Quantitative analysis of p16 methylation in Barrett's carcinogenesis.

p16 hypermethylation in Barrett's carcinogenesis has been evaluated in studies which did not take into account sample heterogeneity and yielded qualitative (methylated/unmethylated) instead of accurate quantitative (percentage of CpG methylation) data. We aimed to measure the degree of p16 methylation in pure samples representing all the steps of Barrett's tumorogenesis and to evaluate the influence of sample heterogeneity in methylation analysis. METHODS: 77 paraffin-embedded human esophageal samples were analyzed. Histological grading was established by two pathologists in: negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Areas of interest were selected by laser-capture microdissection. p16 methylation was quantified by pyrosequencing. An adjacent section of the whole sample was also analyzed to compare methylation data. RESULTS: After microdissection, we obtained 15 samples of squamous epithelium, 36 non-dysplastic Barrett's esophagus, 3 indefinite for dysplasia, 24 low-grade dysplasia, 4 high-grade dysplasia and 12 adenocarcinoma. Squamous epithelium showed the lowest methylation rates: 6% (IQR 5-11) vs. 11%(7-39.50) in negative/indefinite for dysplasia, p<0.01; 10.60%(6-24) in low-grade dysplasia, p<0.05; and 44.50%(9-66.75) in high-grade dysplasia/adenocarcinoma, p<0.01. This latter group also exhibited higher methylation rates than Barrett's epithelium with and without low-grade dysplasia (p<0.05). p16 methylation rates of microdissected and non-microdissected samples did not correlate unless the considered histological alteration comprised >71% of the sample. CONCLUSIONS: p16 methylation is an early event in Barrett's carcinogenesis which increases with the severity of histological alteration. p16 methylation rates are profoundly influenced by sample heterogeneity, so selection of samples is crucial in order to detect differences.

Altered gene expression in human placenta after suspected preterm labour.

INTRODUCTION: Suspected preterm labour occurs in around 9% of pregnancies. However, almost two-thirds of women admitted for threatened preterm labour ultimately deliver at term and are considered risk-free for fetal development. METHODS: We examined placental and umbilical cord blood samples from preterm or term deliveries after threatened preterm labour as well as term deliveries without threatened preterm labour. We quantitatively analysed the mRNA expression of inflammatory markers (IL6, IFNγ, and TNFα) and modulators of angiogenesis (FGF2, PGF, VEGFA, VEGFB, and VEGFR1). RESULTS: A total of 132 deliveries were analysed. Preterm delivery and term delivery after suspected preterm labour groups showed similar increases in TNFα expression compared with the term delivery control group in umbilical cord blood samples. Placental samples from preterm and term deliveries after suspected preterm labour exhibited significantly increased expression of TNFα and IL6 and decreased expression of IFNγ. Suspected preterm labour was also associated with altered expression of angiogenic factors, although not all differences reached statistical significance. DISCUSSION: We found gene expression patterns indicative of inflammation in human placentas after suspected preterm labour regardless of whether the deliveries occurred preterm or at term. Similarly, a trend towards altered expression of angiogeneic factors was not limited to preterm birth. These findings suggest that the biological mechanisms underlying threatened preterm labour affect pregnancies independently of gestational age at birth.

Standardization of skin cleansing in vivo: part II. Validation of a newly developed Automated Cleansing Device (ACiD).

BACKGROUND: Skin cleansers for occupational use are manufactured for different types and degrees of soiling without common, legally binding requirements for product testing. This leads to different, manufacturer-specific test methods and a lack of comparable information on skin cleansing products. OBJECTIVES: The aim of this investigation was to validate a newly developed standardized automated cleansing device (ACiD) for in vivo evaluation of industrial skin cleansers. METHODS: Two ACiD were tested regarding the intra- and inter-device specific reproducibility of test results. RESULTS: Skin cleansing process carried out by the three independent washing modules which constitute an ACiD-unit and two separate ACiD-units led to highly comparable results. There was no significant difference between the washing modules or between the two separate ACiD-units detected. Only different parameter settings resulted in significantly different detergency. CONCLUSIONS: Intra- and inter-device specific test results of an in vivo model of skin cleansing using the automated cleansing device (ACiD) were reproducible. The long-term aim is a standardized classification of occupational skin cleansing products comparing their skin cleansing effectiveness in relation to their skin irritancy. This might then provide the basis for a rational specific product selection by consumers and may be used as a tool for future product development by manufacturers.

Standardization of skin cleansing in vivo: part I. Development of an Automated Cleansing Device (ACiD).

BACKGROUND: To date, there are no legally binding requirements concerning product testing in cosmetics. This leads to various manufacturer-specific test methods and absent transparent information on skin cleansing products. A standardized in vivo test procedure for assessment of cleansing efficacy and corresponding barrier impairment by the cleaning process is needed, especially in the occupational context where repeated hand washing procedures may be performed at short intervals. METHODS: For the standardization of the cleansing procedure, an Automated Cleansing Device (ACiD) was designed and evaluated. Different smooth washing surfaces of the equipment for ACiD (incl. goat hair, felt, felt covered with nitrile caps) were evaluated regarding their skin compatibility. RESULTS: ACiD allows an automated, fully standardized skin washing procedure. Felt covered with nitrile as washing surface of the rotating washing units leads to a homogenous cleansing result and does not cause detectable skin irritation, neither clinically nor as assessed by skin bioengineering methods (transepidermal water loss, chromametry). CONCLUSIONS: Automated Cleansing Device may be useful for standardized evaluation of the cleansing effectiveness and parallel assessment of the corresponding irritancy potential of industrial skin cleansers. This will allow objectifying efficacy and safety of industrial skin cleansers, thus enabling market transparency and facilitating rational choice of products.

[Smoking prevention in schools. A model for success]. / Tabakprävention in Schulen. Ein Erfolgsmodell.

Because of compulsory education and, thus, accessibility to all school-age children, schools are especially good places for tobacco prevention measures. For integrated, successful tobacco prevention in schools, the combination of structural and behavioral topics is necessary ("policy mix"). The Federal Center for Health Education has placed emphasis on school-based smoking prevention at an early stage. Within the framework of the smoke-free campaign, the Federal Center for Health Education created manuals and concepts for pupils, teachers, and parents for nationwide use. Starting in 2003, model projects focusing on smoke-free schools were started by the Federal Center for Health Education. Since 2008, all German federal states have signed smoking bans for schools into law.

[Tobacco prevention. The "smoke-free" youth campaign]. / Tabakprävention der Bundeszentrale für gesundheitliche Aufklärung Die "rauchfrei"-Jugendkampagne.

The sharp increase of adolescent tobacco consumption between 1990 and 2001 and the national health target "reducing tobacco consumption" were two main reasons for the increased prevention measures of the Federal Center for Health Education in promoting non-smoking among young people. This article focuses on the offers and measures of the "smoke-free" youth campaign from the Federal Center for Health Education. To promote non-smoking in adolescence, the Federal Center for Health Education started the "smoke-free" youth campaign in 2002 and has continuously expanded it through the present. The campaign is based on a goal-oriented planning process and is predominantly directed towards adolescents younger than 18 years. To achieve national effects in the target group, concerted measures ranging from mass media (television/cinema spots, advertisement), internet, and face-to-face communication--with a focus on school--were implemented. Simultaneous with the start of the "smoke-free" youth campaign in 2001, there is evidence for continuous reduction of the smoking prevalence of adolescents. The rate of smoking adolescents between 12 and 17 years decreased from 28% in 2001 to 15% in 2008, thus, reaching an all-time low.

Prostaglandin EP2 receptor expression is increased in Barrett's oesophagus and oesophageal adenocarcinoma.

BACKGROUND: Accumulating evidence suggests that cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is involved in oesophageal adenocarcinogenesis. PGE2 exerts its biological action by binding to specific receptors (EP1, EP2, EP3 and EP4). AIM: To investigate which PGE2 receptor subtypes regulate PGE2 signals in the oesophageal adenocarcinoma sequence. METHODS: Expression was determined in oesophageal biopsies from 85 patients with oesophagitis, Barrett's metaplasia, intraepithelial neoplasia, oesophageal adenocarcinoma and normal oesophagus. Levels of mRNA and protein expression were determined by quantitative PCR, immunohistochemistry and western-blot. Expression of EP receptors was also determined in response to acid and bile exposure in the Barrett's adenocarcinoma cell line OE33. RESULTS: All four EP receptors subtypes were expressed in human oesophageal tissues. COX-2 and, especially, EP2 were increased in the Barrett's metaplasia-intraepithelial neoplasia-adenocarcinoma sequence. Expression of the EP4 receptor protein was increased in oesophageal adenocarcinoma. In contrast, expression levels of COX-1 and EP3 receptor were decreased along the sequence. No differences in EP1 expression were found. Treatment with the bile acid deoxycholate increased COX-2, EP1, EP2 and EP4 expression in OE33 cells. CONCLUSIONS: Our data suggest that in addition to COX-2, EP2 and EP4 receptors could be a selective target in the prevention and/or treatment of the Barrett's-associated adenocarcinoma.

Investigación translacional en patología digestiva: del laboratorio a la clínica y viceversa beneficiario: el paciente / No disponible

No disponible

Estudio preliminar de mutaciones germinales en el gen MYH en población aragonesa con cáncer de colon / No disponible

No disponible

TGFB1 gene polymorphisms: their relevance in the susceptibility to Helicobacter pylori-related diseases.

Recent studies have revealed elevated expression of transforming growth factor beta1 (TGF-beta1) in gastric mucosa of patients with gastric cancer (GC) and those undergoing ulcer repair. As production of TGF-beta1 is genetically regulated, we aimed to assess whether functional polymorphisms of the TGFB1 gene are involved in susceptibility to and clinical characteristics of Helicobacter pylori-related diseases. DNA from 142 unrelated Spanish patients with GC, 200 with peptic ulcer and 342 healthy controls was typed for the MspA1I T+869C, and the Sau96I G+915C polymorphisms of the TGFB1 gene using polymerase chain reaction and RFLP analysis. H. pylori infection and CagA/VacA antibody status were determined by Western blot in patients and controls. H. pylori infection (odds ratio (OR): 11.44; 95% confidence interval (CI): 4.45-29.42; P<0.001) and non-steroidal anti-inflammatory drugs (OR: 5.07; 95% CI: 2.53-10.16; P<0.001) were identified as independent risks factors for duodenal ulcer (DU), whereas the TGFB1+869(*)C/C genotype was associated with reduced risk of developing the disease (OR: 0.32; 95% CI=0.15-0.68; P=0.003). Our results show that the TGFB1 T+869C gene polymorphism is involved in the susceptibility to DU and provide further evidence that host genetic factors play a key role in the pathogenesis of H. pylori-related diseases.

Venous pressure dynamics of the healthy human leg. Role of muscle activity, joint mobility and anthropometric factors.

The delicate interplay between the muscle-joint unit and intact venous valves is mandatory for normal venous blood return from the human leg. We have investigated the potential role of (1) muscle activity and joint mobility, and (2) anthropometric factors for peripheral venous pressure physiology. 20 healthy young subjects (10 males, 10 females; mean age: 25.6 +/- 3.1 years) underwent direct venous blood pressure measurements during walking on a treadmill in 12 experimental conditions (duration, 1 min each): these resulted from a combination of two different walking velocities, two different degrees of ascent, and three different walking conditions. Anthropometric parameters (e.g., body height, weight and calf circumference) were also measured in all subjects. Outcome measures were the pressures (mm Hg) during quiet standing (resting pressure, P(0)), the maximal activity-induced pressure reduction (Delta P(max)), i.e., the difference between the resting pressure (P(0)) and minimal pressure during exercise (P(min)), the times (s) to minimal pressure during exercise (T(Pmin)) and to half-maximal recovery of P(0) after exercise (T(max1/2)). We found that the maximal venous pressure reduction (Delta P(max)) during walking increased at the higher walking speed and decreased with restricted joint mobility (p = 0.0001). Taller and heavier subjects, or subjects with a greater calf circumference had higher P(0) (p = 0.0001), showed greater Delta P(max) (p < 0.010), and took longer to achieve minimal pressure during exercise (T(Pmin); p < 0.010) than their corresponding counterparts. Females had lower levels of P(0) (p = 0.0001), but not of Delta P(max), and shorter T(Pmin) (p = 0.0076) than males. These gender effects largely result from differences in body height, weight, and calf circumference (p = 0.0001 for the appropriate ANOVA interaction terms). We conclude that during walking, a higher degree of muscle activity and a greater muscle mass enhance venous emptying of the healthy human leg. Conversely, impairments of joint mobility reduce the efficacy of the muscle-vein pump. Moreover, body height and weight significantly influence venous pressure physiology under both resting and activity-related conditions. These anthropometric factors also largely explain sex differences in peripheral venous hemodynamics.

A new BRCA1 mutation in a Filipino woman with a family history of breast and ovarian cancer.

Mutation of the BRCA1 gene in well-defined breast cancer families has been associated with an 87% lifetime risk for breast cancer and a 44% risk for ovarian cancer. Recent data indicate that the risk associated with these mutations is considerably lower, although still far greater than the risk for disease in the rest of the population. Approximately 81% of the mutations that have been identified have been frameshift (71%) or nonsense (10%) mutations, and either may result in a truncated protein. The protein truncation test (PTT) is often used to screen patients at high risk, because sequencing of this large (100 kb) gene with its 22 coding exons is an arduous task. The PTT was used to analyze genomic DNA and RNA from the peripheral blood of a 31-year-old Filipino woman with a poorly differentiated, stage 2A breast carcinoma and a family history of breast-ovarian cancer. PTT identified the wild-type protein fragment and an additional truncated protein fragment in the patient's sample. Subsequent direct sequencing of the appropriate coding region revealed a point mutation in exon 11 at nucleotide 2178, resulting in a C > T transition that caused a termination (stop codon) in amino acid 687. To our knowledge, this is the first report of mutation of the BRCA1 gene in a Filipino family, and this in-frame stop-codon mutation has not been reported previously.

An isotope-dilution gas chromatography-mass spectrometry method for trace analysis of xylene metabolites in tissues.

A gas chromatography-mass spectrometry (GC-MS) method using isotope dilution was developed to measure trace levels of xylene metabolites in brain tissues. The primary metabolites of xylene are dimethylphenol (DMP), methylbenzyl alcohol (MBA), toluic acid (TA), and methylhippuric acid (MHA). The internal standard was a mixture of deuterated DMP-d3, TA-d7, and MHA-d7. DMP-d3 was commercially available and was used as the internal standard for both DMP and MBA. TA-d7 and MHA-d7 were biosynthesized by administering xylene-d10 to rats and collecting their urine. Based on the noise peaks in 10 blank samples, the on-column limits of quantitation (mean +10 SD of noise peaks) were approximately 305, 1220, 545, and 386 pg for DMP, MBA, TA, and MHA, respectively. Analyte detection and recovery tests from brain tissues of control rats were conducted by spiking the tissues with 32 nmol/g of each analyte, together with the deuterated metabolites. The tissues were homogenized, extracted with ethyl acetate, and derivatized by trimethylsilylation. One microliter of the sample was injected into the GC-MS. The recoveries of the analytes were 104 +/- 8%, 80 +/- 9%, 93 +/- 10%, and 92 +/- 11% (mean +/- SD, n = 7) for DMP, MBA, TA, and MHA, respectively. The tissue preparation efficiency, which was indicated by absolute recoveries of internal standards, was approximately 33% for DMP, MBA, and TA and approximately 80% for MHA. No metabolites were detected in untreated control tissues. This simple and sensitive method to simultaneously detect major xylene metabolites in brain tissues could also be used for the analysis of blood and urine samples from workers to monitor p-xylene exposure.

S-phenylcysteine in albumin as a benzene biomarker.

Biological markers of internal dose are useful for improving the extrapolation of health effects from exposures to high levels of toxic air pollutants in animals to low, ambient exposures in humans. Previous results from our laboratory have shown that benzene is metabolized by humans to form the adduct S-phenylcysteine (SPC). Levels of SPC measured in humans occupationally exposed to benzene were increased linearly relative to exposure concentrations ranging from 0 to 23.1 ppm for 8 hr/day, 5 days/week. However, the method of measurement used was laborious, prone to imprecision and interferences, and insufficiently sensitive for the low-dose exposures anticipated in the United States (100 ppb >). An improved chemical method was necessary before SPC adducts in albumin could be used as a benzene biomarker. A simple, sensitive method to measure SPC adducts is being developed and is based on the cleavage of the cysteine sulfhydryl from blood proteins treated with Raney nickel (RN) in deuterium oxide. The product of the reaction with SPC is monodeuterobenzene. SPC treated with RN released monodeuterobenzene in a concentration-dependent fashion. SPC was measured by RN treatment of globin from rats repeatedly exposed by inhalation to 600 ppm benzene. SPC levels measured using the RN approach were 690 +/- 390 pmol SPC/mg Hb (mean +/- % difference, n = 2), as opposed to 290 +/- 45 pmol SPC/mg Hb (mean +/- SEM, n = 3) as measured by our previous method. This method may facilitate the cost-effective, routine analysis of SPC in large populations of people exposed to ambient levels of benzene.

Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic and cytotoxic effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC). Five complementation groups (A-E) have been distinguished on the basis of somatic cell hybridization experiments, with group FA-A accounting for over 65% of the cases analysed. A cDNA for the group C gene (FAC) was reported and localized to chromosome 9q22.3 (ref.8). Genetic map positions were recently reported for two more FA genes, FAA (16q24.3) and FAD (3p22-26). Here we report the isolation of a cDNA representing the FAA gene, following an expression cloning method similar to the one used to clone the FAC gene. The 5.5-kb cDNA has an open reading frame of 4,368 nucleotides. In contrast to the 63-kD cytosolic protein encoded by the FAC gene, the predicted FAA protein (M(r) 162, 752) contains two overlapping bipartite nuclear localization signals and a partial leucine zipper consensus, which are suggestive of a nuclear localization.

Analysis of butadiene, butadiene monoxide, and butadiene dioxide in blood by gas chromatography/gas chromatography/mass spectroscopy.

A new method was developed to quantify the levels of 1,3-butadiene (BD), butadiene monoxide (BDO), and butadiene diepoxide (BDO2) in blood. The method was based on vacuum distillation of tissues followed by analysis of the distillates using multidimensional GC/MS. Metabolites isolated from blood by vacuum distillation were condensed into a cold trap. After warming the traps to room temperature, BD and BDO were sampled from the trap vapor phase. BDO2 was extracted from the codistilled water phase using ethyl acetate. Samples were analyzed using a multidimensional GC system equipped with a custom-built interface. The method was validated by analysis of 0.75-mL aliquots of mouse blood spiked with 5.0, 3.4, and 0.55 nmol of BD, BDO, and BDO2, respectively. The recoveries of analytes were 96 +/- 18%, 125 +/- 15%, and 98 +/- 12%, respectively (mean +/- SD, n = 6). Kinetic studies indicated no loss of BDO and BDO2 in blood held at room temperature in closed containers for up to 1 h. The method was applied to blood samples from B6C3F1 mice and Sprague-Dawley rats exposed by inhalation (nose-only) to 100 ppm BD for 4 h. Blood levels of BD and BDO in exposed rats were 4.1 +/- 1.0 and 0.10 +/- 0.06 microM, respectively (mean +/- SD, n = 6). Levels of BDO2 were below the limits of detection (0.01 nmol/mL). Blood levels of BD, BDO, and BDO2 in mice exposed to 100 ppm BD for 4 h were 2.9 +/- 1.3, 0.38 +/- 0.14, and 0.33 +/- 0.19 microM, respectively (mean +/- SD, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Species differences in urinary butadiene metabolites: comparisons of metabolite ratios between mice, rats, and humans.

We have previously identified two metabolites, 1,2-dihydroxy-4-(N-acetylcysteinyl-S-)-butane (M-I) and 1-hydroxy-2-(N-acetylcysteinyl-S-)-3-butene (M-II) in the urine of mice, rats, hamsters, and monkeys exposed by inhalation to 8000 ppm [14C]butadiene. The sum of these two metabolites constituted between 50 and 90% of the total urinary [14C]butadiene equivalents. When comparing species, the ratios of excreted M-I relative to the total of M-I + M-II were linearly related to hepatic epoxide hydrolase activities, with mice displaying the lowest ratios and monkeys displaying the highest ratios. Because humans are known to have epoxide hydrolase activities more similar to those of monkeys than mice, we postulated that after inhalation of butadiene, humans would excrete predominantly M-I and little M-II. To address this hypothesis, we measured the two metabolites in the urine of workers occupationally exposed to butadiene. We initially developed an assay to measure the two metabolites in urine using techniques not dependent on radiolabeled compounds. The assay is based on isotope-dilution gas chromatography/mass spectroscopy. After addition of deuterated internal standards, the metabolites were isolated from urine samples by solid-phase extraction and selective precipitation. The metabolites were converted to volatile derivatives by trimethylsilylation prior to analysis. The assay is sensitive down to at least 100 ng/ml of both metabolites in urine. The assay was applied to urine samples of humans occupationally exposed to butadiene in a production plant. M-I, but not M-II, could be readily identified and quantitated in the urine samples at levels frequently greater than 1 microgram/ml, thus supporting our hypothesis. Employees who worked in production areas with historical atmospheric concentrations of 3-4 ppm butadiene could be distinguished as a group from those outside controls. Finally, mice and rats were exposed to 11.7 ppm butadiene for 4 hr, and the ratio of the two metabolites was measured. For mice, the ratios of M-I to M-I + M-II were similar to those reported previously following exposure to 8000 ppm. In contrast, for rats, M-I represented a higher proportion of the excreted metabolites at the lower exposure level. These results confirm earlier in vitro studies that suggested the predominant pathway for clearance of BDO in humans is by hydrolysis rather than direct conjugation with glutathione.