Cédric Hermans

Cloning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family.

Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein, termed B166, with a molecular mass of 17 kDa. Here, we report the cloning of human and rat cDNAs encoding B166, which has been renamed AOEB166 for antioxidantenzyme B166. Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin antioxidant enzymes. Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identifiedSaccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase. Moreover, recombinant AOEB166 expressed in E. coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine synthetase protection assay against dithiothreitol/Fe3+/O2 oxidation. The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene is widely expressed in the body. Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences. Furthermore, human AOEB166 expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles. Finally, acute inflammation induced in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role for AOEB166 in oxidative and inflammatory processes.

Lung hyperpermeability and asthma prevalence in schoolchildren: Unexpected associations with the attendance at indoor chlorinated swimming pools

Aims: To study whether exposure to nitrogen trichloride in indoor chlorinated pools may affect the respiratory epithelium of children and increase the risk of some lung diseases such as asthma. Methods: In 226 healthy children, serum surfactant associated proteins A and B (SP-A and SP-B), 16 kDa Clara cell protein (CC16), and IgE were measured. Lung specific proteins were measured in the serum of 16 children and 13 adults before and after exposure to NCl3 in an indoor chlorinated pool. Relations between pool attendance and asthma prevalence were studied in 1881 children. Asthma was screened with the exercise induced bronchoconstriction test (EIB). Results: Pool attendance was the most consistent predictor of lung epithelium permeability. A positive dose-effect relation was found with cumulated pool attendance and serum SP-A and SP-B. Serum IgE was unrelated to pool attendance, but correlated positively with lung hyperpermeability as assessed by serum SP-B. Changes in serum levels of lung proteins were reproduced in children and adults attending an indoor pool. Serum SP-A and SP-B were already significantly increased after one hour on the pool side without swimming. Positive EIB and total asthma prevalence were significantly correlated with cumulated pool attendance indices. Conclusions: Regular attendance at chlorinated pools by young children is associated with an exposure dependent increase in lung epithelium permeability and increase in the risk of developing asthma, especially in association with other risk factors. We therefore postulate that the increasing exposure of children to chlorination products in indoor pools might be an important cause of the rising incidence of childhood asthma and allergic diseases in industrialised countries. Further epidemiological studies should be undertaken to test this hypothesis.

Transient increase of serum Clara cell protein (CC16) after exposure to smoke.

OBJECTIVES: Smoke inhalation is a well known cause of airways injury in firefighting personnel. The aim of this study was to evaluate whether toxic effects of smoke on the respiratory tract can be detected by measuring Clara cell protein (CC16), a recently described serum marker of lung function. METHODS: CC16 was measured by a sensitive latex immunoassay in the serum of six voluntary firefighters from a chemical plant who had inhaled smoke from the combustion of polypropylene for about 20 minutes. The protein was measured immediately after the fire and 10 days later. The values were compared with those of six control workers examined simultaneously. RESULTS: The mean (SD) concentration of CC16 in the serum of firefighters after the fire (54.4 (34.9) micrograms/l) was significantly higher than that of controls (19.5 (11.7), P = 0.04). 10 days later, serum CC16 from firefighters had returned to the concentrations found in controls (15.9 (2.76) v 17.7 (12.5)). With the values at day 10 as a baseline, the rise of serum CC16 was estimated at 328% on average (range 100%-564%). These changes were found in the absence of any functional sign of lung impairment. CONCLUSION: Acute exposure to smoke results in a transient increase of CC16 in serum due most likely to an increased permeability of the bronchoalveolar/blood barrier. Serum CC16 seems potentially to be a new biomarker for the early detection of acute airways injury caused by smoke.

Clara cell protein (CC16): Characteristics and potential applications as biomarker of lung toxicity

Abstract Most biomarkers of lung toxicity presently available require a bronchoahreolar lavage (BAL). Such a procedure cannot be applied for monitoring populations at risk in the industry or environment nor for a regular follow-up of patients with lung disorders. A lung biomarker, measurable in serum, BAL fluid and sputum has recently been identified. This biomarker is a microptotein initially isolated from urine (Urine Protein 1) and subsequently identified as the major secretory product of lung Clara cells which are non-ciliated cells localized predominantly in terminal bronchioles. This protein called Clara cell protein (CC16) is a homodimer of 15.8 kDA. Several lines of evidence indicate that CC16 is a natural immunoregulator protecting the respiratory tract from unwanted inflammatory reactions. CC16 secreted in the respiratory tract diffuses passively by transudation into plasma from where it is rapidly eliminated by glomerular filtration before being taken up and catabolized in proximal tubule cells. Studies reviewed here suggest that CC16 in BAL fluid or serum is a sensitive indicator of acute or chronic bronchial epithelium injury. A significant reduction of CC16 has been found in serum and BAL fluid of asymptomatic smokers. On average serum CC16 decreases by 15% for each 10 pack-year smoking history. Serum CC16 was also found to be decreased in several occupational groups chronically exposed to different air pollutants (silica, dust, welding fumes). A dose-effect relationship with the intensity of exposure to dust has been found in one study on foundry workers. The concentration of CC16 in serum can also be used to detect an acute or chronic disruption of the bronchoalveolar/blood barrier integrity. While confirming the potential interest of CC16 as a lung biomarker, clinical investigations indicate that CC16 might be an important mediator in the development of lung injury. These findings open new perspectives in the assessment of lung toxicity by suggesting that readily diffusible lung-specific proteins may serve as peripheral markers of pneumotoxicity.

Epidemiology of inhibitor formation with recombinant factor VIII replacement therapy

Summary.u2002 This article reviews the epidemiology of inhibitor formation in patients receiving recombinant factor VIII (rFVIII) replacement therapy for haemophilia A. Data from pivotal trial programmes, post‐marketing surveillance studies, and pharmacovigilance studies for all available rFVIII products were reviewed. To date, no comparative studies have been performed, and in the non‐comparative studies, there were differences in the patient population enrolled and study design (including the number of patient exposure days). In the absence of comparative clinical trials, it is not possible to make comparisons between inhibitor data for the various rFVIII products. This review of the epidemiological data shows that across the pivotal trial programmes for rFVIII products, the observed incidence of inhibitors was in the range of 15–32% in previously untreated patients (PUPs) and 0.9–2.9% in previously treated patients (PTPs). High‐titre inhibitors (peak >5u2003BU) were detected in 10–16% of PUPs and 0–2.3% of PTPs. Several initiatives proposed to help standardize collection and interpretation of inhibitor data for patients receiving rFVIII treatment are described. Such standardization would help to clarify the epidemiology of inhibitor formation across FVIII treatments.

Determinants of serum levels of surfactant proteins A and B and Clara cell protein CC16.

Increased leakage of surfactant proteins A and B (SP-A and SP-B) and Clara cell secretory protein (CC16) from the air spaces into the circulation occurs in a range of respiratory conditions. However, circulating levels depend not only on the rate of entry into the circulation, but also on the rate of clearance. In order to clarify the role of the kidney in the clearance of these proteins, serum levels were related to markers of glomerular filtration in 54 non-smoking patients with varying degrees of renal dysfunction, none of whom had respiratory disease or were receiving dialysis at the time of sampling. Serum SP-A was related to SP-B (r=0.53, p<0.001) and to CC16 (r=0.33, p<0.02). Similarly, SP-B was related to CC16 (r=0.39, p<0.004). Stepwise multiple linear regression analysis suggested that serum SP-A and SP-B are influenced by age (∼20 and ∼25% of variance, respectively), whereas CC16 is determined by renal function and, to a lesser extent, by body weight (∼63% of variance in total). We conclude that CC16 is cleared from blood by the renal route, whereas SP-A and SP-B are not. Serum SP-A and SP-B are influenced by age, which we speculate reflects increased damage to the alveolocapillary barrier.

Dioxin accumulation in residents around incinerators.

To evaluate the human exposure impact of municipal waste incinerators, dioxin and coplanar polychlorinated biphenyl (PCB) concentrations were determined in blood of 84 subjects who resided approximately 18 yr in the vicinity of two old incinerators, one located in a rural area (n = 51) and the other in an industrial area (n = 33). These subjects were compared with 63 controls from an unpolluted area. While no change was found in contaminant levels in residents living around the incinerator in the industrial area, subjects residing around the incinerator in the rural area possessed significantly higher serum levels of dioxins (38 vs. 24 pg TEQ/g fat) and coplanar PCBs (10 vs. 7 pg TEQ/g fat) than controls. These results were confirmed by multipleregression analysis, showing that residence around the incinerator in the rural area (partial r 2 = .18) was the major contributor to dioxin accumulation followed by age (partial r 2 = .07). A two-way analysis of variance (ANOVA) on age-adjusted dioxin levels revealed a significant interaction between residence around incinerators and the consumption of fat from local origin, especially bovine and poultry products. Although age-adjusted dioxin levels in controls did not vary with local animal fat consumption, concentrations of dioxins in subjects living around the incinerators increased proportionally to their intake of local animal fat, with almost a doubling in subjects with a fat intake higher than 150 g fat/wk. Extrapolation from these data suggests that a significant increase of dioxin body burden is likely to occur only when dioxin emissions exceed 5 ng TEQ/Nm 3 , a threshold considerably above most emissions standards currently in force.

Long-acting recombinant factor IX Fc fusion protein (rFIXFc) for perioperative management of subjects with haemophilia B in the phase 3 B-LONG study

In the phase 3 B‐LONG (Recombinant Factor IX Fc Fusion Protein [rFIXFc] in Subjects With Haemophilia B) study, rFIXFc demonstrated a prolonged half‐life compared with recombinant factor IX (rFIX), and safety and efficacy for prophylaxis and treatment of bleeding in subjects with moderately‐severe to severe haemophilia B. In this B‐LONG sub‐analysis, rFIXFc was evaluated for efficacy in subjects requiring major surgery. Dosing was investigator‐determined. Assessments included dosing, consumption, bleeding, transfusions and haemostatic response. A population pharmacokinetics model of rFIXFc was used to predict FIX activity. Twelve subjects underwent 14 major surgeries (including 11 orthopaedic surgeries); most subjects (11/12) received rFIXFc prophylaxis before surgery (range, ~2 weeks–12 months). Investigators/surgeons rated haemostatic responses as excellent (n = 13) or good (n = 1). In most surgeries (85·7%), haemostasis from the pre‐surgical dose until the end of surgery was maintained with a single rFIXFc infusion. Blood loss was consistent with similar surgeries in subjects without haemophilia. The strong correlation (R2 = 0·9586, P < 0·001) between observed and population pharmacokinetic model‐predicted FIX activity suggests surgery did not impact rFIXFc pharmacokinetics. No unique safety concerns or inhibitors were observed. In conclusion, rFIXFc was safe and efficacious, with prolonged dosing intervals and low consumption, when used perioperatively in haemophilia B. Surgery did not appear to alter rFIXFc pharmacokinetics.

European curriculum for thrombosis and haemostasis

J. ASTERMARK,* C. NEGRIER, C. HERMANS, P. A. HOLME,§ R. KLAMROTH,– P. KOTSI,** P. DE MOERLOOSE, J. PASI, A. ROCINO,§§ M. VON DEPKA,–– J. WINDYGA,*** C. A. LUDLAM and ON BEHALF OF THE INTERDISCIPLINARY WORKING GROUP IDWG *Department for Coagulation Disorders, Malmö University Hospital, Malmö, Sweden; Hôpital Edouard Herriot, CRTH Pavillon E, Lyon, France; Haemostasis Department, Clinique Universitaires St Luc, Bruxelles, Belgium; §Section of Hematology, Medical Department, Rikshospitalet University Hospital, Oslo, Norway; –Vivantes Klinikum im Friedrichhain Klinik fuer Innere Medizin, Haemophiliazentrum, Berlin, Germany; **Haemophilia Center, Laikon General Hospital of Athens, Athens, Greece; Unité d Hémostase, Hôpital Cantonal Départment de Médecine Interne, Genève, Switzerland; Centre for Haematology, Institute of Cell and Molecular Science Barts and the London Queen Mary s School of Medicine and Dentistry, London, UK; §§Ospedale San Giovanni Bosco, Centro Emofilia Divisione di Ematologia, Naples, Italy; ––Werlhof Institute for Haemostasis & Thrombosis, Hannover, Germany; ***Department of Haemostasis and Thrombosis, Institute of Hematology and Blood Transfusion, Warsaw, Poland; and Department of Haemophilia and Thrombosis, Centre Royal Infirmary, Edinburgh, Scotland UK

Kinetics and determinants of the changes of CC16, a lung secretory protein in a rat model of toxic lung injury

Purpose. To investigate the kinetics and the determinants of the changes of the Clara cell 16 kDa protein (CC16) as a marker of lung injury following exposure to different toxicants. Methods. Rats were treated with 4-Ipomeanol (IPO), alpha-naphtylthiourea (ANTU), sodium chromate (Na2CrO4) or paraquat-(PQ). The changes of CC16, total protein, albumin, and cystatin-C were determined on BALF supernatan, and CC16, cystatin-C and creatinine levels were also determined in serum. Results. Bronchiolar insult due to IPO or ANTU resulted in an initial transient increase of serum CC16, parallel to that of albumin in BALF, and a subsequent reduction in both BALF and serum. A slight reduction of CC16 in BALF was already apparent one hour following treatment with PQ. In the serum, CC16 increased to 400 % of basal value. With PQ and Na2CrO4, the elevation of CC16 was mainly determined by the degree of renal impairment.