CADASIL brain vessels show a HTRA1 loss-of-function profile.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and a phenotypically similar recessive condition (CARASIL) have emerged as important genetic model diseases for studying the molecular pathomechanisms of cerebral small vessel disease (SVD). CADASIL, the most frequent and intensely explored monogenic SVD, is characterized by a severe pathology in the cerebral vasculature including the mutation-induced aggregation of the Notch3 extracellular domain (Notch3ECD) and the formation of protein deposits of insufficiently determined composition in vessel walls. To identify key molecules and pathways involved in this process, we quantitatively determined the brain vessel proteome from CADASIL patient and control autopsy samples (n = 6 for each group), obtaining 95 proteins with significantly increased abundance. Intriguingly, high-temperature requirement protein A1 (HTRA1), the extracellular protease mutated in CARASIL, was found to be strongly enriched (4.9-fold, p = 1.6 × 10-3) and to colocalize with Notch3ECD deposits in patient vessels suggesting a sequestration process. Furthermore, the presence of increased levels of several HTRA1 substrates in the CADASIL proteome was compatible with their reduced degradation as consequence of a loss of HTRA1 activity. Indeed, a comparison with the brain vessel proteome of HTRA1 knockout mice (n = 5) revealed a highly significant overlap of 18 enriched proteins (p = 2.2 × 10-16), primarily representing secreted and extracellular matrix factors. Several of them were shown to be processed by HTRA1 in an in vitro proteolysis assay identifying them as novel substrates. Our study provides evidence for a loss of HTRA1 function as a critical step in the development of CADASIL pathology linking the molecular mechanisms of two distinct SVD forms.

Heterozygous HTRA1 mutations are associated with autosomal dominant cerebral small vessel disease.

Cerebral small vessel disease represents a heterogeneous group of disorders leading to stroke and cognitive impairment. While most small vessel diseases appear sporadic and related to age and hypertension, several early-onset monogenic forms have also been reported. However, only a minority of patients with familial small vessel disease carry mutations in one of known small vessel disease genes. We used whole exome sequencing to identify candidate genes in an autosomal dominant small vessel disease family in which known small vessel disease genes had been excluded, and subsequently screened all candidate genes in 201 unrelated probands with a familial small vessel disease of unknown aetiology, using high throughput multiplex polymerase chain reaction and next generation sequencing. A heterozygous HTRA1 variant (R166L), absent from 1000 Genomes and Exome Variant Server databases and predicted to be deleterious by in silico tools, was identified in all affected members of the index family. Ten probands of 201 additional unrelated and affected probands (4.97%) harboured a heterozygous HTRA1 mutation predicted to be damaging. There was a highly significant difference in the number of likely deleterious variants in cases compared to controls (P = 4.2 × 10(-6); odds ratio = 15.4; 95% confidence interval = 4.9-45.5), strongly suggesting causality. Seven of these variants were located within or close to the HTRA1 protease domain, three were in the N-terminal domain of unknown function and one in the C-terminal PDZ domain. In vitro activity analysis of HTRA1 mutants demonstrated a loss of function effect. Clinical features of this autosomal dominant small vessel disease differ from those of CARASIL and CADASIL by a later age of onset and the absence of the typical extraneurological features of CARASIL. They are similar to those of sporadic small vessel disease, except for their familial nature. Our data demonstrate that heterozygous HTRA1 mutations are an important cause of familial small vessel disease, and that screening of HTRA1 should be considered in all patients with a hereditary small vessel disease of unknown aetiology.

Cerebral small vessel disease-related protease HtrA1 processes latent TGF-ß binding protein 1 and facilitates TGF-ß signaling.

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor ß (TGF-ß) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-ß signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-ß pathway activation. We identify latent TGF-ß binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-ß bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-ß signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis.

Adolescents' jobs and the course of dermatitis symptoms throughout puberty.

OBJECTIVES: This study evaluated the course of dermatitis symptoms throughout puberty taking into account occupational exposures in a population-based study. METHODS: Participants enrolled in the ISAAC-II (International Study of Asthma and Allergies in Childhood) study in Munich and Dresden in 1995 and 1996 were sent a postal questionnaire in 2002 (age at follow-up 16 to 18 years). The questionnaire included items on atopic diseases, jobs, including holiday jobs and vocational training, and potential confounders. The most recent of the adolescents' jobs held for at least 8 hours a week, and for at least 1 month, were coded according to the ISCO-88 system. RESULTS: Overall, data of 3785 adolescents were included in the analyses. The incidence of dermatitis symptoms during puberty among those without such symptoms at baseline was 7%. Altogether 31% of the participants reported an employment history. Those already employed were more likely to report a new onset of dermatitis symptoms. Jobs associated with a new onset of symptoms were work in the health care sector, vocational training in bakeries, and cleaning. The first 9 months of exposure were particularly relevant for new cases of dermatitis symptoms (odds ratio 3.7, 95% confidence interval 1.5-9.6). CONCLUSIONS: Early occupational exposure is associated with the development of symptoms of dermatitis. The types of skin alterations need to be assessed in the next stage of the study.

Acute blood neutrophilia induced by short-term compost dust exposure in previously unexposed healthy individuals.

OBJECTIVES: Systemic effects of organic dust inhalation have been described in farming environments. The purpose of this study was to examine whether a single exposure at a biowaste composting facility could also exert systemic effects in healthy volunteers not previously exposed to organic dust from such facilities. METHODS: Seventeen subjects (age 20-35 years) were exposed to organic dust for 2 h (exposure day) during moderate exercise; 12 of these subjects also took part in a control experiment (control day). Spirometry was performed before and immediately after the exposure. White blood cell counts and levels of tumor-necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in peripheral blood were determined before and 3 h after exposure. RESULTS: Exposures did not result in significant changes in lung function or blood cytokine levels. In contrast, the number and percentage of neutrophils increased during the exposure day [median (range) percent change of percentages 14 (-2; 67) %; P=0.002], but not during the control day [5 (-22; 35) %; P=0.66). Furthermore, there was a decrease in the number and percentage of eosinophils during the exposure day [-47 (-57; 0.0) %; P=0.002], whereas the change during the control day was smaller [-8 (-56; 71) %; P=0.68]. CONCLUSION: Short-term exposure of healthy, young subjects to organic dust from composting facilities had opposite effects on the numbers of blood neutrophils and blood eosinophils. These effects, though mild, suggest that even during a limited period of moderate work a sufficient amount of bioactive material can be deposited in the lung to elicit acute systemic alterations.

The physical and biological doses of methacholine are different for Mefar MB3 and Jaeger APS sidestream nebulizers.

BACKGROUND: Within a study on respiratory symptoms in rural areas, we used the European Community Respiratory Health Survey methacholine challenge protocol. For quicker and more reliable handling, we had to change the nebulizer in the bronchial challenge system from Mefar model MB3 (Bovezzo, Italy) to Jaeger APS Sidestream (similar to Mefar; Würzburg, Germany). Therefore, we compared the physical properties of the two systems, adapted the challenge protocol, and compared the results of both systems in subjects with and without airway hyperresponsiveness to methacholine. METHOD: The physical properties of both systems were characterized by the residual method indicating a similar particle size distribution and an average output of 6 muL/s for Mefar MB3 and 1.25 muL/s for APS Sidestream. In the comparison study, 34 subjects were included. Airway responsiveness was quantified by provocative dose of methacholine causing a 20% fall in FEV(1). RESULTS: A significant difference was found between the two challenge systems (p =0.004, McNemar test). Nine subjects reached a 20% drop in FEV(1) with the APS Sidestream only. The FEV(1) dropped by > 20% using either system in eight subjects. In 17 subjects, none of the two systems caused a 20% decrease in FEV(1). CONCLUSION: Even if the physical dose is determined with elaborate methods, the biological dose may vary between two nebulizer systems, causing incomparable outcomes for subjects tested with different systems.

Contact urticaria to giraffe hair.

BACKGROUND: Immediate-type hypersensitivity to animal proteins is a common problem in people occupationally exposed to animals. METHODS: A 19-year-old female working as a voluntary zookeeper in her off-time suffered from hives on her forearms following contact to the fur of a giraffe. For diagnostic evaluation, skin prick tests, assessment of specific serum IgE antibodies, and basophil activation tests were performed. RESULTS: Skin prick tests with a standard series of common aeroallergens were positive for various pollens. Prick testing with native materials was positive for extracts of hair from two different giraffe subspecies in the patient, but not in control subjects. By CAP-FEIA, no specific serum IgE antibodies to dander of a large variety of animals were found in the patient. In the basophil activation test, expression of the activation marker CD63 was induced by extract of giraffe hair on the cells from the patient, but not on those from unaffected controls. CONCLUSIONS: This patient suffers from an 'exotic' immediate-type contact allergy to giraffe hair.

Concentrations of dust, allergens and endotoxin in stables, living rooms and mattresses from cattle farmers in southern Bavaria.

Agricultural work is considered to be a major risk factor for occupational diseases. In particular, allergic reactions to cow dander cause numerous cases of airway disorders. We measured the concentration of allergens (e.g. Bos d2, Der p1) and endotoxin in the stables, living-rooms and mattresses of 46 farmers with a diagnosis of occupational asthma or allergic rhinitis caused by cow dander allergen. The concentration of cow dander allergen was highest in stables (median 20,400 microg/g) but also noticeable in dust samples from living-rooms (median 155 microg/g) and mattresses (median 195 microg/g). The sensitization threshold (20-50 microg/g) was exceeded in most cases. Thus, allergen transport from the stables to bed must be prevented by optimizing the hygiene of farmers and family members.

Web-based training in occupational medicine.

OBJECTIVES: The aim of our project was to develop and evaluate an interactive computer-based approach to teach medical students in occupational medicine. To enhance interest in occupational medicine the major focus was on clinical and practical aspects of occupational medicine. METHODS: The computer program was designed in HTML and JavaScript. It presents a guided tour of the patient's case history followed by information on practice and theory of occupational medicine. The program was integrated into the curriculum during the summer term of 1999 and the following winter term. To assess the effectiveness and acceptability of the program we asked students to rate the program on an 18-item questionnaire. RESULTS: Overall, 287 students participated in the evaluation of the program. The participants highly recommended the program structure and had no difficulties in handling the program. This was independent of the computer experience of the students. The evaluation showed that it was possible to enhance interest in occupational medicine by using "virtual patients". CONCLUSION: The program represents a student-orientated learning tool that points out clinical and practical aspects of occupational medicine. The availability via the Internet allows the application as a self-learning tool as well as a teaching tool for the medical curriculum.