An international collaborative study to compare different von Willebrand factor glycoprotein Ib binding activity assays: the COMPASS-VWF study.

Essentials New VWF activity assays are increasingly used but information on their comparability is limited. This is an ISTH SSC-organized study (expert labs, 5 countries) to compare all available assays. VWF activity by six assays correlated well with each other. The new assays show improved characteristics - minor differences are noted. SUMMARY: Background Several new assays have become available to measure von Willebrand factor (VWF) activity. The new assays appear to have improved performance characteristics compared with the old reference standard, ristocetin cofactor activity (VWF:RCo), but information is limited about how they compare with VWF:RCo and each other. Methods The von Willebrand factor Subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) designed a collaborative study involving expert laboratories from several countries to compare available tests with each other and with VWF:RCo. Eight laboratories from five countries were provided with blinded samples from normal healthy individuals and well-characterized clinical cases. Laboratories measured VWF activity using all tests available to them; data from six laboratories, not affected by thawing during transportation, are included in this study. Results All tests correlated well with VWF:RCo activity (r-values ranged from 0.963 to 0.989). Slightly steeper regression lines for VWF:Ab and VWF:GPIbM were clinically insignificant. The new assays showed improved performance characteristics. Of the commercially available assays, the VWF:GPIbR using the AcuStar system was the most sensitive and could reliably detect VWF activity below 1 IU dL-1 . The lower limit of the measuring interval for the VWF:GPIbM and the VWF:GPIbR assays was in the 3-4 and 3-6 IU dL-1 range, respectively. Inter-laboratory variation was also improved for most new assays. Conclusion All VWF activity assays correlated well with each other and the VWF:RCo assay. The slight differences in characteristics found in the COMPASS-VWF study will assist the VWF community in interpreting and comparing activity results.

Early age at smoking initiation and tobacco carcinogen DNA damage in the lung.

BACKGROUND: DNA adducts formed as a consequence of exposure to tobacco smoke may be involved in carcinogenesis, and their presence may indicate a high risk of lung cancer. To determine whether DNA adducts can be used as a "dosimeter" for cancer risk, we measured the adduct levels in nontumorous lung tissue and blood mononuclear cells from patients with lung cancer, and we collected data from the patients on their history of smoking. METHODS: We used the 32P-postlabeling assay to measure aromatic hydrophobic DNA adducts in nontumorous lung tissue from 143 patients and in blood mononuclear cells from 54 of these patients. From the smoking histories, we identified exposure variables associated with increased DNA adduct levels by use of multivariate analyses with negative binomial regression models. RESULTS/ CONCLUSIONS: We found statistically significant interactions for variables of current and former smoking and for other smoking variables (e.g., pack-years [number of packs smoked per day x years of smoking] or years smoked), indicating that the impact of smoking variables on DNA adduct levels may be different in current and former smokers. Consequently, our analyses indicate that models for current and former smokers should be considered separately. In current smokers, recent smoking intensity (cigarettes smoked per day) was the most important variable. In former smokers, age at smoking initiation was inversely associated with DNA adduct levels. A highly statistically significant correlation (r=.77 [Spearman's correlation]; two sided P<.001) was observed between DNA adduct levels in blood mononuclear cells and lung tissue. IMPLICATIONS: Our results in former smokers suggest that smoking during adolescence may produce physiologic changes that lead to increased DNA adduct persistence or that young smokers may be markedly susceptible to DNA adduct formation and have higher adduct burdens after they quit smoking than those who started smoking later in life.

Correlation of DNA adducts in blood mononuclear cells with tobacco carcinogen-induced damage in human lung.

The formation of carcinogen-DNA adducts within the respiratory epithelium is thought to be a critical factor in the induction of lung cancer from tobacco smoke. A reliable surrogate measure of carcinogen damage to the lung would be of great value in molecular epidemiological studies of cancer risk. The validity of measurements of DNA adducts formed from hydrophobic aromatic hydrocarbons in peripheral blood mononuclear cells (MNCs) was investigated by comparing the levels of aromatic DNA adducts detected in lung tissue from 31 lung cancer patients with those detected in MNCs from the same individuals using the 32P-postlabeling assay. The associations of smoking history and intake of dietary antioxidants with adduct levels also were assessed. Tissue-specific, as well as common DNA adducts were detected in lung and blood; total MNC adduct levels were highly correlated with total lung adducts. After smoking cessation, adduct levels appeared to decay in both tissues at similar rates. Multivariate analyses (Poisson regression modeling) indicated that dietary antioxidant intake (carotenoids, vitamin A, and retinol) modified the levels of aromatic DNA adducts in both the lungs and blood. Of all models tested, the optimal one for predicting lung adduct levels included the measure of blood MNC adduct levels only. Therefore, blood MNCs are a valid surrogate tissue for estimating the burden of DNA adducts in respiratory tissue in molecular epidemiological studies.

Study of arsenic mutagenesis using the plasmid shuttle vector pZ189 propagated in DNA repair proficient human cells.

Arsenic is considered a human carcinogen and although it is non-mutagenic in bacterial or human cells, arsenic interacts synergistically with genotoxic agents in the production of mutations. To gain insight into the possible mechanisms of action of arsenic in mutagenesis we studied the effects of sodium arsenite exposure on UV mutagenesis using the pZ189 shuttle vector system in DNA repair proficient GM 637 human fibroblasts. The purpose of the study was to determine whether arsenic alone induces mutations in the supF gene and whether the combination of arsenic and UV irradiation leads to a yield of mutants greater than the sum of the arsenic or UV treatments alone. Treatment of fibroblasts for 72 h with 5.0 microM of sodium arsenite alone produced significant increases in the pZ189 mutant frequency; 1 and 2.5 microM arsenite were not mutagenic. UV irradiation (320 J/m2) increased the yield of mutants 3.5-fold above the background rate. When UV-irradiated plasmid was allowed to replicate in fibroblasts treated with 1, 2.5, or 5.0 microM arsenite, the yields of mutations were significantly greater (p < 0.01) than the yield expected if the effects of each treatment were simply additive. The greatest potentiation of UV-induced mutations (4.9-fold) was observed at 1 microM arsenite, a concentration that was neither mutagenic itself nor cytotoxic. Restriction digest and DNA sequencing analyses indicated that arsenite alone produces both large-scale rearrangements, frameshifts and base substitutions. Hotspots for deletions were observed to be associated with a previously reported deletion hotspot involving 5'-CpC and runs of cytosines. Base substitutions observed involved A:T-->T:A transversions. The results indicate that arsenite alone is mutagenic in human cells using the supF reporter gene. The pZ189 shuttle vector may provide a model to study the molecular nature of co-mutagenesis of arsenic and other environmental agents. Further characterization of arsenic's effects on DNA repair and mutational spectra may be useful in the development of molecular markers in studies of arsenic carcinogenesis in human populations.

Coeliac disease: always something to discover.

The authors present more than 20 years' experience with coeliac disease, with a summary of their published studies. Hair shaft characteristics were determined by scanning electron microscopy. Hair diameter was significantly lower and cuticular erosion scores higher in those who were not on gluten-free diets as compared to controls, showing a tendency towards normal values following start of gluten-free diets. Proton-induced X-ray emission showed significantly lower zinc content of the hair shaft in the group with acute coeliac disease and after a short-term diet, which approached the normal range only after a year-long diet. The serum prolactin levels in healthy controls and in coeliac patients on the diet were within normal limits, whereas in children with coeliac disease taking gluten in their meals, a significant hyperprolactinaemia was found. The erythrocyte glutathione content of coeliac children was elevated, and the glutathione disulfide level was significantly decreased, as compared to values in normal controls. The erythrocyte glutathione disulfide level and glutathione disulfide/erythrocyte glutathione ratio in coeliac children also differed from those in children with iron deficiency. With genotyping, the DQB1*0201/2 (p < 0.00001) and DR3 (p < 0.00001), DR7 (p < 0.01) alleles showed significant positive association with the disease.

[Simultaneous occurrence of selective ACTH deficiency , achalasia, alacrimia and hyperlipoproteinemia]. / Szelektív ACTH érzéketlenség, achalasia és alacrimia együttes elöfordulása hyperlipoproteinaemiával.

An extremely rare clinical syndrome on a 7-year-old girl is presented. Besides isolated glucocorticoid insufficiency, achalasia and alacrimia disturbance of the lipid metabolism was also detected--this is a special feature of this case. The details of the endocrine workup is discussed, providing clues for the possible pathomechanism. The correct diagnosis and specific therapy is of utmost importance in the everyday life of the patient.

[Demonstration of antibodies against gliadin using an immunofluorescence method in childhood celiac disease]. / Gliadin ellenes antitest kimutatás immunofluoreszcens módszerrel gyermekkori coeliákiában.

IgA and IgG type antibodies against gliadin were demonstrated in 396 serum samples of 350 patients with immunofluorescence method. The procedure was used in cases of clinically suspected celiac disease and for the control of the diet of patients with diagnosed celiac disease. The sensitivity of the patient material was 83% in the case of antibodies against IgA and 85% with antibodies against IgG. The specificity value with IgA antibodies was 96% and 91% with IgG antibodies. The authors consider the immunofluorescence technique suitable for the demonstration of immune complexes developed against gliadin and circulating in blood. On this basis the method is recommended both for the screening test of patients with suspected celiac disease and for the control of patients on gluten-free diet.

[Specific enzyme diagnosis in mitochondrial myopathies and encephalomyopathies]. / Mitochondriális myopathiák/encephalomyopathiák specifikus enzymdiagnosztikája.

Mitochondrial enzyme activities (cytochrome c-oxidase = COX, carnitine acyl-transferase = CAT, citrate synthase = CS, lipoamide dehydrogenase = lipDH from the pyruvate-dehydrogenase complex, lactate dehydrogenase = LDH, and malate-dehydrogenase = MDH) were measured from progressive myopathy/encephalomyopathy. Cytochrome oxidase (COX) deficiency was detected from muscle or liver tissues, adult type of COX defectus had been diagnosed in 1 case and infantile type in further 6 cases. The 3 familial atactic children showed decreased activity of carnitine acetyl-transferase, too.

Common large partial VWF gene deletion does not cause alloantibody formation in the Hungarian type 3 von Willebrand disease population.

BACKGROUND: Type 3 von Willebrand disease (VWD) is an autosomal recessive bleeding disorder, characterized by virtually undetectable plasma von Willebrand factor (VWF) and consequently reduced plasma factor VIII levels. Genetic mutations responsible for type 3 VWD are very heterogeneous, scattered throughout the VWF gene and show high variability among different populations. METHODS: Twenty-five severe VWD patients were studied by direct sequencing of the 51 coding exons of the VWF gene. The total number of VWD type 3 families in Hungary is 24, of which 23 were investigated. RESULTS: Fifteen novel mutations were identified in 31 alleles, five being nonsense mutations (p.Q1238X, p.Q1898X, p.Q1931X, p.S2505X and p.S2568X), four small deletions and insertions resulting in frame shifts (c.1992insC, c.3622delT, c.5315insGA and c.7333delG), one a large partial deletion (delExon1-3) of the 5'-region, four candidate missense mutations (p.C35R, p.R81G, p.C295S, p.C623T) and one a candidate splice site mutation (c.1730-10C>A). Six previously described mutations were detected in 17 alleles, including the repeatedly found c.2435delC, p.R1659X and p.R1853X. Only one patient developed alloantibodies to VWF, carrying a homozygous c.3622delT. CONCLUSION: We report the genetic background of the entire Hungarian type 3 VWD population. A large novel deletion, most probably due to a founder effect, seems to be unique to Hungarian type 3 VWD patients with high allele frequency. In contrast to previous reports, none of the five patients homozygous for the large partial deletion developed inhibitors to VWF. This discrepancy raises the possibility of selection bias in some of the reports.