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.

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.

Prevalence of adult-type hypolactasia as diagnosed with genetic and lactose hydrogen breath tests in Hungarians.

The prevalence of adult-type hypolactasia varies ethnically and geographically among populations. A C/T(-13910) single nucleotide polymorphism (SNP), upstream of the lactase gene, is known to be associated with lactase non-persistence. The aim of this study was to determine the prevalence of lactase-persistent and non-persistent genotypes in the Hungarian population, the age at onset and the applicability of the lactose H2 breath test in comparison with genetic screening. The prevalence of the C/C(-13910) genotype among adults was 37%. Hypolactasia starts to appear at around 5 years of age. Over the age of 12 years, almost all of those with a C/C(-13910) genotype have lactase non-persistence. The C/C(-13910) genotype was closely associated with a positive lactose H2 breath test in symptomatic children, whereas the lactase-persistent genotypes correlated better with a negative H2 test in a control group. In conclusion, supplementary non-invasive breath and genotyping tests furnish a perfect clinical diagnosis.

DNA adducts among asphalt paving workers.

OBJECTIVE: Asphalt is used extensively in the highway construction industry and contains a complex mixture of polycyclic aromatic hydrocarbons, some of which are known or suspected to be human carcinogens. Though numerous epidemiologic studies have described an excess cancer risk among asphalt workers, a causal relationship has not been established. Accordingly, the primary objective of this study was to use DNA adducts as a biomarker of biologically effective dose and determine whether DNA damage resulted from occupational exposure to asphalt among paving workers. METHODS: Over a 12 month period, four peripheral blood samples (spring, summer, fall and winter) were obtained from 49 asphalt paving workers (169 samples) and 36 non-paving construction workers (103 samples). The spring, summer and fall samples were collected during the work-season, whereas the winter samples were collected during the off-season (due to the seasonality of paving work). Mononuclear white blood cells were isolated and analyzed for DNA adducts via the (32)P-postlabeling assay and generalized linear models were used to evaluate the DNA adduct data. RESULTS: Among paving workers during the work-season, DNA adducts increased during each day of the workweek such that mean adduct levels were lowest on Mondays (3 adducts per 10(10) nucleotides) and highest on Fridays (46 adducts per 10(10) nucleotides). Additionally, a 3-fold difference in adduct burden was observed by paving task such that mean adduct levels were lowest among roller operators (7 adducts per 10(10) nucleotides) and highest among screedmen (23 adducts per 10(10) nucleotides). Using adducts as a measure of biologically effective dose, these findings (weekday trend and task-based differences) were consistent with a previous evaluation of absorbed dose in the same population. Adduct levels were not, however, higher among paving workers than among non-pavers. Adducts were also highest during the winter months, suggestive of a seasonal effect that has been observed in previous studies. CONCLUSION: These findings indicate that adduct burden increased throughout the workweek among paving workers, suggesting that DNA damage may be associated with occupational exposure to hot-mix asphalt. However, the lack of contrast with non-paving workers, as well as the seasonal variation warrants additional investigation.

Neonatal screening for biotinidase deficiency in Hungary: clinical, biochemical and molecular studies.

From 1989 to 2001, 1,336,145 newborns were screened for biotinidase deficiency in Hungary. Fifty-eight children with the disorder were identified as enzyme-deficient. We have characterized the clinical and biochemical features and mutations of 20 of these children. Eleven children had profound biotinidase deficiency, 7 had partial biotinidase deficiency, and 2 were found to be heterozygous for profound deficiency by mutation analysis. Seventeen different mutations were identified in this population including seven novel mutations. Six of these new mutations are missense, 245C>A, 334G>A, 652G>C, 832C>G, 1253G>C, 1511T>A, and one is a unique allelic double mutation [212T>C;236G>A]. Of five Romanian Gypsies, four were homozygous for the 1595C>T mutation and one was heterozygous for this mutation. Most of the children with profound deficiency have been asymptomatic on therapy; however, four exhibited minimal brain abnormalities, motor delay and abnormal blood chemistries. Compliance with therapy must be questioned in these cases. Of clinical importance, all of the children with partial deficiency exhibited mild symptoms at the time of diagnosis, at several weeks to months of age. These symptoms resolved following biotin therapy. This is in contrast to the experience in the United States, where the children with partial deficiency have been asymptomatic at the time of diagnosis. This finding further indicates that children with partial deficiency should be treated. The incidence of biotinidase deficiency in Hungary is more than twice that observed in a worldwide survey. These results indicate that newborn screening in Hungary is effective and warranted.

Primary lymphoedema associated with xanthomatosis, vaginal lymphorrhoea and intestinal lymphangiectasia.

Primary lymphoedema associated with chylous reflux is a very rare clinical entity. We report a 3-year-old girl with unilateral lymphoedema, xanthomatosis and vaginal lymphorrhoea. Biopsy also revealed intestinal lymphangiectasia. This paper also presents a brief review of the literature and draws attention to the significance of the xanthomatous eruption in the diagnosis of a chylous reflux.

Adenocarcinoma of the colon developing on the basis of Crohn's disease in childhood.

Colorectal carcinoma rarely affects children and has a dismal prognosis with 5-year survival rates as low as 2.5%-7% despite apparently radical surgery. Here we report the case of an adenocarcinoma of the sigmoid colon in a 15-year-old girl preceded by uncertain abdominal complaints of 5 years' duration. Pathological work-up revealed a tumour with lymph node metastases (pT3NI). Immunohistochemical evidence of p53 overexpression by the tumour cells raised the suspicion of an underlying Li-Fraumeni syndrome. In addition, there were aphthoid ulceration, fissuration of the non-tumorous mucosa, along with a mixed transmural infiltrate composed of macrophages, eosinophils, and non-typical giant cells, which were compatible with simultaneous Crohn's disease. Anamnestic data concerning the occurrence of idiopathic inflammatory bowel disease or colorectal carcinoma in the patient's relatives were non-contributory. The present results suggest a possible relationship between Crohn's disease and colon cancer due to the defective p53 gene product.

Effect of simulated microgravity on human lymphocytes.

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).

Polymorphisms in the DNA repair genes XRCC1 and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells.

Polymorphisms in several DNA repair genes have recently been identified, but little is known about their phenotypic significance. To determine whether variation in DNA repair genes is related to host DNA damage, we studied the association between polymorphisms in XRCC1 (codon 399) and ERCC2 (codon 751) and two markers of DNA damage, sister chromatid exchange (SCE) frequencies (n = 76) and polyphenol DNA adducts (n = 61). SCE frequencies were determined using a modified fluorescence-Giemsa method and polyphenol DNA adducts were determined using a P1-enhanced (32)P-post-labeling procedure. XRCC1 and ERCC2 genotypes were identified using PCR-RFLP. Mean SCE frequencies among current smokers who were homozygous carriers of the 399Gln allele in XRCC1 were greater than those in 399Arg/Arg current smokers. We also observed a possible gene-dosage effect for XRCC1 399Gln and detectable DNA adducts, and significantly more adducts among older subjects who were carriers of the 399Gln allele than in younger subjects with the 399Arg/Arg genotype. The polymorphism in ERCC2 was unrelated to SCE frequency or DNA adduct level. Our results suggest that carriers of the polymorphic XRCC1 399Gln allele may be at greater risk for tobacco- and age-related DNA damage.

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.

The effect of simulated microgravity conditions on the TNF-alpha production by human PBMCS.

Our earlier space experiments demonstrated that the interferon production of human lymphocytes in microgravity is 4-8 times higher than those of the synchronous ground controls in vitro (Talas et al. 1983). These data suggested that the microgravity has a significant effect on cells. Since the possibilities to perform space-experiments are very limited and our study raised many interesting questions, we wished to simulate microgravity conditions in our laboratory. For this reason we purchased a Rotary Cell Culture System (RCCS) equipment to study different cell lines and human peripheral blood mononuclear cells (PBMCs) in experimental microgravity conditions. RCCS is a horizontally rotated bubble free culture vessel with membrane diffusion gas exchange. We report here an analysis of TNF-alpha (tumor necrosis factor-alpha) production by human PBMCs (control cultures exposed to simulated microgravity in RCCS). The cells were incubated in the presence or absence of either NDV (Newcastle Disease Virus) or one of the different forms (PHA-M or -P) of Phytohaemagglutinin.

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.

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.

HM-PAO-labeled leukocyte scintigraphy in pediatric patients with inflammatory bowel disease.

Leukocyte scintigraphy (LS) was performed in 20 pediatric patients with inflammatory bowel disease (IBD: 10 with ulcerative colitis, 2 with indeterminate colitis, and 8 with Crohn disease) in different stages of clinical activity. Leukocytes were separated from 15 to 60 ml venous blood and were labeled in vitro with [99mTc]HM-PAO. The segmental extent (small intestine; ascending, transverse, and descending colon; and recto-sigmoideum) of the process was determined by LS. The uptake of each bowel segment was scored in relation to the bone marrow uptake. The scintigraphic activity, calculated by summing the segment scores, was compared with laboratory parameters. The mean labeling efficacy was 76% (60-86%). The segmental extent of the process determined by LS was compared with the results of barium enema or colonoscopy with regard to 32 bowel segments. The sensitivity, specificity, and accuracy of LS were 93, 88, and 91%, respectively. Two extraintestinal manifestations (abdominal abscess and joint involvement) were also detected by LS. These lesions were verified by computed tomography (CT) (abscess) and on the basis of the clinical outcome (arthritis). The scintigraphic activity correlated with the C-reactive protein (CRP) level (r = 0.82, p < 0.001), the alpha 2-globulin level (r = 0.63, p < 0.02), the sedimentation rate (r = 0.51, p < 0.05), and the fS iron level (r = -0.66, p < 0.005). LS is applicable in pediatric patients. The method is an excellent technique for assessment of the extent of IBD in children. Extraintestinal manifestations of IBD can also be investigated by LS. The scintigraphic activity is a useful parameter for determination of the activity of IBD in children.

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.

[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.

"Paralogs", sorbent families for protein separations.

Novel peptide-based (paralog) sorbents are evaluated with respect to performance, reproducibility and reusability in a 96-well test plate screening format, and to utility in protein separations. The results demonstrate that this approach to constructing sorbents provides a new and generally applicable set of tools for separating proteins.

Determination of hair trace elements in childhood celiac disease and in cystic fibrosis.

Applying proton-induced X-ray emission authors investigated the hair trace element contents in 10 children with acute celiac disease after 3 to 6 and 12 months long gluten-free diet; in 9 children with cystic fibrosis and in a control group (6 children) of the same age. There was no difference in Cu, Fe, Ca, Cl values between the examined groups. The Zn contents of the hair are significantly low in the group with acute celiac disease after a short-term diet and also in the group with cystic fibrosis, the data approach the normal range only after a year's diet. The significant rise of hair potassium contents is well indicated in patients with acute celiac disease and this rise may be due to the destruction of cell cuticles. In case of cystic fibrosis there is no significant rise of hair potassium value.

Simultaneous occurrence of selective ACTH insensitivity, achalasia and alacrimia accompanied by hyperlipoproteinaemia.

An extremely rare clinical syndrome on a 7-year-old-girl is presented. Besides isolated glucocorticoid insufficiency, achalasia and alacrima disturbance of the lipid metabolism was also detected--being 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.