days' duration; 5 children had mixed infections. Compared to other organisms found during the same period, EAggEC were isolated as frequently as many other better-known pathogens such as enteropathogenic E. coli, Campylobacter, Salmonella, Shigella, Giardia lamblia, and Cryptosporidium. We conclude that EAggEC may be an important pathogen in developed countries.