comprises newly diagnosed cases of pulmonary sarcoidosis (n = 22, average age 44·7 years, 12 females) recruited at the Clinic of Respiratory Diseases and Allergy at the University Medical Centre, Ljubljana, Slovenia and diagnosed using the European Respiratory Society/American Thoracic Society (ERS/ATS) criteria . Stage II was present in 15 and stage III in seven of the subjects. The average duration of symptoms until final diagnosis and treatment was 5·5 months [median 4·5, standard deviation (s.d.) 3–6]. Extra-pulmonary manifestations were present in seven patients. BAL index mean check details was 7·5 (s.d. 3·0), spirometry vital capacity (VC) 93·8 (s.d. 11) and carbon monoxide diffusing capacity (DLCO) 87% (s.d. 12). There were no differences in immunoglobulin (Ig0A, IgM and IgG antibodies against Aspergillus fumigatus and Candida albicans between controls and sarcoidosis patients. Subjects without pulmonary disease or any respiratory symptoms (n = 20, age 39·9, ±1·8, 13 females) served as controls. All subjects were non-smokers. The study was approved by the Governmental Medical Ethics Committee, Ljubljana (198/05/04) and written, informed consent was obtained. Fluorouracil solubility dmso The clinical stage of the disease was determined using chest X-rays of the lung of subjects with sarcoidosis.
A grading scheme for the
presence of granulomas was used as described previously [11,12,19]. The X-rays were read by two experienced radiologists, unaware of the status of the patient, grading granulomas according to a numerical score (0–4), judging size and extension of the infiltrates (0 = normal, ALOX15 1 = c. 25% of the lung field involved, 2 = up to 50%, 3 = up to 75% and 4 = virtually the whole lung field involved). Repeat evaluations on two successive occasions showed only minor deviations in the classification. Among the subjects with sarcoidosis there were five with X-ray score 1, 13 with score 2 and four with score 3. For ethical reasons, chest X-rays were not performed on controls but were given the value 0. Serum samples were taken and the amounts of TNF-α, IL-2R, IL-6, IL-10 and IL-12 were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Milenyi Biotec, Heidelberg, Germany and Thermo Scientific, San Jose, CA, USA). For the in vitro assay PBMC were incubated with different FCWA or lipopolysaccharide (LPS), as reported previously . Briefly, PBMC were isolated from venous blood samples by density gradient centrifugation and incubated in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mm l-glutamine and 10% heat-inactivated human serum.